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Anti-human serum albumin single-chain antibody and method for connecting polypeptide drugs to carbon end thereof

A single-chain antibody and drug technology, applied in the direction of anti-animal/human immunoglobulin, drug combination, pharmaceutical formula, etc., to achieve the effects of easy large-scale production, avoiding immunogenicity, and weak immunogenicity

Active Publication Date: 2014-12-10
李冉
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The construction of mutants can usually reduce the sensitivity of polypeptides to hydrolytic enzymes and effectively prolong the half-life of polypeptide biopharmaceuticals. However, many mutants with longer half-lives will change the activity of drugs. To obtain a mutant that can meet the half-life requirements and at the same time Mutants that do not alter drug activity are difficult
Therefore, this technology is not suitable for widespread application in the field of protein drugs. PEGylation can improve the stability of protein drugs and reduce their immunogenicity, but it has the potential to affect the biological activity of polypeptide biopharmaceuticals. It is difficult to purify peptide biopharmaceuticals after PEGylation, which increases the difficulty and production cost in the production process
Direct fusion with human proteins with longer half-life is a feasible and effective method to extend the half-life of drugs, but this fusion method greatly increases the molecular weight of polypeptide biopharmaceuticals, which brings many problems to production and purification. Molecular weight The increase of the drug will also change some pharmacokinetic properties of the drug, which may make it difficult to reach the target position, thereby affecting the efficacy of the drug

Method used

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  • Anti-human serum albumin single-chain antibody and method for connecting polypeptide drugs to carbon end thereof
  • Anti-human serum albumin single-chain antibody and method for connecting polypeptide drugs to carbon end thereof
  • Anti-human serum albumin single-chain antibody and method for connecting polypeptide drugs to carbon end thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0029] Example 1, obtaining the single-chain antibody in the present invention:

[0030] 1. Construction of human antibody library

[0031] Screen the variable region sequences of antibodies with specific recognition ability for human serum albumin from the humanized single chain antibody library. The gene fragment is amplified by polymerase chain reaction and spliced ​​into a phage vector. The primers are as follows. An example of primers used to find the light chain CDR: Forward primer [5'-GATATCCAACTTACCCAATCC-3'] Reverse primer [5'-CCTCTTTATTTCTACCTTGG-3'], an example of primers for searching heavy chain CDR: forward primer [5'-GAAGTCCAGCTGCTCG-3'], reverse primer [5'-CGAAGAGACTGTGACTAGCGT-3'].

[0032] Antibody molecular fragments are expressed by phage and displayed on the surface of phage, and human serum albumin (HSA) is used as an antigen to screen the antibodies displayed on the surface of phage to obtain the antibody with the best specificity.

[0033] 2. Antibody...

Embodiment 2

[0039] Example 2, Analysis of the coding sequence of the single-chain antibody, construction of recombinant and expression vectors:

[0040]Propagate the 10D7 strain produced in Example 1 in LB culture medium, then use a DNA extraction kit to purify the plasmid in the cell line, and separate it by restriction endonuclease cutting and 2% agarose gel electrophoresis to obtain The coding sequence of the variable region of the heavy chain, the coding sequence of the variable region of the light chain was obtained by the same method, and the human antibody variable region universal primers were used for PCR amplification, and then the obtained amplification products were sent to Eurofin for sequencing. The results of the obtained DNA sequence and protein sequence are shown in Table 2.1 and Table 2.2, wherein the sequence listed in Table 2.1 is the light chain region (C-SEQ-04) of the humanized anti-human serum albumin single chain antibody, and Its variable region sequence (C-SEQ-0...

Embodiment 3

[0044] Example 3, linking the light and heavy chain sequences of the single-chain antibody and exendin-4, a polypeptide biological drug for the treatment of type 2 diabetes, to form a drug combination:

[0045] The DNA of the light chain region and the heavy chain region of the antibody whose sequence has been determined is linked by direct synthesis or recombinant PCR. The hinge part is the VH chain region of the single-chain antibody with the DNA sequence corresponding to SEQ ID No.2. The operation method of PCR is a conventional method commonly used in the field. The sequencing of the sequence synthesized by Invitrogen Company is completely the same as the sequencing result of the link sequence of the antibody light and heavy chains completed by the method of recombinant PCR. The sequencing results are shown in the table 3.1 shows:

[0046] GAT ATC CAA CTT ACC CAA TCC CCA AGT AGC [V L ] 5 10 CTT TCG GCC TCG ...

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Abstract

The invention discloses an anti-human serum albumin single-chain antibody and a method for connecting polypeptide drugs to a carbon end of the anti-human serum albumin single-chain antibody. According to the anti-human serum albumin single-chain antibody a, a VH (Variable fragments of heavy chain) chain area of the single-chain antibody has a CDR (Complementarity-Determining Region) amino acid sequence of a complementary recognition domain, shown as C-SEQ (Sequence)-05, C-SEQ-06 and C-SEQ-07; a VL (Variable fragments of light chain) chain area of the single-chain antibody has a CDR amino acid sequence of a complementary recognition domain, shown as C-SEQ-08, C-SEQ-09 and C-SEQ-010; and the amino sequence of the single-chain antibody and the amino sequences of polypeptide drugs are connected from a nitrogen end of the single-chain antibody by a protein linking hinge shown as C-SEQ-01 or C-SEQ-03. The single-chain antibody disclosed by the invention has small molecular weight, high antibody specificity, weak immunogenicity and good water solubility, and is easily massively produced through a fermenting way. The invention also discloses a composition manufactured by using the single-chain antibody disclosed by the invention; and the composition can effectively prolong a half-life period of the polypeptide drugs.

Description

technical field [0001] The invention relates to a single-chain antibody, in particular to a method for anti-human serum albumin single-chain antibody and its carbon-terminal-linked polypeptide medicine. Background technique [0002] At present, peptide-based biopharmaceuticals are rapidly being promoted around the world. [0003] Peptide biopharmaceuticals have remarkable effects and can treat many genetic diseases that compound drugs cannot be effective. However, one of the biggest problems in the use of peptide biological drugs is that the attenuation period of such drugs in the human body is generally short, resulting in a relatively high frequency of drug use. Bring larger treatment cost and medication pain to patient. Therefore, prolonging the half-life of polypeptide biopharmaceuticals has become an urgent issue in the field of biopharmaceuticals. [0004] The current methods for prolonging the half-life of polypeptide biopharmaceuticals mainly include constructing...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C07K16/18C07K7/08C07K19/00A61K47/48A61K38/22A61P3/10
Inventor 杨艳坤龙泉张芃芃杨冬刘冰
Owner 李冉
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