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Antibody conjugate, preparation method and application thereof

An antibody conjugate and antibody technology, applied in the field of biomedicine, can solve the problems of CD30 positive tumor drug resistance and high cytotoxicity

Active Publication Date: 2019-08-02
上海复旦张江生物医药股份有限公司 +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0006] The purpose of the present invention is to solve the problem of drug resistance and high cytotoxicity of drugs for treating CD30-positive tumors in the prior art, and provide an anti-human CD30 antibody conjugate with low toxicity and not easy to produce cell drug resistance, and its preparation method and application

Method used

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  • Antibody conjugate, preparation method and application thereof
  • Antibody conjugate, preparation method and application thereof
  • Antibody conjugate, preparation method and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0068] Example 1 Construction and expression of anti-human CD30 antibody cAC10

[0069] Synthesize DNA fragments encoding cAC10 monoclonal antibody heavy chain and light chain from the whole gene (see patent US7090843, B1RECOMBINANT ANTI-CD30ANTIBODIES AND USES THEREOF, Seattle Genetics, Inc., 2006, SEQ ID NO: 1 and SEQ ID NO: 9; and International Nonproprietary Names for Pharmaceutical Substances (INN).WHO Drug Information Vol.24, No.2, 2010), respectively cloned into the pEE12.4 eukaryotic expression vector of Lonza Company, and the operations of enzyme digestion and ligation were carried out according to commercial reagents Kit (DNALigation kit Ver2.0, TAKARA) instructions.

[0070] The constructed heavy chain and light chain expression vectors were respectively transformed into Escherichia coli DH5α, and positive clones were picked and inoculated in 500ml LB medium for amplification. DNA was extracted and purified using Qiagen's Ultrapure Plasmid DNA Purification Kit (Ult...

Embodiment 2

[0073] Embodiment 2 DAR detection method

[0074] The detection of DAR is based on the UV absorption of Ab and DM1 and the calculation of the coupling degree. The average number of DM1 linked to each antibody molecule is determined by measuring the absorbance at 252nm and 280nm. Ultraviolet / visible spectrophotometry (UV / Vis) is a simple and convenient method for determining protein concentration, as well as the average number of antibody-drug conjugates in an antibody drug-conjugate (ADC). DAR can be determined by using ADC absorbance measurements, and the extinction coefficients of the corresponding antibodies and drugs.

[0075] When the absorption coefficient of a certain pure substance under certain conditions is known, the test sample can be made into a solution under the same conditions, and its absorbance can be measured, and the content of the substance can be calculated by the following formula: A=E×c×l, Where A is the absorbance, E is the absorption coefficient, c i...

Embodiment 3

[0086] The preparation of embodiment 3 antibody conjugates

[0087] The invention guarantees the stability of the average coupling ratio (DAR) of the poison antibody by controlling the feeding ratio, pH, temperature, stirring, etc. of the coupling reaction.

[0088] A. Linker SMCC modified antibody:

[0089] Configuration buffer: 50 mM dipotassium hydrogen phosphate-potassium dihydrogen phosphate, 50 mM NaCl, 2 mM EDTA, pH 6.5. The antibody buffer was replaced by 10 times volume ultrafiltration, the final concentration of the antibody was 10 mg / mL, and argon gas was added to fill it. 1.5 ml of 20 mM SMCC (dissolved in DMSO or DMA) was added to 20 ml of cAC10 monoclonal antibody (Brentuximab, namely butuximab) solution, and reacted at room temperature for 4 hours. The reaction mixture was filtered through a Sephadex G25 gel column equilibrated with 5 column volumes of buffer. Collect the characteristic peaks of the antibody at OD280 to obtain the antibody modified by SMCC. ...

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Abstract

The invention discloses an antibody conjugate. A structural formula thereof is Ab-Lm-Yn, wherein Ab is an antihuman CD30 antibody cAC10, an active fragment or a variant thereof; Ab is only connected with L; Y is cytotoxic agent maytenin alkaloid DM1; n is 2-5 and m is more than or equal to n; an amido bond is formed by a left end of L and an amino group of lysine residue of Ab; a sulfur ether bondis formed by a right end of L and S in DM1 sulfydryl. The invention also discloses a preparation method of the antibody conjugate, the antibody conjugate prepared according to the preparation methodand an application of the antibody conjugate in preparing drugs for treating CD30 positive tumors. Main metabolites of the antibody conjugate disclosed by the invention can effectively accumulate in CD30 positive tumor cells. The antibody conjugate has excellent targeting property, tumor-killing effect and safety performance.

Description

technical field [0001] The invention relates to the field of biomedicine, in particular to an antibody conjugate and its preparation method and application. Background technique [0002] Differentiation antigen 30 (CD30), also known as Ki-1, belongs to the tumor necrosis factor receptor superfamily, is a type I transmembrane glycoprotein, and is normally expressed on activated B and T lymphocytes. Overexpressed on a variety of tumor cell membranes, including typical Hodgkin's lymphoma (Hodgkin's lymphoma), anaplastic large cell lymphoma (ALCL), cutaneous T-cell lymphoma (CTCL), primary mediastinal B-cell lymphoma ( PMBCL), diffuse large B-cell lymphoma (DLBCL) (Froese et al., J. Immunol. 139:2081 (1987); Carde et al., Eur. J. Cancer 26:474 (1990)). [0003] It is also expressed on the cell surface of many other tumors, especially those of lymphoid origin. Overexpression of the CD30 receptor on malignant cells is thought to contribute to survival and apoptosis resistance in...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): A61K47/68A61K47/54A61K31/537A61P35/00
CPCA61K31/537
Inventor 杨彤沈毅珺吴劲松赵莉陈奔张一帆曹雪梅徐珺郭青松高贝李华
Owner 上海复旦张江生物医药股份有限公司
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