Human epidermal growth factor receptor 2 (HER2), a well-established oncogenic driver, is overexpressed in approximately 15 to 20 percent of breast cancers, correlating with high aggressiveness and poor clinical outcomes. Current HER2-targeted therapies have improved patient prognosis but often induce severe adverse effects and are prone to resistance mechanisms within tumor cells. To overcome these limitations, the team led by researchers from Weill Cornell Medicine and Memorial Sloan Kettering Cancer Center has innovated a highly sophisticated three-step HER2-targeted ^225Ac-PRIT regimen that maximizes tumor suppression while mitigating systemic toxicity.

Prior clinical attempts employing ^225Ac-labeled antibodies demonstrated promising antitumor activity but were hampered by the retention of alpha-particles in healthy organs, causing significant off-target toxicities. The current research circumvents this by implementing a sequential intravenous protocol starting with a bispecific antibody designed to bind HER2 on tumor cells and a radiometal chelator DOTA. This is followed by administration of a clearing agent that accelerates removal of unbound antibodies from circulation, consequently reducing nonspecific radiation. Finally, the ^225Ac-labeled radiotherapeutic agent is introduced, selectively binding the pretargeted tumor cells and delivering potent alpha radiation exactly where it is needed.

Extensive preclinical evaluation was carried out using the BT-474 human breast cancer xenograft model, which accurately recreates HER2-expressing tumor biology. Dose-finding studies assessed tumor-targeting efficiency, biodistribution, and toxicity profiles, especially nephrotoxicity, a major limiting factor in radionuclide therapy due to kidney accumulation. The researchers applied either one or two treatment cycles spaced by one week, carefully monitoring therapeutic responses and systemic side effects over an extended period.

Remarkably, all treated mice in the BT-474 xenograft model achieved complete tumor responses, with 85% evidencing histologic cures upon microscopic examination. This indicates not just tumor shrinkage but complete pathological eradication. Additionally, one-cycle interventions were as potent as two-cycle regimens, suggesting that treatment intensity can be optimized to reduce exposure without compromising outcomes. Throughout the study, no chronic radiation-induced toxicity was detected in vital organs, underscoring the regimen’s favorable safety profile.

In a compelling extension of the work, the therapy was tested on a patient-derived xenograft (PDX) model, which reproduces the heterogeneity and complexity of human tumors more faithfully. A single cycle of ^225Ac-PRIT elicited complete responses in 60% of PDX-bearing mice and significantly prolonged survival relative to untreated controls. Such translational findings hint at the modality’s potential applicability in clinical settings, offering hope for patients with refractory or advanced HER2-positive breast cancers who have limited therapeutic options.

A critical component of the study was the quantification of nephrotoxic absorbed doses, which delineated upper radiation thresholds to prevent irreversible kidney damage. Understanding these parameters is essential for guiding safe dose escalation in future clinical trials. The precise calibration of dosage and scheduling exemplifies the rational, methodical approach the investigators employed in balancing therapeutic benefit with toxicity risk.

The implications of this research are profound. By integrating molecular targeting with alpha-particle therapy via PRIT, the treatment achieves pinpoint accuracy, concentrating cytotoxic radiation to malignant cells while sparing normal tissues. Alpha-emitters such as ^225Ac deliver high linear energy transfer (LET) radiation that induces double-stranded DNA breaks, causing irreparable tumor cell kill even in radio-resistant cancer subpopulations.

According to Dr. Sarah Cheal from Weill Cornell Medicine, the incorporation of the clearing agent in this multi-step approach was pivotal to minimizing alpha-particle off-target effects, a notorious hurdle in traditional alpha-radioimmunotherapy. This innovation enhances the therapeutic index and opens new avenues for applying alpha emitters beyond hematologic malignancies to solid tumors—especially those driven by HER2.

Dr. Nai Kong Cheung of Memorial Sloan Kettering Cancer Center highlights this therapy’s promise not only in breast cancer but also in other HER2-expressing solid tumors, which include subsets of gastric, ovarian, and lung cancers. As HER2 remains a prominent oncogenic driver across multiple cancers, this PRIT platform could serve as a versatile and transformative treatment modality.

Future directions involve advancing this proof-of-concept into clinical trials with rigorously designed protocols to evaluate safety, dosing, and efficacy in human subjects. Given the favorable preclinical safety profile and high curative potential, ^225Ac-PRIT could redefine the therapeutic landscape for patients with HER2-positive malignancies, reducing reliance on chemotherapies and mitigating the pervasive problem of therapeutic resistance.

This study embodies the convergence of molecular biology, radiochemistry, immunology, and oncology to produce a next-generation therapeutic that exemplifies precision medicine’s core principles. The strategic sequencing of antibody targeting, clearing agent clearance, and alpha-radioisotope delivery lays the foundation for bespoke cancer treatments tailored to molecular tumor markers.

In summary, the advent of HER2-targeted ^225Ac-PRIT represents a paradigm shift in cancer radioimmunotherapy. It shows compelling preclinical evidence of durable tumor control and histologic cure with negligible chronic toxicity risks. Such a potent combination of efficacy and safety could soon translate into clinical breakthroughs that improve survival and quality of life for patients battling aggressive HER2-positive breast cancer.

Subject of Research: HER2-targeted alpha-emitter radioimmunotherapy for breast cancer

Article Title: 225Ac α-Pretargeted Radioimmunotherapy of Human Epidermal Growth Factor Receptor 2–Expressing Breast Cancer

News Publication Date: November 3, 2025

Web References:
https://doi.org/10.2967/jnumed.125.269601
JNM website

Image Credits: Image created by S. Rinne et al., Weill Cornell Medicine, New York, NY.

Keywords: Molecular imaging, Personalized medicine, Breast cancer

At the heart of this innovation is the targeting of the NR4A2 gene locus to drive IL-12 expression in CAR T cells. By engineering human anti-LeY CAR T cells to express IL-12 under the control of the NR4A2 promoter, the researchers observed a remarkable elevation in key cytokines such as interferon gamma (IFNγ), tumor necrosis factor (TNF), and interleukin-2 (IL-2) upon in vitro stimulation. This intracellular rewiring allowed the modified CAR T cells to expand robustly and maintain functionality, a feat that is critical for sustained antitumor activity.

In vivo experiments using OVCAR-3 xenograft models further underscored the therapeutic potential of NR4A2/IL-12 CAR T cells. These engineered cells mediated superior tumor regression compared to mock-edited counterparts and, strikingly, cured mice exhibited durable immunity against secondary tumor challenges. This phenomenon indicates that the rewired CAR T cells not only eradicate existing tumors but also contribute to long-lasting immunological memory, a vital component for preventing relapse.

Importantly, the NR4A2-driven IL-12 expression occurred without observable toxicities, setting these cells apart from previous CAR T models that utilized PD-1 locus editing for IL-12 delivery, which resulted in rapid weight loss and systemic adverse effects. This safety profile highlights the advantage of harnessing endogenous regulatory elements, which appear to limit cytokine expression to the tumor microenvironment, thereby avoiding harmful systemic exposure.

A closer examination of the tumor-infiltrating CD8+ CAR T cells revealed heightened expression of effector molecules including granzyme B and proliferative marker Ki67, confirming enhanced cytotoxic potential and proliferation. Transcriptomic analyses via RNA sequencing demonstrated that NR4A2/IL-12 CAR T cells distinctly upregulated gene pathways involved in IL-12 signaling, TNF-related cascades, and STAT family transcription factors (STAT3/4/5), underpinning their robust effector functions and persistence within the hostile tumor milieu.

While IL-12 proved highly effective when driven by NR4A2, the researchers probed whether a different cytokine could be similarly harnessed. Anti-LeY CAR T cells engineered to express IL-2 under the NR4A2 promoter only showed mild tumor control enhancements. This observation prompted exploration of the RGS16 promoter, a gene known to support stronger intratumoral transgene expression. Indeed, RGS16/IL-2 CAR T cells significantly outperformed NR4A2/IL-2 in tumor control and cytokine production, without eliciting toxicity.

Mechanistic studies on RGS16/IL-2 CAR T cells revealed elevated Ki67, IFNγ, and TNF expression alongside increased CD8+ and CD4+ CAR T cell frequencies in tumor, spleen, and peripheral blood. These data suggest that the promoter choice for therapeutic payloads must be carefully matched to the biological properties of the payload itself, balancing efficacy with safety to maximize clinical utility.

Extending their findings to an immunocompetent model, murine anti-hHer2 CAR T cells driven by the RGS16/IL-2 construct demonstrated strong therapeutic responses against E0771-hHer2 tumors without increasing the frequency of immunosuppressive regulatory T cells (Tregs) within the tumor. This result alleviates concerns about inadvertent immune dampening that could offset the benefits of cytokine secretion.

The researchers also validated these gene modulation strategies in patient-derived diffuse large B-cell lymphoma (DLBCL) CAR T cells. CRISPR-mediated knock-in of IL-12 or IL-2 reporter constructs into NR4A2 or RGS16 loci led to controlled cytokine expression after stimulation, confirming the approach’s translatability to human clinical contexts.

Importantly, comparison between traditional retroviral transduction methods and precise TRAC-targeted knock-ins underscored the superiority of endogenous gene rewiring. Knock-in CAR T cells exhibited regulated expression patterns and potent antitumor activity, affirming the promise of CRISPR-based genome editing in generating safer and more efficacious next-generation cell therapies.

This study’s findings open new horizons for reprogramming CAR T cells by integrating synthetic biology with deep understanding of endogenous gene regulation. By strategically repurposing native promoters like NR4A2 and RGS16, therapeutic payloads can now be precisely delivered within tumor sites, achieving potent antitumor immunity while minimizing systemic toxicity. This approach potentially lays the groundwork for a new class of “smart” CAR T therapies tailored for personalized cancer treatment.

With clinical translation in mind, these results highlight the need for further exploration of endogenous regulatory elements to control diverse cytokines or immunomodulators. Such advances could enable combinatorial payload delivery tailored to distinct tumor types or immune environments, leveraging the full arsenal of immune biology to eradicate refractory malignancies safely.

In summary, the engineered NR4A2/IL-12 and RGS16/IL-2 human CAR T cells represent a major breakthrough in immunotherapy design. Leveraging endogenous gene promoters to modulate therapeutic payload expression ushers in a new era of precision-engineered cell therapies with enhanced efficacy and safety profiles. The work by Chen and colleagues moves us closer to realizing the full potential of cellular immunotherapy against cancer.

Subject of Research: Engineering endogenous gene promoters in human CAR T cells to achieve tumor-restricted cytokine delivery for enhanced antitumor efficacy and reduced systemic toxicity.

Article Title: Rewiring endogenous genes in CAR T cells for tumour-restricted payload delivery.

Article References: Chen, A.X.Y., Yap, K.M., Kim, J.S. et al. Rewiring endogenous genes in CAR T cells for tumour-restricted payload delivery. Nature (2025). https://doi.org/10.1038/s41586-025-09212-7

Image Credits: AI Generated