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Method used for producing humanized antibodies or antigen combination fragments

A technology for humanized antibodies and binding fragments, which is applied in the field of production of humanized antibodies or antigen-binding fragments, can solve the problems of difficulty and wide application limitations, and achieve the effects of improving success rate, avoiding dependence and maintaining affinity

Active Publication Date: 2014-02-19
NANJING LEGEND BIOTECH CO LTD
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Problems solved by technology

In addition, since the technique relies on structural biology, which is not easy for many laboratories, its widespread application is limited

Method used

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  • Method used for producing humanized antibodies or antigen combination fragments
  • Method used for producing humanized antibodies or antigen combination fragments
  • Method used for producing humanized antibodies or antigen combination fragments

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Embodiment 1

[0040] 1.1. Cloning and sequence determination of parent monoclonal antibody

[0041] Anti-c-Myc polypeptide murine hybridoma monoclonal antibody samples are from GenScript Inc (Cat. No. RP11731). This murine mAb is hereinafter abbreviated as aM.

[0042] Total RNA was extracted from aM hybridoma cells aM using TRIzol (Invitrogen, Carlsbad, CA). cDNA was generated by reverse transcription using the Omniscript reverse transcription kit (QIAGEN, Shanghai, China). Using the obtained cDNA as a template, the V of aM was amplified by PCR with the following primers: H and V L Gene: for V L The forward primer of gene amplification is 5'-TTATTTACTCGCGGCCCAGCCGGCC-3' (SEQ ID NO.1), and the reverse primer is 5'-GGTGCAGCCACCGTACGTTTGATTTC-3' (SEQ ID NO.2); for V H The forward primer for gene amplification is 5'-CATGGCCGAGGTGCAGCTGGCTAGC-3' (SEQ ID NO.3), and the reverse primer is 5'-TGCGGCCCCATTTGCGGCCGCAGAG-3' (SEQ ID NO.4). Then V H and V L The PCR product of the gene was cloned...

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Abstract

The invention discloses a method used for producing humanized antibodies or antigen combination fragments. The method comprises following steps: antibody heavy chain variable region and light chain variable region sequences of a non-human organism specific to a certain antigen are obtained; the sequences are compared with amino acid sequences of human embryonal system antibodies; human embryonal system amino acid sequences, which possesses highest homology with the heavy chain variable region and the light chain variable region of the non-human organism, are selectively respectively; human embryonal system antibody heavy chain and light chain variable region frame libraries are constructed, and a complete frame assembly library is assembled; the frame assembly library is expressed so as to obtain humanized heavy chain variable region and light chain variable region, which are capable of combining with the antigen, by screening, and realize preparation of the humanized antibodies or the antigen combination fragments of the humanized heavy chain variable region and light chain variable region. The method is capable of avoiding dependence on antibody structure analysis in antibody engineering processes, at the same, realizing screening on antibody expression and stability, and maintaining affinity of the humanized antibodies as far as possible.

Description

technical field [0001] The invention belongs to the field of biological immunity and relates to a method for producing humanized antibodies or antigen-binding fragments. Background of the invention [0002] hybridoma technology [1] Since its establishment, a large number of murine monoclonal antibodies (mAbs) have been produced and characterized. Many of these are used in the diagnosis of human diseases such as cancer, infectious diseases, autoimmune diseases and others. However, due to its ability to broadly elicit human anti-mouse antibody (HAMA) responses in human patients [2] , so that its clinical application in the treatment of the above diseases is limited. HAMA reactions occur in up to 50% of patients following administration of murine hybridoma-derived antibodies [3] , which seriously jeopardizes the safety, efficacy and biological half-life of these antibody drugs. In addition, the constant regions of murine antibodies cannot directly induce human immune effec...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K16/18C12N15/70
Inventor 张剑冰武术章方良
Owner NANJING LEGEND BIOTECH CO LTD
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