The Society of Toxicology (SOT) hosted its 63rd Annual Meeting and ToxExpo from March 10 to 14, 2024, at the Salt Palace Convention Center in Salt Lake City, Utah. This event brought together over 5,000 toxicologists and professionals in related fields to share the latest advancements in toxicology. The conference featured more than 70 scientific sessions, over 2,000 poster presentations, and a three-day ToxExpo with 250 exhibiting companies.
Kevin Simpson1, Megan Terral1, Eva Schmidt1, Wesley Anderson1, Lowry Curley1, Lise Harbom1
1 AxoSim Inc., New Orleans, LA, USA
Background and Purpose:
Antibody-drug conjugates (ADCs) utilize the combination of a chemotherapeutic drug attached to an antibody (via linker) for targeted delivery of highly potent payloads to cancer cells. Unfortunately, ADCs may also cause peripheral neuropathy (PN) by a variety of mechanisms, including a bystander effect, in which cell-permeable payloads may be released in a non-targeted way into adjacent tissue. The stability and specificity of the ADC unit is critical for targeted cell dosing and to avoid associated side effects of PN. When developing ADC-based therapeutics, screening in the most relevant cell systems is vital for translatable data before clinical applications. This includes the need for inclusion of supporting cells, in addition to neuronal cells, within the test system to evaluate potential PN. As such, we have characterized a novel, fully human cell-based, co-culture system comprised of induced pluripotent stem cell-derived sensory neurons (hSNs) and primary human Schwann cells (hSCs) for screening cancer therapeutic candidates for off-target effects.
Methods:
The co-culture system of hSNs and hSCs was plated into a 96-well format for high throughput screening capabilities and dosed with monomethyl auristatin E (MMAE) and a vehicle control in a range of concentrations for a predetermined length of time. MMAE is a synthetic antineoplastic agent which is too toxic to use as a drug itself but has been coupled into the ADC format for developing therapeutics. Widely cited as a cause of chemotherapy-induced peripheral neuropathy (CIPN), MMAE was used as a free agent in solution to validate the efficacy of the system as a platform for toxicity screening. Standard immunocytochemistry (ICC) techniques were employed to identify individual cells, neuron and Schwann cell numbers and several outgrowth metrics for neurons. In addition, development of a shape factor analysis (Circularity Index) for hSCs allowed characterization of hSC health/stress as a function of change in morphology after test article (TA) exposure. Specifically, healthy hSC bodies were spread out in culture, with some population associating along the length of healthy hSN axonal outgrowth; while in the presence of toxic agents, the hSCs retracted into a more spherical morphology before eventual death.
Results:
The co-culture system of hSNs and hSCs showed consistent cell seeding density across the imaging frames for the ICC control well group(s), indicating healthy and predictable cell adhesion before exposure to TAs. MMAE dose response curves indicated toxicity in both hSN and hSC cell populations. Comparison of IC50 results across various metrics highlighted the effects of MMAE on cell bodies and specific
processes, with neuron-positive cells and neurite branches exhibiting IC50 values of 5.627 nM and 0.796 nM, respectively. Data indicated stress and toxicity for the hSC population as well, with hSC shape factor producing an IC50 value of 1.025 nM.
Conclusions:
In summary, this fully human co-culture cell system of hSNs and hSCs provides a fast, cost-effective pathway to relevant human-based data for the evaluation of potential ADC and other CIPN effects. The variety of metrics allows for detailed insights into mechanistic and cell-specific compound effects with additional value in evaluating multiple ADC candidates for off-target toxicity as compared to one another and/or to MMAE toxicity when narrowing the candidate field during development. Current efforts are focused on further characterizing the system with other representative chemotherapeutic drug classes which have been shown to cause CIPN through various mechanisms of action: oxaliplatin (platinum-based antineoplastics), vincristine (vinca alkaloids), paclitaxel (taxanes) and bortezomib (proteasome inhibitors).
Tyler Rodriguez1, Neki Patel5, Mattia Cocco5, Luke Masterson5, Jay Harper6, Megan Terral1, Eva Schmidt1, Lowry Curley1, Michael J Moore1, 2, 3, Edward Spack1, Catherine Rodger4, Mary McFarlane4, and Corey Rountree1
1 AxoSim Inc., New Orleans, LA, USA
2 Department of Biomedical Engineering, Tulane University, New Orleans, LA, USA
3 Brain Institute, Tulane University, New Orleans, LA, USA
4 Oncology Safety, Clinical Pharmacology and Safety Sciences, R&D, AstraZeneca, Cambridge, UK
5 Tumor Targeted Delivery, Oncology R&D, AstraZeneca, London, UK
6 Tumor Targeted Delivery, Oncology R&D, AstraZeneca, Gaithersburg, MD, USA
Background and Purpose:
Development of pharmaceutical treatments for neurological disease is stifled by high cost and low success rate in clinical trials. A more predictive human pre-clinical model would significantly reduce the barrier to entry while accelerating the drug discovery pipeline. Microphysiological systems (MPS) are in vitro model systems with in vivo-like physiology potentially filling this need. We have developed a novel MPS human Nerve-on-a-Chip, or NerveSim®, comprised of an iPSC-derived neuron and primary human Schwann cell 3D coculture system that forms a morphological and electrophysiological simulacrum of a human nerve. Neuronal processes are grown down a channel containing embedded electrodes capable of stimulating and recording from 10 locations along the nerve. This system is employed in a 24-well format to provide high-throughput electrophysiological characterization amenable to testing multi-drug panels for neurotoxicity on a clinically relevant nerve model. We have also developed a live-cell automated imaging analysis technique to detect neurotoxicity via morphological changes in human NerveSim® over time. Here, we tested several chemotherapeutic compounds with known neurotoxicities by correlating changes in compound action potentials (CAPs) and axonal morphology for multidimensional non-invasive longitudinal functional characterization.
Methods:
CAPs are evoked with a series of current steps (1 µA, 5 µA, 10 µA, 24 µA, and 48 µA) from 6 different electrode locations sequentially in the NerveSim® channel while recording from 10 electrodes simultaneously. The average of 12 trials at each current step for all 45 electrode combinations is converted from the time domain to the velocity domain and the envelope is calculated to produce a purely positive signal. The velocity envelope signals for each current step are aligned in velocity and the maximal velocity projection (MVP) is calculated to collapse the 45 individual recordings into a single measurement. The area under the curve (AUC) of the MVP is calculated to produce the velocity density index (VDI) metric that is then compared, on a well-by-well basis, before and after dosing with chemotherapeutics to measure functional changes from neurotoxicity. To measure morphological changes associated with dosing, we took images through the dose week and performed automated segmentation to quantify fiber length, fiber number, and object number producing a nerve degeneration index (NDI). IC50’s were then calculated for each assay type using appropriate positive and negative controls to give a multidimensional assessment of toxicity. Chemotherapeutic compounds were selected from auristatins, maytansinoids, vinca alkaloids, and antibody drug conjugates (ADCs) thereof.
Results:
Of the auristatins, MMAF was less cytotoxic than MMAE producing an IC50 approximately three thousand-fold higher as measured by electrophysiology (62 nM and 0.02 nM respectively). The NDI, derived from fiber length analysis, showed IC50s with a similar trend between MMAF and MMAE but with lower sensitivity than electrophysiology (150 nM and 4.3 nM respectively). This growth metric estimates the amount of fragmentation as a reduction in fiber length. Visually, MMAF and MMAE produced clear fragmentation in human NerveSim®. The ADC HER2-MMAE DAR4 proved less toxic than its payload alone with an electrophysiology IC50 of 17,524 ng/mL and an imaging IC50 of 24,161 ng/mL. These IC50s contrast with literature reports testing N87 cancer cells where HER2-MMAE inhibits proliferation with an IC50 of 95 ng/mL. The antibody maytansine conjugate (AMC), trastuzumab-DM1 (T-DM1), and the AMC metabolite, DM4-SMe, both showed potent neurotoxicity (103 ng/mL and 0.93 nM respectively) as determined by electrophysiology. However, while the IC50 from the growth assay for DM4 SMe agreed with the electrophysiology IC50 (3.84 nM), treatment of NerveSim® cultures with T-DM1 resulted in relatively little visible fragmentation and an IC50 of 27,542 ng/mL. This growth IC50 for T-DM1 was above the maximum dose tested whereas electrophysiology detected toxicity near the lowest dose. Together, this suggests morphology was relatively unaffected in contrast to inducing significant functional detriments. The vinca alkaloids, vincristine and vinblastine, were also potent neurotoxic compounds when assayed by electrophysiology (IC50s of 1.43 nM and 7.58 nM respectively). They produced clearly visible neuronal fragmentation and growth IC50s of similar trend to electrophysiology (IC50s of 7.68 nM and 45 nM respectively). This data supports the utility of human NerveSim® in testing novel ADCs for off-target effects on the nervous system while highlighting the necessity for functional assessment.
Conclusions:
The NerveSim® platform is a valuable pre-clinical model for sensitively testing novel therapeutics for potential functional and morphological effects on the peripheral nervous system. Our 24-well NerveSim® EEA plate is 1,200-fold more efficient than field recordings, enabling generation of high through-put functional data in combination with novel imaging metrics to investigate multi-drug panels for safety and efficacy on human cells.
