Translational PK/PD framework for antibody-drug conjugates to inform drug discovery and development.

ADCs represent a transformative class of medicine that combines the specificity of monoclonal antibodies with the potency of highly cytotoxic agents through linkers, aiming to enhance the therapeutic index of cytotoxic drugs. Given the complex molecular structures of ADCs, combining the molecular characteristics of small-molecule drugs and those of large-molecule biotherapeutics, there are several unique considerations when designing nonclinical-to-clinical PK/PD translation strategies.This complexity also demands a thorough understanding of the ADC's components - antibody, linker, and payload - to the overall toxicological, PK/PD, and efficacy profile. ADC development is a multidisciplinary endeavour requiring a strategic integration of nonclinical safety, pharmacology, and PK/PD modelling to translate from bench to bedside successfully.The ADC development underscores the necessity for a robust scientific foundation, leveraging advanced analytical and modelling tools to predict human responses and optimise therapeutic outcomes.This review aims to provide an ADC translational PK/PD framework by discussing unique aspects of ADC nonclinical to clinical PK translation, starting dose determination, and leveraging PK/PD modelling for human efficacious dose prediction and potential safety mitigation.

PEGylation of anti-MerTK Antibody Modulates Ocular Biodistribution.

Here, we explore whether PEGylation of antibodies can modulate their biodistribution to the eye, an organ once thought to be immune privileged but has recently been shown to be accessible to IV-administered large molecules, such as antibodies. We chose to PEGylate an anti-MerTK antibody, a target with known potential for ocular toxicity, to minimize biodistribution to retinal pigment epithelial cells (RPEs) in the eye by increasing the hydrodynamic volume of the antibody. We used site-specific conjugation to an engineered cysteine on anti-MerTK antibody to chemically attach 40-kDa branched or linear PEG polymers. Despite reduced binding to MerTK on cells, site-specifically PEGylated anti-MerTK retained similar potency in inhibiting MerTK-mediated macrophage efferocytosis of apoptotic cells. Importantly, we found that PEGylation of anti-MerTK significantly reduced MerTK receptor occupancy in RPE cells in both naïve mice and MC-38 tumor-bearing mice, with the branched PEG exhibiting a greater effect than linear PEG. Furthermore, similar to unconjugated anti-MerTK, PEGylated anti-MerTK antibody triggered type I IFN response and exhibited antitumor effect in syngeneic mouse tumor studies. Our results demonstrate the potential of PEGylation to control ocular biodistribution of antibodies.

Novel Anti-LY6G6D/CD3 T-Cell-Dependent Bispecific Antibody for the Treatment of Colorectal Cancer.

New therapeutics and combination regimens have led to marked clinical improvements for the treatment of a subset of colorectal cancer. Immune checkpoint inhibitors have shown clinical efficacy in patients with mismatch-repair-deficient or microsatellite instability-high (MSI-H) metastatic colorectal cancer (mCRC). However, patients with microsatellite-stable (MSS) or low levels of microsatellite instable (MSI-L) colorectal cancer have not benefited from these immune modulators, and the survival outcome remains poor for the majority of patients diagnosed with mCRC. In this article, we describe the discovery of a novel T-cell-dependent bispecific antibody (TDB) targeting tumor-associated antigen LY6G6D, LY6G6D-TDB, for the treatment of colorectal cancer. RNAseq analysis showed that LY6G6D was differentially expressed in colorectal cancer with high prevalence in MSS and MSI-L subsets, whereas LY6G6D expression in normal tissues was limited. IHC confirmed the elevated expression of LY6G6D in primary and metastatic colorectal tumors, whereas minimal or no expression was observed in most normal tissue samples. The optimized LY6G6D-TDB, which targets a membrane-proximal epitope of LY6G6D and binds to CD3 with high affinity, exhibits potent antitumor activity both in vitro and in vivo. In vitro functional assays show that LY6G6D-TDB-mediated T-cell activation and cytotoxicity are conditional and target dependent. In mouse xenograft tumor models, LY6G6D-TDB demonstrates antitumor efficacy as a single agent against established colorectal tumors, and enhanced efficacy can be achieved when LY6G6D-TDB is combined with PD-1 blockade. Our studies provide evidence for the therapeutic potential of LY6G6D-TDB as an effective treatment option for patients with colorectal cancer.

Multiplexed Quantitative Analysis of Antibody-Drug Conjugates with Labile CBI-Dimer Payloads In Vivo Using Immunoaffinity LC-MS/MS.

Quantitative analysis of antibody-drug conjugates (ADCs) involves cleavage of ADCs into smaller analytes representing different components and subsequent measurements from multiple assays for a more comprehensive pharmacokinetic (PK) assessment. Multiple PK analytes including the drug remaining conjugated to the antibody (or antibody-conjugated drug, acDrug) and total antibody can be accessed simultaneously using a multiplex assay by proteolytic digestion of an ADC, if the sites of conjugation are homogeneous for an ADC and the linker drug is stable to proteases. Herein, a multiplexed immunoaffinity liquid chromatography-mass spectrometry (LC-MS)/MS PK assay is described involving immunoaffinity enrichment, enzymatic conversion of prodrug, trypsin digestion, and LC-MS/MS as applied to next-generation ADCs constructed from linker drugs bearing dimeric cyclopropabenzindole (CBI) payloads (duocarmycin analogues). The cytotoxic payload is chemically labile, requiring extensive optimization in sample preparation steps to stabilize the drug without ex vivo modification and to convert the prodrug into a single active form of the drug. The qualification data for this assay format showed that this approach provides robust acDrug and total antibody data and can be extended to ADCs with different monoclonal antibody frameworks and linker chemistries. Applications of this multiplexed assay to support preclinical studies are presented.

Calicheamicin Antibody-Drug Conjugates with Improved Properties.

Calicheamicin antibody-drug conjugates (ADCs) are effective therapeutics for leukemias with two recently approved in the United States: Mylotarg (gemtuzumab ozogamicin) targeting CD33 for acute myeloid leukemia and Besponsa (inotuzumab ozogamicin) targeting CD22 for acute lymphocytic leukemia. Both of these calicheamicin ADCs are heterogeneous, aggregation-prone, and have a shortened half-life due to the instability of the acid-sensitive hydrazone linker in circulation. We hypothesized that we could improve upon the heterogeneity, aggregation, and circulation stability of calicheamicin ADCs by directly attaching the thiol of a reduced calicheamicin to an engineered cysteine on the antibody via a disulfide bond to generate a linkerless and traceless conjugate. We report herein that the resulting homogeneous conjugates possess minimal aggregation and display high in vivo stability with 50% of the drug remaining conjugated to the antibody after 21 days. Furthermore, these calicheamicin ADCs are highly efficacious in mouse models of both solid tumor (HER2+ breast cancer) and hematologic malignancies (CD22+ non-Hodgkin lymphoma). Safety studies in rats with this novel calicheamicin ADC revealed an increased tolerability compared with that reported for Mylotarg. Overall, we demonstrate that applying novel linker chemistry with site-specific conjugation affords an improved, next-generation calicheamicin ADC.

Antibody-mediated delivery of chimeric protein degraders which target estrogen receptor alpha (ERα).

Chimeric molecules which effect intracellular degradation of target proteins via E3 ligase-mediated ubiquitination (e.g., PROTACs) are currently of high interest in medicinal chemistry. However, these entities are relatively large compounds that often possess molecular characteristics which may compromise oral bioavailability, solubility, and/or in vivo pharmacokinetic properties. Accordingly, we explored whether conjugation of chimeric degraders to monoclonal antibodies using technologies originally developed for cytotoxic payloads might provide alternate delivery options for these novel agents. In this report we describe the construction of several degrader-antibody conjugates comprised of two distinct ERα-targeting degrader entities and three independent ADC linker modalities. We subsequently demonstrate the antigen-dependent delivery to MCF7-neo/HER2 cells of the degrader payloads that are incorporated into these conjugates. We also provide evidence for efficient intracellular degrader release from one of the employed linkers. In addition, preliminary data are described which suggest that reasonably favorable in vivo stability properties are associated with the linkers utilized to construct the degrader conjugates.

Antibody Conjugation of a Chimeric BET Degrader Enables in†vivo Activity.

The ability to selectively degrade proteins with bifunctional small molecules has the potential to fundamentally alter therapy in a variety of diseases. However, the relatively large size of these chimeric molecules often results in challenging physico-chemical properties (e. g., low aqueous solubility) and poor pharmacokinetics which may complicate their in vivo applications. We recently discovered an exquisitely potent chimeric BET degrader (GNE-987) which exhibited picomolar cell potencies but also demonstrated low in vivo exposures. In an effort to improve the pharmacokinetic properties of this molecule, we discovered the first degrader-antibody conjugate by attaching GNE-987 to an anti-CLL1 antibody via a novel linker. A single IV dose of the conjugate afforded sustained in vivo exposures that resulted in antigen-specific tumor regressions. Enhancement of a chimeric protein degrader with poor in vivo properties through antibody conjugation thereby expands the utility of directed protein degradation as both a biological tool and a therapeutic possibility.

Preclinical pharmacokinetics and pharmacodynamics of DCLL9718A: An antibody-drug conjugate for the treatment of acute myeloid leukemia.

Few treatment options are available for acute myeloid leukemia (AML) patients. DCLL9718A is an antibody-drug conjugate that targets C-type lectin-like molecule-1 (CLL-1). This receptor is prevalent on monocytes, neutrophils, and AML blast cells, and unlike CD33, is not expressed on hematopoietic stem cells, thus providing possible hematopoietic recovery. DCLL9718A comprises an anti-CLL-1 IgG1 antibody (MCLL0517A) linked to a pyrrolobenzodiazepine (PBD) dimer payload, via a cleavable disulfide-labile linker. Here, we characterize the in vitro and in vivo stability, the pharmacokinetics (PK) and pharmacodynamics (PD) of DCLL9718A and MCLL0517A in rodents and cynomolgus monkeys. Three key PK analytes were measured in these studies: total antibody, antibody-conjugated PBD dimer and unconjugated PBD dimer. In vitro, DCLL9718A, was stable with most (> 80%) of the PBD dimer payload remaining conjugated to the antibody over 96 hours. This was recapitulated in vivo with antibody-conjugated PBD dimer clearance estimates similar to DCLL9718A total antibody clearance. Both DCLL9718A and MCLL0517A showed linear PK in the non-binding rodent species, and non-linear PK in cynomolgus monkeys, a binding species. The PK data indicated minimal impact of conjugation on the disposition of DCLL9718A total antibody. Finally, in cynomolgus monkey, MCLL0517A showed target engagement at all doses tested (0.5 and 20 mg/kg) as measured by receptor occupancy, and DCLL9718A (at doses of 0.05, 0.1 and 0.2 mg/kg) showed strong PD activity as evidenced by notable reduction in monocytes and neutrophils.

Modulating Therapeutic Activity and Toxicity of Pyrrolobenzodiazepine Antibody-Drug Conjugates with Self-Immolative Disulfide Linkers.

A novel disulfide linker was designed to enable a direct connection between cytotoxic pyrrolobenzodiazepine (PBD) drugs and the cysteine on a targeting antibody for use in antibody-drug conjugates (ADCs). ADCs composed of a cysteine-engineered antibody were armed with a PBD using a self-immolative disulfide linker. Both the chemical linker and the antibody site were optimized for this new bioconjugation strategy to provide a highly stable and efficacious ADC. This novel disulfide ADC was compared with a conjugate containing the same PBD drug, but attached to the antibody via a peptide linker. Both ADCs had similar efficacy in mice bearing human tumor xenografts. Safety studies in rats revealed that the disulfide-linked ADC had a higher MTD than the peptide-linked ADC. Overall, these data suggest that the novel self-immolative disulfide linker represents a valuable way to construct ADCs with equivalent efficacy and improved safety. Mol Cancer Ther; 16(5); 871-8. ©2017 AACR.

Development and Translational Application of an Integrated, Mechanistic Model of Antibody-Drug Conjugate Pharmacokinetics.

Antibody drug conjugates (ADC), in which small molecule cytotoxic agents are non-specifically linked to antibodies, can enable targeted delivery of chemotherapeutics to tumor cells. ADCs are often produced and administered as a mixture of conjugated antibodies with different drug to antibody ratios (DAR) resulting in complex and heterogeneous disposition kinetics. We developed a mechanism-based platform model that can describe and predict the complex pharmacokinetic (PK) behavior of ADCs with protease-cleavable valine-citrulline (VC) linker linked to Monomethylmonomethyl auristatin F/E by incorporating known mechanisms of ADC disposition. The model includes explicit representation of all DAR species; DAR-dependent sequential deconjugation of the drug, resulting in the conversion of higher DAR to lower DAR species; and DAR-dependent antibody/ADC clearance. PK profiles of multiple analytes (total antibody, drug-conjugated antibody, and/or antibody-conjugated drug) for different ADC molecules and targets in rodents and cynomolgus monkeys were used for model development. The integrated cross-species model was successful in capturing the multi-analyte PK profiles after administration of purified ADCs with defined DAR species and ADCs with mixtures of DAR. Human PK predictions for DSTP3086S (anti-STEAP1-vc-MMAE) with the platform model agreed well with PK (total antibody and antibody-conjugated drug concentrations) measurements in the dose-ranging phase I clinical study. The integrated model is applicable to various other ADCs with different formats, conjugated drugs, and linkers, and provides a valuable tool for the exploration of mechanisms governing disposition of ADCs and enables translational predictions.