DNA-Model-Based Design and Execution of Some Fused Benzodiazepine Hybrid Payloads for Antibody-Drug Conjugate Modality.

A new series with the tetrahydroisoquinoline-fused benzodiazepine (TBD) ring system combined with the surrogates of (1-methyl-1H-pyrrol-3-yl)benzene ("MPB") payloads were designed and executed for conjugation with a monoclonal antibody for anticancer therapeutics. DNA models helped in rationally identifying modifications of the "MPB" binding component and guided structure-activity relationship generation. This hybrid series of payloads exhibited excellent in vitro activity when tested against a panel of various cancer cell lines. One of the payloads was appended with a lysosome-cleavable peptide linker and conjugated with an anti-mesothelin antibody via a site-specific conjugation method mediated by the enzyme bacterial transglutaminase (BTGase). Antibody-drug conjugate (ADC) 50 demonstrated good plasma stability and lysosomal cleavage. A single intravenous dose of ADC 50 (5 or 10 nmol/kg) showed robust efficacy in an N87 gastric cancer xenograft model.

Design, Synthesis, and Structure-Activity Relationships of Novel Tetrahydroisoquinolino Benzodiazepine Dimer Antitumor Agents and Their Application in Antibody-Drug Conjugates.

A series of tetrahydroisoquinoline-based benzodiazepine dimers were synthesized and tested for in vitro cytotoxicity against a panel of cancer cell lines. Structure-activity relationship investigation of various spacers guided by molecular modeling studies helped to identify compounds with picomolar activity. Payload 17 was conjugated to anti-mesothelin and anti-fucosylated monosialotetrahexosylganglioside (FucGM1) antibodies using lysosome-cleavable valine-citrulline dipeptide linkers via heterogeneous lysine conjugation and bacterial transglutaminase-mediated site-specific conjugation. In vitro, these antibody drug conjugates (ADCs) exhibited significant cytotoxic and target-mediated selectivity on human cancer cell lines. The pharmacokinetics and efficacy of these ADCs were further evaluated in gastric and lung cancer xenograft models in mice. Consistent pharmacokinetic profiles, high target specificity, and robust antitumor activity were observed in these models after a single dose of the ADC-46 (0.02 µmol/kg).

High-Throughput Platform to Identify Antibody Conjugation Sites from Antibody-Drug Conjugate Libraries.

Antibody-drug conjugates (ADCs) are a therapeutic modality that traditionally enable the targeted delivery of highly potent cytotoxic agents to specific cells such as tumor cells. More recently, antibodies have been used to deliver molecules such as antibiotics, antigens, and adjuvants to bacteria or specific immune cell subsets. Site-directed mutagenesis of proteins permits more precise control over the site and stoichiometry of their conjugation, giving rise to homogeneous chemically defined ADCs. Identification of favorable sites for conjugation in antibodies is essential as reaction efficiency and product stability are influenced by the tertiary structure of immunoglobulin G (IgG). Current methods to evaluate potential conjugation sites are time-consuming and labor intensive, involving multistep processes for individually produced reactions. Here, we describe a highly efficient method for identification of conjugatable genetic variants by analyzing pooled ADC libraries using mass spectrometry. This approach provides a versatile platform to rapidly uncover new conjugation sites for site-specific ADCs.

Design, synthesis and biological evaluation of phenol-linked uncialamycin antibody-drug conjugates.

Uncialamycin is one of the structurally simpler and newer members of enediyne family of natural products. It exhibits highly potent activity against several types of bacteria and cancer cells. Described herein is a strategy for the targeted delivery of this cytotoxic agent to tumors using an antibody-drug conjugate (ADC) approach. Central to the design of ADC were the generation of potent and chemically stable uncialamycin analogues and attachment of protease cleavable linkers to newly realized phenolic handles to prepare linker-payloads. Conjugation of the linker-payloads to tumor targeting antibody, in vitro activity and in vivo evaluation are presented.

Universal Affinity Capture Liquid Chromatography-Mass Spectrometry Assay for Evaluation of Biotransformation of Site-Specific Antibody Drug Conjugates in Preclinical Studies.

Antibody drug conjugates (ADCs) can undergo in vivo biotransformation (e.g., payload metabolism, deconjugation) leading to reduced or complete loss of activity. The location/site of conjugation of payload-linker can have an effect on ADC stability and hence needs to be carefully optimized. Affinity capture LC-MS of intact ADCs or ADC subfragments has been extensively used to evaluate ADC biotransformation. However, the current methods have certain limitations such as the requirement of specific capture reagents, limited mass resolution of low mass change metabolites, low sensitivity, and use of capillary or nanoflow LC-MS. To address these challenges, we developed a generic affinity capture LC-MS assay that can be utilized to evaluate the biotransformation of any site-specific ADC independent of antibody type and site of conjugation (Fab and Fc) in preclinical studies. The method involves a combination of some or all of these steps: (1) "mono capture" or "dual capture" of ADCs from serum with streptavidin magnetic beads coated with a generic biotinylated antihuman capture reagent, (2) "on-bead" digestion with IdeS and/or PNGase F, and (3) reduction of interchain disulfide bonds to generate ∼25 kDa ADC subfragments, which are finally analyzed by LC-HRMS on a TOF mass spectrometer. The advantages of this method are that it can be performed using commercially available generic reagents and requires sample preparation time of less than 7 h. Furthermore, by reducing the size of intact ADC (∼150 kDa) to subfragments (∼25 kDa), the identification of conjugated payload and its metabolites can be achieved with excellent sensitivity and resolution (hydrolysis and other small mass change metabolites). This method was successfully applied to evaluate the in vitro and in vivo biotransformation of ADCs conjugated at different sites (LC, HC-Fab, and HC-Fc) with various classes of payload-linkers.

Uncialamycin as a novel payload for antibody drug conjugate (ADC) based targeted cancer therapy.

Uncialamycin analogs were evaluated as potential cytotoxic agents in an antibody-drug conjugate (ADC) approach to treating human cancer. These analogs were synthesized using Hauser annulations of substituted phthalides as a key step. A highly potent uncialamycin analog 3c with a valine-citrulline dipeptide linker was conjugated to an anti-mesothelin monoclonal antibody (mAb) through lysines to generate a meso-13 conjugate. This conjugate demonstrated subnanomolar potency (IC50 = 0.88 nM, H226 cell line) in in vitro cytotoxicity experiments with good immunological specificity to mesothelin-positive lung cancer cell lines. The potency and mechanism of action of this uncialamycin class of enediyne antitumor antibiotics make them attractive payloads in ADC-based cancer therapy.

Macrocyclic pyrrolobenzodiazepine dimers as antibody-drug conjugate payloads.

Macrocyclic pyrrolobenzodiazepine dimers were designed and evaluated for use as antibody-drug conjugate payloads. Initial structure-activity exploration established that macrocyclization could increase the potency of PBD dimers compared with non-macrocyclic analogs. Further optimization overcame activity-limiting solubility issues, leading to compounds with highly potent (picomolar) activity against several cancer cell lines. High levels of in vitro potency and specificity were demonstrated with an anti-mesothelin conjugate.

Validation of an integrated series of ligand-binding assays for the quantitative determination of antibody-drug conjugates in biological matrices.

BACKGROUND: The bioanalytical strategy for antibody-drug conjugates (ADC) includes multiple integrated measurements of pharmacologically relevant ADC. METHODS & RESULTS: Three ligand-binding assays were validated for the measurement of total antibody, active ADC and total ADC. Accuracy and precision demonstrate %bias from -8 to 14%, %CV from 3 to 11% and total error from 3 to 21%, with >98% samples meeting incurred sample reanalysis criteria. Each assay met stability, selectivity, dilutional integrity, carry over and specificity criteria with no interference from associated metabolite/impurity. Given the active ADC assay sensitivity to payload, active ADC was used to assess drug to antibody ratio. DISCUSSION & CONCLUSION: Implementation of a microfluidic automated platform enabled high throughput sample analysis of multiple analytes with minimal sample processing.

Pharmacokinetic characterization of BMS-936561, an anti-CD70 antibody-drug conjugate, in preclinical animal species and prediction of its pharmacokinetics in humans.

CD70 is a tumor necrosis factor (TNF)-like type II integral membrane protein that is transiently expressed on activated T- and B-lymphocytes. Aberrant expression of CD70 was identified in both solid tumors and haematologic malignancies. BMS-936561 (αCD70_MED-A) is an antibody-drug conjugate composed of a fully human anti-CD70 monoclonal antibody (αCD70) conjugated with a duocarmycin derivative, MED-A, through a maleimide-containing citrulline-valine dipeptide linker. MED-A is a carbamate prodrug that is activated by carboxylesterase to its active form, MED-B, to exert its DNA alkylation activity. In vitro serum stability studies suggested the efficiencies of hydrolyzing the carbamate-protecting group in αCD70_MED-A followed a rank order of mouse>rat > >monkey>dog~human. Pharmacokinetics of αCD70_MED-A was evaluated in mice, monkeys, and dogs after single intravenous doses. In mice, αCD70_MED-A was cleared rapidly, with no detectable exposures after 15 min following dosing. In contrast, αCD70_MED-A was much more stable in monkeys and dogs. The clearance of αCD70_MED-A in monkeys was 58 mL/d/kg, ~2-fold faster than that in dogs (31 mL/d/kg). The human PK profiles of the total αCD70 and αCD70_MED-A were predicted using allometrically scaled monkeys PK parameters of αCD70 and the carbamate hydrolysis rate constant estimated in dogs. Comparing the predicted and observed human PK from the phase I study, the dose-normalized concentration-time profiles of αCD70_MED-A and the total αCD70 were largely within the 5(th)-95(th) percentile of the predicted profiles.

Bioanalysis of antibody-drug conjugates: American Association of Pharmaceutical Scientists Antibody-Drug Conjugate Working Group position paper.

Antibody-drug conjugates (ADCs) typically consist of a cytotoxic drug covalently bound to an antibody by a linker. These conjugates have the potential to substantially improve efficacy and reduce toxicity compared with cytotoxic small-molecule drugs. Since ADCs are generally complex heterogeneous mixtures of multiple species, these novel therapeutic products present unique bioanalytical challenges. The growing number of ADCs being developed across the industry suggests the need for alignment of the bioanalytical methods or approaches used to assess the multiple species and facilitate consistent interpretation of the bioanalytical data. With limited clinical data, the current strategies that can be used to provide insight into the relationship between the multiple species and the observed clinical safety and efficacy are still evolving. Considerations of the bioanalytical strategies for ADCs based on the current industry practices that take into account the complexity and heterogeneity of ADCs are discussed.

Novel detection of DNA-alkylated adducts of antibody-drug conjugates with potentially unique preclinical and biomarker applications.

BACKGROUND: MDX-1203 is an antibody-drug conjugate (ADC) currently in clinical trials for the treatment of renal carcinoma. The active ingredient of MDX-1203 is a DNA minor groove-binding cytotoxic drug that forms a covalently linked adduct with an adenine base. Formation of this adenine adduct prevents DNA replication, thus triggering cell death. RESULTS: A method has been developed to successfully isolate, identify and quantitate the adenine adduct using LC-MS/MS. The method is highly useful to validate the mode of action of this class of ADCs. Additionally, we have demonstrated that this method could potentially be utilized to assess the efficacy of the ADC in in vitro studies by measuring the amount of adenine adduct in various cells expressing the antigen. CONCLUSION: Upon validation, this method could serve as an invaluable tool to evaluate compounds in preclinical in vivo models and in utilizing the DNA adduct as a potential biomarker.