Reprinted from the October 2015 Issue of Contract Pharma contractpharma.com

AIT BiosciencesFranklin Spriggs, Ligand Binding Assay Group Leader

Stephanie Mowery, Senior Scientist Bo Kowalcyk, Vice President of Business Development

Partnering with CrOs tO DevelOP high-Quality aDCs

Key Bioanalytical StrategiesaDCs are a breakthrOugh CanCer treatment that Can Deliver CytOtOxins DireCtly tO CanCer Cells

withOut the sweePing COllateral Damage Of traDitiOnal ChemOtheraPy.

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 bio-analytical strategies used to support ADC pro-grams, the critical challenges associated with this development and subsequent validations, and hurdles that biopharma companies and CROs may neglect to consider.

ADCs Are ConCeptuAlly simpleTraditional 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 explo-sion 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 mono-clonal antibody (mAb) and a cytotoxic agent conjugated via a stable linker2 (Figure 2). 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 1).

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 DevelopmentThough conceptually simple, ADC development is intri-cate because linker chemistry is complex. The ADC linker must be stable or risk pre-maturely releasing its agent, resulting in widespread cell death. Not only must the conjugation chemistry pro-vide stability but so must the mechanism of linker cleavage. Current and future ADCs use

figure 1. Mechanism of ADC receptor-mediated endocytosis.2

figure 2. Schematic of an antibody drug conjugate.2

Developing ADCs

Reprinted from the October 2015 Issue of Contract Pharma contractpharma.com

either the cysteine’s thiol or the lysine’s amine to conjugate the linker and the mAB.1,4

Conjugation of the mAbs ly-sine residues results in a more heterogeneous ADC because there are many possible lysine residues available for conjuga-tion. 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 ho-mogeneity are directly related to the drug-to-antibody ra-tio (DAR) of the antibody. The DAR, as discussed below, is one important consideration when developing the best ADC bio-analytical strategy.

pArtnering With A Cro for ADC BioAnAlysisTo 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 develop-ment, validation, and execution. It is the heterogenic nature of antibody drug conjugates that presents many hurdles for char-acterization (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 exper-tise 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 pharmaceuti-cal company and the CRO. Working together, these individuals should be empowered to make decisions critical to ADC devel-opment, 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 ex-pertise of both large molecule and small molecule labs, each of which employs unique approaches to quantifying and character-izing investigational drugs. The marriage of these two historically divergent bioanalytical departments calls for a level of collabora-tion not typically needed for discovery and development of tradi-tional therapeutics.

Due to the complexity of ADC analysis, it is important for pharmaceutical compa-nies 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 determin-ing 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 num-ber 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 ob-tain 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 pharma-cokinetics and immunogenicity of the dosed ADC.

importAnt ConsiDerAtions for ADC BioAnAlysisThe bioanalytical team first needs to collaborate in determining what analyte(s) should be measured and with what bioanalyti-cal platform. A 2013 publication outlines a basic strategy for bio-analysis 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 predict-ing 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 ef-fect the DAR has on the accurate quantitation of the ADC (and/

figure 3. ADC Analyte permeations. (A) Heterogeneous ADC and possible analytes; (B) permutations of 1 DAR; (C) drug conjugated to antibody; (D) unconjugated and conjugated species that make up total antibody; (E) unconjugated antibody; (F) free cytotoxic drug. Reproduced from Bioanlysis. (2013) 5(9), 997–1006 with permission of Future Medicine Ltd.

Developing ADCs

Reprinted from the October 2015 Issue of Contract Pharma contractpharma.com

or related analyte) and the detection of the Anti-Drug Antibod-ies (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 conju-gated, 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, individ-ually 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 quan-titation of the ADC in matrix. Depending on format (such as gde-tecting with an anti-chemotoxic agent), the assay can generate significantly different results based on the DAR number.6,8

ADA methoDologiesWhen 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 mol-ecule ADA testing plan outlined by Mire-Sluis et. al.11 as well as a method to determine domain specificity, allowing for the correla-tion 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 cre-ated 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.

ConClusionAntibody 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 bio-analytical capabilities can prove to be advantageous since both technologies are needed to fully characterize the pharmacokinet-ics/toxicokinetics of an ADC. More importantly, the drug devel-opers and bioanalytical scientists must collaborate and determine the most efficient path forward to enable quick turn-around of high quality data. Cp

References1. Rostami, S., Qazi, I., & Sikorski, R. (2014). The Clinical Landscape of Anti-

body-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 develop-ing 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 Conju-gates. 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-Conju-gated 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 in-terference: 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 Genera-tion and Next Generation Biotherapeutics: What Do We Expect to Change Going Forward? San Diego, CA, USA: American Association of Pharma-ceutical 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., Tanigu-chi, 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.

“Due to the complexity of ADC analysis,

it is important for pharmaceutical compa-

nies outsourcing bioanalysis of their ADCs

to identify a CRO that specializes in both

large and small molecule analysis.”