Multivalent CD301 fusion protein based on optimized peptide linker, functionalized magnetic bead and application of multivalent CD301 fusion protein and functionalized magnetic bead 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 of the functionalized magnetic bead in CTC capture, the protein is of a single polypeptide chain structure and comprises a plurality of CD301 carbohydrate recognition domains which are connected in series through the peptide linker, and preferably, the protein is in a tetramer form. According to the invention, peptide linkers connecting each recognition domain are specially designed, including optimized flexible linkers or rigid linkers rich in O-glycosylation sites (such as hCG [beta] CTP), so as to regulate the spatial orientation of the domains. By immobilizing the directionally arranged multivalent protein on the surface of the magnetic particle, the binding affinity with a Tn antigen on the surface of a tumor cell is remarkably enhanced by utilizing a multivalent synergistic effect. The method provided by the invention can effectively capture the low EpCAM expression CTC with epithelial-mesenchymal transition (EMT), and has extremely high binding stability under the shear force of blood fluid.

Description

Technical Field

[0001] This invention relates to the field of biomedical detection technology, specifically to a multivalent CD301 fusion protein based on optimized peptide linkers, functionalized magnetic beads, and their application in CTC capture. Background Technology

[0002] Cancer is one of the leading causes of death worldwide, and tumor metastasis is a core factor contributing to cancer patient mortality. Circulating tumor cells (CTCs) are tumor cells that detach from primary or metastatic tumor lesions and enter the peripheral blood circulation; they are considered key biomarkers for tumor "liquid biopsies." The capture, counting, and molecular subtyping of CTCs have irreplaceable clinical value for early cancer screening, real-time monitoring of treatment efficacy, and prognostic assessment.

[0003] Currently, mainstream CTC enrichment technologies, such as the FDA-approved CellSearch® system, primarily rely on magnetic beads coated with anti-epithelial cell adhesion molecule (EpCAM) antibodies for positive enrichment. However, this epithelial marker-based capture strategy has a significant inherent flaw: tumor cells often undergo epithelial-mesenchymal transition (EMT) during invasion and metastasis. During EMT, EpCAM expression on the surface of CTCs is significantly downregulated or even completely absent, while simultaneously gaining stronger migration and anti-apoptotic abilities. This makes existing EpCAM-dependent technologies highly susceptible to "missing" this most invasive and metastatic mesenchymal CTC segment, resulting in false negative results and misleading clinical judgment.

[0004] To overcome these limitations, researchers began searching for broad-spectrum tumor markers independent of EpCAM. Abnormal changes in cell surface glycosylation are a common feature of tumorigenesis. Among them, Tn antigen (GalNAcα1-Ser / Thr) is a truncated O-glycan that is almost not expressed in normal tissues but is highly specifically expressed in various malignant tumors such as breast cancer, colorectal cancer, and gastric cancer, making it an ideal target for CTC capture. CD301 (also known as macrophage mannose lectin, MGL / CLEC10A) is a type C lectin receptor in the human body that specifically recognizes Tn antigen. Theoretically, CD301 could be used to achieve broad-spectrum capture of CTCs with different phenotypes.

[0005] Despite the recognition specificity of CD301, developing it into a highly efficient CTC capture agent faces significant biophysical challenges, primarily in the following two aspects: 1. Weak monomer affinity: Unlike the nanomolar (nM) level binding affinity between antibodies and antigens, the interaction between natural lectin monomers (CRD domains) and glycoantigens is typically weak, with dissociation constants (Kd) mostly in the micromolar (μM) range. Under the shear forces of blood flow, CD301-modified magnetic beads struggle to firmly grasp rare CTCs and are prone to detachment. 2. Complexity of polymerization and linker design: To improve binding affinity, multivalent effects must be utilized, i.e., multiple CRDs must simultaneously bind to glycoclusters on the cell surface. However, simply tandemly connecting multiple CRDs through genetic engineering does not necessarily produce the desired effect. If the peptide linkers connecting the various domains are too short, adjacent CRDs will be unable to freely adjust their angles due to space congestion, resulting in the inability to simultaneously bind to antigen sites on the cell surface. If excessively long conventional flexible linkers are used, disordered entanglement can easily occur within the protein molecule, obscuring the active site. In addition, excessively long unstructured sequences are easily degraded by proteases in plasma, leading to reagent inactivation.

[0006] Currently, there is a lack of a precisely engineered multivalent CD301 trapping molecule with optimal spatial orientation and stability. In particular, there is a lack of research and products that optimize the flexibility and rigidity of the linker to balance spatial degrees of freedom and structural stability.

[0007] Therefore, developing a multimeric CD301 capture system that utilizes optimized linkers to generate a strong multivalent chelation effect and is stable in the blood environment is of great significance for overcoming the current technical bottlenecks in CTC detection. Summary of the Invention

[0008] The present invention aims to provide a multivalent CD301 fusion protein based on optimized peptide linkers, functionalized magnetic beads, and their application in CTC capture, with the goal of providing a multimeric CD301 capture system that generates a strong multivalent chelation effect and is stable in the blood environment.

[0009] To achieve the above objectives, the present invention adopts the following technical solution: a multivalent CD301 fusion protein based on an optimized peptide linker, wherein the protein is a single polypeptide chain structure and the polypeptide chain contains at least two human CD301 protein carbohydrate recognition domains connected in series through peptide linkers.

[0010] Preferably, as an improvement, the peptide linker is a flexible linker or a rigid linker.

[0011] Preferably, as an improvement, the peptide linker is a flexible linker, the amino acid sequence of which contains a repeating glycine-serine motif, with the general formula (GGGGS)n, where n is an integer from 2 to 4.

[0012] Preferably, as an improvement, the flexible joint is (GGGGS)3.

[0013] Preferably, as an improvement, the peptide linker is a rigid linker, and the amino acid sequence of the rigid linker is derived from the C-terminal peptide hCGβ CTP of the human chorionic gonadotropin β subunit.

[0014] Preferably, as an improvement, the amino acid sequence of the rigid linker is SSSKAPPPSLPSPSRLPGPSDTPILPQ, or a sequence that has at least 90% homology with it and retains the O-glycosylation site.

[0015] Preferably, as an improvement, the N-terminus or C-terminus of the polypeptide chain is also attached with an affinity tag for site-specific modification, which is a biotin receptor peptide Avi-tag, a histidine tag His-tag, or an Fc fragment.

[0016] Preferably, as an improvement, a functionalized magnetic bead has the aforementioned recombinant fusion protein immobilized on its surface via covalent bonds or affinity interactions.

[0017] Preferably, as an improvement, the application of functionalized magnetic beads in the preparation of tumor liquid biopsy kits.

[0018] Preferably, as an improvement, the application of functionalized magnetic beads in the preparation of a system for enriching and separating circulating tumor cells involves mixing the biological fluid sample to be tested with functionalized magnetic beads, incubating it to allow the multivalent recombinant fusion protein on the surface of the magnetic beads to specifically bind to the Tn antigen on the surface of the circulating tumor cells; using a magnetic field to separate and collect the magnetic bead-cell complex, and washing to remove background cells that have not specifically bound.

[0019] The principle and advantages of this solution are as follows: In practical applications, this technical solution addresses the problems of existing technologies by using genetic engineering to tandem multiple CD301 CRD domains onto a single polypeptide chain. The peptide linkers connecting these domains are meticulously engineered to achieve optimal spatial conformation matching. During the technology development phase, the peptide linkers connecting adjacent CRD domains play a crucial regulatory role, and the type and length of the peptide linkers have a significant impact on the performance of the recombinant fusion protein, making them one of the key aspects of this technical solution's development. If the peptide linker is too short, it will lead to steric hindrance and failure of the multivalent effect. In the early stages of development, the linker length used was insufficient (e.g., less than 10 amino acids), causing adjacent CRD domains to adhere tightly together, resulting in significant steric hindrance. This tight structure means that when one CRD binds to an antigen, the other CRD cannot bind to a neighboring antigen due to angular limitations, preventing the designed "multivalent effect" from being achieved and thus affecting the binding efficiency of the fusion protein. Conversely, if the peptide linker is too long, it will cause disordered entanglement within the molecule and masking of the active site. Simply lengthening the flexible linker (e.g., by more than 25 amino acids) to increase flexibility can lead to excessive flexibility and disordered entanglement of the polypeptide chain within the molecule. This entanglement not only obscures the protein's active sites, rendering it unable to function, but also makes the loose structure more easily recognized and cleaved by proteases in plasma, resulting in inactivation. Therefore, finding an "optimal distance" that ensures flexibility while avoiding entanglement is crucial. Based on this, our research and screening revealed that using three repeating GGGGS units (i.e., (GGGGS)3, 15 amino acids) is the preferred approach. This length provides a theoretical extension distance of approximately 50-60 angstroms, which precisely matches the common spacing of glycoantigen clusters on mucin on the surface of tumor cells, ensuring both flexibility and avoiding entanglement.

[0020] Furthermore, during the research and development process, the stability of the fusion protein, characterized by easy degradation and short half-life in complex whole blood environments, was addressed. To prevent protein degradation, this approach introduces a rigid linker (hCGβ CTP, sequence: SSSKAPPPSLPSPSRLPGPSDTPILPQ) as a linker. The CTP sequence is rich in proline (Pro), and in eukaryotic expression systems, multiple serine (Ser) sites on it undergo high-level O-glycosylation modification. These modified glycans increase the rigidity and hydrodynamic radius of the linker, effectively physically separating adjacent CRD domains like a "scaffold." The introduction of CTP significantly prolongs the half-life of the fusion protein and prevents protein degradation, which has unexpected technical benefits for maintaining the stability of the capture reagent in complex whole blood environments. This design overcomes the problem of insufficient hydrodynamic radius, significantly prolongs the half-life of the fusion protein, and solves the challenge of maintaining the stability of the capture reagent in complex whole blood environments.

[0021] In summary, the beneficial effects of this technical solution are as follows: 1. This technical solution achieves ultra-high capture efficiency through multivalent effect and optimized connector design: By precisely designing the connector length and type, it avoids the spatial steric hindrance of short connectors and solves the entanglement problem of long ordinary connectors, allowing multiple CD301CRD domains to efficiently bind to Tn antigen clusters on the surface of tumor cells, activating a strong multivalent chelation effect. Experimental data in Example 1 show that the capture efficiency of experimental group A, using the optimized flexible connector ((GGGGS)3), reached 88.2%±2.5%, while that of experimental group B, using the rigid connector (CTP), reached 92.1%±1.8%, and that of the short connector control group C was only 42.5%±3.8%.

[0022] 2. The rigid linker glycosylation modification in this technical solution significantly improves stability in the blood environment: By introducing the hCGβCTP rigid linker, its O-glycosylation modification acts like a "scaffold + shield," maintaining the protein's extended conformation and resisting degradation by plasma proteases, thus solving the problem of easy inactivation of existing reagents in complex blood environments. In the stability test of Experiment Example 1, after incubation in human plasma at 37°C for 4 hours, the residual activity rate of experimental group B (CTP rigid linker) still reached 85.3%±2.4%, while that of experimental group A (flexible linker) was only 60.5%±2.1%.

[0023] 3. This technical solution abandons EpCAM dependence and targets a broad spectrum of tumor-specific Tn antigens, successfully solving the problem of missed detection of EMT-type CTCs and achieving full phenotypic coverage of CTCs (including EMT mesenchymal type). Experimental Example 2 results show that the traditional EpCAM magnetic beads had a capture rate of only 16.5%±3.2% for mesenchymal MDA-MB-231 cells (low EpCAM, high Tn antigen), while the magnetic beads of this solution achieved a capture rate of 92.8%±1.5%, and also maintained a high capture rate of 91.5%±1.8% for epithelial MCF-7 cells. Attached Figure Description

[0024] Figure 1 This is a comparison chart of the capture performance and stability of CD301 functionalized magnetic beads with different connector designs in embodiments of the present invention.

[0025] Figure 2 This is a comparison diagram of the capture performance of multivalent CD301 magnetic beads and traditional EpCAM magnetic beads for tumor cells of different phenotypes in embodiments of the present invention.

[0026] Figure 3 This is a statistical chart showing the CTC capture performance of Tetra-CD301-CTP magnetic beads in clinical blood samples in an embodiment of the present invention. Detailed Implementation

[0027] The following detailed description provides further details on specific embodiments, but the embodiments of the present invention are not limited thereto. Unless otherwise specified, the technical means used in the following embodiments are conventional means well known to those skilled in the art; the experimental methods used are all conventional methods; and the materials, reagents, etc. used, unless otherwise specified, can be obtained commercially.

[0028] Overview of the plan: A multivalent CD301 fusion protein based on an optimized peptide linker is disclosed. This protein is a single polypeptide chain containing multiple CD301 carbohydrate recognition domains tandemly linked by a linker, preferably in a tetrameric form. The linker is selected from flexible or rigid linkers. An affinity tag for site-directed modification is also attached to the N-terminus or C-terminus of the polypeptide chain; the affinity tag is selected from biotin receptor peptides (Avi-tag), histidine tags (His-tag), or Fc fragments.

[0029] The flexible linker amino acid sequence contains a repeating glycine-serine motif, with the general formula (GGGGS)n, where n is an integer from 2 to 4; preferably (GGGGS)3 (i.e., 15 amino acid lengths) to achieve a balance between avoiding steric hindrance and preventing intramolecular entanglement.

[0030] The rigid linker amino acid sequence is derived from the C-terminal peptide (hCGβ CTP) of the β subunit of human chorionic gonadotropin; preferably (SSSKAPPPSLPSPSRLPGPSDTPILPQ), or a sequence that has at least 90% homology with it and retains the O-glycosylation site.

[0031] A functionalized magnetic bead, wherein the recombinant fusion protein described above is immobilized on the surface of the magnetic bead through covalent bonds or affinity interactions.

[0032] SEQ ID NO: 1 Flexible Linker: GGGGSGGGSGGGGS; SEQ ID NO: 2 Rigid Linker - hCGβ CTP: SSSKAPPPSLPSPSRLPGPSDTPILPQ; Example of CD301 CRD structure field: SEQ ID NO: 3 CRD sequence of CD301: (UniProt No: Q8IUN9, aa163-316) LTCQVATLNNNASTEGTCCPVNWVEHQDSCYWFSHSGMSWAEAEKYCQLKNAHLVVINSREEQNFVQKYLGSAYTWMGLSDPEGAWKWVDGTDYATGFQNWKPGQPDDWQGHGLGGGEDCAHFHPDGRWNDDVCQRPYHWVCEAGLGQTSQESH Core protein protective sequence aa188-306: SEQ ID NO: 4 HQDSCYWFSHSGMSWAEAEKYCQLKNAHLVVINSREEQNFVQKYLGSAYTWMGLSDPEGAWKWVDGTDYATGFQNWKPGQPDDWQGHGLGGGEDCAHFHPDGRWNDDVCQRPYHWVCE SEQ ID NO: 5 Biotinylated receptor peptide Avi: GLNDIFEAQKIEWHE Example 1 Preparation and modification of an engineered single-chain tetramer CD301 protein (Tetra-CD301): In order to obtain a recombinant protein with high purity and directional binding function, this embodiment uses genetic engineering technology to express the fusion protein in mammalian cells and achieves biotinylation of Avi-tag through two optional routes (in vitro enzymatic modification or in vivo co-expression modification).

[0033] S1. Gene Construction and Vector Design: A fusion gene sequence was designed based on the extracellular CRD region of the human CD301 gene (GenBank Accession No. NM_006344). It contains the following elements sequentially from the N-terminus to the C-terminus: (a) Signal peptide: The mouse Igκ chain guide sequence (Amino Acid Sequence: METDTLLLWVLLLWVPGSTG) is used to guide the efficient secretion of the fusion protein into the cell culture supernatant. This signal peptide will be cleaved by intracellular signal peptidase during protein secretion. (b) Purification tag: 8×His tag (HHHHHHHHH), used for nickel column affinity purification; (c) Flexible connector: (GGGGS)2, used to connect the label to the functional area; (d) Targeted modification tag: Avi-tag (sequence: GLNDIFEAQKIEWHE), as a substrate for biotin ligase BirA; (e) Core functional area: single-chain tetramer CD301, containing 4 CRD structural domains in series, with adjacent structural domains connected by optimized joints (such as flexible joints or rigid joints).

[0034] Component A (Optimized Flexible): Tetra-CD301-Flex General structural formula: N-terminus-[signal peptide]-[His]-[Avi]-[CRD]-(G4S)3-[CRD]-(G4S)3-[CRD]-(G4S)3-[CRD]-C-terminus Component B (Rigid Extension): Tetra-CD301-Rigid General structural formula: N-terminus-[signal peptide]-[His]-[Avi]-[CRD]-(CTP)-[CRD]-(CTP)-[CRD]-(CTP)-[CRD]-C-terminus The above-mentioned fusion gene was fully synthesized and cloned into the mammalian expression vector pcDNA3.1 to construct the recombinant plasmid pVector-TetraCD301.

[0035] S2, Protein Expression and Biotinylation Modification This invention provides two feasible modification schemes, both of which can be implemented according to production needs: Cell line: Expi293F™ Cells (Thermo Fisher) Culture medium: Expi293™ Expression Medium (serum-free) Plasmids: pcDNA3.1-His-Avi-TetraCD301, pcDNA3.1-BirA-ER Transfection reagents: ExpiFectamine™ 293 or PEI MAX (MW 40,000) Purification packing material: Ni-NTA Agarose In vitro modification reagents: BirA enzyme, ATP, D-Biotin (10mM Stock in DMSO) Option A: In vitro enzymatic modification method (preferred option) pVector-TetraCD301 plasmid was transfected into Expi293F cells and cultured in suspension for 5-7 days. The cell culture supernatant was collected by centrifugation and the target protein was captured using an N-terminal His tag via Ni-NTA affinity chromatography, followed by elution with imidazole-containing buffer. The purified protein was then transferred to reaction buffer, and recombinant BirA enzyme, ATP, Mg2+, and D-biotin were added. The mixture was incubated at 30°C for 1 hour to overnight to complete site-directed biotinylation of the Avi-tag. Free biotin, ATP, and BirA enzyme were removed by Superdex 200 molecular sieve chromatography (SEC), and the target monomer peak was collected.

[0036] Option B: In vivo co-expression modification method The pVector-TetraCD301 plasmid and the codon-optimized BirA enzyme expression plasmid (pVector-BirA) were co-transfected into Expi293F cells at a specific ratio (e.g., 4:1 to 9:1). Simultaneously with or 24 hours after transfection, D-biotin was added to the culture medium to a final concentration of 50-100 μM. Cells expressed the BirA enzyme concurrently with the target protein, utilizing intracellular ATP and supplemented biotin to complete Avi-tag modification before protein secretion. The supernatant was collected and purified directly using Ni-NTA affinity chromatography and molecular sieve chromatography to obtain the biotinylated recombinant protein.

[0037] Experiment Example 1: Performance Comparison of Different Linker Constructs To verify the impact of linker design (short chain vs. flexible long chain vs. rigid long chain) on the capture efficiency of Tn antigen-positive tumor cells in multivalent tandem CD301 proteins. To evaluate the functional stability of different constructs in a simulated human blood environment (37°C plasma).

[0038] 1. Experimental Groups: Experimental Group A (Flex-Long): Tetra-CD301-(G4S)3 (Flexible Optimization Group) Experimental group B (Rigid-Long): Tetra-CD301-CTP (rigid / glycosylated group) Control group C (Short): Tetra-CD301-(G4S)1 (short chain control group) Negative control group D (Beads-Only): Streptavidin magnetic beads that are blocked but not coupled with the protein. 2. Reagents: Magnetic beads: Dynabeads™ MyOne™ Streptavidin T1.

[0039] Cell line: MDA-MB-231-GFP (breast cancer cells, known to express high levels of Tn antigen, low levels of EpCAM, and GFP).

[0040] Blood sample: Fresh peripheral blood from healthy volunteers.

[0041] Buffer solution: Coupling buffer: PBS (pH 7.4) + 0.1% BSA.

[0042] Capture / wash buffer: PBS (pH 7.4) + 1% BSA + 2 mM EDTA (to prevent blood clotting) + 0.05% Tween-20.

[0043] 3. Experimental methods, specifically including the following steps: Step 1: Functional Coupling of Magnetic Beads (1) Take 4 portions of magnetic beads, each 1 mg. Separate them using a magnetic rack, remove the preservation solution, and wash 3 times with 500 μL of coupling buffer.

[0044] (2) Add 200 pmol of recombinant protein to every 1 mg of magnetic beads, and add coupling buffer to 200 μL.

[0045] (3) Incubate at room temperature (25℃) using a vertical rotary mixer (15 rpm) for 45 minutes. Remove the supernatant magnetically. Add PBS containing 10 μg / mL free biotin and incubate for 10 minutes (to block unbound avidin sites). Wash four times with capture buffer and resuspend in 100 μL capture buffer (final concentration 10 mg / mL). Store at 4℃.

[0046] Step 2: Simulated Sample Preparation (1) Take fresh anticoagulated peripheral blood and mix it with an equal volume of PBS. Carefully add the mixture to the surface of the lymphocyte separation medium (Ficoll) and centrifuge (400g, 20-30 minutes, acceleration / deceleration set to 0). Aspirate the intermediate white membrane layer (i.e., MNC / PBMC) and wash twice with PBS. Count the cells and adjust the concentration to approximately 1×10⁻⁶. 6 per mL.

[0047] (2) Collect MDA-MB-231-GFP cells in the logarithmic growth phase and dilute the cells to a working solution of 5,000 cells / mL.

[0048] (3) Construct a simulated sample, taking 1×10 6 One MNC, each containing 100 μL of cell working solution MDA-MB-231-GFP.

[0049] Step 3: Capture Performance Test Add 20 μL (0.2 mg) of the prepared magnetic beads to each of the above test tubes containing 1 mL of simulated blood sample. Incubate at room temperature (25°C) with vertical rotation (10 rpm) for 30 minutes. Place on a magnetic rack and let stand for 2 minutes, then aspirate the supernatant. Add 1 mL of washing buffer and gently invert 5 times to mix. Aspirate magnetically and discard the supernatant. Repeat the washing process 3 times.

[0050] The magnetic beads were resuspended in 100 μL of PBS and transferred to a 96-well plate. The entire well was scanned under a fluorescence microscope (FITC channel) to count the number of green fluorescent cells encapsulated by the magnetic beads.

[0051] Data calculation: Capture efficiency (%) = (Number of fluorescent cells captured by magnetic beads / Average number of cells added) × 100% Step 4: Stability Testing Prepared Group A (Flex) and Group B (Rigid) magnetic beads. Take 0.2 mg of each magnetic bead and soak it in 100% fresh human plasma. Incubate at 37°C for 4 hours. After 4 hours, remove the plasma by magnetic adsorption and wash once with buffer. Using the pressure-tested magnetic beads, capture freshly prepared simulated blood samples (containing 500 cells) according to the method in "Step Three".

[0052] Calculation of active residue rate: Residual activity rate (%) = (Capture efficiency of magnetic beads after pressure test / Initial capture efficiency of untreated magnetic beads) × 100% Test results are as follows Figure 1As shown: (A) Initial capture efficiency of magnetic beads in different experimental groups for Tn antigen-positive tumor cells (MDA-MB-231-GFP). Data are expressed as mean ± standard deviation (n=5). (B) Residual activity of magnetic beads in different experimental groups after incubation in human plasma at 37°C for 4 hours.

[0053] like Figure 1 As shown in Figure A, the capture efficiency of control group C (short chain group) was significantly low, at only 42.5% ± 3.8%. This confirms that when the linker is too short (only 5 amino acids), there is severe steric hindrance between adjacent CRD domains, preventing the tetramer from simultaneously binding to multiple antigen sites on the cell surface, and thus the multivalent chelation effect is not effectively activated. In contrast, experimental groups A (flexible long chain) and B (rigid long chain) both exhibited excellent capture performance, with capture efficiencies of 88.2% ± 2.5% and 92.1% ± 1.8%, respectively. This indicates that increasing the length of the linker (whether flexible or rigid) can provide sufficient spatial freedom to the CRD domain, thereby adapting to the spacing of Tn antigen clusters on the tumor cell surface and achieving efficient capture. The capture rate of the negative control group D was close to zero, ruling out the influence of non-specific adhesion of magnetic beads.

[0054] like Figure 1 As shown in Figure B: After a stress test involving incubation in human plasma at 37°C for 4 hours, the two groups of long-chain constructs exhibited significant differences in stability. The residual activity of experimental group A (flexible group) decreased to 60.5% ± 2.1%. This may be because the (G4S)3 flexible sequence is prone to disordered entanglement in the complex plasma environment, obscuring some active sites, and the unstructured flexible peptide chain is more easily exposed to the attack of plasma proteases and undergoes degradation.

[0055] Experimental group B (rigid / CTP group) maintained an activity residue rate as high as 85.3% ± 2.4%. This unexpectedly excellent performance is mainly attributed to the O-glycosylation modification on the CTP sequence. These glycans not only maintain the rigid extended structure of the protein like a scaffold (preventing entanglement), but also act as a shield to prevent proteases from cleaving the peptide backbone, thereby significantly improving the tolerance and half-life of the capture reagent in in vitro diagnostic environments.

[0056] Although flexible long linkers (experimental group A) can solve the capture efficiency problem, the introduction of rigid glycosylated linkers (experimental group B, i.e. the preferred solution of this invention) can simultaneously achieve high capture efficiency and high stability, and is the best design for clinical complex blood sample testing.

[0057] Experiment Example 2: Broad-spectrum capture ability and specificity test of tumor cells with different phenotypes To verify whether the Tetra-CD301-CTP rigid tetramer magnetic beads (experimental group) constructed in this invention can solve the problem of missed detection by existing Anti-EpCAM magnetic beads (control group) when facing tumor cells undergoing epithelial-mesenchymal transition (EMT), we conducted a comparative capture experiment: 1. Cell model: MCF-7-GFP: A human breast cancer cell line stably transfected with green fluorescent protein. Its molecular characteristics include high EpCAM expression (++++) and Tn antigen expression (+). This cell line represents conventional epithelial tumor cells that are easily captured by existing technologies.

[0058] MDA-MB-231-GFP: A stable human triple-negative breast cancer cell line that transfects green fluorescent protein. Its molecular characteristics include low or negative EpCAM expression (+ / -) and high Tn antigen expression (+++). This cell line represents stromal tumor cells that undergo EMT, are highly invasive, and are often missed by current techniques.

[0059] 2. Capture reagent: Experimental group: Tetra-CD301-CTP rigid connector functionalized magnetic beads prepared in this invention.

[0060] Control group: Commercially available mainstream anti-EpCAM immunomagnetic beads.

[0061] 3. Experimental Methods (1) Take fresh anticoagulated peripheral blood and mix it with an equal volume of PBS. Carefully add the mixture to the surface of the lymphocyte separation medium (Ficoll) and centrifuge (400g, 20-30 minutes, acceleration / deceleration set to 0). Aspirate the intermediate white membrane layer (i.e., MNC / PBMC) and wash it twice with PBS. Count the cells and adjust the concentration to approximately 1×10⁻⁶. 4 per mL.

[0062] (2) Collect MCF-7-GFP cells and MDA-MB-231-GFP cells in the logarithmic growth phase and dilute the cells to a working solution of 1×104 cells / mL.

[0063] (3) Precisely incorporate approximately 1000 MCF-7-GFP cells (simulating epithelial CTCs) or 1000 MDA-MB-231-GFP cells (simulating mesenchymal CTCs) into each 1 mL of whole blood.

[0064] (4) Add the experimental group magnetic beads (0.2 mg) and the control group magnetic beads (0.2 mg) respectively, and incubate vertically at room temperature for 30 minutes.

[0065] (5) Place the sample in a magnetic rack for separation, remove the supernatant, and wash three times with PBS buffer to remove non-specifically adhered blood cells (white blood cells).

[0066] The magnetic bead resuspension was transferred to a 96-well plate, and the number of tumor cells effectively captured was counted under a fluorescence microscope based on green fluorescence.

[0067] Test results are as follows Figure 2 As shown: (A) is a capture microscopic image of MCF-7-GFP cells (epithelial type) with high EpCAM expression and positive Tn antigen. The left side is the bright field, and the right side is the green fluorescent field. The experimental group used the Tetra-CD301-CTP magnetic beads of this invention, and the control group used commercially available Anti-EpCAM magnetic beads. It can be seen that both groups can enrich a large number of green fluorescent cells. Scale bar = 100 μm. (B) is a capture microscopic image of MDA-MB-231-GFP cells (mesenchymal type) with low EpCAM expression and positive Tn antigen. It can be seen that the field of view of the experimental group is full of captured cells, while the field of view of the control group has very few cells. (C) is a quantitative statistical graph of the capture efficiency of the two magnetic beads for different cell lines (n=5). Data are expressed as Mean±SD. Circular scatter points represent data of the experimental group, and triangular scatter points represent data of the control group. The results show that the magnetic beads of this invention maintain a high capture rate of >90% for both cell lines, while the capture rate of MDA-MB-231 in the control group is significantly reduced.

[0068] Capture performance against epithelial tumor cells (MCF-7) Figure 2 A and Figure 2 (C, left side): MCF-7 cells represent traditional, non-metastatic tumor cells that simultaneously highly express EpCAM and Tn antigens. From Figure 2 As can be seen from Figure A, dense green fluorescent cells were bound to the surface of the magnetic beads in both the experimental and control groups. Quantitative statistics ( Figure 2 C) shows that the capture efficiency of the experimental group was 91.5%±1.8%, while that of the control group was 90.2%±2.1%. For conventional epithelial CTCs, the capture performance of this invention is comparable to the existing clinical gold standard technology, demonstrating that this invention has excellent basic capture capabilities.

[0069] Capture performance against mesenchymal tumor cells (MDA-MB-231) Figure 2 B and Figure 2 (C right side): MDA-MB-231 cells represent highly invasive tumor cells undergoing EMT, characterized by significantly downregulated or absent EpCAM expression, but with maintained high Tn antigen expression. From Figure 2 B revealed highly different results. In the experimental group, a large number of captured cells were still visible in the field of view, with a density similar to the MCF-7 group; while in the control group, only a few scattered cells were visible, with most of the field of view being blank. Quantitative statistics ( Figure 2C) This difference was further confirmed. The capture efficiency of MDA-MB-231 in the experimental group remained at a high level of 92.8%±1.5%, while the capture efficiency of the control group dropped sharply to 16.5%±3.2%.

[0070] In summary, this invention utilizes the more stable broad-spectrum target Tn antigen, combined with the strong grasping effect of rigid tetramers, to successfully overcome the problem of biomarker loss caused by EMT, and achieves full coverage capture of CTCs of different molecular subtypes.

[0071] Experiment 3: Enrichment of CTCs from Patient Blood To verify the effectiveness of the Tetra-CD301-CTP rigid tetrameric magnetic beads constructed in this invention in a real clinical setting, we conducted a double-blind test on 15 clinical samples (10 cancer patients + 5 healthy patients): 1. Subject Recruitment and Sample Collection Experimental group: 10 patients with stage III or IV colorectal cancer (CRC) were recruited.

[0072] Control group: Five healthy volunteers (HC) were recruited to evaluate the specificity of the method.

[0073] Peripheral venous blood was collected from each subject using vacuum blood collection tubes containing EDTA anticoagulant. Samples were processed within 4 hours of collection.

[0074] 2. Sample preprocessing Transfer 7.5 mL of whole blood to a 50 mL centrifuge tube, add 4 volumes of erythrocyte lysis buffer (ACK Lysis Buffer), and incubate at room temperature for 5–8 minutes until the erythrocytes are completely lysed. Centrifuge at 300 × g for 5 minutes and discard the red supernatant. Resuspend the white blood cell / tumor cell pellet at the bottom with PBS buffer, and centrifuge and wash again. Resuspend the cell pellet in 1 mL of capture buffer (PBS + 1% BSA) to prepare the cell suspension for testing.

[0075] 3. Magnetic bead enrichment Add 20 μL (approximately 0.2 mg) of Tetra-CD301-CTP functionalized magnetic beads to 1 mL of the cell suspension. Place the tube on a vertical rotary mixer and gently incubate at 10 rpm for 45 minutes at room temperature to allow the magnetic beads to fully bind to the Tn antigen on the surface of CTCs. Place the tube on a magnetic rack and let it stand for 2 minutes until the magnetic beads are completely adsorbed onto the tube wall. Slowly aspirate the supernatant (to remove non-specific cells). Add 1 mL of washing buffer, gently invert to mix, and perform magnetic separation. Repeat the washing process three times to remove background leukocytes as much as possible.

[0076] 4. Immunofluorescence staining and identification To distinguish between captured tumor cells and residual leukocytes, the classic “CK+ / CD45- / DAPI+” triple labeling method was used for in situ identification: the magnetic bead-cell complex was fixed with 4% paraformaldehyde (PFA) for 10 minutes, followed by permeabilization with 0.1% Triton X-100 for 5 minutes.

[0077] Add the staining mixture containing the following reagents and incubate in the dark for 1 hour: Pan-CK Antibody - FITC marker (green): Specifically recognizes epithelial-derived tumor cells. Anti-CD45 Antibody - PE marker (red / orange): Specifically recognizes leukocytes. DAPI (blue): Stains cell nuclei, indicating nucleated cells.

[0078] After washing, the sample was spread onto a glass slide and scanned and counted using a fluorescence microscope.

[0079] 5. Interpretation criteria CTC (circulating tumor cells): cells that meet the criteria of being CK positive (+), DAPI positive (+), CD45 negative (-), and have intact cell morphology and abnormal nucleocytoplasmic ratio.

[0080] WBC (white blood cells): cells that meet the criteria of being CD45 positive (+), DAPI positive (+), and CK negative (-).

[0081] Test results are as follows Figure 3 The bar chart shows the number of circulating tumor cells (CTCs) detected in 7.5 mL peripheral blood samples from 5 healthy volunteers (HC01-HC05) and 10 colorectal cancer patients (CRC-01 to CRC-10). The horizontal axis (X-axis) represents the subject number. HC represents the healthy control group, and CRC represents the colorectal cancer group. The vertical axis (Y-axis) represents the absolute count of CTCs detected per 7.5 mL of blood. The number of CTCs detected in the healthy control group was extremely low (close to 0), while a significant number of CTCs were detected in all cancer patient samples, with individual differences among patients.

[0082] Data from the healthy control group (HC01-HC05) on the left side of the figure shows that the vast majority of samples (4 / 5) had a detection result of 0, with only one sample detecting one incidental cell. This indicates that the magnetic beads of the present invention have extremely low non-specific adsorption for non-tumor components such as leukocytes and erythrocytes in a normal blood background. This "clean" background is crucial for clinical diagnosis and can effectively avoid false positive diagnoses. Data from the cancer patient group (CRC-01 to CRC-10) on the right side of the figure shows that the method of the present invention successfully captured CTCs in the blood of all 10 confirmed patients. The detection range is quite wide, from a minimum of 3 (CRC-04, CRC-09) to a maximum of 48 (CRC-03). The CRC-03 sample showed an extremely high CTC count (48 / 7.5mL), and this patient was clinically diagnosed as a stage IV patient with liver metastasis, suggesting a positive correlation between CTC count and tumor burden and metastatic status.

[0083] The fluctuations in the bar chart objectively reflect the differences in CTC load among different patients. The high sensitivity of the method of this invention ensures that even patients with low load (such as CRC-04) can be accurately detected without any missed detections, further confirming that the Tetra-CD301-CTP magnetic beads maintain excellent capture performance in complex whole blood environments.

[0084] The above descriptions are merely embodiments of the present invention, and common knowledge such as specific technical solutions and / or characteristics are not described in detail here. It should be noted that those skilled in the art can make various modifications and improvements without departing from the technical solutions of the present invention, and these should also be considered within the scope of protection of the present invention. These modifications and improvements will not affect the effectiveness of the implementation of the present invention or the practicality of the patent. The scope of protection claimed in this application should be determined by the content of its claims, and the specific embodiments described in the specification can be used to interpret the content of the claims.

Claims

1. A multivalent CD301 fusion protein based on an optimized peptide linker, characterized in that: The protein is a single polypeptide chain structure containing at least two human CD301 protein carbohydrate recognition domains linked in tandem by peptide linkers.

2. The multivalent CD301 fusion protein based on an optimized peptide linker according to claim 1, characterized in that: The peptide linker can be a flexible linker or a rigid linker.

3. The multivalent CD301 fusion protein based on an optimized peptide linker according to claim 2, characterized in that: The peptide linker is a flexible linker, and the amino acid sequence of the flexible linker contains a repeating glycine-serine motif, with the general formula (GGGGS)n, where n is an integer from 2 to 4.

4. A multivalent CD301 fusion protein based on an optimized peptide linker according to claim 3, characterized in that: The flexible joint is (GGGGS)3.

5. A multivalent CD301 fusion protein based on an optimized peptide linker according to claim 2, characterized in that: The peptide linker is a rigid linker, and the amino acid sequence of the rigid linker is derived from the C-terminal peptide hCGβ CTP of the human chorionic gonadotropin β subunit.

6. A multivalent CD301 fusion protein based on an optimized peptide linker according to claim 5, characterized in that: The amino acid sequence of the rigid linker is SSSKAPPPSLPSPSRLPGPSDTPILPQ, or a sequence that has at least 90% homology with it and retains the O-glycosylation site.

7. A multivalent CD301 fusion protein based on an optimized peptide linker according to claim 1, characterized in that: The N-terminus or C-terminus of the polypeptide chain is also connected to an affinity tag for site-specific modification. The affinity tag is a biotin receptor peptide Avi-tag, a histidine tag His-tag, or an Fc fragment.

8. A functionalized magnetic bead, characterized in that: The surface of the magnetic beads is fixed with the recombinant fusion protein according to any one of claims 1 to 7 via covalent bonds or affinity.

9. The application of the functionalized magnetic beads according to claim 8 in the preparation of a tumor liquid biopsy kit.

10. The application of the functionalized magnetic beads according to claim 8 in the preparation of a system for enriching and separating circulating tumor cells, characterized in that: The biological fluid sample to be tested was mixed with functionalized magnetic beads and incubated to allow the multivalent recombinant fusion protein on the surface of the magnetic beads to specifically bind to the Tn antigen on the surface of circulating tumor cells. The magnetic bead-cell complex was separated and collected using a magnetic field, and background cells that did not bind specifically were washed away.

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