Combination of tucatinib and neural stem cells secreting anti-HER2 antibody prolongs survival of mice with metastatic brain cancer

Brain metastases are a leading cause of death in patients with breast cancer. The lack of clinical trials and the presence of the blood–brain barrier limit therapeutic options. Furthermore, overexpression of the human epidermal growth factor receptor 2 (HER2) increases the incidence of breast cancer brain metastases (BCBM). HER2-targeting agents, such as the monoclonal antibodies trastuzumab and pertuzumab, improved outcomes in patients with breast cancer and extracranial metastases. However, continued BCBM progression in breast cancer patients highlighted the need for novel and effective targeted therapies against intracranial metastases. In this study, we engineered the highly migratory and brain tumor tropic human neural stem cells (NSCs) LM008 to continuously secrete high amounts of functional, stable, full-length antibodies against HER2 (anti-HER2Ab) without compromising the stemness of LM008 cells. The secreted anti-HER2Ab impaired tumor cell proliferation in vitro in HER2+ BCBM cells by inhibiting the PI3K-Akt signaling pathway and resulted in a significant benefit when injected in intracranial xenograft models. In addition, dual HER2 blockade using anti-HER2Ab LM008 NSCs and the tyrosine kinase inhibitor tucatinib significantly improved the survival of mice in a clinically relevant model of multiple HER2+ BCBM. These findings provide compelling evidence for the use of HER2Ab-secreting LM008 NSCs in combination with tucatinib as a promising therapeutic regimen for patients with HER2+ BCBM.

Breast cancer metastasis is one of the leading causes of cancer-related deaths among women worldwide (1). This is especially true in the context of the brain, where the presence of the blood–brain barrier (BBB) significantly decreases the efficacy of the existing systemic therapies (2). The burden of brain metastatic breast cancer is further compounded by the fact that the current standard treatment is palliative and primarily local, whereby surgical resection, stereotactic radiosurgery, and/or whole-brain radiation therapy achieve limited survival benefits (3). In addition, some intracranial lesions, such as diffused multiple micrometastases or metastases close to the eloquent areas in the brain, are not suitable for surgical resection (4).The overexpression of the human epidermal growth factor receptor 2 (HER2), a tyrosine kinase receptor, is observed in about 30% of patients with breast cancer and is known to be associated with advanced disease and decreased overall survival (5). In addition, up to 50% of patients with HER2+ overexpressing breast cancer will develop central nervous system (CNS) metastases, resulting in a median survival of 11 to 18 mo after diagnosis (6–9). Trastuzumab (Herceptin), a humanized monoclonal antibody (mAb) targeting HER2, was the first clinically approved targeted therapy for the treatment of HER2+ overexpressing breast cancer and is now used routinely as the first-line therapy (10, 11). The antitumor mechanisms of trastuzumab therapy are complex, which include antibody-dependent cell-mediated cytotoxicity, inhibition of cleavage of the extracellular domain of HER2, inhibition of ligand-independent HER2 receptor dimerization, impaired activation of HER2 downstream pathways, induction of cell cycle arrest and apoptosis, inhibition of angiogenesis, and interference with DNA repair (12–14). Pertuzumab is another mAb that binds to the HER2 dimerization domain, inhibiting its heterodimerization with other HER family receptors (15, 16). In combination with trastuzumab, pertuzumab further improves invasive disease-free survival among patients with HER2+ breast cancer (17). More recently, the combination of both agents was reported to improve the response rate in patients with HER2+ metastatic breast cancer and progressive CNS metastases (18). However, the large molecular sizes of trastuzumab or pertuzumab and their weak permeability through the BBB require high dosages that can lead to toxicity. A clinical trial that evaluated the effect of tucatinib in combination with trastuzumab and capecitabine in patients with HER2+ metastatic breast cancer (HER2CLIMB) showed promising results in patients with brain metastases (19). Tucatinib (Tukysa) is a Food and Drug Administration (FDA)-approved oral tyrosine kinase inhibitor (TKI) that is highly selective for the kinase domain of HER2 with minimal inhibition of the epidermal growth factor receptor, limited low-grade toxicity, strong ability to cross the BBB, and notable antitumor activity in heavily pretreated HER2+ metastatic breast cancer patients (19–22). The risk of disease progression or death was decreased by 52% in patients with HER2+ breast cancer brain metastasis (BCBM) receiving the tucatinib combination treatment compared to those patients in the placebo combination group (23). Nevertheless, the limited efficacy of tucatinib as a monotherapy for HER2+ BCBM (24) underscores the need for innovative therapeutic regimens and delivery platforms that can improve clinical outcomes in patients with brain metastases.Human neural stem cell (NSC)-based therapies have emerged in the last few years as promising strategies for the treatment of CNS malignancies. Proof-of-concept preclinical and clinical studies have demonstrated the efficacy and feasibility of these NSCs for targeted delivery of therapeutic agents and oncolytic viruses (25–28). Our group has previously demonstrated the ability of the v-MYC immortalized HB1.F3 NSC line to deliver functional anti-HER2 antibodies (anti-HER2Ab) when injected directly in the CNS, improving significantly the survival of mice bearing breast cancer cells in the brain (4). The present study utilized the L-MYC–immortalized human NSC line LM-NSC008 (LM008), previously described as nontumorigenic in vivo and with tumor cell tropism and high migratory properties (29). Transduction of NSCs with L-MYC reduces the risk of oncogenic transformation, enhances their in vivo engraftment and migration capabilities, and results in a complete absence of tumorigenicity for up to 9 mo when injected in mouse brains (30, 31). After modifying the LM008 cells to secrete stable and high amounts of anti-HER2Ab (LM008–HER2Ab cells), we analyzed their efficacy when delivered locally in the brain and systemically in a model of multiple HER2+ BCBM. Our results demonstrate a significant survival benefit in mice injected with LM008–HER2Ab cells that was further improved when these mAb-secreting NSCs were used in combination with tucatinib. Thus, this study provides compelling evidence for the use of LM008–HER2Ab NSCs in combination with tucatinib for the treatment of HER2+ overexpressing BCBM.