Antibody drug conjugates (ADCs) have grown extremely popular in the pharma industry with 238 clinical trials testing 47 unique ADCs in 2014¹, but these are complex molecules that need careful development of a bioanalytical strategy to inform the clinical team as to the exposure, safety, and risk profiles of the dosed molecule.

This article will highlight some of the key bioanalytical strategies used to support ADC programs, the critical challenges associated with this development and subsequent validations, and hurdles that biopharma companies and CROs may neglect to consider.

ADCs are Conceptually Simple
Traditional chemotherapeutics work like dropping a carpet bomb. The highly cytotoxic compounds kill fast growing cells indiscriminately. Cancer cells die, but healthy cells get caught in the explosion too, and patients often experience fatigue, pain and nausea. ADCs have the advantage of precisely targeting cancer cells with a monoclonal antibody (mAB).

Conceptually, ADCs are simple two-part structures: a monoclonal antibody (mAb) and a cytotoxic agent conjugated via a stable linker² (Figure 1). This antibody binds to specifically to a cancer cell surface receptor and causes the cell to engulf it. Once inside, the ADC releases its toxins and kills the cell without extra casualties2-4 (Figure 2).

Additionally, the mAb’s long circulating half-life and low immunogenicity make it the ideal delivery system for powerful cytotoxic compounds such as monomethyl auristatin E (MMAE) and calicheamicin.

Complexity of ADC Development
Though conceptually simple, ADC development is intricate because linker chemistry is complex. The ADC linker must be stable or risk prematurely releasing its agent, resulting in widespread cell death. Not only must the conjugation chemistry provide stability but so must the mechanism of linker cleavage. Current and future ADCs use either the cysteine’s thiol or the lysine’s amine to conjugate the linker and the mAB.^1,4 

Conjugation of the mAbs lysine residues results in a more heterogeneous ADC because there are many possible lysine residues available for conjugation. For example, although IgG has 20 readily available lysines, there are only eight possible cysteine conjugation sites. Thus using the antibody’s cysteine-containing disulfide bridges results in a more homogeneous ADC.
Heterogeneity and homogeneity are directly related to the drug-to-antibody ratio (DAR) of the antibody. The DAR, as discussed below, is one important consideration when developing the best ADC bioanalytical strategy.

Partnering With a CRO for ADC Bioanalysis
To help mitigate the challenges of analysis, early and in-depth strategy conversations are needed to prevent potential program delays due to complications with bioanalytical method development, validation, and execution. It is the heterogenic nature of antibody drug conjugates that presents many hurdles for characterization (Figure 3). For this reason, both ligand binding and liquid chromatography-mass spectrometry (LC-MS) assays are employed for these unique marriages of small and large molecule therapeutics. 

Bioanalysis requires highly specialized equipment and expertise that are often only found at a contract research organization (CRO). Arguably, the most important consideration is assembling a collaborative development team with diverse knowledge and technical expertise comprising members from the pharmaceutical company and the CRO. Working together, these individuals should be empowered to make decisions critical to ADC development, from discovery through clinical trials. A well-rounded team should include an overall lead, bioanalytical scientist(s) with LBA (ligand binding assay) as well as LC-MS/MS expertise, pharmacokineticists, drug safety representative (if non-clinical), and a clinical pharmacologist (if clinical). 

The ADC analytes that are frequently considered for analysis are: total antibody, unconjugated antibody, antibody with cleaved payload, free cytotoxic drug, and drug bound to linker.^3,5 The wide variety of analytes needing assessment requires the expertise of both large molecule and small molecule labs, each of which employs unique approaches to quantifying and characterizing investigational drugs. The marriage of these two historically divergent bioanalytical departments calls for a level of collaboration not typically needed for discovery and development of traditional therapeutics. 

Due to the complexity of ADC analysis, it is important for pharmaceutical companies outsourcing bioanalysis of their ADCs to identify a CRO that specializes in both large and small molecule analysis. Partnering with a bioanalytical CRO can assist in determining an assay’s sensitivity to the DAR and what ADC forms will be evaluated in the study – a decision that should occur as early as possible. These points will be further discussed below (see “Important considerations for ADC bioanalysis”).

Co-locating the CRO’s and sponsor’s bioanalytical teams and equipment is crucial to a successful partnership, both in transferring critical knowledge and in efficient use of study samples. Due to the high number of analytes measured at each time point and small original matrix volume available for dividing for one specific analyte evaluation, there is a tangible risk that one or more of the samples will have insufficient volume for its designated assay. Not co-locating the assays might result in the samples being sent to many different locations, potentially across the country. Therefore, if one lab runs low on a sample volume, a high level of coordination and cost is required to obtain additional volumes necessary to fully evaluate the ADC and its effects. Having both bioanalytical labs in the same building minimizes this problem by allowing less division of the sample or allowing the reallocation of leftover sample volumes. This results in better data, enabling the sponsor to characterize the pharmacokinetics and immunogenicity of the dosed ADC.

Important Considerations for ADC Bioanalysis
The bioanalytical team first needs to collaborate in determining what analyte(s) should be measured and with what bioanalytical platform. A 2013 publication outlines a basic strategy for bioanalysis of ADCs using both LBA and LC-MS/MS techniques.³ A pharmacokineticist can outline details needed from non-clinical pharmacokinetic (PK) and toxicology studies to include predicting human exposure, devising dosing strategies, and prioritizing method development and validation. A clinical pharmacologist can use the non-clinical toxicology studies to identify the form(s) of the ADC most likely to be associated with toxicity in humans. This input can help guide the team in crafting accurate timelines to complete analytical method development while targeting the sensitivity essential to patient safety in phase 1 clinical trials—and beyond.

Once the appropriate analyte(s) have been identified, another crucial question for the bioanalytical lab(s) to consider is the effect the DAR has on the accurate quantitation of the ADC (and/or related analyte) and the detection of the Anti-Drug Antibodies (ADAs) using an LBA. The DAR can make a difference to the performance of an assay in multiple ways, depending on the drug substance and the reagents chosen for the ligand binding assay. 

The DAR levels present in reference material frequently are a mixture of proteins containing unconjugated, moderately conjugated, and highly conjugated antibodies.^4,6 When developing LBA methods, the difference in DAR can negatively affect the drug’s ability to bind an immobilized antigen.⁷ As a result, Gorovits et al recommend that to understand and ideally minimize the effect drug-to-antibody ratio has on the bioanalytical method, individually prepared ADC materials containing a single DAR fraction be generated and evaluated during method development.³ These single DAR reference material preparations can be used to select and test a detection reagent to identify the most suitable format of an LBA method, i.e. one that results in the most accurate quantitation of the ADC in matrix. Depending on format (such as gdetecting with an anti-chemotoxic agent), the assay can generate significantly different results based on the DAR number.^6,8 

ADA Methodologies
When developing a robust risk-based ADA testing strategy, an ADA assay should minimally be able to detect antibodies formed to the antibody backbone, the linker, cytotoxic “payload”, or any number of other permutations of this complex modality.⁹ This is due to the heterogeneity of the drug as well as the ADA response. Additional characterization to determine specific ADC portion(s) against which ADA have been formed may then be correlated to any adverse effects observed in the PK or safety profiles.³ Hock et. al.¹⁰ describe a testing plan for evaluating and characterizing ADA responses for ADCs that includes the traditional large molecule ADA testing plan outlined by Mire-Sluis et. al.¹¹ as well as a method to determine domain specificity, allowing for the correlation of different ADA reactivity to potential safety, efficacy, and effect on PK. 

Sauerborn and van Dongen identified one safety finding due to ADAs to an ADC domain: the linker-to-antibody coupling site generated a new epitope primed for ADA formation leading to ADC off-target toxicity.5 Specifically, the ADAs formed created large immune complexes readily identified and subsumed by the immune system, resulting in the unintentional delivery of cytotoxic “payload” to healthy cells, ultimately inducing cell death.

Conclusion
Antibody drug conjugates are one of the more promising safe and effective cancers treatments. As with any innovation, the benefits are not without a learning curve and growing pains. Partnering with a CRO that has collocated ligand binding and LC-MS bioanalytical capabilities can prove to be advantageous since both technologies are needed to fully characterize the pharmacokinetics/toxicokinetics of an ADC. More importantly, the drug developers and bioanalytical scientists must collaborate and determine the most efficient path forward to enable quick turn-around of high quality data. 

References

1. Rostami, S.,  Qazi, I., & Sikorski, R. (2014). The Clinical Landscape of Antibody-drug Conjugates. ADC Review.
2. Jain, N., Smith, S. W., Ghone, S., & Tomczuk, B. (2015). Current ADC Linker Chemistry. Pharm Res, doi:10.1007/s11095-015-1657-7.
3. Gorovits, B., Alley, S. C., Bilic, S., Booth, B., Kaur, S., Oldfield, P., . . . Siguenza, P. (2013). Bioanalysis of antibody-drug conjugates: American Association of Pharmaceutical Scientists Antibody-Drug Conjugate Working Group position paper. Bioanalysis, 5(9), 997-1006.
4. Stephan, J. P., Kozak, K. R., & Wong, W. L. (2011). Challenges in developing bioanalytical assay for characterization of antibody-drug conjugates. Bioanalysis, 3(6), 677-700.
5. Sauerborn, M., & van Dongen, W. (2014). Practical Considerations for the Pharmacokinetic and Immunogenic Assessment of Antibody–Drug Conjugates. BioDrugs, 28:383–391.
6. Kozak, K. R., Tsai, S. P., Fourie-O’Donohue, A., Chuh, J. d., Roth, L., Cook, R., . . . Wong, W. L. (2013). Total Antibody Quantification for MMAE-Conjugated Antibody-Drug Conjugates: Impact of Assay Format and Reagents. Bioconjugate Chemistry, 24, 772-779.
7. Stephan, J.-P., Chan, P., Lee, C., Nelson, C., Elliott, J. M., Bechtel, C., . . . Ebens, A. (2008). Anti-CD22-MCC-DM1 and MC-MMAF Conjugates: Impact of Assay Format on Pharmacokinetic Parameters Determination. Bioconjugate Chemistry, 19(8), 1673-1683.
8. Spriggs, F. P., Duriga, N., Rathi, A., & Qu, Q. (2014). Resolution of matrix interference: quantitative and quasi-quantitative ligand-binding assay case studies. Bioanalysis, 6(8), 1091-1101.
9. Swanson, S. J. (2014, May 20). AAPS National Biotechnology Conference Podium Presention. Lessons Learned from Standard of Care, First Generation and Next Generation Biotherapeutics: What Do We Expect to Change Going Forward? San Diego, CA, USA: American Association of Pharmaceutical Scientists. Retrieved August 27, 2015, from https://cms.psav.com/efb7b65/public/download_uploaded_media/presentation/5
10. Hock, M.B., Thudium, K.E., Carrasco-Triguero, M. & Schwabe, N.F. (2015). Immunogenicity of antibody drug conjugates: bioanalytical methods and monitoring strategy for a novel therapeutic modality. AAPS Journal. 17(1): 35-43.
11. Mire-Sluis, A.R., Barrett, Y.C., Devanarayan, V., Koren, E., Liu, H. Maia, M., Parish, T., Scott, G., Shankar, G., Shores, E., Swanson, S.J., Taniguchi, Gl, Wierda, D., & Zuckerman, L.A. (2004). Recommendations for the design and optimization of immunoassays used in the detection of host antibodies against biotechnology products. J. Immunol. Methods. 289(1-2): 1-16.