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Humanized anti-PD-1 and c-MET bispecific antibody, and preparation method and application thereof

A bispecific antibody, PD-1 technology, applied in the direction of antibodies, anti-receptor/cell surface antigen/cell surface determinant immunoglobulin, anti-tumor drugs, etc., can solve problems such as toxic reactions and side effects, and achieve High activity, simple preparation process, and low immunogenicity

Inactive Publication Date: 2015-11-25
FUDAN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] Antineoplastic drugs, because their targets are usually expressed in normal tissues, are likely to cause toxic reactions in normal tissues, resulting in serious side effects

Method used

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  • Humanized anti-PD-1 and c-MET bispecific antibody, and preparation method and application thereof
  • Humanized anti-PD-1 and c-MET bispecific antibody, and preparation method and application thereof
  • Humanized anti-PD-1 and c-MET bispecific antibody, and preparation method and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0029] 1) Sequence design of humanized anti-PD-1 and anti-c-MET bispecific antibody and construction of expression plasmid

[0030] By comparing human antibody libraries, computer molecular structure simulation, molecular docking simulation and sequence analysis, design recombinant humanized anti-PD-1 and anti-c-MET bispecific antibody molecules (including affinity maturation, structure optimization, humanization) and codon optimization), and on the basis of optimizing the signal peptide sequence and plasmid expression vector replicon, construct the expression plasmid of recombinant humanized anti-PD-1 and anti-c-MET bispecific antibody;

[0031] 2) Expression of humanized anti-PD-1 and c-MET bispecific antibody

[0032] Acclimate 293E and CHO cells to adapt to serum-free suspension culture;

[0033] Recombinant humanized anti-PD-1 and anti-c-MET bispecific antibody expression plasmids were transiently transfected into 293E cells, and protein expression and purification condi...

Embodiment 2

[0037] Determination of humanized anti-PD-1 and c-MET bispecific antibody

[0038] 1) Specificity and affinity analysis and determination

[0039] Use biomacromolecule interaction analyzers (Biacore, ITC, etc.) to track the interaction between recombinant humanized anti-PD-1 and anti-c-MET bispecific antibodies and the extracellular domain of target receptors in real time, and analyze their binding specificity Sex, affinity and kinetic properties;

[0040] The binding affinity and specificity of recombinant humanized anti-PD-1 and anti-c-MET bispecific antibodies to target receptors (PD-1, c-MET) on the surface of living cells were analyzed by flow cytometry;

[0041] 2) Stimulate T cell proliferation and cytokine secretion

[0042] Human peripheral blood was cultured from whole blood, pretreated with recombinant humanized anti-PD-1 and anti-c-MET bispecific antibodies, and then stimulated with leucoagglutinin (PHA-L) to evaluate recombinant humanized anti-PD-1 1 and the ab...

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PUM

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Abstract

The invention belongs to the biotechnical field, and concretely relates to a recombinant humanized anti-PD-1 and c-MET bispecific antibody, and a preparation method and an application thereof. Humanized antibody database comparison, computer molecular structure simulation, molecular docking simulation and sequence analysis are carried out, and recombinant humanized anti-PD-1 and c-MET bispecific antibody molecule designing comprising affinity maturation, structure optimization, humanization and codon optimization is carried out, and an expression plasmid of the recombinant humanized anti-PD-1 and c-MET bispecific antibody is constructed on the basis of an optimized signal peptide sequence and a plasmid expression vector replicon. The bispecific antibody has the characteristics of low immunogenicity, simple and safe preparation process, high yield, high purity and high activity, and can be used to further prepare new specific drugs for treating non-small cell lung cancer.

Description

technical field [0001] The invention belongs to the field of biological technology, and in particular relates to a humanized anti-PD-1 and c-MET bispecific antibody and its preparation method and application. Background technique [0002] The prior art discloses that monoclonal antibodies have been widely used in tumor therapy. Studies have shown that traditional monoclonal antibodies kill tumor cells mainly by recruiting immune effector cells with Fcγ receptors, activating complement, inducing apoptosis, and inhibiting growth factors; however, because T cells do not have Fcγ receptors on the surface, traditional monoclonal antibodies Monoclonal antibodies cannot recruit T cells for treatment, thus limiting the therapeutic effect of monoclonal antibodies. [0003] Studies have shown that T cells play an important role in tumor therapy, and inducing T cell responses in animal models can cause tumor regression and inhibit tumor recurrence. Some studies have reported that inf...

Claims

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Application Information

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IPC IPC(8): C07K16/28A61K39/395A61P35/00
Inventor 于敏吴毅
Owner FUDAN UNIV
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