Newsletter Archive >> Spring 2012 >> Critical Aspects of Antibody Drug Conjugates

Critical Aspects of Antibody Drug Conjugates: Structure and Analysis

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by Robert J. Duff, Ph.D., manager, Biochemistry Group

Targeted drug therapies for diseased cells (i.e. cancer) using monoclonal antibodies has been a topic of growing interest and an area of continued development. The promise of this approach has been to deliver a therapeutic (or cytotoxic) agent with greater selectivity thereby reducing toxic side effects. Antibodydrug conjugates (ADC) are an emerging class of targeted agents demonstrating tremendous potential both in vitro and in vivo. These rationally designed conjugates, formed through the chemical linkage of a potent small molecule cytotoxin (drug) to a monoclonal antibody (mAb), have more complex and heterogeneous structures than the corresponding antibodies.

The mechanism of action is believed to be, first antigen recognition and binding, followed by endocytosis whereby the cell’s lysosomal enzymes release the cytotoxin. The molecular diversity of the cytotoxins is remarkable. Most notably, microtubule disruptors have been recognized, which are represented by two major classes: maytansinoids and auristatins. The newer classes of cytotoxins are at least two orders of magnitude more potent with in vitro studies against tumor cell lines.

The rationale development of the conjugate is crucial to success. Each part (the mAb, the drug and the linker) must be carefully chosen in order to provide the best therapeutic index. Antibodies have targeted antigens (e.g. CD22, HER2, PSMA, CD70, EphA2) that are known to play a key role in cancer cell growth and survival. The commercial pipeline of antibody-based therapeutics continues to grow and now totals nearly 350 candidates. With respect to ADCs, the FDA recently, in August 2011, approved Adcetris (brentuximab vedotin) for Hodgkin’s Lymphoma. Currently, 20 new ADCs are currently in clinical development.

Conjugates must employ the right linker with the method of attachment as an essential part of the ADC program. Many areas around the process have improved, however, the linker strategy for ADC manufacturing and their application has certainly been primary for clinical advancement. The choice of a linker is influenced by which toxin is used, as each toxin has different chemical constraints. The creation of linkers that are stable in circulation but labile upon binding of the ADC to its target has resulted in the current generation of ADCs having better stability and lower systemic toxicity. Early versions of ADCs suffered from instability of the linkage between the mAb and the cytotoxic small molecule. Endogenous proteases in the blood caused the premature release of the cytotoxin and resulted in side-effect profiles similar to that of an unconjugated chemotherapeutic. The new generation of linkers is more resistant to degradation in the blood while still allowing release of the payload at the target.

Regulatory agencies are primarily concerned with the selection of the most appropriate analytical methods for an ADC – which is the same as for any other biopharmaceutical or pharmaceutical product. These methods must discern the identity, potency and heterogeneity of the biodegradable linker, the cytotoxic drug and the choice of mAb attachment sites (lysines, inter-chain cysteines, Fc glycans). Heterogeneity depends wholly on the selectivity of the chemical reaction between the linker and mAb and the cytotoxin.

Improvements in analytical techniques such as protein mass spectrometry and capillary electrophoresis have significantly increased the quality of information that can be obtained for use in product and process characterization and for routine lot release and stability testing. Analytical methodologies need to discern primary structure (intact mass, peptide mapping [sequencing], NMR, FTIR and drug linkage discernment), secondary/tertiary structure (circular dichroism, X-ray), drug-antibody ratio (UV), fragments/aggregates (AUC, SEC-MALS, SE-HPLC), charge variants (CE, iCE, IEX, MS), glycosylation and other post translational modifications (LC-MS/ MS). Antigen binding, biological activities and effector function are used as appropriate (ELISA, SPR [BiaCore]). Assays for free drug and (bio)process impurities such as synthetic impurities or host cell proteins should be included. Overall, the ADC should be considered a new molecular entity and not a combination product. ADCs show tremendous promise for the future and now better methodologies are available to prove structure-function relationship through site-specific mAbs and non-natural amino acids.