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CN-121991229-A - Nanometer antibody targeting PODXL protein and application thereof

CN121991229ACN 121991229 ACN121991229 ACN 121991229ACN-121991229-A

Abstract

The invention relates to the field of biotechnology and biological medicine, and discloses a nano antibody targeting PODXL protein and application thereof, wherein the nano antibody comprises complementarity determining regions CDR1-CDR3 shown in SEQ ID NO. 4-SEQ ID NO. 6, specifically binds DHLM core epitopes of 207 th-210 th positions in an amino acid sequence shown in SEQ ID NO.2 in the PODXL protein, and is obtained by transfecting HEK293F cells with recombinant expression plasmids, and purifying through suspension culture, centrifugal filtration, ni-NTA affinity chromatography and gel filtration chromatography. The invention utilizes the single domain structural characteristics of the nano antibody to specifically recognize the DHLM epitope of the PODXL protein, reduces the risk of cross reaction, combines with the strategy of fusing heterologous polypeptide or coupling effector molecules, and realizes the application of the nano antibody in the preparation of diagnostic reagents and therapeutic drugs for PODXL protein positive tumors.

Inventors

  • HUANG DEGAO
  • WANG XIA
  • WANG PEIPEI
  • LIAO LINCHUAN

Assignees

  • 四川大学

Dates

Publication Date
20260508
Application Date
20260305

Claims (10)

  1. 1. The nano antibody targeting the PODXL protein is characterized by comprising a complementarity determining region, wherein the amino acid sequences of CDR1, CDR2 and CDR3 of the complementarity determining region are respectively shown as SEQ ID NO. 4, SEQ ID NO. 5 and SEQ ID NO. 6; the nano antibody specifically binds to DHLM core epitopes from 207 th to 210 th positions in an amino acid sequence shown as SEQ ID NO. 2 in PODXL protein.
  2. 2. The nanobody targeting PODXL protein according to claim 1, wherein the amino acid sequence of the nanobody is shown as SEQ ID No. 1; the 5 'end of the nano antibody coding sequence is connected with a signal peptide sequence which is shown as SEQ ID NO. 3, and the 3' end of the nano antibody coding sequence is connected with a sequence which is used for coding a 6xHis tag; The nanobody is obtained by constructing a recombinant expression plasmid pNCMO-Nb-PODXL-C01 containing the nanobody coding sequence and converting the recombinant expression plasmid into host cell expression.
  3. 3. The nanobody targeting PODXL protein of claim 2, wherein the nanobody is prepared by: Regulating HEK293F cells cultured in suspension to a preset cell density, and transfecting the recombinant expression plasmid pNCMO-Nb-PODXL-C01 into the HEK293F cells by using a liposome transfection reagent to obtain a transfected cell system; Performing suspension culture on the transfected cell system under the conditions of preset culture temperature, CO 2 concentration and rotation speed of a shaking table, centrifuging after the culture is finished to remove cells and fragments, and filtering supernatant to obtain clear feed liquid; Balancing a Ni-NTA agarose affinity chromatography column by using a binding buffer, loading the clarified feed liquid to the Ni-NTA agarose affinity chromatography column, and washing by using the binding buffer to remove the impurity protein; Eluting the Ni-NTA agarose affinity chromatographic column by using an elution buffer solution, collecting effluent liquid, concentrating, loading the effluent liquid to a Superdex 75 increasing 10/300 GL gel filtration chromatographic column, eluting by using a mobile phase, and collecting main peak components to obtain the purified nano antibody.
  4. 4. A nanobody targeting PODXL protein according to claim 3, wherein said predetermined cell density is 2.0 x 10 6 cells/mL-3.0×10 6 cells/mL, the volume mass ratio of said liposome transfection reagent to said recombinant expression plasmid pNCMO-Nb-PODXL-C01 is 2:1-4:1, and incubation is performed at room temperature for 10min-20min at transfection; The preset culture temperature is 36.5-37.5 ℃, the concentration of CO 2 is 7.5-8.5%, the rotating speed of the shaking table is 120-130 rpm, the suspension culture time is 5-7 days, the centrifugation is carried out for 15-25 min under the centrifugal force condition of 3800-4200 g, and the pore diameter of a filter membrane used for filtering is 0.22 mu m; The binding buffer solution comprises sodium phosphate with the concentration of 15mM-25mM, naCl with the concentration of 450mM-550mM and imidazole with the concentration of 15mM-25mM, the pH value of the binding buffer solution is 7.2-7.6, the loading flow rate is 1.0mL/min-2.0mL/min, and the washing volume is 8-12 times of the column volume; The elution buffer comprises sodium phosphate with the concentration of 15mM-25mM, naCl with the concentration of 450mM-550mM and imidazole with the concentration of 300mM-600mM, the pH value of the elution buffer is 7.2-7.6, the mobile phase is PBS buffer with the pH value of 7.2-7.6, the elution flow rate is 0.4mL/min-0.6mL/min, and the main peak with the retention volume in the range of 13mL-15mL is collected.
  5. 5. The nanobody targeting PODXL protein of claim 2, wherein said nanobody is fused to a heterologous polypeptide sequence by peptide bond 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 signaling domain of a chimeric antigen receptor.
  6. 6. The nanobody targeting PODXL protein of claim 2, wherein said nanobody is conjugated to an effector molecule via a chemical bond to form an immunoconjugate; The effector molecule is selected from the group consisting of a fluorescent dye, an enzyme, biotin, a radioisotope, a magnetic bead, a cytotoxic drug, a protein toxin, or a radionuclide.
  7. 7. An isolated nucleic acid molecule, expression vector or host cell encoding a PODXL protein-targeting nanobody of any one of claims 1-5; the expression vector comprises the isolated nucleic acid molecule; The host cell comprises the expression vector or the genome with the isolated nucleic acid molecule integrated therein; The host cells include engineered HEK293F cells, engineered T lymphocytes, or engineered natural killer cells.
  8. 8. A pharmaceutical composition comprising an active ingredient and a pharmaceutically acceptable carrier; the active ingredient is selected from a nanobody targeting PODXL protein according to any one of claims 1-6 or the host cell according to claim 7; wherein, when the active ingredient is an immunoconjugate, the immunoconjugate comprises a cytotoxic drug, a protein toxin, or a radionuclide; when the active ingredient is the host cell, the host cell is selected from an engineered T lymphocyte or an engineered natural killer cell.
  9. 9. A test kit comprising a core recognition element and a solid support; the core recognition element is a nanobody targeting PODXL protein according to any one of claims 1-6; Wherein, when the core recognition element is an immunoconjugate, the immunoconjugate comprises a fluorescent dye, an enzyme, biotin, a radioisotope, or a magnetic bead; The solid phase carrier is selected from a microwell plate, magnetic beads or a chromatographic test strip.
  10. 10. Use of a nanobody targeting PODXL protein according to any one of claims 1-6, a host cell according to claim 7, a pharmaceutical composition according to claim 8 or a detection kit according to claim 9 for the manufacture of a diagnostic reagent for detecting PODXL protein expression or a medicament for treating PODXL protein positive tumors; The detection method for PODXL protein expression comprises enzyme-linked immunosorbent assay, immunohistochemistry, flow cytometry or in vivo imaging.

Description

Nanometer antibody targeting PODXL protein and application thereof Technical Field The invention relates to the field of biotechnology and biological medicine, in particular to a nano antibody targeting PODXL protein and application thereof. Background Podocalyxin (PODXL) is a highly glycosylated type I transmembrane protein, is expressed in various malignant tumors such as oral squamous carcinoma, breast cancer, lung cancer and the like, is related to the growth, invasion and metastasis processes of tumor cells, and is used as a tumor marker and a treatment target. The existing research shows that DHLM region from 207 th to 210 th of amino acid sequence of PODXL protein is functional epitope of the protein, and the traditional monoclonal antibody aiming at the epitope has been used for research and development of related diseases. However, conventional monoclonal antibodies generally have a large molecular weight and a complex quaternary structure, resulting in limited penetration ability in solid tumor tissues, and difficult effective enrichment in deep focal tissues. The traditional antibody preparation process often involves complex cell culture and purification processes to maintain its activity, and its structural features limit the flexibility of genetic engineering, which is unfavorable for fusion with heterologous polypeptides or coupling with effector molecules to construct diverse diagnostic preparations. Currently, no nanobody molecule against the DHLM epitope of PODXL protein has been developed in the art, and the lack of such small molecule antibody tools limits the efficient targeted delivery against PODXL positive tumors and the construction of multifunctional diagnostic products. Therefore, obtaining a nanobody that specifically targets the PODXL protein DHLM epitope and has good tissue penetration ability and engineering potential is a problem that needs to be solved in the art. Disclosure of Invention Aiming at the defects of the prior art, the invention provides a nano antibody targeting PODXL protein and application thereof, and solves the problems that the existing antibody targeting the DHLM epitope of the PODXL protein has larger molecular weight, limited tissue penetration capacity and difficult diversified engineering transformation. In order to solve the problems, the invention provides the following technical scheme: The invention provides a nano antibody targeting PODXL protein, which adopts the following technical scheme: A nano antibody for targeting PODXL protein comprises a complementarity determining region, wherein the amino acid sequences of CDR1, CDR2 and CDR3 of the complementarity determining region are respectively shown as SEQ ID NO. 4, SEQ ID NO. 5 and SEQ ID NO. 6, and the nano antibody specifically binds to DHLM core epitopes from 207 th to 210 th positions in the amino acid sequence shown as SEQ ID NO. 2 in the PODXL protein. By adopting the technical scheme, due to the fact that the CDR1, CDR2 and CDR3 sequence combination which are specially designed are adopted, the nano-antibody can form a specific antigen binding groove, is matched with DHLM core tetrapeptide epitope of 207 th-210 th positions of PODXL protein, is DHLM epitope located on the exposed surface of a specific structural domain of the PODXL protein, can bind with a target antigen with higher affinity and specificity through targeting the region, reduces the risk of non-specific cross reaction with homologous proteins, and has tissue penetrating capacity superior to that of a traditional IgG antibody due to the characteristics of a single domain of the nano-antibody, and is beneficial to enrichment in the deep part of tumor tissues, so that the targeting recognition efficiency is improved. Preferably, the amino acid sequence of the nano antibody is shown as SEQ ID NO. 1, the 5 'end of the nano antibody coding sequence is connected with a signal peptide sequence which is coded as shown as SEQ ID NO.3, the 3' end of the nano antibody coding sequence is connected with a sequence which is coded with a 6xHis tag, and the nano antibody is obtained by constructing a recombinant expression plasmid pNCMO-Nb-PODXL-C01 containing the nano antibody coding sequence and converting host cell expression. By adopting the technical scheme, the full-length sequence shown in SEQ ID NO.1 comprises a CDR region responsible for specific binding and a framework region optimized through humanized design, is favorable for maintaining the structural stability of the protein under physiological conditions and reducing immunogenicity, and the signal peptide sequence shown in SEQ ID NO. 3 is introduced, so that a newly synthesized peptide chain can be guided into an endoplasmic reticulum cavity and finally secreted out of cells through a Golgi body to realize secretory expression, simplify the subsequent protein harvesting step, reduce host protein pollution caused by intracellular cleavage, and the 6xHis tag at the C end provi