In the preliminary stages of research a cytotoxic drug is often added to an antibody candidate to enable confirmation that a prototype ADC is binding to the target of choice. Popular choices are DM1, DM4, vcMMAE and vcMMAF – and these links are for the oYo labelling versions of these drugs, that are site specific antibody labelling reagents enabling quick screening of antibodies that are being delivered and internalised by cells. For fluorescent or biotin detection of antibody binding there are a wide range of fluorescent and or biotinylated labelling reagents available with different functional groups.

Although the specificity of the target for antibody binding is the obvious first consideration, the isotype is important too.   

Different isotypes have different Fc portions that can elicit different responses from the immune system, such as has the potential to start antibody-dependent cellular cytotoxicity, but can also affect re-cycling and thus the pharmacokinetics of the ADC. The presence of the linker may or may not interfere with the antibody Fc’s interaction with the immune system, but this will need assessing and controlling for pharmaceuticals. Gamma Proteins offer a range of FcR at high quality, but budget friendly prices, to enable binding studies for monitoring and control of this aspect of design.

It’s likely not desirable that the antibodies might be recognised as ‘foreign’ and elicit the immune system to activate against the ADC, however most antibodies are raised in animal models first and then may need to be humanised. Antibody engineering from suppliers such as Absolute Antibody can humanise antibodies with species and/or isotype switching of their recombinant antibodies. In addition, recombinant antibodies can be manufactured consistently over time and be designed to include functional groups in specific locations for linker attachment.

Antibody glycosylation might also be a feature to consider. Most therapeutic antibodies are currently not glycosylated, and this may be a hard production step to control to ensure a homogenous product. The Lectenz GlycoSense platform can facilitate quick assessment of glycosylation, or enzymes such as PNGase F-II etc to cleave off potential unwanted glycan motifs.

3. Drug

There are a number of different chemistries that can be used to label an antibody. Whilst targeting amino acids with reagents such as maleimides or NHS Esters that target amino groups can ensure a density of attachment, the unspecific nature of these conjugations reduces control. A heterogenous mix will be the result and although antibodies may have the same antibody drug ratio, they may perform differently should the conjugation have occurred in the antibody binding site, or at a location that then prevents interaction of the FC domain.

As shown, functional groups can be engineered into an antibody.

Click chemistry motifs can also be added – with or without a copper catalyst depending on the linkers selected.

Copper based click chemistry is between Azide and Alkyne groups and can the reactions can be improved with accelerating ligands THPTA (tris-hydroxypropyltriazolylmethylamine) or BTTAA a next generation water soluble accelerating ligand, and both these reagents can help reduce the cytotoxicity and cell perturbations caused by the copper catalyst.

The design of the linker can help to overcome the hydrophobicity of the drug and prevent aggregation, can increase the achievable drug antibody ratio and can include cleavable sites for selective degradation.

Polyethylene glycol (PEG) is a popular choice of linker. The length of the PEG chain can be used to selectively set the spatial arrangement and accessibility of sites, and flexibility of the chain can allow movement and interactions between molecules – including shielding cleavable sites until the desired destination is reached.

PEG chains can be used to attach more drug molecules to each antibody conjugation site. This can either be branching or conjugated to functional groups along a PEG backbone as in the Sidewinder ™ linkers.  

Location of the drug conjugation sites to the linker can also be an important consideration as protecting distal ends of PEG spacers can protect payloads and modify performance, that might not be an available option if the drug was conjugated here.

Linkers can have a substantial effect on the therapeutic window, and thus it is important for therapeutic use that linkers be produced homogenously. Through careful selection of functional and active groups on each component of the ADC, it is possible to create high-purity compounds with reproducible purity profiles.

Vector Laboratories offer a wide range of monodisperse PEG linkers with a wide range of functional chemistries. They also offer technical assistance to assist in creation of linkers to meet your needs through their BioDesign service.