Format-tuning of in vivo-launched bispecific T cell engager enhances efficacy against renal cell carcinoma.

BACKGROUND: Advanced clear cell renal cell carcinoma (ccRCC) is a prevalent kidney cancer for which long-term survival rates are abysmal, though immunotherapies are showing potential. Not yet clinically vetted are bispecific T cell engagers (BTEs) that activate T cell-mediated cancer killing through intercellular synapsing. Multiple BTE formats exist, however, with limited cross-characterizations to help optimize new drug design. Here, we developed BTEs to treat ccRCC by targeting carbonic anhydrase 9 (CA9) while characterizing the persistent BTE (PBTE) format and comparing it to a new format, the persistent multivalent T cell engager (PMTE). These antibody therapies against ccRCC are developed as both recombinant and synthetic DNA (synDNA) medicines. METHODS: Antibody formatting effects on binding kinetics were assessed by flow cytometry and intercellular synaptic strength assays while potency was tested using T-cell activation and cytotoxicity assays. Mouse models were used to study antibody plasma and tumor pharmacokinetics, as well as antitumor efficacy as both recombinant and synDNA medicines. Specifically, three models using ccRCC cell line xenografts and human donor T cells in immunodeficient mice were used to support this study. RESULTS: Compared with a first-generation BTE, we show that the PBTE reduced avidity, intercellular synaptic strength, cytotoxic potency by as much as 33-fold, and ultimately efficacy against ccRCC tumors in vivo. However, compared with the PBTE, we demonstrate that the PMTE improved cell avidity, restored intercellular synapses, augmented cytotoxic potency by 40-fold, improved tumor distribution pharmacokinetics by 2-fold, and recovered synDNA efficacy in mouse tumor models by 20-fold. All the while, the PMTE displayed a desirable half-life of 4 days in mice compared with the conventional BTE's 2 hours. CONCLUSIONS: With impressive efficacy, the CA9-targeted PMTE is a promising new therapy for advanced ccRCC, which can be effectively delivered through synDNA. The highly potent PMTE format itself is a promising new tool for future applications in the multispecific antibody space.

Prevalence of intratumoral regulatory T cells expressing neuropilin-1 is associated with poorer outcomes in patients with cancer.

Despite the success of immune checkpoint blockade therapy, few strategies sufficiently overcome immunosuppression within the tumor microenvironment (TME). Targeting regulatory T cells (Tregs) is challenging, because perturbing intratumoral Treg function must be specific enough to avoid systemic inflammatory side effects. Thus, no Treg-targeted agents have proven both safe and efficacious in patients with cancer. Neuropilin-1 (NRP1) is recognized for its role in supporting intratumoral Treg function while being dispensable for peripheral homeostasis. Nonetheless, little is known about the biology of human NRP1+ Tregs and the signals that regulate NRP1 expression. Here, we report that NRP1 is preferentially expressed on intratumoral Tregs across six distinct cancer types compared to healthy donor peripheral blood [peripheral blood lymphocyte (PBL)] and site-matched, noncancer tissue. Furthermore, NRP1+ Treg prevalence is associated with reduced progression-free survival in head and neck cancer. Human NRP1+ Tregs have broad activation programs and elevated suppressive function. Unlike mouse Tregs, we demonstrate that NRP1 identifies a transient activation state of human Tregs driven by continuous T cell receptor (TCR) signaling through the mitogen-activated protein kinase pathway and interleukin-2 exposure. The prevalence of NRP1+ Tregs in patient PBL correlates with the intratumoral abundance of NRP1+ Tregs and may indicate higher disease burden. These findings support further clinical evaluation of NRP1 as a suitable therapeutic target to enhance antitumor immunity by inhibiting Treg function in the TME.

Neuropilin-1: a checkpoint target with unique implications for cancer immunology and immunotherapy.

Checkpoint blockade immunotherapy established a new paradigm in cancer treatment: for certain patients curative treatment requires immune reinvigoration. Despite this monumental advance, only 20%-30% of patients achieve an objective response to standard of care immunotherapy, necessitating the consideration of alternative targets. Optimal strategies will not only stimulate CD8+ T cells, but concomitantly modulate immunosuppressive cells in the tumor microenvironment (TME), most notably regulatory T cells (Treg cells). In this context, the immunoregulatory receptor Neuropilin-1 (NRP1) is garnering renewed attention as it reinforces intratumoral Treg cell function amidst inflammation in the TME. Loss of NRP1 on Treg cells in mouse models restores antitumor immunity without sacrificing peripheral tolerance. Enrichment of NRP1+ Treg cells is observed in patients across multiple malignancies with cancer, both intratumorally and in peripheral sites. Thus, targeting NRP1 may safely undermine intratumoral Treg cell fitness, permitting enhanced inflammatory responses with existing immunotherapies. Furthermore, NRP1 has been recently found to modulate tumor-specific CD8+ T cell responses. Emerging data suggest that NRP1 restricts CD8+ T cell reinvigoration in response to checkpoint inhibitors, and more importantly, acts as a barrier to the long-term durability of CD8+ T cell-mediated tumor immunosurveillance. These novel and distinct regulatory mechanisms present an exciting therapeutic opportunity. This review will discuss the growing literature on NRP1-mediated immune modulation which provides a strong rationale for categorizing NRP1 as both a key checkpoint in the TME as well as an immunotherapeutic target with promise either alone or in combination with current standard of care therapeutic regimens.

CD56dim CD16- Natural Killer Cell Profiling in Melanoma Patients Receiving a Cancer Vaccine and Interferon-α.

Natural killer (NK) cells are innate cytotoxic and immunoregulatory lymphocytes that have a central role in anti-tumor immunity and play a critical role in mediating cellular immunity in advanced cancer immunotherapies, such as dendritic cell (DC) vaccines. Our group recently tested a novel recombinant adenovirus-transduced autologous DC-based vaccine that simultaneously induces T cell responses against three melanoma-associated antigens for advanced melanoma patients. Here, we examine the impact of this vaccine as well as the subsequent systemic delivery of high-dose interferon-α2b (HDI) on the circulatory NK cell profile in melanoma patients. At baseline, patient NK cells, particularly those isolated from high-risk patients with no measurable disease, showed altered distribution of CD56dim CD16+ and CD56dim CD16- NK cell subsets, as well as elevated serum levels of immune suppressive MICA, TN5E/CD73 and tactile/CD96, and perforin. Surprisingly, patient NK cells displayed a higher level of activation than those from healthy donors as measured by elevated CD69, NKp44 and CCR7 levels, and enhanced K562 killing. Elevated cytolytic ability strongly correlated with increased representation of CD56dim CD16+ NK cells and amplified CD69 expression on CD56dim CD16+ NK cells. While intradermal DC immunizations did not significantly impact circulatory NK cell activation and distribution profiles, subsequent HDI injections enhanced CD56bright CD16- NK cell numbers when compared to patients that did not receive HDI. Phenotypic analysis of tumor-infiltrating NK cells showed that CD56dim CD16- NK cells are the dominant subset in melanoma tumors. NanoString transcriptomic analysis of melanomas resected at baseline indicated that there was a trend of increased CD56dim NK cell gene signature expression in patients with better clinical response. These data indicate that melanoma patient blood NK cells display elevated activation levels, that intra-dermal DC immunizations did not effectively promote systemic NK cell responses, that systemic HDI administration can modulate NK cell subset distributions and suggest that CD56dim CD16- NK cells are a unique non-cytolytic subset in melanoma patients that may associate with better patient outcome.