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Anti-CD20 antibody-monomethyl auristatin E conjugate, preparation method and application thereof

A technology of dolastatin and conjugates, applied in the fields of biotechnology and medicine, can solve problems such as cell damage, reduce toxic side effects, and enhance anti-tumor effects

Active Publication Date: 2014-05-21
ZHEJIANG UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, because it has strong non-selective toxicity and can cause damage to normal cells, it cannot be used as a medicine by itself.

Method used

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  • Anti-CD20 antibody-monomethyl auristatin E conjugate, preparation method and application thereof
  • Anti-CD20 antibody-monomethyl auristatin E conjugate, preparation method and application thereof
  • Anti-CD20 antibody-monomethyl auristatin E conjugate, preparation method and application thereof

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Experimental program
Comparison scheme
Effect test

Embodiment 12F2

[0057] Embodiment 12 Construction of F2 mutant-dolastatin conjugate

[0058] Construction of 12F2 Antibody Heavy Chain and Light Chain Expression Vectors

[0059] (1) Synthesis of 2F2 antibody variable region sequence

[0060] According to the patent document with publication number US2004 / 0167319A15, the variable region DNA sequences of the heavy chain and light chain of the 2F2 antibody were synthesized as follows:

[0061] Light chain variable region (V L ) (including the signal peptide sequence and restriction site, the underlined part is the restriction site, and the lowercase letter part is the signal peptide sequence):

[0062] 5'- atggaagccccagctcagcttctcttcctcctgctactctggctcccagataccaccggaGAAATTGTGTTGACACAGTCTCCAGCCACCCTGTCTTTGTCTCCAGGGGAAAGAGCCACCCTCTCCTGCAGGGCCAGTCAGAGTGTTAGCAGCTACTTAGCCTGGTACCAACAGAAACCTGGCCAGGCTCCCAGGCTCCTCATCTATGATGCATCCAACAGGGCCACTGGCATCCCAGCCAGGTTCAGTGGCAGTGGGTCTGGGACAGACTTCACTCTCACCATCAGCAGCCTAGAGCCTGAAGATTTTGCAGTTTATTACTGTCAGCAGCGTAGCAACT...

Embodiment 2

[0117] The biological activity of embodiment 2 conjugates

[0118] Next, the CD20 positive cells Ramos, Raji, Daudi and WIL2-S were used as objects to detect the biological activity of the 2F2 mutant and the 2F2 mutant-vcMMAE conjugate.

[0119] 1 Flow affinity assay

[0120] (1) Take 1×10 6 Ramos cells were treated with different concentrations (10, 3.33, 1.11, 0.37, 0.12 μg / mL) of 2F2 antibody, 2F2 mutant (Thio-2F2), 2F2 mutant-vcMMAE (Thio-2F2-vcMMAE) at 1% Incubate in BSA (dissolved in PBS) solution at 4°C for 30 minutes;

[0121] (2) After washing twice with PBS, add FITC-labeled goat anti-human IgG (H+L) polyclonal antibody (diluted 1:200) and incubate at 4°C for 30 min; after washing with PBS, measure the average fluorescence intensity of the cells by flow cytometry (MFI).

[0122] Flow cytometry was used to detect the binding of 2F2 antibody, 2F2 mutant, and 2F2 mutant-vcMMAE to CD20 positive cell Ramos, and the average fluorescence intensity of FITC after secondar...

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Abstract

The invention discloses an anti-CD20 antibody-monomethyl auristatin E (MMAE) conjugate, its preparation method and application. The anti-CD20 antibody-monomethyl auristatin E conjugate is formed by coupling of monomethyl auristatin E to the anti-CD20 antibody through a linking arm. Specifically, the light chain amino acid sequence of the anti-CD20 antibody is shown as SEQ ID No.1, and its heavy chain amino acid sequence is shown as SEQ ID No.2. The anti-CD20 antibody-monomethyl auristatin E conjugate can be applied in preparation of antitumor drugs. The conjugate provided in the invention has the biological functions of resisting both the CD20 antibody and MMAE, and under cooperation of the two, the anti-tumor effect can be significantly enhanced.

Description

technical field [0001] The invention belongs to the fields of biotechnology and medicine, and in particular relates to an anti-CD20 antibody-dolastatin conjugate and a preparation method and application thereof. Background technique [0002] CD20 is a unique membrane surface protein on the surface of B lymphocytes and B lymphoma cells, which is not expressed in hematopoietic stem cells, primitive B cells, normal plasma cells and other normal tissues (Bubien J K et al., J Cell Biol, 1993, 121 (5): 1121-1132). Therefore, the development of monoclonal antibodies against CD20 antigen can specifically kill B cells and B lymphoma cells and achieve good therapeutic effects. [0003] Ofatumumab (2F2) is a fully humanized targeting anti-CD20 monoclonal antibody, which was approved by the FDA in 2009 for refractory chronic lymphocytic leukemia (CLL) ineffective in the treatment of fludarabine and alemtuzumab (CASTILLO J. Expert Opin Investing Drμgs, 2009, 18(4):491-500.). 2F2 binds...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C07K19/00A61K38/07A61K47/48A61P35/00C07K1/107
Inventor 陈枢青李朝辉张骞徐晓倩
Owner ZHEJIANG UNIV
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