Secondary antibody-drug conjugates: a potent tool for ADC candidate monoclonal antibody screening

Antibody drug conjugates combine the specificity of a monoclonal antibody with the cytotoxicity of a payload that has become a new strategy against various diseases including cancers. In ADC development, identifying successful antibodies is one of the most critical principles. Identification of ADC candidate monoclonal antibodies(mAbs) capable of internalizing following antigen binding has been accomplished by methods including flow cytometry-based internalization analyses and more recently, pH sensitive dyes. However, these methods may not be sufficiently sensitive for screening nor wholly predictive of the mAbs' capacity for a specific drug delivery. Moreover, in order to test the function of ADCs, mAb is typically conjugated with payloads, which requires many mAbs and time consuming.

Prior studies of ADC demonstrated internalization of a primary ADC leads to trafficking to lysosomes, drug release by lysosomal cathepsin B, and ensuing cell death. A secondary antibody-drug conjugate capable of binding primary mAbs bound to the surface of antigen-positive cells has comparable drug delivery capability. The system is sufficiently sensitive to detect internalizing mAbs in nonclonal hybridoma supernatants and is predictive of the activity of subsequently produced primary ADC. Because of their high extracellular stability, the noninternalized secondary antibody-drug conjugate are 100--1000-fold less toxic to cells over extended periods of time, permitting an assay in which components can be added without the need for separate wash steps. This homogeneous screening system is amenable to medium-throughput screening applications and enables the early identification of mAbs capable of intracellular trafficking for drug delivery and release. What's more, the secondary ADCs can also be applied to recombinant protein ligands with Fc fusion for targeting cell-surface receptors.

Fig. 1 Using secondary ADCs for pre-screening ADC candidates.

Creative Diagnostics is dedicated to easing the ADC candidate mAb search process and we have developed a series of secondary antibody-drug conjugates as tools for screening ADC-suitable internalizing antibodies under "load-bearing" conditions to facilitate your ADC development research.

We have developed multiple panels of secondary ADCs linked to many highly sought-after cytotoxic small molecules designed with the following concepts:

1. The secondary antibodies recognize either the Fc or Fab portion of human/mouse/rat/ rabbit IgGs as well as isotype controls typically found in the discovery of therapeutic antibodies.
2. The small molecule toxins include the well-known tubulin inhibitors, potent DNA/RNA damaging reagents, and DNA/ RNA synthesis inhibitors
3. Either cleavable or non-cleavable linkers, the linkers connecting the secondary antibodies to the small molecule drugs are either non-cleavable or cleavable by endosome cathepsins or free thiol reducing molecules.

Fig. 2 Secondary ADC panels