Nanometer antibody targeting PODXL protein and application thereof

Abstract

The invention relates to the field of biotechnology and biomedicine, and discloses a PODXL protein targeting nano antibody and application thereof.The PODXL protein targeting nano antibody comprises complementary determining regions CDR1-CDR3 shown in SEQ ID NO: 4-SEQ ID NO: 6, DHLM core epitopes at the 207th-210th sites in the amino acid sequence shown in SEQ ID NO: 2 in PODXL protein are specifically combined, and the PODXL protein targeting nano antibody is obtained. The nano antibody is obtained by transfecting HEK293F cells with recombinant expression plasmids and carrying out suspension culture, centrifugal filtration, Ni-NTA affinity chromatography and gel filtration chromatography purification. According to the invention, the PODXL protein DHLM epitope is specifically recognized by utilizing the single-domain structural characteristics of the nano antibody, so that the risk of cross reaction is reduced; and by combining with a strategy of fusing heterologous polypeptide or coupling effector molecules, the application of the nano antibody in preparation of diagnostic reagents and therapeutic drugs for PODXL protein positive tumors is realized.

Description

Technical Field

[0001] This invention relates to the fields of biotechnology and biomedicine, specifically to a nanobody targeting the PODXL protein and its applications. Background Technology

[0002] Podocalyxin (PODXL) is a highly glycosylated type I transmembrane protein expressed in various malignant tumors, including oral squamous cell carcinoma, breast cancer, and lung cancer. It is associated with tumor cell growth, invasion, and metastasis, and is therefore considered a tumor marker and therapeutic target. Current research indicates that the DHLM region from amino acid positions 207 to 210 of the PODXL protein is its functional epitope, and traditional monoclonal antibodies targeting this epitope have been used in research and development related to these diseases.

[0003] However, traditional monoclonal antibodies typically have large molecular weights and complex quaternary structures, which limits their penetration into solid tumor tissues and makes it difficult to effectively accumulate in deep lesions. The preparation of traditional antibodies often involves complex cell culture and purification processes to maintain their activity, and their structural characteristics restrict the flexibility of genetic engineering, hindering their fusion with heterologous peptides or conjugation with effector molecules to construct diverse therapeutic agents.

[0004] Currently, no nanobody molecules targeting the DHLM epitope of the PODXL protein have been developed in this field. The lack of such small-molecule antibody tools limits the efficient targeted delivery of PODXL-positive tumors and the development of multifunctional diagnostic and therapeutic products. Therefore, obtaining a nanobody that specifically targets the DHLM epitope of the PODXL protein and possesses good tissue penetration and engineering potential is a problem that needs to be solved in this field. Summary of the Invention

[0005] To address the shortcomings of existing technologies, this invention provides a nanobody targeting the PODXL protein and its application, solving the problems of large molecular weight, limited tissue penetration, and difficulty in diverse engineering modifications of existing antibodies targeting the DHLM epitope of the PODXL protein.

[0006] To address the above problems, the present invention provides the following technical solution: This invention provides a nanobody targeting the PODXL protein, employing the following technical solution: A nanobody targeting the PODXL protein, the nanobody comprising a complementarity-determining region, wherein the amino acid sequences of CDR1, CDR2, and CDR3 of the complementarity-determining region are shown in SEQ ID NO:4, SEQ ID NO:5, and SEQ ID NO:6, respectively; the nanobody specifically binds to the DHLM core epitope at positions 207-210 of the amino acid sequence shown in SEQ ID NO:2 in the PODXL protein.

[0007] By employing the above technical solution, the nanobody can form a specific antigen-binding groove by using a specially designed combination of CDR1, CDR2, and CDR3 sequences, which fits the DHLM core tetrapeptide epitope at positions 207-210 of the PODXL protein. The DHLM epitope is located on the exposed surface of a specific structural domain of the PODXL protein. By targeting this region, the nanobody can bind to the target antigen with high affinity and specificity, reducing the risk of non-specific cross-reaction with homologous proteins. The single-domain characteristic of the nanobody gives it superior tissue penetration ability compared to traditional IgG antibodies, which is beneficial for enriching deep within tumor tissue, thereby improving the efficiency of targeted recognition.

[0008] Preferably, the amino acid sequence of the nanobody is as shown in SEQ ID NO:1; the 5' end of the nanobody encoding sequence is connected to a signal peptide sequence as shown in SEQ ID NO:3, and the 3' end of the nanobody encoding sequence is connected to a sequence encoding a 6xHis tag; the nanobody is obtained by constructing a recombinant expression plasmid pNCMO2-Nb-PODXL-C01 containing the nanobody encoding sequence and transforming it into host cells for expression.

[0009] By adopting the above technical solutions, the full-length sequence shown in SEQ ID NO:1 includes the CDR region responsible for specific binding and the humanized and optimized framework region, which is beneficial to maintaining the structural stability of the protein under physiological conditions and reducing immunogenicity. The introduction of the signal peptide sequence shown in SEQ ID NO:3 can guide the newly synthesized peptide chain into the endoplasmic reticulum lumen and finally secrete it into the extracellular space through the Golgi apparatus, realizing secretory expression, simplifying the subsequent protein harvesting steps, and reducing host protein contamination caused by intracellular lysis. The C-terminal 6xHis tag utilizes the coordination binding principle of histidine and nickel ions to provide a molecular basis for affinity chromatography.

[0010] Preferably, the nanobody is prepared by the following steps: HEK293F cells in suspension culture were adjusted to a predetermined cell density, and the recombinant expression plasmid pNCMO2-Nb-PODXL-C01 was transfected into the HEK293F cells using liposome transfection reagent to obtain the transfected cell system. The transfected cell system was cultured in suspension at a predetermined culture temperature, CO2 concentration and shaker speed. After the culture was completed, the cells and debris were removed by centrifugation, and the supernatant was filtered to obtain a clear liquid. The Ni-NTA agarose affinity chromatography column was equilibrated with binding buffer, the clarified solution was loaded onto the Ni-NTA agarose affinity chromatography column, and the column was washed with the binding buffer to remove impurities. The Ni-NTA agarose affinity chromatography column was eluted with elution buffer, and the eluent was collected, concentrated, and loaded onto a Superdex 75 Increase 10 / 300 GL gel filtration chromatography column. The column was eluted with the mobile phase, and the main peak fraction was collected to obtain the purified nanobody.

[0011] By adopting the above technical solution and using the HEK293F expression system in mammalian cells, which possesses a complete post-translational modification mechanism, the nanobody is facilitated to form correct disulfide bonds and spatial folding, thereby maintaining its biological activity. The preparation process employs a two-step purification strategy combining affinity capture and fine separation. Ni-NTA affinity chromatography captures the target protein based on the His tag and removes most of the host contaminant proteins. Superdex 75 Increase 10 / 300 GL gel filtration chromatography further removes any possible protein aggregates or degradation fragments based on the difference in molecular hydrodynamic radii, thereby improving the uniformity and purity of the final product.

[0012] Preferably, the predetermined cell density is 2.0 × 10⁻⁶. 6 cells / mL - 3.0 × 10 6 The volume-to-mass ratio of the liposome transfection reagent to the recombinant expression plasmid pNCMO2-Nb-PODXL-C01 is 2:1-4:1, and the transfection is performed at room temperature for 10-20 minutes. The predetermined culture temperature is 36.5℃-37.5℃, the CO2 concentration is 7.5%-8.5%, the shaking speed is 120rpm-130rpm, and the suspension culture time is 5d-7d; The centrifugation is carried out under a centrifugal force of 3800g-4200g for 15min-25min, and the filter membrane used for filtration has a pore size of 0.22μm; The binding buffer contains sodium phosphate at a concentration of 15 mM-25 mM, NaCl at a concentration of 450 mM-550 mM, and imidazole at a concentration of 15 mM-25 mM, and the pH of the binding buffer is 7.2-7.6. The loading flow rate is 1.0 mL / min-2.0 mL / min, and the washing volume is 8-12 times the column volume; The elution buffer contains sodium phosphate at a concentration of 15 mM-25 mM, NaCl at a concentration of 450 mM-550 mM, and imidazole at a concentration of 300 mM-600 mM, and the pH of the elution buffer is 7.2-7.6. The mobile phase is PBS buffer with a pH of 7.2-7.6, the elution flow rate is 0.4 mL / min-0.6 mL / min, and the main peak with a retention volume in the range of 13 mL-15 mL is collected.

[0013] By adopting the above technical solution, the cell density was controlled at 2.0 × 10⁻⁶. 6 cells / mL - 3.0 × 10 6 The use of cells / mL and a transfection reagent to plasmid ratio of 2:1-4:1 can balance cell transfection efficiency and metabolic load, reduce premature cell apoptosis, and thus increase protein yield. The binding buffer contains a low concentration of imidazole of 15mM-25mM, which can inhibit the weak binding of non-specific proteins to the nickel column, while the elution buffer contains a high concentration of imidazole of 300mM-600mM, which allows the target nanobody to be eluted. By controlling the retention volume of the Superdex 75 chromatography column to be within the range of 13mL-15mL to collect the main peak, the monomeric form of the nanobody can be intercepted, and impurities with molecular weight deviations can be excluded.

[0014] Preferably, the nanobody is fused with a heterologous polypeptide sequence via peptide bonds to form a fusion protein; the heterologous polypeptide sequence is selected from the Fc domain of an antibody, the variable region of another specific antibody, or the transmembrane region and intracellular signal transduction domain of a chimeric antigen receptor.

[0015] By adopting the above technical solutions, the modular fusion design of the nanobody expands its functional properties; the fusion of the Fc domain can extend the in vivo half-life of the nanobody by utilizing the neonatal Fc receptor-mediated recycling mechanism and induce antibody-dependent cell-mediated cytotoxicity; the fusion of another specific antibody can construct a bispecific antibody to achieve synergistic binding to two targets; the fusion of a chimeric antigen receptor-related domain can be used to construct CAR-T or CAR-NK cells targeting PODXL, transforming the recognition ability of the nanobody into a killing command for immune cells.

[0016] Preferably, the nanobody is chemically coupled with an effector molecule to form an immunoconjugate; the effector molecule is selected from fluorescent dyes, enzymes, biotin, radioisotopes, magnetic beads, cytotoxic drugs, protein toxins, or radionuclides.

[0017] By employing the above technical solution, using the nanobody as a carrier to conjugate cytotoxic drugs or toxins, antibody-drug conjugates can be constructed. These conjugates deliver toxic molecules into PODXL-positive tumor cells through receptor-mediated endocytosis, reducing toxic side effects on normal tissues. Conjugation with fluorescent dyes or radionuclides can enable in vitro or in vivo tracking and imaging of tumor tissues.

[0018] This invention also provides an isolated nucleic acid molecule, expression vector, or host cell, employing the following technical solution: An isolated nucleic acid molecule, expression vector, or host cell, wherein the isolated nucleic acid molecule encodes the aforementioned nanobody targeting the PODXL protein; the expression vector contains the isolated nucleic acid molecule; the host cell contains the expression vector or has the isolated nucleic acid molecule integrated into its genome; the host cell includes engineered HEK293F cells, engineered T lymphocytes, or engineered natural killer cells.

[0019] By adopting the above technical solution, the genetic material basis and production and application carrier of the nanobody are provided; the engineered HEK293F cells are used to secrete and express the nanobody protein; the engineered T lymphocytes or engineered natural killer cells use the nanobody structural domain expressed on the cell surface to recognize the PODXL antigen on the tumor surface, activate downstream signaling pathways, and kill tumor cells.

[0020] The present invention also provides a pharmaceutical composition, which adopts the following technical solution: A pharmaceutical composition comprising an active ingredient and a pharmaceutically acceptable carrier; the active ingredient being selected from the aforementioned nanobody targeting the PODXL protein or the aforementioned host cell; wherein, when the active ingredient is the immunoconjugate, the immunoconjugate comprises a cytotoxic drug, a protein toxin, or a radionuclide; and when the active ingredient is the host cell, the host cell is selected from engineered T lymphocytes or engineered natural killer cells.

[0021] By adopting the above technical solution, the solubility, stability and bioavailability of the active ingredients are improved by combining the nanobody or the engineered cell with a pharmaceutically acceptable carrier, making it a usable drug formulation and providing a specific drug form for the treatment of PODXL positive tumors.

[0022] The present invention also provides a detection kit, which adopts the following technical solution: A detection kit comprising a core recognition element and a solid-phase carrier; the core recognition element being the aforementioned nanobody targeting the PODXL protein; wherein, when the core recognition element is the immunoconjugate, the immunoconjugate comprises a fluorescent dye, an enzyme, biotin, a radioactive isotope, or magnetic beads; the solid-phase carrier is selected from microplates, magnetic beads, or chromatographic test strips.

[0023] By adopting the above technical solution, a detection tool is constructed by utilizing the specific binding ability of the nanobody to the DHLM epitope of PODXL protein; the solid-phase carrier enables the antigen-antibody complex to be separated or enriched from the sample matrix, and in conjunction with an enzyme or fluorescent labeling signal amplification system, quantitative or qualitative analysis of PODXL expression levels in serum or tissue samples can be achieved.

[0024] The present invention also provides the application of the aforementioned nanobody targeting PODXL protein, the aforementioned host cell, the aforementioned pharmaceutical composition, or the aforementioned detection kit in the preparation of diagnostic reagents for detecting PODXL protein expression or drugs for treating PODXL protein-positive tumors, using the following technical solution: The methods for detecting PODXL protein expression include enzyme-linked immunosorbent assay (ELISA), immunohistochemistry, flow cytometry, or in vivo imaging.

[0025] By adopting the above technical solution, the pharmaceutical applications of the product have been clarified. Based on the physicochemical properties and binding activity of the nanobody, the nanobody can be used to develop therapeutic biological products, and can also be applied to detection platforms for enzyme-linked immunosorbent assay (ELISA), immunohistochemistry, and flow cytometry to meet the needs of clinical pathological diagnosis and companion diagnostics.

[0026] This invention provides a nanobody targeting the PODXL protein and its application. It has the following beneficial effects: 1. This invention employs a complementary determinant region containing the CDR sequence shown in SEQ ID NO:4 to SEQ ID NO:6, enabling the nanobody to specifically recognize and bind to the DHLM core epitope at positions 207-210 of the amino acid sequence of PODXL protein as shown in SEQ ID NO:2. By utilizing the single-domain structural characteristics of the nanobody, the risk of cross-reaction with non-target proteins is reduced, thereby improving the recognition efficiency of the nanobody for PODXL protein-positive tumors in vivo or in vitro.

[0027] 2. This invention utilizes the HEK293F cell expression system and a preparation method combining Ni-NTA agarose affinity chromatography and Superdex 75 Increase 10 / 300 GL gel filtration chromatography. By leveraging the post-translational modification mechanism of the eukaryotic expression system, the correct spatial folding of the nanobody is ensured. Furthermore, by controlling the volume-to-mass ratio during transfection and the imidazole concentration gradient during chromatography, protein aggregates and host contaminants are removed, thereby obtaining a purified nanobody with biological activity suitable for subsequent drug development.

[0028] 3. This invention constructs fusion proteins that can guide engineered cells to kill tumors by using chimeric antigen receptor domains to fuse heterologous polypeptide sequences with the nanobody as the core recognition element through peptide bonds or couple effector molecules through chemical bonds, as well as immunoconjugates that can achieve targeted delivery using cytotoxic drugs or antigen detection using fluorescent dyes in conjunction with solid-phase carriers. This enables the nanobody to be used in the preparation of diagnostic reagents and therapeutic drugs for PODXL protein-positive tumors in a variety of applications. Attached Figure Description

[0029] Figure 1 This is a structural diagram of the DHLM epitope antigen peptide generated by AlphaFold3 in an embodiment of the present invention. Figure 2 This is a schematic diagram illustrating the extraction of the VHH skeleton from the three-dimensional conformation of the PDB ID:7EOW complex in an embodiment of the present invention. Figure 3 This is a sequencing result diagram of the recombinant expression plasmid pNCMO2-Nb-PODXL-C01 in an embodiment of the present invention; Figure 4 This is a graph showing the protein purification effect of the nanobody Nb-PODXL-C01 in this embodiment of the invention; Figure 5 This is a Western Blot result of the experiment verifying the specific binding of the nanobody Nb-PODXL-C01 to the PODXL protein in an embodiment of the present invention. Detailed Implementation

[0030] The technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings, preparation examples, implementation examples, comparative examples, test examples, and application examples. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0031] Preparation Examples 1-2: Preparation Example 1: This preparation example provides a method for molecular design and sequence acquisition of nanobodies targeting the DHLM epitope of the PODXL protein, including the following steps: First, modeling of the target epitope and antigen structure was performed. The amino acid sequence 200-219 of the PODXL protein (SEQ ID NO:2) was selected as the target site, with the core epitope being DHLM at positions 207-210. Since the three-dimensional structure of this peptide was not resolved, AlphaFold3 was used to predict the three-dimensional structure of this 20-peptide (e.g.,...). Figure 1 As shown), the predicted peptide conformation is used as the spatial coordinate input for the target epitope during design. Simultaneously, the clinically validated humanized nanobody backbone Caplacizumab is selected, and the variable region backbone (e.g., 7EOW) is extracted from the three-dimensional conformation of the Caplacizumab / vWF complex in the PDB database. Figure 2 (As shown), serving as a fixed framework for the design; Computational design was then performed. The framework sequence and 3D structure of the humanized Caplacizumab variable region backbone were used as fixed templates and input into the RFantibody platform. Simultaneously, the 3D structure of the antigenic peptide containing the DHLM epitope, predicted by AlphaFold3, was used as the target input. On the target antigenic peptide structure, core epitope residues D207, H208, L209, and M210 were designated as key residues that must be bound to the design, guiding the algorithm to target this region with the newly generated CDR loop. The RFantibody platform ran the finely tuned RFdiffusion model, generating a novel 3D structure of the complementarity-determining region loop while maintaining the input variable region framework unchanged. Simultaneously, it sampled various rigid docking postures of the nanobody relative to the target epitope, producing a binding interface with good complementarity. Next, sequence generation and screening were performed on the generated backbone. For the nanobody backbone with binding posture generated by RFdiffusion, ProteinMPNN was used to generate amino acid sequences for the newly designed CDR loops to stabilize the nanobody folding and facilitate interaction with the DHLM epitopes. The generated design models were re-predicted and screened using AlphaFold3, with designs showing higher AF3 prediction interface template model scores and predictive binding conformations consistent with the original design model selected as candidate sequences. The final sequence obtained was Nb-PODXL-C01 (SEQ ID NO:1), which contains complementarity-determining regions CDR1 (SEQ ID NO:4), CDR2 (SEQ ID NO:5), and CDR3 (SEQ ID NO:6), for subsequent preparation.

[0032] Preparation Example 2: This preparation example provides a recombinant expression plasmid pNCMO2-Nb-PODXL-C01 for expressing the above-mentioned nanobody, which is prepared through the following steps: First, gene sequence design was performed. Mammalian cell expression optimization was conducted based on the amino acid sequence of Nb-PODXL-C01 obtained in Preparation Example 1. A DNA sequence encoding the mouse immunoglobulin κ chain signal peptide (SEQ ID NO:3) was added to the 5' end of the coding sequence to achieve secretory protein expression; a sequence encoding a 6×His tag was added to the 3' end of the coding sequence to facilitate subsequent purification. Then, synthesis and vector construction were performed. The codons of the above fusion gene were optimized using human embryonic kidney cells, and the full sequence was synthesized. The synthesized double-stranded DNA fragment contained BamHI and XhoI restriction enzyme sites at both ends that matched the vector. The purified DNA fragment was ligated into the mammalian expression vector pNCMO2, which had been digested with the same enzymes, and the ligation product was transformed into competent E. coli. Finally, plasmid verification was performed. Positive clones were initially screened by colony PCR and plasmid restriction enzyme digestion, and plasmid DNA from the positive clones was extracted and sequenced. The sequencing results were consistent with the designed optimized sequence alignment results (e.g., Figure 3 As shown in the figure, no frameshift or mutation was found, and the recombinant expression plasmid was obtained.

[0033] Examples 1-3: Example 1: This embodiment provides a nanobody targeting the PODXL protein, which is prepared through the following steps: S01. Construction of cell transfection and expression system: HEK293F cells in suspension culture were adjusted to a cell density of 2.0 × 10⁶ cells / year. 6 Cells / mL were used as the expression host. Using the plasmid constructed in Preparation Example 2, a transfection complex was prepared at a ratio of 2:1 of liposome transfection reagent (μL) to plasmid DNA (μg). After incubation at room temperature for 10 minutes, the complex was added to the cell culture medium. S02. Secretory Expression Culture: Transfected cells were placed in a constant temperature shaking incubator for suspension culture at a temperature of 36.5℃, a CO2 concentration of 7.5%, and a shaking speed of 120 rpm. The cells were cultured under these conditions for 5 days without additional culture medium, relying on intracellular metabolic mechanisms to secrete and express the target nanobody. S03. Harvesting the culture supernatant: After the culture is completed, the cell culture medium is transferred to a centrifuge tube and centrifuged at 3800×g for 15 minutes at 4℃. The supernatant is collected, and the precipitated cells and debris are discarded. The supernatant is filtered through a 0.22μm filter membrane to obtain a clear solution containing nanoantibodies. S04. Affinity Chromatography Capture: Equilibrate the Ni-NTA agarose affinity chromatography column using binding buffer (15 mM sodium phosphate, 450 mM NaCl, 15 mM imidazole, pH 7.2). Load the filtered sample into the column at a flow rate of 1.0 mL / min. After loading, wash with 8 column volumes of the binding buffer to remove any non-specifically bound proteins. S05, Elution and Collection: Elution was performed using elution buffer (15 mM sodium phosphate, 450 mM NaCl, 300 mM imidazole, pH 7.2). The elution solution corresponding to the elution peak was collected, which is the crude pure nanobody solution. S06. Fine Purification and Replacement: The eluent was concentrated to 1 mL using an ultrafiltration centrifuge tube with a molecular weight cutoff of 3 kDa, and then loaded onto a Superdex 75 Increase 10 / 300 GL gel filtration chromatography column. PBS buffer (pH 7.2) was used as the mobile phase, and elution was performed at a flow rate of 0.4 mL / min. Based on the UV absorption peak at 280 nm, the main peak fraction with a retention volume in the range of 13-15 mL was collected; this is the purified nanobody.

[0034] Example 2: This embodiment provides a nanobody targeting the PODXL protein, which is prepared through the following steps: S01. Construction of cell transfection and expression system: HEK293F cells in suspension culture were adjusted to a cell density of 2.5 × 10⁻⁶. 6 Cells / mL were used as the expression host. Using the plasmid constructed in Preparation Example 2, a transfection complex was prepared at a ratio of 3:1 between liposome transfection reagent (μL) and plasmid DNA (μg). After incubation at room temperature for 15 minutes, the complex was added to the cell culture medium. S02. Secretory Expression Culture: Transfected cells were placed in a constant temperature shaking incubator for suspension culture at a temperature of 37.0℃, a CO2 concentration of 8.0%, and a shaking speed of 125 rpm. The cells were cultured under these conditions for 6 days without additional culture medium, relying on intracellular metabolic mechanisms to secrete and express the target nanobody. S03. Harvesting the culture supernatant: After the culture is completed, the cell culture medium is transferred to a centrifuge tube and centrifuged at 4000×g for 20 minutes at 4℃. The supernatant is collected, and the precipitated cells and debris are discarded. The supernatant is filtered through a 0.22μm filter membrane to obtain a clear solution containing nanoantibodies. S04. Affinity Chromatography Capture: Equilibrate the Ni-NTA agarose affinity chromatography column using binding buffer (20 mM sodium phosphate, 500 mM NaCl, 20 mM imidazole, pH 7.4). Load the filtered sample into the column at a flow rate of 1.5 mL / min. After loading, wash with 10 column volumes of the binding buffer to remove any non-specifically bound proteins. S05, Elution and Collection: Elution was performed using elution buffer (20 mM sodium phosphate, 500 mM NaCl, 500 mM imidazole, pH 7.4). The elution solution corresponding to the elution peak was collected, which is the crude pure nanobody solution. S06. Fine Purification and Replacement: The eluent was concentrated to 1 mL using an ultrafiltration centrifuge tube with a molecular weight cutoff of 3 kDa, and then loaded onto a Superdex 75 Increase 10 / 300 GL gel filtration chromatography column. PBS buffer (pH 7.4) was used as the mobile phase, and elution was performed at a flow rate of 0.5 mL / min. Based on the UV absorption peak at 280 nm, the main peak fraction with a retention volume in the range of 13-15 mL was collected; this is the purified nanobody.

[0035] Example 3: This embodiment provides a nanobody targeting the PODXL protein, which is prepared through the following steps: S01. Construction of cell transfection and expression system: The HEK293F cells in suspension culture were adjusted to a cell density of 3.0 × 10⁶ cells / year. 6 Cells / mL were used as the expression host. Using the plasmid constructed in Preparation Example 2, a transfection complex was prepared at a ratio of 4:1 of liposome transfection reagent (μL) to plasmid DNA (μg). After incubation at room temperature for 20 minutes, the complex was added to the cell culture medium. S02. Secretory Expression Culture: Transfected cells were placed in a constant temperature shaking incubator for suspension culture at a temperature of 37.5℃, a CO2 concentration of 8.5%, and a shaking speed of 130 rpm. The cells were cultured under these conditions for 7 days without additional culture medium, relying on intracellular metabolic mechanisms to secrete and express the target nanobody. S03. Harvesting the culture supernatant: After the culture is completed, the cell culture medium is transferred to a centrifuge tube and centrifuged at 4200×g for 25 minutes at 4℃. The supernatant is collected, and the precipitated cells and debris are discarded. The supernatant is filtered through a 0.22μm filter membrane to obtain a clear solution containing nanoantibodies. S04. Affinity Chromatography Capture: Equilibrate the Ni-NTA agarose affinity chromatography column using binding buffer (25 mM sodium phosphate, 550 mM NaCl, 25 mM imidazole, pH 7.6). Load the filtered sample into the column at a flow rate of 2.0 mL / min. After loading, wash with 12 column volumes of the binding buffer to remove any non-specifically bound proteins. S05, Elution and Collection: Elution was performed using elution buffer (25 mM sodium phosphate, 550 mM NaCl, 600 mM imidazole, pH 7.6). The elution solution corresponding to the elution peak was collected, which is the crude pure nanobody solution. S06. Fine Purification and Replacement: The eluent was concentrated to 1 mL using an ultrafiltration centrifuge tube with a molecular weight cutoff of 3 kDa, and then loaded onto a Superdex 75 Increase 10 / 300 GL gel filtration chromatography column. PBS buffer (pH 7.6) was used as the mobile phase, and elution was performed at a flow rate of 0.6 mL / min. Based on the UV absorption peak at 280 nm, the main peak fraction with a retention volume in the range of 13-15 mL was collected; this is the purified nanobody.

[0036] Comparative Examples 1-3: Comparative Example 1: Compared with Example 2, the difference is that the expression plasmid used in step S01 is pNCMO2-Nb-Scramble, and the amino acid sequence of the CDR3 region of the nanobody encoded by this plasmid is a randomized arrangement (Scramble sequence) of SEQ ID NO:6 in Example 2. All other parameters and steps are the same.

[0037] Comparative Example 2: Compared with Example 2, the difference is that the expression plasmid used in step S01 is pNCMO2-Nb-Mutant, and the arginine (Arg) at the key site of the CDR3 region of the nanobody encoded by this plasmid is mutated to alanine (Ala). All other parameters and steps are the same.

[0038] Comparative Example 3: Compared with Example 2, the difference is that the anti-PODXL monoclonal antibody purchased from Thermofisher is used directly as the finished antibody, and steps S01 to S06 in Example 2 are not involved. The buffer system used for subsequent tests is the same.

[0039] Test Example 1-3: Test Example 1: Protein Yield and Purity Analysis Experimental description: This test aims to examine the expression level of nanobodies in mammalian cell systems and the quality of the final product, while comparing the preparation effects of different sequence designs (the calculated design sequence in Example 2 and the mutated / randomized sequence in the comparative example). All samples were prepared strictly according to the gene synthesis, HEK293F cell transfection, and purification process described in Example 2.

[0040] Experimental steps: First, the expression vector was constructed and cells were transfected. The nanobody sequences to be tested (Examples 1, 2, 3 and Comparative Examples 1, 2) were fused with a mouse immunoglobulin κ chain signal peptide and a C-terminal 6×His tag, respectively, and constructed into the pNCMO2 vector. The plasmids were transfected into HEK293F cells in logarithmic growth phase using liposome transfection reagent and cultured at 37°C and 8% CO2 for 5 to 7 days. Protein purification was then performed. Cell culture supernatant was collected, filtered, and loaded onto a Ni-NTA affinity chromatography column. The target protein was eluted with 500 mM imidazole. The eluent was concentrated by ultrafiltration and then further purified using a Superdex 75 Increase gel filtration chromatography (SEC) column, and the main peak fraction was collected. Finally, concentration and purity were determined. Protein concentration was determined using the BCA method, and the total yield was calculated based on the elution volume. Equal amounts of sample were subjected to reducing SDS-PAGE electrophoresis. After Coomassie brilliant blue staining and destaining, the grayscale ratio of the main bands was analyzed using a gel imaging system and ImageJ software.

[0041] Experimental data: The yield and purity test results for each group of samples are shown in the table below: Table 1. Summary of protein yield and purity test data for different experimental groups

[0042] Experimental conclusion: According to the data analysis in Table 1, the sequences designed in this invention exhibit good drug-like properties in the HEK293F eukaryotic expression system.

[0043] Example 2 (Nb-PODXL-C01) was purified in two steps by Ni-NTA affinity chromatography and gel filtration chromatography, resulting in a total protein yield of 12.15 mg and a purity of 97.83%. The protein exhibited a single monomer peak on the SEC spectrum (SDS-PAGE electrophoresis results are shown below). Figure 4 As shown in the data (corresponding to purity data), this sequence design demonstrates significant expression stability and anti-aggregation ability. Although the process parameters or sequence details of Examples 1 and 3 differ slightly, both achieved high yields and purity standards.

[0044] Data from Comparative Examples 1 and 2 show that the protein yield and purity are at the same level as in Example 2. This result indicates that sequence perturbations in the CDR3 region (Comparative Example 1) or mutations at key sites (Comparative Example 2) did not affect the folding and secretion efficiency of the nanobody backbone in mammalian cells. This indirectly confirms that the differences in biological activity among the different groups are entirely due to the specific recognition of the target by the sequence, rather than differences in the quality of protein preparation.

[0045] Test Example 2: Detection of Specificity and Applicability of Western Blot Experimental description: This test aims to verify the binding specificity and applicability of the nanobody Nb-PODXL-C01 prepared in Example 2 against the natural full-length PODXL protein. Six human cell lines with different expression backgrounds (NTERA-2, HEK-293, HeLa, Hep G2, Jurkat, and PANC-1) were selected as test samples. A commercial antibody (Thermofisher:39-3800, i.e., the anti-PODXL antibody, corresponding to Comparative Example 3) was introduced as a positive control. Comparative Example 1 and Comparative Example 2 were also set up as antibody structure controls to evaluate the molecular recognition ability of Nb-PODXL-C01.

[0046] Experimental steps: NTERA-2, HEK-293, HeLa, Hep G2, Jurkat and PANC-1 cells in logarithmic growth phase were collected, and total protein was extracted by adding RIPA lysis buffer. An equal amount of protein sample was added to reducing loading buffer and denatured in boiling water for 5 minutes. After separation by 10% SDS-PAGE, the samples were transferred to a PVDF membrane by wet transfer and blocked at room temperature for 1 hour using TBST buffer containing 5% skim milk. The membrane was incubated with the test antibody (concentration 1 μg / mL) of Example 2, Comparative Example 1, Comparative Example 2, or the reference antibody of Comparative Example 3. The nanobody group used HRP-conjugated anti-His tag antibody as the secondary antibody, and the reference antibody group used HRP-conjugated anti-mouse IgG antibody as the secondary antibody. The signal intensity of the target protein band at approximately 58 kDa was recorded using ECL chemiluminescent substrate for color development.

[0047] Experimental data: Figure 5The following are some representative electrophoresis patterns from this experiment. The left side shows the detection results of the reference anti-PODXL antibody (Comparative Example 3) against the lysates of six cell lines. The right side shows the detection results of the nanobody Nb-PODXL-C01 prepared in Example 2 against four representative cell lines (NTERA-2, HEK-293, HeLa, and PANC-1). This experiment selected the detection data of HeLa and PANC-1, two representative cell lines, for relative signal intensity statistical analysis. The results for each experimental group are shown in Table 2. Table 2. Statistical data on specific detection signal intensity in Western Blot

[0048] Note: ND in the table represents no specific band detected.

[0049] Experimental conclusion: Table 2 Data and corresponding representative electrophoresis patterns (e.g.) Figure 5 As shown in the figure, the nanobody of the present invention can recognize the endogenous PODXL protein.

[0050] The Nb-PODXL-C01 prepared in Example 2 was able to recognize the native PODXL protein with an apparent molecular weight of approximately 58 kDa in various human cell lines, and its main band position was consistent with the detection results of Comparative Example 3. In HeLa and PANC-1 cell lines, the relative signal intensity of Example 2 reached 8.4 × 10⁻⁶. 4 At the AU level, it exhibits a clear detection signal and demonstrates binding ability comparable to Comparative Example 3.

[0051] In contrast, Comparative Example 1 showed no signal (ND) at the target site in either cell line, indicating that the nanobody scaffold itself does not generate nonspecific adhesion. Comparative Example 2 showed only a weak signal close to the background in HeLa and no signal was detected at all in PANC-1. These results validate that arginine residues at specific sites play an important role in maintaining the stability of the antigen-antibody complex.

[0052] Test Example 3: ELISA Affinity and Specificity Assay Experimental description: This assay uses enzyme-linked immunosorbent assay (ELISA) to quantitatively assess the binding affinity of nanobodies to synthetic peptides containing a core epitope (DHLM). The antigenic peptides were immobilized using a streptavidin and biotin system to maintain the native conformation of the peptides and simulate the display of linear epitopes.

[0053] Experimental steps: First, antigen fixation was performed. The biotinylated antigen peptide (SEQ ID NO:2) was diluted to 2 µg / mL with PBS, and 100 µL was added to each well of a 96-well plate pre-coated with streptavidin. The plate was incubated at room temperature for 1 hour, washed with PBS, and then blocked with 3% BSA. Antibody incubation was then performed. The nanobodies to be tested (Examples 2, Comparative Examples 1 and 2) and commercial antibodies (Comparative Example 3) were serially diluted (starting at 500 nM, 3-fold dilution) and added to well plates, then incubated at room temperature for 1.5 hours. Next, the detection was performed. HRP-conjugated anti-His-tagged antibody (for nanobodies) or anti-mouse IgG antibody (for commercial antibodies) was added, and after incubation for 1 hour, TMB was added for color development. The reaction was terminated with sulfuric acid. Finally, data analysis was performed. OD450 absorbance values ​​were measured, and a four-parameter logistic regression model was used to fit the curve and calculate EC50.

[0054] Experimental data: The absorbance values ​​and kinetic parameters of each experimental group at different concentrations are shown in the table below: Table 3. Raw data and kinetic parameters of ELISA binding affinity assay

[0055] Note: N / A in the table means the data cannot be fitted; >500 means that saturation has not been reached within the test concentration range.

[0056] Experimental conclusion: ELISA data further confirmed the high affinity and specificity of the Nb-PODXL-C01 nanobody in Example 2.

[0057] The sample in Example 2 exhibited a standard S-shaped dose-dependent curve, with a calculated EC50 value of 4.12 nM, indicating a tight binding to the target linear peptide. This affinity value is superior to the control commercial antibody (7.85 nM), demonstrating the spatial advantage of small molecular weight nanobodies in recognizing linear epitopes.

[0058] Comparative Example 1 maintained its OD value at the baseline level (N / A) throughout, confirming that binding depends on a specific CDR3 sequence. Comparative Example 2, at its highest concentration of 500 nM, had an OD value of only 0.210, far from saturation (>500 nM), indicating that mutations in key residues led to a decrease in affinity of at least two orders of magnitude. Combined with the results of Test Examples 2 and 3, this fully demonstrates that the sequences computationally designed in this invention can accurately recognize and bind to the PODXL protein and its core epitopes through specific intermolecular forces.

[0059] Application example: This application example provides an isolated nucleic acid molecule, an expression vector, and a host cell.

[0060] The isolated nucleic acid molecule encodes a nanobody targeting the PODXL protein. The isolated nucleic acid molecule is cloned into an expression vector, such that the expression vector contains the isolated nucleic acid molecule. The expression vector is then introduced into a host cell, or the isolated nucleic acid molecule is integrated into the genome of a host cell, such that the host cell contains the expression vector or the genome in which the isolated nucleic acid molecule is integrated.

[0061] The host cells include engineered HEK293F cells, engineered T lymphocytes, or engineered natural killer cells. Specifically, engineered HEK293F cells can be used for recombinant protein expression; an expression vector can be constructed by using the nucleic acid sequence encoding the nanobody as the extracellular antigen recognition domain of the chimeric antigen receptor, and then introduced into lymphocytes or natural killer cells to prepare engineered T lymphocytes or engineered natural killer cells that express nanobodies targeting the PODXL protein on their surface.

[0062] This application example provides a pharmaceutical composition and its use in the preparation of a medicament for treating PODXL protein-positive tumors.

[0063] The pharmaceutical composition comprises an active ingredient and a pharmaceutically acceptable carrier. Due to the small molecular weight and significant tissue penetration of nanobodies, the active ingredient is selected from nanobodies targeting the PODXL protein or host cells.

[0064] When the active ingredient is an immunoconjugate, the immunoconjugate contains a cytotoxic drug, a protein toxin, or a radionuclide. For example, a nanobody targeting the PODXL protein can be conjugated with a cytotoxic drug, a protein toxin, or a radionuclide to form an antibody-drug conjugate or a radioimmunoconjugate, used to target and deliver toxic molecules or radionuclides to tumor tissue; when the active ingredient is a host cell, the host cell is selected from engineered T lymphocytes or engineered natural killer cells.

[0065] This application example provides a detection kit and its use in the preparation of diagnostic reagents for detecting PODXL protein expression.

[0066] The assay kit contains a core recognition element and a solid-phase carrier. The core recognition element is a nanobody targeting the PODXL protein; when the core recognition element is an immunoconjugate, the immunoconjugate contains a fluorescent dye, enzyme, biotin, radioactive isotope, or magnetic beads, i.e., the nanobody targeting the PODXL protein forms a conjugate complex with the above-mentioned labeling to achieve the reading of the detection signal; the solid-phase carrier is selected from microplates, magnetic beads, or chromatography strips.

[0067] Nanobodies, host cells, pharmaceutical compositions, or detection kits targeting the PODXL protein show promise in the preparation of diagnostic reagents for detecting PODXL protein expression or drugs for treating PODXL protein-positive tumors. Methods for detecting PODXL protein expression include enzyme-linked immunosorbent assay (ELISA), immunohistochemistry, flow cytometry, or in vivo imaging.

[0068] Appendix: Nucleotide sequence of nanobody Nb-PODXL-C01: SEQ ID NO:1: EVQLVESGGGLVQPGGSLRLSCAASGADFSTGPMGWFRQAPGKGRELVAAITADGSGTYYPDSVEGRFTISRDNAKRMVYLQMNSLRAEDTAVYYCAARGEDASGSPYLLTESAFTGWGQGTQVTVSS.

[0069] The amino acid sequence of the PODXL protein from position 200 to 219: SEQ ID NO:2: TPTSSGHDHLMKISSSSSTV.

[0070] Mouse immunoglobulin κ chain signal peptide nucleotide sequence: SEQ ID NO:3: METDTLLLWVLLLWVPGSTG.

[0071] CDR1 nucleotide sequence: SEQ ID NO:4: GRTFSYNPMG.

[0072] CDR2 nucleotide sequence: SEQ ID NO:5: ISRTGGSTYYPDSVEG.

[0073] CDR3 nucleotide sequence: SEQ ID NO:6: AAAGVRAEDGRVRTLPSEY.

Claims

1. A nanobody targeting the PODXL protein, characterized in that, The nanobody includes a complementarity-determining region, and the amino acid sequences of the complementarity-determining regions CDR1, CDR2 and CDR3 are shown in SEQ ID NO:4, SEQ ID NO:5 and SEQ ID NO:6, respectively. The nanobody specifically binds to the DHLM core epitope at positions 207-210 within the amino acid sequence shown in SEQ ID NO:2 of the PODXL protein.

2. The nanobody targeting PODXL protein according to claim 1, characterized in that, The amino acid sequence of the nanobody is shown in SEQ ID NO:1; The 5' end of the nanobody encoding sequence is connected to a signal peptide sequence as shown in SEQ ID NO:3, and the 3' end of the nanobody encoding sequence is connected to a sequence encoding a 6xHis tag. The nanobody was obtained by constructing a recombinant expression plasmid pNCMO2-Nb-PODXL-C01 containing the encoding sequence of the nanobody and transforming it into host cells for expression.

3. The nanobody targeting the PODXL protein according to claim 2, characterized in that, The nanobody is prepared by the following steps: HEK293F cells in suspension culture were adjusted to a predetermined cell density, and the recombinant expression plasmid pNCMO2-Nb-PODXL-C01 was transfected into the HEK293F cells using liposome transfection reagent to obtain the transfected cell system. The transfected cell system was cultured in suspension at a predetermined culture temperature, CO2 concentration and shaker speed. After the culture was completed, the cells and debris were removed by centrifugation, and the supernatant was filtered to obtain a clear liquid. The Ni-NTA agarose affinity chromatography column was equilibrated with binding buffer, the clarified solution was loaded onto the Ni-NTA agarose affinity chromatography column, and the column was washed with the binding buffer to remove contaminating proteins. The Ni-NTA agarose affinity chromatography column was eluted with elution buffer, and the eluent was collected, concentrated, and loaded onto a Superdex 75 Increase 10 / 300 GL gel filtration chromatography column. The column was eluted with the mobile phase, and the main peak fraction was collected to obtain the purified nanobody.

4. The nanobody targeting PODXL protein according to claim 3, characterized in that, The predetermined cell density is 2.0 × 10⁻⁶. 6 cells / mL - 3.0 × 10 6 The volume-to-mass ratio of the liposome transfection reagent to the recombinant expression plasmid pNCMO2-Nb-PODXL-C01 is 2:1-4:1, and the transfection is performed at room temperature for 10-20 minutes. The predetermined culture temperature is 36.5℃-37.5℃, the CO2 concentration is 7.5%-8.5%, the shaking speed is 120rpm-130rpm, and the suspension culture time is 5-7 days; the centrifugation is carried out at a centrifugal force of 3800g-4200g for 15min-25min, and the filter membrane used for filtration has a pore size of 0.22μm; The binding buffer contains sodium phosphate at a concentration of 15 mM-25 mM, NaCl at a concentration of 450 mM-550 mM, and imidazole at a concentration of 15 mM-25 mM; the pH of the binding buffer is 7.2-7.6; the loading flow rate is 1.0 mL / min-2.0 mL / min; and the washing volume is 8-12 times the column volume. The elution buffer contains 15mM-25mM sodium phosphate, 450mM-550mM NaCl, and 300mM-600mM imidazole, with a pH of 7.2-7.

6. The mobile phase is PBS buffer with a pH of 7.2-7.6, and the elution flow rate is 0.4mL / min-0.6mL / min, collecting the main peak with a retention volume in the range of 13mL-15mL.

5. The nanobody targeting PODXL protein according to claim 2, characterized in that, The nanobody is fused with a heterologous polypeptide sequence via peptide bonds to form a fusion protein; The heterologous polypeptide sequence is selected from the Fc domain of an antibody, the variable region of another specific antibody, or the transmembrane region and intracellular signal transduction domain of a chimeric antigen receptor.

6. The nanobody targeting PODXL protein according to claim 2, characterized in that, The nanobodies are chemically coupled with effective molecules to form immunoconjugates. The effector molecules are selected from fluorescent dyes, enzymes, biotin, radioactive isotopes, magnetic beads, cytotoxic drugs, protein toxins, or radionuclides.

7. An isolated nucleic acid molecule, expression vector, or host cell, characterized in that, The isolated nucleic acid molecule encodes a nanobody targeting the PODXL protein as described in any one of claims 1-5; The expression vector contains the isolated nucleic acid molecules; The host cell contains the isolated nucleic acid molecule integrated into the expression vector or genome; The host cells include engineered HEK293F cells, engineered T lymphocytes, or engineered natural killer cells.

8. A pharmaceutical composition, characterized in that, The pharmaceutical composition comprises an active ingredient and a pharmaceutically acceptable carrier; The active ingredient is selected from the nanobody targeting the PODXL protein as described in any one of claims 1-6 or the host cell as described in claim 7; When the active ingredient is an immunoconjugate, the immunoconjugate contains cytotoxic drugs, protein toxins, or radionuclides. When the active ingredient is the host cell, the host cell is selected from engineered T lymphocytes or engineered natural killer cells.

9. A test kit, characterized in that, The kit contains a core recognition element and a solid-phase carrier; The core recognition element is a nanobody targeting the PODXL protein as described in any one of claims 1-6; When the core recognition element is an immunoconjugate, the immunoconjugate contains fluorescent dyes, enzymes, biotin, radioactive isotopes, or magnetic beads. The solid support is selected from microplates, magnetic beads, or chromatography strips.

10. The use of a nanobody targeting PODXL protein as described in any one of claims 1-6, the host cell as described in claim 7, the pharmaceutical composition as described in claim 8, or the detection kit as described in claim 9 in the preparation of a diagnostic reagent for detecting PODXL protein expression or a medicament for treating PODXL protein-positive tumors; The methods for detecting PODXL protein expression include enzyme-linked immunosorbent assay (ELISA), immunohistochemistry, flow cytometry, or in vivo imaging.

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