Development of a Highly Sensitive Hybrid LC/MS Assay for the Quantitative Measurement of CTLA-4 in Human T Cells.

Cytotoxic T-lymphocyte-associated protein 4 (CTLA-4) is a check point protein expressed on the surface of T cells and plays a central role in regulating the immune response. In recent years, CTLA-4 has become a popular target for cancer immunotherapy in which blocking CTLA-4 can restore T-cell function and enhance the immune response against cancer. Currently, there are many CTLA-4 inhibitors in a variety of modalities, including cell therapies, which are being developed in both preclinical and clinical stages to further harness the potential of the target for the treatment of certain types of cancer. In drug discovery research, measuring the level of CTLA-4 in T cells is important for drug discovery and development because it provides key information for quantitative assessment of the pharmacodynamics, efficacy, and safety of the CTLA-4-based therapies. However, to our best knowledge, there is still no report of a sensitive, specific, accurate, and reliable assay for CTLA-4 measurement. In this work, an LC/MS-based method was developed to measure CTLA-4 in human T cells. The assay demonstrated high specificity with an LLOQ of 5 copies of CTLA-4 per cell when using 2.5 million T cells for analysis. As shown in the work, the assay was successfully used to measure CTLA-4 levels in subtype T-cell samples from individual healthy subjects. The assay could be applied in supporting the studies of CTLA-4-based cancer therapies.

An Automated Multicycle Immunoaffinity Enrichment Approach Developed for Sensitive Mouse IgG1 Antibody Drug Analysis in Mouse Plasma Using LC/MS/MS.

In the immuno-oncology field, surrogate mouse monoclonal antibodies are often preferred in establishing proper PK/PD/efficacy correlations as well as supporting anticipated mouse to human translation. Thus, a highly sensitive and specific bioanalytical method is needed in quantifying those surrogate mouse antibodies after dosing in mice. Unfortunately, when specific reagents, such as recombinant target antigen and anti-idiotypic antibody, are not available, measuring mouse surrogate antibody drugs in mice is very challenging for ligand binding assay (LBA) due to the severe cross reactivity potential. Different from LBA, if at least one unique surrogate peptide can be identified from the surrogate antibody sequence, the immunoaffinity enrichment based LC/MS/MS assay may be able to differentiate the analyte response from the high endogenous immunoglobulin background and provide adequate sensitivity. Herein, a new automated multicycle immunoaffinity enrichment method was recently developed to extract a surrogate mouse IgG1 (mIgG1) antibody drug from mouse plasma using a commercially available antimouse IgG1 secondary antibody. In the assay, reuse of the capture antibody up to six times mostly resolved the binding capacity issue caused by the abundant endogenous mIgG1 and made the immunoaffinity enrichment step more cost-effective. Combined with a unique surrogate peptide identified from the antibody, the LC/MS/MS assay achieved a limit of quantitation of 5 ng/mL with satisfactory assay precision, accuracy, and dynamic range. The successful implementation of this novel approach in discovery pharmacokinetic (PK) studies eliminates the dependence on specially generated immunoaffinity capturing reagents.

A Citrulline-Based Translational Population System Toxicology Model for Gastrointestinal-Related Adverse Events Associated With Anticancer Treatments.

Gastrointestinal (GI)-related adverse events (AEs) are commonly observed in the clinic during cancer treatments. Citrulline is a potentially translatable biomarker of GI AEs. In this study, irinotecan-induced citrulline changes were studied for a range of doses and schedules in rats. A translational system toxicology model for GI AEs using citrulline was then developed based on new experimental data and parameters from a literature intestinal cell dynamic model. With the addition of feedback-development and tolerance-development mechanisms, the model well captured the plasma citrulline profiles after irinotecan treatment in rats. Subsequently, the model was translated to humans and predicted the observed GI AE dynamics in humans including dose-scheduling effect using the cytotoxic and feedback parameters estimated in rats with slight calibrations. This translational toxicology model could be used for other antineoplastic drugs to simulate various clinical dosing scenarios before human studies and mitigate potential GI AEs.

An LC/MS based method to quantify DNA adduct in tumor and organ tissues.

Highly potent DNA damaging agents have become a key class of toxins for antibody-drug conjugate (ADC) based targeted therapy. However, until recently, no quantitative bioanalytical method was available to measure the toxin in the form of DNA adducts. In this work, a novel microwave assisted organic solvent extraction and LC-MS/MS based bioanalytical method was developed to extract and quantify DNA-bound toxin IGN-P1 in tissue samples. Using ADC-1 as the model ADC, the method was orthogonally checked with a radioactive method for the recovery of free toxins from DNA adducts in biological matrices. It was found that the bioanalytical method can achieve a high recovery of the IGN-P1 toxin from DNA adducts. In further assessment, tumor and organ tissue samples collected at multiple time points from in vivo studies after dosing with two other ADCs, ADC-2 and ADC-3, were measured by the method. Given the generic nature of the established bioanalytical method without the need of radiolabels, the methodology could be broadly utilized to quantitatively assess the relationship between DNA adduct levels and pharmacological/toxicological effects.

A generic approach for simultaneous measurements of total antibody and cleavable antibody-conjugated drug by LC/MS/MS.

The current industry practice for antibody-drug conjugate (ADC) bioanalysis includes quantification of total antibody and antibody-conjugated drug. Here, we report a novel 2-in-1 approach for measuring total antibody and protease-cleavable conjugated drug Monomethyl Auristatin E (MMAE) concurrently. This allows for the determination of the DAR (Drug Antibody Ratio) for in vivo samples, with a 3-orders linear range based on total antibody concentration from 0.1 to 100 µg/mL. Our generic, concurrent method has been cross-validated with the previously established methods in an animal study. This novel approach is applicable to all human IgG1 ADCs with papain cleavable conjugated drug in preclinical studies.

Mechanistic evaluation of substrate inhibition kinetics observed from aldehyde oxidase-catalyzed reactions.

While most enzyme-catalyzed reactions are adequately described by Michaelis-Menten kinetics, Aldehyde Oxidase (AOX) metabolism might exhibit atypical kinetics due to possible substrate inhibition. Ignoring this phenomenon may lead to erroneous estimates of kinetic parameters and over simplification of the enzyme mechanism. In this study, in vitro metabolism data for 3 AOX substrates exhibiting varying degrees of substrate inhibition were analyzed with the following kinetic models: A) Michaelis-Menten (naïve) model; B) Substrate inhibition (empirical) model; and C) Twobinding site (mechanistic) model. The application of this mechanistic model is a novel interpretation for kinetic analysis of AOX metabolism whereby substrate can presumably bind to two enzymes' active site(s). Unlike the other models, this mechanistic model quantitatively captures the degree of substrate inhibition observed. Analysis by this model showed: A) All tested substrates have simultaneous access to the metabolic and inhibitory site of the enzyme with Ks (binding affinity for inhibitory site) greater (1.3- to 28-fold) than Km (binding affinity for metabolic site); B) Dissociation constants for binding of a second substrate in either the productive and nonproductive enzyme conformations decreased with factor α ranging from 2.58 to 15.6 between compounds; and C) In addition, a drastic decrease (from 64%-98%) in the metabolism rates between compounds was exhibited by factor ß (ranging from 0.02-0.36). Overall, the mechanistic two-binding site model best fitted the experimental data. Moreover, the observed differences between kinetic parameters generated by these models highlight the importance of appropriate model selection to adequately fit the substrate inhibition kinetics of AOX metabolism.