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Humanized bispecific nano antibody targeting EGFR (epidermal growth factor receptor) dimer interface

A bispecific, nanobody technology, applied in the field of genetic engineering, can solve the problem that the anti-tumor activity is difficult to reach conventional antibodies, etc., and achieve the effect of enhancing the inhibitory effect

Active Publication Date: 2020-03-20
GUANGDONG PHARMA UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Since a single nanobody does not have the ADCC effect of conventional antibodies, the in vivo anti-tumor activity of monospecific antibodies targeting signaling pathways may hardly reach the level of conventional antibodies

Method used

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  • Humanized bispecific nano antibody targeting EGFR (epidermal growth factor receptor) dimer interface
  • Humanized bispecific nano antibody targeting EGFR (epidermal growth factor receptor) dimer interface
  • Humanized bispecific nano antibody targeting EGFR (epidermal growth factor receptor) dimer interface

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0037]Example 1 Gene design of humanized bispecific nanobody targeting EGFR dimer interface

[0038] According to Genbank’s published nanobody targeting human serum albumin HSA pm6A6, anti-NK cell surface Fc receptor FcγRIIIa (CD16) nanobody and the laboratory’s existing nanobody targeting EGFR dimer interface EGFR dimer Nb77, construction of a humanized bispecific nanobody targeting the EGFR dimer interface.

[0039] Wherein, the amino acid sequence of HSA pm6A6 is:

[0040] AVQLVESGGGLVQPGNSLRLSCAASGFTFRSFGMSWVRQAPGKEPEWVSSISGSGSDTLYADSVKGRFTISRDNAKTTLYLQMNSLKPEDTAVYYCTIGGSLRSSQGTQVTVSS (the amino acid sequence is shown in SEQ ID NO.3);

[0041] The amino acid sequence of the Nanobody against Fc receptor FcγRIIIa (CD16) on the surface of NK cells is:

[0042] EVQLVESGGELVQAGGSLRLSCAASGLTFSSYNMGWFRRAPGKEREFVASITWSGRDTFYADSVKGRFTISRDNAKNTVYLQMSSLKPEDTAVYYCAANPWPVAAPRSGTYWGQGTQVTVS (the amino acid sequence is shown in SEQ ID NO.4);

[0043] The amino acid sequence of the nan...

Embodiment 2

[0048] Example 2 Construction of a recombinant plasmid containing a humanized bispecific nanobody targeting the EGFR dimer interface

[0049] 1. Experimental method

[0050] Primers were designed using Primer Premier 6.0 software, and the primers were synthesized by Wuhan Jinkairui Bioengineering Co., Ltd. The design is shown in Table 1.

[0051] Table 1 Primer Design Sequence:

[0052]

[0053] F: upstream primer; R: downstream primer

[0054] Using the recombinant vector pUC57-NK-77 obtained in Example 1 as a template, the target gene was amplified by PCR, and the pGAPZα-A plasmid was extracted at the same time, followed by enzyme digestion and ligation of the target gene and the vector.

[0055] 2. Experimental results

[0056] The target gene and the expression vector pGAPZαA were digested with Xho I and Xba I at the same time. After the plasmid was recovered, the results of agarose electrophoresis identification were shown in figure 2 . The size of the vector is ...

Embodiment 3

[0057] Example 3 Transformation and expression of a recombinant plasmid containing a humanized bispecific nanobody targeting the EGFR dimer interface

[0058] 1. Experimental method

[0059] The positive transformant NK-77-pGAPZαA-DH5α obtained in Example 2 was expanded and cultivated, and a large amount of recombinant plasmid NK-77-pGAPZαA was extracted, and the NK-77-pGAPZαA of the recombinant plasmid was linearized. The reaction system was: ddH 2 O 30 μl, 10×K buffer 20 μl, BlnⅠ 10 μl, and recombinant plasmid 140 μl were centrifuged briefly for 10 seconds, digested in a water bath at 37°C for 2 hours to linearize the plasmid NK-77-pGAPZαA.

[0060] Take the current Pichia X-33 competent cells, add linearized NK-77-pGAPZαA plasmid for electrotransformation, use Zeocin YPD solid medium plate containing 1000 μg / mL to screen highly resistant transformants, and colony PCR to identify highly resistant recombinants Substitutes, SDS-PAGE electrophoresis to identify whether the cor...

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Abstract

The invention discloses a humanized bispecific nano antibody targeting an EGFR (epidermal growth factor receptor) dimer interface. The amino acid sequence of the humanized bispecific nano antibody isshown as SEQ ID NO: 1. The antibody is combined with PBMCs (peripheral blood mononuclear cells) in an in-vitro test, so that the inhibition effect on tumor cells with EGFR overexpression can be effectively enhanced. The humanized bispecific nano antibody is a bispecific nano antibody capable of targeting the EGFR dimer interface, an NK (natural killer) cell Fc gamma RIIIa receptor and human serumalbumin, and has tumor inhibition activity. The humanized bispecific nano antibody has the potential of developing a therapeutic nano antibody drug which has an anti-EGFR homologous and anti-heterodimerization function, a conventional antibody ADCC (antibody dependent cytotoxicity) effect and a long half-life period, and a novel method is provided for solving the drug resistance problem of an EGFRtargeting drug.

Description

technical field [0001] The invention relates to the technical field of genetic engineering, and more specifically, relates to a humanized bispecific nanobody targeting the EGFR dimer interface. Background technique [0002] Epidermal growth factor receptor (EGFR or Her 1 / ErbB 1) is an important member of the human epidermal receptor family, plays an important role in the growth and differentiation of normal epidermal cells, and is an effective target for the treatment of many epithelial cancers. EGFR, as a receptor tyrosine kinase (RTK), consists of a glycosylated extracellular domain, a single hydrophobic transmembrane segment, and an intracellular portion (comprising a membrane-proximal segment, a protein kinase domain, and a carboxyl terminus). Binding of ligands such as epidermal growth factor (EGF) to cell surface EGFRs results in exposure of the dimer interface and homodimerization with another EGFR or heterodimerization with other family members. Dimerization leads t...

Claims

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Application Information

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IPC IPC(8): C07K16/46C12N15/13C12N15/81C12N1/19A61K39/395A61P35/00C12R1/84
CPCA61K2039/505A61P35/00C07K16/18C07K16/283C07K16/2863C07K16/32C07K16/40C07K2317/24C07K2317/31C07K2317/569C07K2317/732C07K2317/94C12N15/815
Inventor 李黄金赵林温碧燕
Owner GUANGDONG PHARMA UNIV
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