Antibody-drug conjugates are an emerging class of cancer therapeutics constructed from monoclonal antibodies conjugated with small molecule effectors. First-generation molecules of this class often employed heterogeneous conjugation chemistry, but many site-specifically conjugated ADCs have been described recently.Here, we undertake a systematic comparison of ADCs made with the same antibody and the same macrocyclic maytansinoid effector but conjugated either heterogeneously at lysine residues or site-specifically at cysteine residues. Characterization of these ADCs in vitro reveals generally similar properties, including a similar catabolite profile, a key element in making a meaningful comparison of conjugation chemistries. In a mouse model of cervical cancer, the lysineconjugated ADC affords greater efficacy on a molar payload basis. Rather than making general conclusions about ADCs conjugated by a particular chemistry, we interpret these results as highlighting the complexity of ADCs and the interplay between payload class, linker chemistry, target antigen, and other variables that determine efficacy in a given setting.

Despite an improving therapeutic landscape, significant challenges remain in treating the majority of patients with advanced ovarian or renal cancer. We identified the cell-cell adhesion molecule cadherin-6 () as a lineage gene having significant differential expression in ovarian and kidney cancers. HKT288 is an optimized CDH6-targeting DM4-based antibody-drug conjugate (ADC) developed for the treatment of these diseases. Our study provides mechanistic evidence supporting the importance of linker choice for optimal antitumor activity and highlights CDH6 as an antigen for biotherapeutic development. To more robustly predict patient benefit of targeting CDH6, we incorporate a population-based patient-derived xenograft (PDX) clinical trial (PCT) to capture the heterogeneity of response across an unselected cohort of 30 models-a novel preclinical approach in ADC development. HKT288 induces durable tumor regressions of ovarian and renal cancer models , including 40% of models on the PCT, and features a preclinical safety profile supportive of progression toward clinical evaluation. We identify CDH6 as a target for biotherapeutics development and demonstrate how an integrated pharmacology strategy that incorporates mechanistic pharmacodynamics and toxicology studies provides a rich dataset for optimizing the therapeutic format. We highlight how a population-based PDX clinical trial and retrospective biomarker analysis can provide correlates of activity and response to guide initial patient selection for first-in-human trials of HKT288. .

Directed cell migration and process outgrowth are vital to proper development of many metazoan tissues. These processes are dependent on reorganization of the actin cytoskeleton in response to external guidance cues. During development of the nervous system, the MIG-10/RIAM/Lamellipodin (MRL) signaling proteins are thought to transmit positional information from surface guidance cues to the actin polymerization machinery, and thus to promote polarized outgrowth of axons. In C. elegans, mutations in the MRL family member gene mig-10 result in animals that have defects in axon guidance, neuronal migration, and the outgrowth of the processes or ‘canals’ of the excretory cell, which is required for osmoregulation in the worm. In addition, mig-10 mutant animals have recently been shown to have defects in clustering of vesicles at the synapse. To determine additional molecular partners of MIG-10, we conducted a yeast two hybrid screen using isoform MIG-10A as bait and isolated Abelson-interactor protein-1 (ABI-1). ABI-1, a downstream target of Abl non-receptor tyrosine kinase, is a member of the WAVE regulatory complex (WRC) involved in the initiation of actin polymerization. Further analysis using a co-mmunoprecipitation system confirmed the interaction of MIG-10 and ABI-1 and showed that it requires the SH3 domain of ABI-1. Single mutants for mig-10 and abi-1 displayed similar phenotypes of incomplete migration of the ALM neurons and truncated outgrowth of the excretory cell canals, suggesting that the ABI-1/MIG-10 interaction is relevant in vivo. Cell autonomous expression of MIG-10 isoforms rescued both the neuronal migration and the canal outgrowth defects, showing that MIG-10 functions autonomously in the ALM neurons and the excretory cell. These results suggest that MIG-10 and ABI-1 interact physically to promote cell migration and process outgrowth in vivo. In the excretory canal, ABI-1 is thought to act downstream of UNC-53/NAV2, linking this large scaffolding protein to actin polymerization during excretory canal outgrowth. abi-1(RNAi) enhanced the excretory canal truncation observed in mig-10 mutants, while double mutant analysis between unc-53 and mig-10 showed no increased truncation of the posterior canal beyond that observed in mig-10 mutants. Morphological analysis of mig-10 and unc-53 mutants showed that these genes regulate canal diameter as well as its length, suggesting that defective lumen formation may be linked to the ability of the excretory canal to grow out longitudinally. Taken together, our results suggest that MIG-10, UNC-53, and ABI-1 act sequentially to mediate excretory cell process outgrowth.

Antibody-drug conjugates (ADCs) are of great interest as targeted cancer therapeutics. Preparation of ADCs for early stage screening is constrained by purification and biochemical analysis techniques that necessitate burdensome quantities of antibody. Here we describe a method, developed for the maytansinoid class of ADCs, enabling parallel conjugation of antibodies in 96-well format. The method utilizes ∼ 100 µg of antibody per well and requires <5 µg of ADC for characterization. We demonstrate the capabilities of this system using model antibodies. We also provide multiple examples applying this method to early-stage screening of maytansinoid ADCs. The method can greatly increase the throughput with which candidate ADCs can be screened in cell-based assays, and may be more generally applicable to high-throughput preparation and screening of different types of protein conjugates.

Background: Genotoxic conditioning prior to allogeneic and autologous bone marrow transplantation (BMT) limits the use of these potentially curative treatments due to risks of regimen-related morbidities and mortality, including risks of organ toxicity, infertility, and secondary malignancies. CD117, which is specifically expressed on hematopoietic stem cells (HSCs) and progenitors is rapidly internalized and is an ideal target for an antibody drug conjugate (ADC) based approach to conditioning. We have previously shown that a single dose of an anti-CD117 ADC depleted >95% of bone marrow HSCs in a humanized mouse model and reduced disease burden while extending survival in an AML tumor model (Hartigan et al., Blood 2017 130:1894). The aim of this translational study was to develop a potent anti-CD117 ADC highly effective in eliminating host HSCs with a short half-life and minimal adverse side effects in a non-human primate (NHP) model. Methods: Three different DNA-damaging cytotoxic payloads and amanitin (AM) were site specifically conjugated to an anti-CD117 antibody. The ADCs were titrated and evaluated for in vitro cytotoxicity using human bone marrow CD34+ cells. The ADCs were administered in ascending doses to humanized NSG mice. HSC depletion and immunophenotype of the human cells in the peripheral blood was determined by flow cytometry. For amanitin conjugates, NHP HSC depletion was evaluated in male cynomolgus monkeys in single ascending doses (3/group). HSC content in the bone marrow was monitored by flow cytometry and colony-forming unit (CFU) analysis on day 7 or 14 and 56 post dosing. Hematology and clinical chemistries were evaluated throughout the two-month study. Results: Of the toxins evaluated, only anti-CD117 conjugated with the RNA polymerase II inhibitor amanitin resulted in >90% depletion of human HSCs in humanized NSG mice at 0.3 mg/kg. The AM-conjugates also demonstrated a broad therapeutic window in this model (therapeutic index of >120). As a proof-of-concept for the depletion of HSCs in large animals, a single i.v. dose escalation study was performed with the cross-reactive anti-CD117-AM in NHP. On-target, dose-dependent decreases in phenotypic HSCs and CFUs were observed in the bone marrow at day 7 post anti-CD117-AM dosing with >95% HSC depletion observed with a single dose of 0.3 mg/kg (Fig. 1). In the periphery, a dose-dependent transient decrease in reticulocytes was observed at day 4 with a neutrophil and monocyte nadir at day 18. The depth and duration of the depletion was also dose-dependent. The anti-CD117-AM induced depletion was on target and amanitin dependent as the unconjugated antibody and isotype-AM had no effect. Notably, white blood cell and lymphocyte counts were stable through day 56, demonstrating that this strategy will spare the adaptive immune system. Thrombocytopenia occurred 4-8 days post infusion and was dose-dependent, transient and reversible. This also occurred with the isotype-AM, suggesting the effect was off-target. Because the half-life of the anti-CD117-AM was 5 days, a second dose escalation study with anti-CD117-AM engineered to have a short half-life (~18 h) was performed in NHPs. The short half-life anti-CD117-AM demonstrated similar potency on all cell parameters at 0.3 mg/kg and was well tolerated at the effective dose. As expected, the short half-life anti-CD117-AM was rapidly cleared with a half-life of 15-18 h. In both studies, a transient dose dependent elevation of liver enzymes was observed in groups treated with the highest doses of isotype-AM, anti-CD117-AM, and the short half-life anti-CD117-AM. Conclusions: Anti-CD117-AM exhibited potent elimination of NHP HSCs and progenitors in vivo. The potency of short half-life anti-CD117-AM was comparable, providing a model for target cell depletion and rapid clearance prior to BMT. Both ADCs were well tolerated at the efficacious doses. This strategy preserves the adaptive immune system with delayed onset of neutrophil nadir (18 days), potentially shortening the period of neutropenia. Targeted depletion of hematopoietic cell subtypes with limited off-target effects could provide a significant improvement in standard-of-care approaches to patient preparation prior to HSC transplant. Disclosures Pearse: Magenta Therapeutics: Employment, Equity Ownership, Patents & Royalties. McDonough:Magenta Therapeutics: Employment, Equity Ownership. Proctor:Magenta Therapeutics: Employment, Equity Ownership. Panwar:Magenta Therapeutics: Employment, Equity Ownership, Patents & Royalties. Sarma:Magenta Therapeutics: Employment, Equity Ownership. McShea:Magenta Therapeutics: Employment, Equity Ownership. Kien:Magenta Therapeutics: Employment, Equity Ownership. Dushime:Magenta Therapeutics: Employment, Equity Ownership. Adams:Magenta Therapeutics: Employment, Equity Ownership. Hyzy:Magenta Therapeutics: Employment, Equity Ownership. Brooks:Magenta Therapeutics: Employment, Equity Ownership. Palchaudhuri:Magenta Therapeutics: Employment, Equity Ownership, Patents & Royalties; Harvard University: Patents & Royalties. Li:Magenta Therapeutics: Employment, Equity Ownership. Kallen:Magenta Therapeutics: Employment, Equity Ownership. Sawant:Magenta Therapeutics: Employment, Equity Ownership. McDonagh:Magenta Therapeutics: Employment, Equity Ownership, Patents & Royalties. Boitano:Magenta Therapeutics: Employment, Equity Ownership, Patents & Royalties. Cooke:Magenta Therapeutics: Employment, Equity Ownership, Patents & Royalties.