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Intermediate for preparing antibody and drug conjugate (ADC) and preparation method and application thereof

A technology of conjugated body and antibody, applied in the field of medicine and biology

Pending Publication Date: 2021-12-28
DARTSBIO PHARM LTD +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Up to now, other ADC molecules targeting Trop-2 are still under development. However, whether ADC technology can be successfully applied to the targeted therapy of Trop-2 positive tumors, the key issue is to screen out the best linker and Combination of payloads to achieve the highest therapeutic index

Method used

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  • Intermediate for preparing antibody and drug conjugate (ADC) and preparation method and application thereof
  • Intermediate for preparing antibody and drug conjugate (ADC) and preparation method and application thereof
  • Intermediate for preparing antibody and drug conjugate (ADC) and preparation method and application thereof

Examples

Experimental program
Comparison scheme
Effect test

preparation example Construction

[0208] (b) Preparation of ADC and detection of DAR value

[0209] The reduction reaction was carried out by adding 6 molar equivalents of TCEP (1 mg / mL) to 8 mg / mL antibody solution containing 9% sucrose, 10 mM acetate, 0.01% Tween-20, pH 5.0 at 37°C for 2 hours. After reduction, 20 molar equivalents of the linker payload were added per antibody sulfhydryl group and incubated at 4°C for more than 16 hours. The solution mixture was concentrated by centrifugation (4000 rpm, 15 minutes, Thermo Fisher ST40R TX-1 ) with an Amicon Ultracontainer (50,000 MWCO, Millipore Corporation). The concentration of ADC was measured by UV detector (Nanodrop 100, Thermo Fisher Scientific Inc.) under absorption at 280 nm. The DAR value of the final product was confirmed by reverse phase (RP)-HPLC and QTOF mass spectrometer.

[0210] (c) RP-HPLC analysis of DAR value of ADC

[0211] RP-HPLC analysis is carried out under the following conditions on 1260Infinity UHPLC (Agilent Technologies): 1) Ag...

Embodiment 1

[0234] Antibody preparation

[0235] In this example, hRS7 antibody was expressed and purified to obtain hRS7 antibody. Methods as below:

[0236] The amino acid sequences of hRS7 light and heavy chains were obtained from US Patent No. 9931417B2, and then reverse-transcribed into cDNA sequences by Vector NTI software. The DNA sequence was synthesized and subcloned into the pcDNA3.1 vector and amplified in E. coli. The purified plasmid was transfected into HEK293 cells by PEI. Then suspend the cells in FreeStyle TM293 expression medium. After 5 days of culture, cell culture supernatants were harvested and antibodies were affinity purified by protein A.

Embodiment 2

[0238] Preparation of Compound VK-PTX

[0239] The structure of compound VK-PTX is shown below:

[0240]

[0241] Paclitaxel was reacted with the VK linker to form the compound VK-PTX, namely

[0242] Mal-peg4-Val-Lys(peg24)-PAB-paclitaxel (VK-PTX), the method is as follows:

[0243]

[0244] Step 1: Synthesis of Compound 3 (Fmoc-Val-Lys(Trt)-PAB-PTX)

[0245] Fmoc-Val-Lys(Trt)-PAB-PNP (purchased from Levena Biopharma, USA) and PTX (purchased from Levena Biopharma, USA) were dissolved in anhydrous DMF, and DIEA was added to the solution, and the mixture was heated at room temperature at 22°C The reaction was stirred for 2 hours, and the target product Fmoc-Val-Lys(Trt)-PAB-PTX was directly purified by reverse HPLC, and compound 3 was formed as a white powder after lyophilization.

[0246] Step 2: Synthesis of compound 4 (Fmoc-Val-Lys-PAB-PTX TFA salt)

[0247] The compound 3 obtained in step 1 was dissolved in 10% TFA / DCM solution and stirred at room temperature for ...

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Abstract

The invention relates to an intermediate (a combination of a connexon and a load) for preparing an antibody and a drug conjugate (ADC) and a preparation method and application thereof. Specifically, the combination of the connexon and the load has a structure shown as a formula I, the compound shown as the formula I can be applied to coupling of high-hydrophobicity compound loads such as paclitaxel (PTX') and the like which are not easy to couple, and the prepared ADC molecule has good hydrophilicity and thermal stability and can reach a relatively high drug-to-antibody ratio (DAR) value. According to the intermediate, the Trop-2 targeting ADC prepared by using the connexon represented by the formula I shows the strong in vitro drug effect in the low expression cell line in the Trop-2 compared to the Trop-2 targeting ADC with the effective loads of MMAE and SN38, and shows the strong tumor inhibition ability in the mouse in vivo xenotransplantation tumor model, and the bystander effect is proved. Experimental results show that the problem that the paclitaxel compound is used as an ADC effective load can be solved by utilizing the hydrophilic connexon, and ADC molecules which are expected to be used for treating various human cancers are obtained.

Description

technical field [0001] The invention belongs to the field of medical biology, and in particular relates to an intermediate (combination of a linker and a load) used for the preparation of an antibody and drug conjugate (ADC) and a preparation method and application thereof. Background technique [0002] Antibody-drug conjugates (ADCs) are a type of tumor-targeted therapy. Antibodies targeting tumor antigens are covalently coupled to cytotoxic payloads through chemically synthesized linkers. The choice of drug linker and payload plays a key role in the therapeutic efficacy and safety of ADCs. [0003] Currently, most ADC molecules entering clinical development utilize cleavable linkers, including: acid-labile linkers, peptide linkers, and disulfide linkers. A cleavable dipeptide linker formed by linking valine-citrulline (Val-Cit) to a self-cleavable spacer benzyl hydroxyl (PAB) is widely used in the clinical development of ADCs. However, due to the strong hydrophobicity of...

Claims

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Application Information

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IPC IPC(8): A61K47/68A61K47/54A61K31/337A61P35/00A61P37/02
CPCA61K47/6889A61K47/6803A61K47/545A61K31/337A61P35/00A61P37/02A61K47/68031A61K47/68037A61K47/6851C07K16/30A61K47/60A61K47/6855
Inventor 王春河邵婷陈田志王琪陈艺丽林以均
Owner DARTSBIO PHARM LTD
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