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The cysteine conjugation technology provides a flexible way for site-specific and non-specific ADCs development and production. BOC Sciences has built a complete and state-of-the-art technology platform that offers customized antibody-drug conjugate (ADC) services using cysteine conjugation. With a well-established profession in antibody engineering and pharmaceuticals, we are capable of providing comprehensive one-stop services for ADC development.
Stochastic cysteine conjugation and rebridging are techniques that exploit the natural presence of cysteine residues inside the antibody structure. Both methods are adopted on native mAbs. However, considerations regarding these strategies include the heterogeneity caused by the stochastic cysteine approach and the mAb fragmentation formation when using the rebridging method, especially during the conjugation with hydrophobic drugs.
Fig. 1. Cysteine-based conjugation strategies
The conjugation site significantly impacts ADC stability and pharmacokinetics-pharmacodynamics profile. High drug loading often leads to rapid plasma clearance, and ADCs with low DAR (drug-to-antibody ratio) demonstrate weak activity. Albeit improvement with uniformity was achieved using the cysteine conjugation method compared to the lysine conjugation method, the control of DAR value and drug load distribution (DLD) still needs to be strengthened. Because DAR and DLD optimization are strongly related to the pharmacokinetics of ADCs; thus, BOC Sciences is dedicated to helping developers and manufacturers improve the homogeneity of ADCs and ensure product safety by developing new methods to guide attachment sites, as well as optimizing DAR.
Currently, the development of ADCs relies on inter-chain disulfide cysteine as a conjugation site. Among these, four (IgG1 and IgG4) or six (IgG2) inter-chain disulfide bonds are reduced by excessive reducing agents such as hydrogen phosphate or dithiothreitol. This method avoids the destruction of the intrachain disulfide bonds and releases sulfhydryl from the cysteine residues involved in the interchain disulfide bonds. Moreover, this conjugation method produces mixed ADC products with a drug-to-antibody ratio (DAR) value of 0, 2, 4, 6, 8, 10, and 12. Our technical team has years of experience performing inter-chain cysteines conjugation and can quickly achieve effective control of DAR and product uniformity to shorten the research cycle for our customers.
With the development of genetic and site-specific conjugation technologies, active amino acid groups were introduced into antibodies through genetic engineering and conjugate toxins through amino or sulfhydryl groups site specifically to achieve effective control of DAR and products uniformity. For example, Thiemab technology enables the desired site-selectivity and uniformity modification on antibodies by utilizing engineered reactive cysteines that do not involve structural disulfide bonds. BOC Sciences can support your project research by producing ADCs with a single DAR value through the novel site-specific conjugation technology.
The bridging conjugation technology bridges four pairs of interchain disulfide bonds through linkers and reconnects the opened disulfide bonds. This conjugation method can achieve ADC with a relatively high DAR value and fixed conjugation position. The bridging technique can also maintain the intrachain disulfide bond and improve overall ADC stability. Furthermore, the bridging-conjugated ADC retains high antigen-binding capacity, plasma stability, efficiency, and antigen-selectivity to induce cell death in vitro and in vivo. Based on the most comprehensive linker development library, BOC Sciences can provide our customers with the most advanced inter-chain cysteine bridging conjugation technologies.
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