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An antibody-dolastatin conjugate and its preparation method and application

A technology of dolastatin and conjugates, which is applied in the preparation methods of peptides, chemical instruments and methods, anti-animal/human immunoglobulins, etc., can solve the problems of antibody stability or decreased targeting, and achieve uniformity. The effect of good performance and good tumor killing ability

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

[0006] Monoclonal antibodies have a stable structure and good targeting, but common coupling methods such as amino and thiol coupling methods may reduce the stability or targeting of antibodies

Method used

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  • An antibody-dolastatin conjugate and its preparation method and application
  • An antibody-dolastatin conjugate and its preparation method and application
  • An antibody-dolastatin conjugate and its preparation method and application

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0036] Preparation of CLA12 HL-vcMMAE conjugate.

[0037]The monoclonal antibody targeting the EAAGIGILTV / HLA-A2 complex is CLA12, whose sequence comes from US patent: US20100158927A1. Antibody light and heavy chain variable region gene sequence (the heavy chain variable region gene sequence is shown in SEQ ID No.5, and the light chain variable region gene sequence is shown in SEQ ID No.6) from Sangon Bioengineering (Shanghai) Co., Ltd. Co., Ltd. on behalf of the synthesis, the antibody light and heavy chain constant region sequence is preserved in the laboratory, the variable region of the light and heavy chain is connected to the constant region of the human Lamda light chain and the constant region of the human IgG1 type heavy chain by PCR method, and the C-terminal of the light and heavy chain The recombinant antibody obtained by linking the LPETG sequence and expressing it was named CLA12 HL.

[0038] (1) Modification of the C-terminals of the light and heavy chains of t...

Embodiment 2

[0043] Identification of CLA12 HL-vcMMAE conjugates.

[0044] (1) High performance liquid phase identification of CLA12 HL-vcMMAE conjugated drug, take 20 μg each of CLA12 HL-vcMMAE conjugated drug and CLA12 HL monoclonal antibody, add a little TCEP powder, mix with a pipette gun, and incubate at 37°C for 10 minutes. with PLRP-S The reverse column was used for analysis, and the detection wavelength was 280nm.

[0045] Test results such as figure 1 As shown, CLA12 HL monoclonal antibody has two peaks of light chain (L0) and heavy chain (H0), and CLA12HL-vcMMAE also has two peaks of light chain (L0) and heavy chain (H0), but compared with CLA12 HL monoclonal antibody The positions of the two peaks of the light chain and the heavy chain of the cloned antibody are obviously shifted backwards, and there is no overlap with the positions of the two peaks of the light chain and the heavy chain of the CLA12 HL monoclonal antibody, indicating that the CLA12 HL-vcMMAE light chain and ...

Embodiment 3

[0060] Biological activity detection of CLA12 HL-vcMMAE conjugate.

[0061] 1. Flow affinity detection

[0062] (1) Take 5×10 5 MEL 624.38 cells were incubated with 5 μg / mL CLA12 monoclonal antibody and CLA12 HL-vcMMAE in 1% BSA in PBS solution for 30 min at 4°C;

[0063] (2) After washing twice with PBS, add goat anti-human IgG (H+L) polyclonal antibody (1:200 dilution), and incubate at 4°C for 30 minutes; after washing twice with PBS, detect the average fluorescence intensity of the cells with a shedding cell analyzer (MFI).

[0064] The combination of CLA12 and CLA12 HL-vcMMAE with MART-1, HLA-A2 double-positive cells MEL624.38 was detected by flow cytometry, and the strength of the binding force was shown by the average fluorescence intensity of FITC labeled with the secondary antibody.

[0065] Test results such as Figure 8 As shown, CLA12 monoclonal antibody and CLA12 HL-vcMMAE have almost the same affinity for the same amount of MART-1, HLA-A2 double positive cells...

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Abstract

The invention discloses a preparation method for an antibody-monomethyl auristatin E (MMAE) conjugate. The method comprises the following steps: (1) a monoclonal antibody with an LPXTG sequence at each C terminus of an anti-MART-1 protein presentation peptide EAAGIGILTV / HLA-A2 composite, and monomethyl auristatin E or a monomethyl auristatin E derivative with an oligo-glycine joint are provided; (2) under the catalysis of a Sortase A enzyme, the LPXTG sequences and the oligo-glycine joint are subjected to a transpeptidation reaction, and the monoclonal antibody is coupled with and the monomethyl auristatin E or the monomethyl auristatin E derivative with the oligo-glycine joint; and (3) after the reaction is completed, separation purification is performed, and therefore the antibody-monomethyl auristatin E conjugate is obtained. Through site-direct coupling, the method provided by the invention makes per molecule of the antibody have four molecules of the MMAE, has good uniformity, andexhibits good tumor killing capacity in vivo experiments; and compared with current target spots of ADCs (antibody-drug conjugates), CLA12-vcMMAE ADCs expand the selection areas of the current targetspots of the ADCs, demonstrate the view that an intracellular protein can also be taken as the target spot of the ADCs through presentation of MHC (major histocompatibility complex) I molecules.

Description

technical field [0001] The invention relates to the technical field of biopharmaceuticals, in particular to an antibody-dolastatin conjugate and its preparation method and application. Background technique [0002] MART-1 protein is a melanoma-associated antigen that is only expressed in melanocytes and most melanoma cells, and is highly expressed in melanoma cells. According to incomplete statistics, 100% of melanoma samples have Expression of MART-1 protein (Barrow C et al., Clin Cancer Res. 2006 Feb 1;12(3 Pt 1):764-771). Therefore, drugs designed to target the MART-1 protein will be widely used in melanoma. [0003] In the past 20 years, the rapid development of the field of monoclonal antibodies has played a pivotal role in the field of clinical tumor treatment, such as cetuximab, trastuzumab, rituximab and a series of monoclonal antibodies are in They have shown good clinical therapeutic effect on their respective tumor indications. They usually inhibit the growth o...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C07K16/30C07K1/107A61K38/08A61K47/68A61K47/65A61P35/00
CPCA61K38/08C07K16/3053
Inventor 来骏吴姗姗王赟陈枢青
Owner ZHEJIANG UNIV
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