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Camelid-derived nanobodies capable of specifically binding to carbonic anhydrase IX and applications thereof

A nano-antibody and carbonic anhydrase technology, applied in the direction of antibodies, applications, specific peptides, etc., can solve problems such as low concentration, restrictions on the clinical application of ultrasonic molecular imaging, and strong immunogenicity of monoclonal antibodies

Active Publication Date: 2021-09-24
中国人民解放军陆军特色医学中心
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] The targeted ultrasonic microbubbles modified by monoclonal antibodies have some unsatisfactory features, such as strong immunogenicity of monoclonal antibodies, poor stability, large relative molecular mass of monoclonal antibody-microparticle complexes, weak tissue penetration, and poor ability to actually reach the target. Low concentrations in tissues, etc., which restrict the clinical application of ultrasound molecular imaging

Method used

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  • Camelid-derived nanobodies capable of specifically binding to carbonic anhydrase IX and applications thereof
  • Camelid-derived nanobodies capable of specifically binding to carbonic anhydrase IX and applications thereof
  • Camelid-derived nanobodies capable of specifically binding to carbonic anhydrase IX and applications thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0068] Example 1 Eukaryotic expression of CA IX extracellular region

[0069]Using the primer pair CA IXECD-VF (5'-gatcagctagcacagaggttgccccggatgca-3', as shown in SEQ ID No.3) and CA IXECD-VR (5'-catgactcgaggtcaccagcagccaggcag-3', as shown in SEQ ID No.4) from the template plasmid A DNA fragment containing the region from Gln38 to Asp414 of the extracellular region of CA IX (hereinafter referred to as CA IX protein) was amplified from pMDCAIX, and inserted into the eukaryotic expression vector pRAG2a using NheI and XhoI restriction endonucleases. The constructed recombinant plasmid was transformed into competent cells E.coli Top 10, positive clones were identified by colony PCR, and finally the correct plasmid pRAG2a-huCA IXECD was obtained through sequencing verification. After extracting a large amount of the resulting plasmid, use 293fectin to transfect the plasmid DNA into HEK 293F cells for suspension culture and transient expression of the target protein. After the cel...

Embodiment 2

[0071] Example 2 Construction of immune nanobody library

[0072] Two male Xinjiang Bactrian camels about two years old were used for immunization, and the immunization injections were carried out 8 times for a duration of 105 days. The time points of the first two immunizations were day 0 and day 17, respectively, and the amount of antigen per injection per camel was 50 μg and 100 μg, respectively. The subsequent immunization time points were respectively on the 40th, 59th, 74th day and the 87th day, and the amount of antigen injected was 300 μg each time. The last two booster immunizations were completed within 1 week, and the time points were the 102nd and 105th days respectively. One week later, 200 mL of peripheral blood was collected from each camel for immune library construction.

[0073] The total RNA of camel peripheral blood lymphocytes was extracted by T R Izol reagent, the mRNA was extracted, and the mRNA was reverse transcribed into cDNA. Then, the heavy chain...

Embodiment 3

[0075] Example 3 Panning of Nanobodies against CA IX Extracellular Region

[0076] The phage display VHH immune library constructed above was used. Three rounds of in vitro directed screening were performed against the CA IX protein to isolate Nanobodies that specifically bind to CA IX. Briefly, approximately 4 × 10 10 The transformed TG1 cells were inoculated into 2×TY medium containing 100 μg / mL ampicillin and 1% glucose for culture. After 2h, add 4×1010 helper phage M13KO7 and incubate at room temperature for 30min for infection. The infected cells were collected by centrifugation, resuspended in 2×TY medium containing 100 μg / mL ampicillin and 50 μg / mL kanamycin, and cultured overnight at 37°C and 220 r / min. Centrifuge the culture supernatant, add PEG 6 000 / NaCl (20% PEG 6 000 and 2.5mol / L NaCl) solution to precipitate and concentrate the phage particles, and finally resuspend in sterile and pre-cooled PBS buffer.

[0077] The directional screening experiment used the s...

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Abstract

The invention relates to the field of biomedicine, in particular to a camel-derived nanobody capable of specifically binding to carbonic anhydrase IX (CA IX) and an application thereof. The invention expresses the extracellular region of the CA IX antigen through eukaryotic expression, immunizes camels with it, prepares and identifies nanobodies against the extracellular region of CA IX, and lays the foundation for the next step of targeted imaging and treatment.

Description

technical field [0001] The invention relates to the field of biomedicine, in particular to a camel-derived nanobody capable of specifically binding to carbonic anhydrase IX (CA IX) and an application thereof. Background technique [0002] Kidney cancer-associated antigen CA IX was discovered in 1986 and has been systematically and deeply studied. CA IX antigen has good specificity for kidney cancer. Immunohistochemical studies show that: more than 85% of primary renal cancer and metastatic renal cancer express CAIX antigen, and renal clear cell carcinoma, which accounts for 90% of renal cancer, almost all express this antigen, and normal renal tissue does not express CA IX antigen. CA IX is a single transmembrane protein located downstream of the VHL tumor suppressor gene and activated by the hypoxia-inducible factor-1 pathway. CA IX itself is a transmembrane glycoprotein composed of acidic amino acids, as CO 2 Catalyst for the reversible reaction of hydration, can cataly...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C07K16/40C12N15/13G01N33/573A61K39/395A61P35/00
CPCA61K2039/505A61P35/00C07K16/3038C07K16/40C07K2317/22C07K2317/569G01N33/573
Inventor 王洛夫胡明
Owner 中国人民解放军陆军特色医学中心
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