Drug development continues to evolve with the emergence of novel therapeutic modalities, including antibody-drug conjugates (ADCs) and RNA-based drugs. These drug categories offer promising therapeutic possibilities but come with unique pharmacokinetic (PK), bioanalytical, and toxicity-related challenges that result in a narrow therapeutic window. These challenges make optimizing their safety profiles especially critical.
A crucial step in developing new modalities is determining each compound’s Maximum Tolerated Dose (MTD), an important preclinical threshold that informs dose selection and risk mitigation strategies. MTD testing helps establish the safety parameters for advancing these therapies into clinical trials. By understanding how MTD testing is applied to ADCs and RNA-based drugs, developers and sponsors can better navigate the intricate balance between efficacy and safety. MTD testing’s role in optimizing safety profiles for these innovative therapies and its significance in dose selection and risk mitigation highlights its immense value in drug development.
Understanding MTD Testing
MTD testing identifies the highest dose of a drug that can be administered without causing unacceptable toxicity. The primary objective of MTD testing is to define a therapeutic window where the drug is effective while minimizing adverse effects. By determining the MTD, researchers can set a baseline for further toxicological studies, ensuring that subsequent trials are conducted within safe limits. It’s also noteworthy that MTD testing is usually not a regulatory requirement, but it does play a critical role in risk assessment and other IND-enabling studies.
MTD testing is generally conducted stepwise, starting with a low dose and gradually increasing until severe toxicity is observed. This method identifies the highest tolerable dose while minimizing morbidity and mortality.
Preclinical Versus Clinical Use
While MTDs are determined in both preclinical and clinical trials, this article focuses on preclinical settings involving animal models. This approach allows for assessing a drug’s toxicity profile before human exposure, providing critical data necessary for the design of clinical trials. The insights gained from these studies will eventually help determine safe starting doses and monitoring parameters during human trials.
Endpoints and Study Design
MTD studies typically involve observing severe clinical signs and mortality as endpoints. These endpoints are used to determine the dose at which toxicity becomes unacceptable. The study design often involves single-dose escalation, which informs dose range finding studies and longer-term safety assessments, such as 14-day and 28-day studies, which are essential for IND-enabling submission.
MTD is often compared to other dose-related parameters but serves a distinct function in preclinical drug evaluation. The no-observed-adverse-effect level (NOAEL) is the highest dose at which no significant adverse effects are observed. While MTD testing identifies the toxicity threshold, NOAEL defines the upper safety boundary. MTD studies often provide critical information to select the high dose level that is used in pivotal toxicology studies. The high dose level is key to selecting low and mid dose levels for IND-enabling studies. Proper dose selection will ensure that a NOAEL can be identified to assign a starting dose in clinical settings that is not toxic but will show efficacy.
Considerations for MTD Testing in ADCs
ADCs are rapidly expanding cancer therapies, combining monoclonal antibodies’ (mAbs) specificity with the potency of cytotoxic payloads. This unique structure makes MTD testing for ADCs more complex than for small molecules. Small molecules typically have well-defined pharmacokinetics and toxicity profiles, but that’s not true for ADCs. The mAb, cytotoxic payload, and linker all contribute to the drug’s safety and efficacy, requiring a nuanced approach to MTD testing.
Immunotoxicity
The potential for cytokine release syndrome (CRS) is the primary challenge ADCs face regarding immunotoxicity. ADCs engage the immune system directly or through antigen targeting, which can lead to inflammatory responses and severe systemic reactions. These are especially relevant for ADCs designed to activate T cells, as these therapies can induce rapid cytokine surges, resulting in dangerous immune overactivation. To mitigate this risk, MTD studies for ADCs must incorporate cytokine profiling and biomarker analysis to detect early signs of immune activation.
Drug-antibody ratio (DAR)
A higher DAR increases potency but elevates systemic toxicity risks, while a lower DAR may be safer but less effective. Advances in site-specific conjugation have improved DAR consistency, but MTD testing remains essential in determining the optimal balance between efficacy and safety.
Linker stability
Cleavable linkers rely on enzymatic or environmental triggers to release the cytotoxic payload inside tumor cells. This leaves them vulnerable to premature release during circulation, leading to unintended systemic toxicity. Conversely, non-cleavable linkers depend on lysosomal degradation within the tumor cell to activate the payload - a more controlled release that can reduce drug efficacy. MTD testing compares cleavable versus non-cleavable linkers, measuring payload release kinetics and determining whether systemic exposure remains within acceptable safety limits.
Delayed toxicity
The most challenging aspect of ADC toxicity assessment is delayed toxicity, arising from prolonged tissue retention and slow clearance. Unlike small molecules that are rapidly metabolized and excreted, ADCs can accumulate in the liver, spleen, and bone marrow, leading to long-term toxicity.
To address these risks, MTD studies for ADCs extend further than standard acute toxicity assessments. The testing regimen incorporates longer monitoring periods, repeated-dose escalation studies, and histopathological analysis to capture delayed-onset adverse effects. Comprehensive MTD studies during preclinical development can mitigate these risks and allow ADCs to achieve their full therapeutic potential.
Considerations for MTD Testing in RNA-Based Drugs
RNA-based therapies include small interfering RNA (siRNA), antisense oligonucleotides (ASOs), and messenger RNA (mRNA)-based drugs, each of which presents unique challenges in MTD testing. Unlike small molecules or even ADCs, these therapies often rely on specialized delivery systems, exhibit distinct PK properties, and can elicit complex immune responses. As a result, establishing MTD for RNA-based drugs requires a careful approach that considers the effects of the drug itself and its delivery mechanism.
Delivery system toxicity
Lipid nanoparticles (LNPs) present one of the biggest challenges for MTD testing in RNA therapeutics. LNPs enable efficient cellular uptake but can also cause hepatic stress and immune activation, leading to adverse effects that are sometimes unrelated to the RNA payload itself. The primary site of LNP metabolism is the liver, which is particularly vulnerable to inflammatory responses and lipid accumulation. Too many LNPs can overwhelm the organ and trigger acute and chronic toxicity. To distinguish between RNA-induced toxicity and delivery system-related effects, MTD studies must carefully evaluate hepatic biomarkers, cytokine levels, and immune activation markers.
Off-target effects
ASO and siRNA therapies function by silencing specific genes, but when the sequencing misfires, it can lead to off-target gene silencing and unpredictable consequences. Chemical modifications and improved sequence design can mitigate these risks, but early toxicology studies must assess unintended gene interactions to ensure that the therapies do not interfere with biological pathways.
PK challenges
Unlike small molecules, siRNA and ASO drugs have short plasma half-lives but are retained in the liver, kidney, and spleen for abnormally long durations. Thus, plasma clearance does not always correlate with drug persistence in tissues, making it difficult to predict long-term toxicity. Accumulation of RNA-based drugs in tissues can also lead to delayed toxicity, requiring longer MTD studies. Additional research on tissue-specific biomarkers, histopathology, and prolonged dosing will help scientists understand RNA-drug behavior over time.
Regulatory Considerations
From a regulatory perspective, MTD testing is integral to IND-enabling studies, which are required for IND applications. Global regulatory agencies rely on IND-enabling studies to assess the safety of new drug candidates before they enter clinical trials. These studies are part of a comprehensive package that includes pharmacology, toxicology, and manufacturing information, which are important in determining safe and efficacious starting doses for human studies. While MTD testing is not a clinical trial endpoint, it informs the design of subsequent clinical trials by providing essential safety parameters. Regulatory guidelines, including ICH M3(R2), emphasize the importance of preclinical safety assessments, including MTD testing, in supporting the transition from preclinical to clinical drug development.
A Final Word
MTD testing is critical in optimizing drug safety and guiding dose selection for ADCs and RNA-based therapies. As these novel modalities introduce unique toxicity challenges, a well-executed MTD study can help refine dosing strategies and mitigate risks early in development.
However, given the complexity of MTD assessment - particularly for ADCs and RNA-based drugs - many organizations may lack the in-house capabilities, expertise, or resources to conduct these studies effectively. For those without internal capacity, enlisting the expertise of a trusted lab testing partner ensures that MTD studies are performed with precision, adherence to best practices, and regulatory rigor.
Looking ahead, drug developers and sponsors must prioritize rigorous and strategically designed MTD studies to de-risk development, optimize therapeutic potential, and bring life-saving medicines to market with confidence.
Author Details
Tina Rogers, Ph.D., MBA, DABT, Senior Technical Director, WuXi AppTec
Tina Rogers is an expert in preclinical drug development services. Her leadership positions include senior technical director at WuXi AppTec, vice president of preclinical sciences at Altasciences, executive vice president, and director of research at M.P.I. Research (now Charles River), and vice president of drug development at Southern Research Institute. She has served as an advisor and driven growth and profitability in all her leadership roles. Dr. Rogers holds a doctoral degree in molecular and cellular biology and pathobiology from the Medical University of South Carolina and an MBA from Auburn University. She has a broad technical background, including cell biology, immunology, toxicology, cell and gene therapy, sepsis, inflammation, BL-3 and select agents, flow cytometry and predictive/in vitro toxicology. Dr. Rogers has served as a board member for several biotech, academic and not-for-profit institutions and is a Diplomate of the American Board of Toxicology (DABT).
Publication Details
This article appeared in Pharmaceutical Outsourcing:Vol. 26, No.2 Apr/May/June 2025Pages: 13-15