ADC Affinity Measurement

Creative biolabs is a leader in antibody-drug conjugate (ADC) development and we have established a comprehensive analytical platform to ensure high-quality and high-accuracy biochemical and in vitro efficacy characterization of a customized ADC. We offer a customarily tailored analysis package to evaluate the binding affinity of an ADC towards its designated target antigen and Fc receptors using various approaches including surface plasmon resonance (SPR), enzyme-linked immunosorbent assay (ELISA), and fluorescence-activated cell sorting (FACS) flow cytometry assay.

The elegant design of an antibody-drug conjugate is designated to achieve targeted delivery of the conjugated cytotoxic agents to tumor cells and drug release upon antigen binding and internalization, thus maximizing the antitumor effects while minimizing cytotoxicity to normal tissues. The efficacy of an ADC greatly depends on the specific antigen binding activities of the monoclonal antibody (mAb) portion of the molecule.

In theory, antibodies with higher antigen binding affinity may exert greater uptake and internalization, increasing this unique therapeutic advantage. However, the higher uptake and internalization rate of an antibody and the corresponding ADC will also result in a reduced mAb/ADC penetration and distribution within a ‘bulky tumor’, for instance a solid tumor, resulting in a negative effect on ADC efficacy in these cases. Thus, there is an intricate balance between the mAb/ADC binding affinity and the rate of internalization. Creative biolabs provides a variety of means to measure the target binding affinity of a mAb and in combination of the internalization analysis module, we are confident in assisting our clients with the selection of the most appropriate mAb for ADC development.

As mentioned earlier, the preparation of an ADC involves the chemical conjugation of small molecular entities comprised of payload drugs and chemical linkers (drug-linker complexes) to a mAb. Depending on the site of conjugation and chemistry applied, this chemical conjugation treatment could affect the antigen binding affinity of the mAb portion and subsequently result in an altered ADC binding pattern. Thus, the binding affinity of an ADC need to be reevaluated before further downstream analyses. Using the same analytical platform, scientists from Creative biolabs provide accurate ADC binding affinity measurements to ensure its appropriate binding affinity using unconjugated mAb as control.

In some cases, the cytotoxicity mediated from the mAb Fc region is desired in an ADC as a “secondary efficacy”. In these cases of scenarios, the Fc receptor binding affinity of an ADC should be equivocal to that of the unconjugated mAb. Utilizing proper approaches, Creative biolabs also provides services to assess the Fc receptor binding affinity of an ADC using the unconjugated mAb as control.

Examples of the methods and assay services we offer to evaluate ADC affinity to target antigen and Fc receptors (not limited to):

• Surface plasmon resonance (SPR)
• Target antigen or Fc receptor expression
• Sensor coating with antigen or Fc receptor
• ADC affinity measurement using unconjugated mAb as control
• Data analysis

• Enzyme-linked immunosorbent assay (ELISA)
• Plates coating with antigen or Fc receptor
• ADC/mAb binding (concentration gradient)
• ADC/mAb binding with HRP-tagged secondary antibodies
• Detection with chromogenic substrates
• Data analysis

• Fluorescence-activated cell sorting (FACS) assay
• Cell binding with ADC/mAb.
• Cell labeling with fluorescent secondary antibodies
• Flow cytometry measurement
• Data analysis

ADC affinity analysis. The binding affinity of an ADC towards the targeted antigen and Fc receptors is measured by approaches such as ELISA (A, PNAS, 2012), FACS folw cytometry (B, Cancer Sci., 2015) and fluorescent competitive flow cytometry (C, J Natl Cancer Inst., 2012).

Creative biolabs is dedicated to provide customized analytical services to characterize various aspects of ADC in vitro efficacy. Please contact us for more information and a detailed quote.

References:

1. Axup, J.Y.; et al. Synthesis of site-specific antibody-drug conjugates using unnatural amino acids. PNAS. 2012, 109(40): 16101–16106.
2. Yamamoto, Y.; et al. Enhanced antitumor effect of anti-tissue factor antibody-conjugated epirubicin-incorporating micelles in xenograft models. Cancer Sci. 2015, 106(5): 627-634.
3. Moldenhauer, G.; et al. Therapeutic potential of amanitin-conjugated anti-epithelial
4. Gerhard M.; et al. Therapeutic potential of amanitin-conjugated anti-Epithelial cell adhesion molecule monoclonal antibody against pancreatic carcinoma. J Natl Cancer Inst. 2012, 104:622–634.