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Human source antibody and humanization remolding method thereof

A humanized antibody and antibody technology, applied in chemical instruments and methods, biochemical equipment and methods, antibodies, etc., can solve the problems of complex high-affinity antibody methods, time-consuming and labor-intensive methods, and different antibodies are not optimal

Inactive Publication Date: 2010-12-29
UNIV OF SCI & TECH OF CHINA
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Compared with the first method, this method can obtain the optimized framework sequence more quickly, and can further improve the antigen affinity of the antibody on this basis; It may not be optimal, and the method of screening high-affinity antibodies by constructing a phage library is also complicated, time-consuming and labor-intensive

Method used

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  • Human source antibody and humanization remolding method thereof
  • Human source antibody and humanization remolding method thereof
  • Human source antibody and humanization remolding method thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0088] Example 1. Humanized transformation of chimeric antibody chA21

[0089] The sequence of the chimeric antibody chA21 has already been described by L.S.Cheng et al. (Chen L.S., Liu A.P., Liu.J. Construction, expression and characterization of the engineered antibody against tumor surface antigen P185 c-erbb-2 .Cell Res.2003, 13:35-48), such as figure 1 shown. Structure of chimeric antibody chA21: two identical protein chains joined by a disulfide bond formed in the Fc region. Each protein chain in turn consists of a light chain variable region (V L ), linker, heavy chain variable region (V H ) and the Fc region are sequentially connected, and its amino acid sequence is shown in SEQ ID NO: 9, wherein the 1-114th position is V L , the 115th-134th bit is linker, the 135th-253rd bit is V H , the 254th-489th position is Fc.

[0090] 1. Selection of Human Antibody Template

[0091] Sequence similarity comparison: compare the amino acid sequence similarity between the li...

Embodiment 2

[0114] Example 2. Construction of expression vectors for various antibodies

[0115] 1. Construction of pSectag2A-dFc vector

[0116] The pSectag2A vector was purchased from Invitrogen. Use PCR to amplify the Fc fragment, design primers to add EcoRI and XhoI restriction sites at the N-terminal and C-terminal of the Fc fragment, respectively, then double-digest the Fc fragment with EcoRI and XhoI, and recover the double-digested fragment; at the same time, pSectag2A plasmid Carry out double digestion with EcoRI and XhoI, and recover the double-digested fragment; connect the double-digested Fc fragment and pSectag2A vector with T4-DNA ligase, transform the ligated product into Top10 competent cells, and extract the plasmid for double-digestion. Restriction identification and sequencing verification, as a result, the gene sequence inserted between the EcoRI and XhoI restriction sites of pSectag2A is shown in nucleotides 760-1470 in SEQ ID NO: 2 (ie the Fc fragment), indicating t...

Embodiment 3

[0156] Example 3, Transient 293T cell expression of humanized antibody and purification of expression product

[0157] (1) Expression and purification of antibodies H1-1, H1-2, H2-1, H1-V1, ChA21

[0158] 293T cells were purchased from ATCC, catalog number CRL-11268.

[0159] DMEM medium was purchased from GIBCO, the product catalog number is 11960.

[0160] Cultivate a sufficient amount of 293T cells (DMEM / 10% serum / 1% double antibody) in advance (double antibody is penicillin and streptomycin, purchased from Shanghai Sangong, product number BS732), when the cells reach about 80% fullness, in the right Several growth phases were used to transfect recombinant antibody plasmid DNA.

[0161]The transfection steps are as follows: (1) Add 20ul Lipofectamine 2000 to 1ml DMEM, mix well with fingers or a pipette tip, and let stand at room temperature for 5 minutes. (2) Then add 10ug of plasmid DNA ready for transfection to 1ml DMEM and mix well. (3) Mix (1) and (2) (need to trans...

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Abstract

The invention discloses a human source antibody and a humanization remolding method thereof. The antibody is one of the following proteins: a) protein consisting of amino acid sequences from 1st to 253rd locus from the N end in a sequence 3 in a sequence table; and b) protein consisting of amino acid sequences shown in the sequence 3 in the sequence table. When the remolded antibody structure is compared with the initial chimeric antibody, the affinity of the remolded antibody with antigen is approximate, and the remolded antibody reaches complete humanization, and has better clinical application prospect.

Description

technical field [0001] The invention relates to a humanized antibody and a humanized transformation method thereof. Background technique [0002] P185 is an important receptor tyrosine kinase on the cell surface encoded by the oncogene Her2 / erbB-2, which belongs to the epidermal growth factor receptor family. Similar to other members of this family, the P185 / Her2 receptor consists of three domains: the extracellular region, the transmembrane region and the intracellular region, and the extracellular region (ECD) is divided into four subregions, among which I, III The subregion is a leucine-rich region, and the II and IV subregions are a cysteine-rich region (Carpenter G. (1987) Receptors of epidermal growth factor and other polypeptide mitogens.Annu.Rev.Biochem.56:881- 914). Epidermal growth factor family receptors are widely distributed in various tissues, especially expressed in epithelial tissue, mesenchymal tissue and neurogenesis tissue, and play an important role in ...

Claims

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Application Information

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IPC IPC(8): C07K16/32C07K16/28C12N15/13C12N15/63C12N1/15C12N1/19C12N1/21C12N5/10C12N7/01A61K39/395A61K48/00A61P35/00
CPCC07K2317/92C07K16/32C07K2317/24A61P35/00
Inventor 肖卫华常亮郭雨刚胡思怡刘兢
Owner UNIV OF SCI & TECH OF CHINA
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