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Antibody conjugated medicine and preparation method and application thereof

An antibody-conjugated drug and anti-tumor drug technology, applied in the field of biotechnology and medicine, can solve the problems of improving purification difficulty, activity restricted by endocytosis, low homogeneity, etc., avoiding the use of organic reagents, high body Internal and external antitumor activity and homogeneity improvement effect

Active Publication Date: 2016-12-21
ZHEJIANG UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, both targets are antigens that are easily endocytosed by target cells, and the existing technology still cannot solve the problem of using Sortase A to prepare antibody-conjugated drugs (ADCs) for receptors or antigens that cannot be endocytosed
Moreover, antigens or receptors with poor endocytosis include important antibody drug targets, such as CD20, CD21, and CD71, etc., which severely restrict the target range of antibody-drug conjugates
[0011] In addition, due to the poor water solubility of commonly used drugs for conjugation, such as the dolastatin derivative Maleimidocaproyl-valine-citrulline-p-aminobenzoyloxycarbonyl-monomethyl auristatin E (abbreviated as vcMMAE), it is necessary to add organic reagents to the coupling reaction between it and the antibody. Increase its solubility, so it may affect the antibody structure and increase the difficulty of subsequent purification
[0012] In summary, the activity of existing ADCs targeting non-endocytic targets such as CD20 is restricted by endocytosis, and due to the use of cysteine ​​or lysine residues of the antibody itself to couple drugs, resulting in its Low homogeneity; drugs used for chemical coupling are usually poor in water solubility, which is not conducive to subsequent production and purification

Method used

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  • Antibody conjugated medicine and preparation method and application thereof
  • Antibody conjugated medicine and preparation method and application thereof
  • Antibody conjugated medicine and preparation method and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0070] Example 1 Preparation of OFA-HL Antibody

[0071] 1. Construct the expression vector of anti-CD20 monoclonal antibody with LPETG tag on the heavy chain

[0072] A plasmid carrying the anti-CD20 monoclonal antibody gene was constructed using the expression vector of the heavy chain anti-CD20 monoclonal antibody with LPETG (Ofatumumab-HeavyChain-LPETG, hereinafter abbreviated as OFA-HL). For the specific construction method, refer to Chinese invention patent ZL201310046396.9 and ZL201310170344.2.

[0073] Using the above plasmid as a template and P1 and P2 as primers, the expression gene of LPETG was fused to the 3' end of the antibody heavy chain gene by PCR method to obtain the anti-CD20 monoclonal antibody heavy chain gene with LPETG tag. After double digestion, the gene obtained above was subcloned into the original pFUSE-CHIg-hG1 expression vector to obtain the expression vector IgH-OFA-HL expressing OFA-HL heavy chain. The vector for expressing the OFA-HL light ch...

Embodiment 2

[0089] Example 2 Preparation and further purification and analysis of anti-CD20 monoclonal antibody-dolastatin conjugate

[0090] 1. Expression and purification of Sortase A enzyme and its mutants

[0091] First, the genome of Staphylococcus aureus was extracted using the Takara Bacterial Genome Extraction Kit, and then the wild-type Sortase A enzymes of different lengths were amplified with the following primers (as shown in Table 1) by PCR. The genes for the different Sortase A enzymes were inserted between the NcoI and XhoI restriction sites on the pET28(+) plasmid.

[0092] After the correct sequence was verified by sequencing, pET28-SrtA(M4) and pET28-SrtA(M3) were constructed using Agilent's point mutation kit (QuikChange IISite-Directed Mutagenesis Kit) using the pET28-SrtA(△N59) expression vector as a template Expression vector, the primers used are as follows (as shown in Table 1).

[0093] Schematic diagram of the length and mutation of Sortase A enzymes of differe...

Embodiment 3

[0130] Example 3 Study on the Affinity of ADC Prepared by Sortase A Enzyme and CD20+ Tumor Cells

[0131] The OFA, OFA-HL, OFA-HL-MMAE and OFA-HL-vcMMAE of each concentration gradient were mixed with ice-cold incubation buffer (10% BSA in PBS, pH 7.4) containing 106 Ramos cells, and placed on ice Place on ice for 30 min; wash twice with pre-cooled PBS (pH 7.4), then add FITC-labeled goat anti-human secondary antibody diluted in PBS containing 1% BSA, and incubate on ice for 30 min; after washing twice, use flow cytometer Cytomics FC 500MCL (Beckman Coulter) detects mean optical density (MFI). The sample concentration is taken as the abscissa, and the corresponding MFI is drawn as the ordinate.

[0132] attached Figure 14 is the affinity of the anti-CD20 monoclonal antibody OFA and its conjugates to Ramos cells. attached Figure 14 It can be seen that adding an LPETG tag to the C-terminus of OFA's heavy chain will slightly reduce its affinity for Ramos cells, and using Sor...

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Abstract

The invention discloses an antibody conjugated medicine and a preparation method and application thereof. The antibody conjugated medicine is formed by connecting an antibody and a medicine through a connecting arm. The antibody is an anti-CD20 monoclonal antibody with heavy chain containing LPXTG sequence. The medicine is aplysiatoxin or a derivative thereof. The connecting arm contains a short-peptide linker for connecting the antibody and a self-elimination linker for connecting the medicine. The short-peptide linker contains at least 1-3 continuous glycines. The antibody conjugated medicine has high uniformity and can achieve higher in-vitro and in-vivo antineoplastic activity with less drug loading capacity (DAR) than CD20 targeting ADC prepared by other chemical methods. IC50 (median inhibitory concentration) for Ramos cells can reach 0.005 nanogram / milliliter.

Description

technical field [0001] The invention relates to the fields of biotechnology and medicine, in particular to an antibody-coupled drug and its preparation method and application. Background technique [0002] The B lymphocyte antigen CD20 is a kind of phosphoprotein without glycosylation, and no natural ligand has been found for it. It can be expressed in all stages of B cells except pre-B cells and mature B cells, and almost all B-cell tumors express CD20, so it becomes an ideal target for antibody therapy. [0003] Therefore, various companies and research institutions have developed many anti-CD20 monoclonal antibodies against CD20, and many of them have been approved by the FDA, such as rituximab (Rituximab), ofatumumab (Ofatumumab) and obinutuzumab (commodity Name: Gazyva) and so on. They mainly use programmed cell death (PCD), complement-dependent cytotoxicity (complement-dependent cytotoxicity, CDC) or antibody-dependent cell-mediated cytotoxicity (antibody-dependent c...

Claims

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Application Information

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IPC IPC(8): A61K47/48A61K38/15A61P35/00
CPCA61K47/6889A61K47/6811A61K47/6817A61K47/6849A61K38/15
Inventor 潘利强陈枢青
Owner ZHEJIANG UNIV
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