Antigen-binding fragment of an antibody-dolastatin conjugate and its preparation method and application

A technology combining fragments and dolastatin, which is applied in the direction of antibody mimics/scaffolds, chemical instruments and methods, medical preparations of non-active ingredients, etc., can solve the problem of narrow antibody therapeutic window, difficulty in evaluating metabolic properties, and poor therapeutic effect. Good and other questions

Active Publication Date: 2021-03-30
ZHEJIANG UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] The DAR of antibody-conjugated drugs obtained by traditional chemical conjugation methods has poor uniformity, and it is difficult to evaluate the metabolic properties in vivo. Moreover, the heterogeneity of antibodies will result in a narrow therapeutic window and poor therapeutic effect.

Method used

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  • Antigen-binding fragment of an antibody-dolastatin conjugate and its preparation method and application
  • Antigen-binding fragment of an antibody-dolastatin conjugate and its preparation method and application
  • Antigen-binding fragment of an antibody-dolastatin conjugate and its preparation method and application

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Embodiment 1

[0030] The expression and purification of SortaseA include:

[0031] (1) The gene sequence encoding SortaseA enzyme was obtained by PCR from the genome of Staphylococcus aureus, and a His6 tag was added to its N-terminus, constructed in the expression vector PET28a, and expressed by Escherichia coli Rosetta plyss2 (DE3). The expression strain of sortaseA was inoculated into 1L of LB medium at a final concentration of 1% and cultured in a shaker at 37°C at 180rpm for about 6h. When the OD value reached about 0.6, 1mM IPTG was added to induce expression at 30°C for about 12h.

[0032] (2) Collect the bacterial liquid by centrifugation, and dissolve the bacterial liquid in 150mM NaH 2 PO 4 , 300mM NaCl, use French press (Thermo) to break the wall, centrifuge to collect the supernatant, pass through a water membrane with a pore size of 0.45μM to sterilize, and use a HisTrap HP (5ml, GE) column to purify, and the eluted effluent is ultrafiltered with a 10kDa millipore Tubes were ...

Embodiment 2

[0034] Fab or Fab-CH3mut monoclonal antibody preparation.

[0035]The miniaturized anti-CD20 antibody is modified on the basis of ofatumumab (OFA) monoclonal antibody. OFA monoclonal antibody can specifically bind to CD20 antigen molecules, thereby specifically killing B lymphoma cells.

[0036] Fab represents an antigen-binding fragment, including a heavy chain (H) and a light chain (L), the amino acid sequence of the heavy chain is shown in SEQ ID No.1, the amino acid sequence of the light chain is shown in SEQ ID No.2, and the gene encoding the heavy chain The sequence is shown in SEQ ID No.3, and the light chain coding gene sequence is shown in SEQ ID No.4. In the present invention, the Fab is also modified on the basis of the antigen-binding fragment of the original OFA monoclonal antibody for coupling with the drug. The modification method is to have a polypeptide sequence of LPETGGHHHHHH at the CH1 end of the heavy chain, and LPETG is the SortaseA enzyme Recognition se...

Embodiment 3

[0040] Preparation of Fab-vcMMAE and Fab-CH3mut-vcMMAE conjugates. The principle of the coupling reaction is that the sortaseA enzyme attacks the LPETG fragment at the end of the antibody, specifically cuts the amino acid residues T and G, and adds GGG-vcMMAE to the end of LPET through dehydration condensation to form a peptide bond, and finally forms the present invention The Fab-vcMMAE and Fab-CH3mut-vcMMAE conjugates.

[0041] Fab-vcMMAE, the preparation method of Fab-CH3mut-vcMMAE conjugate:

[0042] (1) Add 6 μM Fab or Fab-CH3mut monoclonal antibody (dissolved in 50mM Tris-HCl, 150mM NaCl, pH7.4) to the 10ml reaction system respectively, and use buffer solution (50mM tris-HCl, 150mM NaCl, pH7.4 ) dissolved 300 μM GGG-vcMMAE (synthesized by Nanjing Lianning Biotechnology Co., Ltd., in GGG-vcMMAE, GGG represents three glycine residues, vc represents the dipeptide linker (valine- Citrulline), MMAE is highly toxic small molecule drug dolastatin.), 50mM sortaseA, the CaCl of...

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Abstract

The invention discloses an antibody antigen-binding fragment-aplysiatoxin conjugate and a preparation method and application thereof. The preparation method includes steps: (1) providing an antigen-binding fragment of an anti-CD20 monoclonal antibody with an LPXTG sequence at a C-terminal and aplysiatoxin or derivatives thereof with oligonucleotide linkers; (2) under Sortase A enzyme catalysis, the LPXTG sequence and the oligonucleotide linkers have peptide transferase reaction to enable coupling of the antigen-binding fragment with the aplysiatoxin or derivatives thereof with the oligonucleotide linkers; (3) after reaction is completed, separating and purifying to obtain the antigen-binding fragment-aplysiatoxin conjugate. The antigen-binding fragment-aplysiatoxin conjugate has advantagesthat specificity, affinity and CD20 positive tumor cell killing effects of whole antigen-aplysiatoxin conjugates are retained, and high tumor penetrating rate and high tumor position clustering quantity are realized.

Description

technical field [0001] The invention relates to the technical field of biopharmaceuticals, in particular to an antibody antigen-binding fragment-dolastatin conjugate and a preparation method and application thereof. Background technique [0002] Antibodies are a class of protein drugs with a relatively large molecular weight (~150kDa), and it is difficult to reach tumor cells in the deep part of solid tumors through the vascular endocortex and intercellular space. Therefore, few antibodies reach the tumor site. Studies by Sedlacek et al. have shown that antibodies can only be injected 0.01% of the dose reaches the tumor site (Sedlacek H-H, et.al, Antibodies ascarriers of cytotoxicity, Contributions to Oncology, 1992; 43:1-145), which greatly limits the application of whole antibodies in solid tumors. Therefore, the miniaturization of the total resistance has become a trend, regardless of the economic cost or safety considerations. [0003] In the initial stage of miniaturiz...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C07K19/00A61K47/68A61K38/07A61P35/00
CPCA61K38/00C07K5/0205C07K16/2887C07K2319/00
Inventor 刘文慧赵文彬陈枢青
Owner ZHEJIANG UNIV
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