Conceptual illustration of laboratory glassware transforming into stylized antibodies, symbolizing the evolution of biopharmaceutical safety testing.

Monoclonal Antibody Toxicity: Are Traditional Tests Enough?

Mira Elwood in Health & Wellness August 2025 • 4 min read.

"Exploring the limitations of standard in vitro assays for assessing adverse effects of mAb drugs like Rituximab and Trastuzumab, and how to improve safety testing."

Monoclonal antibody (mAb) therapeutics have revolutionized the treatment of various diseases, including cancer and autoimmune disorders. These engineered antibodies are designed to target specific molecules in the body, offering a precise and often less toxic approach compared to traditional therapies. However, the development and approval of mAb drugs have been hampered by manufacturing challenges and unexpected adverse effects. Therefore, it's important to ensure that safety testing methods are able to accurately evaluate potential toxicity.

Traditional in vitro toxicity tests, which involve studying the effects of substances on cells in a laboratory setting, have long been a cornerstone of drug development. However, the applicability of these tests to mAbs has come under scrutiny. mAbs are complex molecules that can interact with the immune system and trigger a variety of cellular responses, making it difficult to predict their effects using simple in vitro assays.

A recent study published in the journal Antibodies delves into the effectiveness of traditional in vitro toxicity tests for assessing the adverse effects of mAbs. The researchers focused on two widely used mAbs, Rituximab and Trastuzumab, and investigated their potential off-target effects on specific organ systems using hepatocarcinoma cell line (HepG2) and human dermal fibroblasts neonatal (HDFn).

Traditional Toxicity Tests: Are They Up to the Challenge?

Conceptual illustration of laboratory glassware transforming into stylized antibodies, symbolizing the evolution of biopharmaceutical safety testing.

The study explored three key mechanisms of mAb-induced toxicity: antibody-dependent cytotoxicity (ADCC), complement-dependent cytotoxicity (CDC), and complement-dependent cellular cytotoxicity (CDCC). ADCC occurs when mAbs bind to target cells and recruit immune cells to destroy them. CDC is triggered when mAbs activate the complement system, a part of the immune system that can directly kill cells. CDCC involves both complement activation and immune cell involvement.

The researchers used two different cell lines to represent potential off-target effects in different organ systems. HepG2 cells, derived from a liver tumor, were used to assess potential hepatotoxicity. HDFn cells, which are skin cells, were used to evaluate potential dermal toxicity.

The study's findings revealed some limitations of traditional in vitro toxicity tests for assessing mAb safety: HepG2 Cells: No apparent ADCC, CDCC, or CDC-mediated decrease in cell viability was observed for HepG2 cells. HDFn Cells: Although ADCC or CDCC-mediated decreases in cell viability weren't detected, a CDC-mediated decrease in cell viability was observed. Donor Variability: The use of peripheral blood mononuclear cells (PBMCs) from different donors introduced variability in the results, highlighting the influence of individual immune responses. Technical Issues: In the case of traditional toxicity tests used to assess off target effects, a continuous maintenance of PBMCs would confound the ability of the test to detect any decrease in cell viability.

These results suggest that traditional in vitro toxicity tests may not be sensitive enough to detect subtle or complex off-target effects of mAbs. The researchers emphasize that several considerations must be taken into account when developing in vitro assays for mAb toxicity, including: co-incubation of cell line of interest with immune responsive cells, optimisation of cell density and incubation times; and choice of off-target organ system and assay endpoint.

The Future of mAb Safety Testing

The study highlights the need for more sophisticated in vitro assays that can better mimic the complexity of the human immune system and capture the potential off-target effects of mAbs. As the field of mAb therapeutics continues to grow, advancements in non-clinical safety testing strategies, such as incorporating immune cell co-cultures, advanced imaging techniques, and computational modeling, will be essential to ensure the development of safe and effective mAb drugs.

About this Article -

This article was crafted using a human-AI hybrid and collaborative approach. AI assisted our team with initial drafting, research insights, identifying key questions, and image generation. Our human editors guided topic selection, defined the angle, structured the content, ensured factual accuracy and relevance, refined the tone, and conducted thorough editing to deliver helpful, high-quality information.See our About page for more information.

This article is based on research published under:

DOI-LINK: 10.3390/antib7030030, Alternate LINK

Title: Applicability Of Traditional In Vitro Toxicity Tests For Assessing Adverse Effects Of Monoclonal Antibodies: A Case Study Of Rituximab And Trastuzumab

Subject: Drug Discovery

Journal: Antibodies

Publisher: MDPI AG

Authors: Arathi Kizhedath, Simon Wilkinson, Jarka Glassey

Published: 2018-08-17

Everything You Need To Know

How do monoclonal antibodies (mAbs) such as Rituximab and Trastuzumab cause toxicity, and why are traditional tests potentially insufficient?

Monoclonal antibodies (mAbs) like Rituximab and Trastuzumab are designed to target specific molecules, but they can trigger antibody-dependent cytotoxicity (ADCC), complement-dependent cytotoxicity (CDC), and complement-dependent cellular cytotoxicity (CDCC). These mechanisms may lead to off-target effects. Traditional in vitro toxicity tests, may not fully capture the complexity of these immune-mediated responses, leading to potential gaps in safety assessment.

What are traditional in vitro toxicity tests, and why is there concern about their applicability to assessing the safety of monoclonal antibody (mAb) drugs?

Traditional in vitro toxicity tests evaluate the effects of substances on cells in a lab. However, their effectiveness for mAbs is debated because mAbs are complex molecules that can interact with the immune system, potentially causing cellular responses that simple in vitro assays might miss. The research using HepG2 and HDFn cell lines highlights these limitations, as not all toxicity mechanisms were detected effectively.

What were the specific findings regarding HepG2 and HDFn cells in the study, and what do these results suggest about the limitations of traditional in vitro toxicity tests for mAbs?

In the study, HepG2 cells (derived from a liver tumor) showed no ADCC, CDCC, or CDC-mediated decrease in cell viability when exposed to mAbs. HDFn cells (skin cells) only showed a CDC-mediated decrease in cell viability, but no ADCC or CDCC effects. This indicates that traditional in vitro tests may not be sensitive enough to detect all potential off-target toxicities of mAbs across different organ systems.

What is 'donor variability' in the context of mAb toxicity testing, and why is it a significant concern?

Donor variability arises from using peripheral blood mononuclear cells (PBMCs) from different donors. This variability significantly impacts the results of in vitro toxicity tests, emphasizing that individual immune responses can influence mAb toxicity. This highlights the need for more standardized and controlled in vitro assays that account for these individual differences to ensure consistent and reliable safety assessments.

What advancements are necessary for more effective monoclonal antibody (mAb) safety testing, and what are the potential implications of not improving these methods?

To improve mAb safety testing, more sophisticated in vitro assays are needed to mimic the complexity of the human immune system. These assays should incorporate immune cell co-cultures, advanced imaging techniques, and computational modeling. By optimizing cell density, incubation times, and selecting appropriate off-target organ systems and assay endpoints, researchers can better capture the potential off-target effects of mAbs, leading to safer and more effective therapeutics. Failure to improve safety testing may result in unexpected adverse effects during clinical development or post-market surveillance.