Systematic LC/MS/MS Investigations for the IND-Enabling Extended Characterization of Antibody-Drug Conjugate Modifications.

We hypothesized that systematic liquid chromatography-tandem mass spectrometry investigations of an antibody-drug conjugate (ADC), its small and large molecular components, and surrogate small-molecule conjugates might comprise a simple and efficient approach for the extended characterization of ADCs. Furthermore, we envisioned that results from this work might allow us to assign specific composition changes in the ADC based on monoisotopic mass shifts of conjugatable modifications as detected in the surrogate small-molecule conjugates. We tested our hypothesis with a case study using an aldehyde-tag-based ADC conjugated to a noncleavable linker bearing a maytansine payload. Nearly quantitative bioconversion from cysteine to formylglycine was observed in the monoclonal antibody, and bioorthogonal conjugation was detected only on the formylglycine residues in the ADC. Using our method, both conjugatable and nonconjugatable modifications were discovered in the linker/payload; however, only conjugatable modifications were observed on the ADC. Based on these results, we anticipate that our approach to systematic mass spectrometric investigations can be successfully applied to other ADCs and therapeutic bioconjugates for investigational new drug (IND)-enabling extended characterization.

CAT-02-106, a Site-Specifically Conjugated Anti-CD22 Antibody Bearing an MDR1-Resistant Maytansine Payload Yields Excellent Efficacy and Safety in Preclinical Models.

Hematologically derived tumors make up ∼10% of all newly diagnosed cancer cases in the United States. Of these, the non-Hodgkin lymphoma (NHL) designation describes a diverse group of cancers that collectively rank among the top 10 most commonly diagnosed cancers worldwide. Although long-term survival trends are improving, there remains a significant unmet clinical need for treatments to help patients with relapsed or refractory disease, one cause of which is drug efflux through upregulation of xenobiotic pumps, such as MDR1. CD22 is a clinically validated target for the treatment of NHL, but no anti-CD22 agents have yet been approved for this indication. Recent approval of an anti-CD22 antibody-drug conjugate (ADC) for the treatment of relapsed/refractory ALL supports the rationale for targeting this protein. An opportunity exists for a next-generation anti-CD22 antibody-drug conjugate (ADC) to address unmet medical needs in the relapsed/refractory NHL population. We describe a site-specifically conjugated antibody-drug conjugate, made using aldehyde tag technology, targeted against CD22 and bearing a noncleavable maytansine payload that is resistant to MDR1-mediated efflux. The construct was efficacious against CD22+ NHL xenografts and could be repeatedly dosed in cynomolgus monkeys at 60 mg/kg with no observed significantly adverse effects. Exposure to total ADC at these doses (as assessed by AUC0-inf) indicated that the exposure needed to achieve efficacy was below tolerable limits. Together, the data suggest that this drug has the potential to be used effectively in patients with CD22+ tumors that have developed MDR1-related resistance to prior therapies. Mol Cancer Ther; 17(1); 161-8. ©2017 AACR.

A simple LC/MRM-MS-based method to quantify free linker-payload in antibody-drug conjugate preparations.

Antibody-drug conjugates represent a growing class of biologic drugs that use the targeted specificity of an antibody to direct the localization of a small molecule drug, often a cytotoxic payload. After conjugation, antibody-drug conjugate preparations typically retain a residual amount of free (unconjugated) linker-payload. Monitoring this free small molecule drug component is important due to the potential for free payload to mediate unintended (off-target) toxicity. We developed a simple RP-HPLC/MRM-MS-based assay that can be rapidly employed to quantify free linker-payload. The method uses low sample volumes and offers an LLOQ of 10nM with 370pg on column. This analytical approach was used to monitor free linker-payload removal during optimization of the tangential flow filtration manufacturing step.