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Progress made in antibody-mediated drug delivery with homogeneous ADC products

Antibody-drug conjugates (ADCs) are monoclonal antibodies coupled to cytotoxic agents. They travel to target cells, where the antibody binds to its antigen expressed on the cell surface. Upon binding, the ADCs are internalized by receptor-mediated endocytosis. Following lysosomal degradation of the ADCs, the cytotoxic agents are released inside the cells, which ultimately lead to the death of the target cells by apoptosis. A well-known example of ADC is ADCETRIS®, which targets CD30-positive cancer cells. It is also the first ADC to be approved by the US Food and Drug Administration for the treatment of relapsed or refractory cases of classical Hodgkin lymphoma. Successful development of an ADC requires optimization of several different components such as the structure of the antibody, the potency of the cytotoxic drug, the stability of the linker, the conjugation site and the drug-antibody ratio (DAR). Typically, drugs can be linked to antibodies through cysteine or lysine residues, which results in ADCs with a heterogeneous number of drug molecules ranging from 0 to 11 per antibody. Antibodies without drugs are ineffective and compete with ADCs for binding to antigen-expressing cells. Conversely, products with a high DAR have less favorable pharmacokinetics, resulting in less favorable in vivo efficacy. There is, consequently, a need for new technologies capable of producing ADCs with a defined number of drugs per antibody.

Innate Pharma, a biopharmaceutical company specialized in developing first-in-class immunotherapy drugs for cancer and inflammatory diseases, worked on a new ADC-coupling technology. The latter relies on the use of bacterial transglutaminase to conjugate in a site-specific and homogenous manner variants of the anti-CD30 antibody cAC10 (Brentuximab) with a toxin called monomethyl auristatin E (MMAE) and yield ADC products with a defined DAR (Dennler et al., 2014). Evaluating the stability of those ADC products in vitro is paramount to validating the technology concept for clinical applications. Using Enzo’s PROTEOSTAT Thermal Shift stability assay kit, they were able to determine the aggregation propensity of their ADC products generated by either one-step enzymatic or two-step chemo-enzymatic reactions. Results confirmed the initial hypothesis that the greater the number of MMAE toxins coupled to the monoclonal antibody and the more lipophilic the linker between the monoclonal antibody and the toxins correlated with lower temperatures of aggregation and therefore, higher propensities for aggregation. Using Enzo’s PROTEOSTAT Protein aggregation assay kit and LC-MS, they demonstrated that physical stress tests and long-term storage at 5°C had little to no effect on the percentage of aggregates and DAR of their ADC products, respectively. Conversely, ADC products showed a significant increase in aggregation after storage conditions of accelerated degradation although DAR remained unaffected validating the stability and robustness of the linkers developed by Innate Pharma even when stored at 40°C (Schneider N et al., 2014).

More recently, they released new data in Molecular Pharmaceutics where they investigated the pharmacokinetics and therapeutic efficacies of these ADC products. In vitro cell toxicity with CD30-positive cells demonstrated similar EC50-values between ADCETRIS® and the four cAC10 variants conjugated with MMAE. Upon injection of iodinated labeling of antibodies and ADCs to xenografted and non-xenografted SCID mice, in vivo biodistribution studies were undertaken and showed a higher uptake by the tumor of the enzymatically conjugated cAC10 than ADCETRIS® with lesser non-targeted and non-specific uptakes by the liver and the spleen. Male Wistar rats were then used to conduct pharmacokinetics studies. These experiments established that the DAR of chemo-enzymatically conjugated cAC10 remained stable in vivo over the tested period and that the terminal half-lives of ADC products and ADCETRIS® were comparable. Clearance was, however, largely in favor of the ADC products developed by Innate Pharma. Finally, the maximum tolerated dose was also determined and results showed that 60mg/kg of bacterial transglutaminase-conjugated antibody could be injected to male Wistar rats with no signs of toxicity or weight loss, namely more than three times the maximum tolerated dose of ADCETRIS®. Altogether, these data highlight the in vitro and in vivo stability of bacteria transglutaminase-coupled antibodies and could go a long way towards proving their therapeutic utility and advancing their clinical development.

Enzo Life Sciences offers a comprehensive bioprocess portfolio to optimize and monitor product integrity, purity and efficiency including protein aggregation and thermal shift stability kits; some of which are listed below:

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  1. P. Dennler, et al. Transglutaminase-based chemo-enzymatic conjugation approach yields homogeneous antibody-drug conjugates. Bioconj. Chem. (2014) 25:569.
  2. N. Schneider, et al. Predicting aggregation propensity and monitoring aggregates in ADCs. BioProcess Int. (2014) 12:32.
  3. F. Lhospice, et al. Site-specific conjugation of monomethyl auristatin E to anti-CD30 antibodies improves their pharmacokinetics and therapeutic index in rodent models. Mol. Pharm. (2015) 12:1863.

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