Roger Hayes, PhD, Senior Vice President, General Manager, Laboratory Sciences
Chemotherapy is a mainstay of a standardized treatment regimen for cancer. However, the nonspecific targeting of healthy cells as well as tumor cells by cytotoxic small molecule drugs often results in intolerable side effects. These side effects compromise the efficacy of the treatment regimen and dramatically decrease the quality of life for cancer patients. Antibody drug conjugates (ADCs) are a newer class of chemotherapeutics comprised of monoclonal antibodies that selectively bind to tumor-associated antigens and have an associated cytotoxic small-molecule payload. Much effort has been made to identify highly tumor-specific monoclonal antibodies (mAbs), cytotoxic payloads with maximum efficacy, and linkers that are stable in circulation but allow for rapid cleavage to release the cell killing drugs following intracellular uptake of the ADCs.
1. Assay Formats for Pharmacokinetic Methods
Ideally, the assay methods for nonclinical PK bioanalysis should be developed during the early stages of ADC development and characterization. Evaluating the assays could be done with the recovery of enriched or purified drug antibody ratio (DAR) fractions compared with the average DAR standard to ensure that the different assay formats recover drug equally. If this analysis is not possible, detailed information of the ADC’s mechanism of action, targeted tumor antigen, type of linker, drug antibody ratio, cytotoxic drug, etc., are necessary for the bioanalytical method design. In addition to LBA methods, hydrophilic interaction liquid chromatography (HILIC), HPLC, and LC-MS are being used to quantify ADCs. These methods, however, are beyond the scope of the current discussion and are reported elsewhere 1, 3, 4.
2. Immunogenicity of ADC
Although the same methods used for determining the immunogenicity of general therapeutic antibodies can be used to determine the immunogenicity of ADCs, further characterization of anti-ADC antibodies for the targeting antibody, the linker, and the drug components are required to address the specificity of positive samples. The complexity of the ADC structure raises additional questions not previously encountered in the analysis of monoclonal antibody therapeutics or small molecule drugs. For instance, does an antibody response against the linker or the cytotoxic drug affect ADC internalization? Alternatively, do only neutralizing antibodies against the complementarity determining region (CDR) of the targeting antibody reduce the efficacy of the ADC?
3. Matrix Selection
Antibodies are commonly recognized as stable; therefore, most bioanalytical assays for therapeutic monoclonal antibodies are established in serum. This blanket approach, however, is not appropriate for the bioanalysis of ADCs where the conjugation of small molecule drug to the antibody via a linker creates a molecule whose overall stability depends upon the least stable of the three components. To this end, plasma is suggested as being the preferred matrix used for PK, and immunogenicity sample analysis because the inhibition of the clotting cascade in plasma results in much less proteolysis than in serum. Moreover, protease inhibitors could be added during sample collection to further stabilize the ADCs in plasma.
4. Critical Reagents
The LBA has been the primary analysis platform used for ADC bioanalysis because of its many advantages, including the ability to measure the test article in matrix without further sample extraction, its high-throughput nature, the broad dynamic range and high sensitivity that can be achieved, and the requirement of minimal sample volume. However, the availability of critical reagents is a key component to the development of highly specific and sensitive LBA methods. Much time and effort is usually taken to create anti-idiotype antibodies against the carrier antibody or the cytotoxic drug. Because of the low immunogenic nature of cytotoxic drugs, conjugation with keyhole limpet hemocyanin (KLH) or an adjuvant may be required for antibody creation. The tight timelines for most IND-enabling studies necessitate early planning during the design of bioanalytical assays and must factor in the timelines for generation of critical reagents.
In conclusion, ADC technology has become increasingly prevalent, it is imperative that new and reliable methods are developed to better characterize different ADCs both in vitro and in vivo. Because of the complexity of ADCs, many LBA formats are available for the same bioanalytical purpose.
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