CAR T-cell therapy, often called a “living drug,” is revolutionizing cancer treatment. By engineering a patient’s own T lymphocytes to express chimeric antigen receptors (CARs), these therapies deliver targeted cytotoxicity against tumor cells and have already produced durable remissions in B-cell malignancies and multiple myeloma.

Great promise, however, carries great risk. Before reaching patients, CAR T constructs must undergo rigorous preclinical testing to ensure safety.

Why Preclinical Testing for CAR T Therapy Is Essential

Unlike traditional drugs, CAR T-cells are living therapies that expand, persist, and interact dynamically with the immune system. This complexity introduces risks not seen with small molecules or antibodies. Preclinical models help answer three key questions:

1. Do CAR T-cells target only tumor cells, or also healthy tissues?
2. What systemic toxicities could emerge?
3. How long do they persist and where do they travel in the body?

Although human CAR-Ts cannot be perfectly modeled in preclinical models, regulators require a weight-of-evidence approach: in vitro specificity assays combined with xenograft or transgenic models or other in-vivo test systems to capture whole-body effects.

Off-Target Toxicity in CAR T Preclinical Studies

CAR-Ts are engineered to recognize tumor-associated antigens, but many of these markers are also present at low levels in normal tissues. Preclinical safety studies help de-risk:

• On-target, off-tumor toxicity; CD19 CAR-Ts eliminate both malignant and healthy B cells, leading to long-term B-cell aplasia. While manageable with immunoglobulin replacement, it underscores unavoidable risks of shared antigen expression.
• Cross-reactivity; A well-documented HER2-targeted CAR-T case demonstrated fatal lung toxicity when CARs attacked healthy tissue expressing low levels of HER2. This highlights the importance of cross-reactivity screening in CAR T safety testing.

Systemic Toxicity: Managing CRS and ICANS

Even when tumor targeting is correct, systemic toxicities can emerge:

• Cytokine Release Syndrome (CRS): Characterized by fever, hypotension, and organ dysfunction due to massive cytokine secretion. Preclinical cytokine monitoring and mitigation strategies like IL-6 blockade (tocilizumab) are now standard.
• Neurotoxicity (ICANS): Patients may develop confusion, seizures, or cerebral edema. While mechanisms remain unclear, neurotoxicity monitoring in CAR T preclinical studies provides early warning.
• Organ-specific inflammation: Biodistribution studies reveal potential for localized injury in tissues where the target antigen is present.

CAR T Preclinical Safety Priorities

Successful CAR-T development requires robust assessment across four safety domains:

• Toxicity characterization – profiling cytokine release, neurotoxicity markers, and tissue cross-reactivity.
• Mechanistic insight – studying receptor specificity and immune activation kinetics.
• Controlled deactivation – testing engineered features that allow CAR-T activity to be shut down if needed.
• Vector risk analysis – weighing viral vs. non-viral delivery platforms and monitoring genomic stability.

Preclinical models typically include xenograft mice (NSG/NOG), human antigen transgenic mice, and other test systems when target epitopes are conserved in that species.

Expanding Beyond Oncology: CAR T Safety in New Indications

CAR-Ts are now being explored for autoimmune diseases, fibrotic conditions, and chronic infections. These programs expand the promise of the platform but also demand even more rigorous preclinical evaluation, since risks of immune misfiring may differ outside oncology.

Why Choose Attentive Science?

No single model fully predicts human responses, but together they provide a risk benefit profile strong enough to justify first-in-human trials. In vitro assays are critical for characterizing CAR-T cells, but they cannot fully capture the complexity of an entire organism. In vivo studies help answer questions such as:

1. Do CAR-T cells persist and expand in a living system?
2. How do they distribute to different tissues?
3. What risks of off-target or systemic toxicity may emerge?

Preclinical safety is not only a regulatory milestone; it is the foundation of therapeutic success. When partnering with Attentive Science you will receive:

• Scientific Rigor – High-resolution safety assessments tailored to your programs.
• Agility – Flexible study designs with efficient initiation and execution.
• Regulatory Alignment – Data packages built to meet IND, FDA, TGA and global expectations.

Attentive Science integrates deep scientific expertise with operational precision, ensuring that sponsors move forward with confidence in both their data and their development path.

Contact us to experience the Attentive difference!