HPN328: An Anti-DLL3 T Cell Engager for Treatment of Small Cell Lung Cancer Delta-like protein 3 (DLL3) is a Notch inhibitory ligand is expressed in more than 70% of small cell lung cancers (SCLCs), while there is little to no surface expression in normal adult tissues outside of the CNS *1. SCLC is an aggressive neuroendocrine tumor that represents about 15 percent of all lung cancers. Although often responsive to standard of care treatment, relapse is common, with a median progression-free survival of 2–3 months and median overall survival (OS) of 8–13 months and 5-year OS rate of <5%*2. DLL3 thus represents an attractive potential target for T cell–redirecting immunotherapy in SCLC. HPN328 is a tri-specific T cell activating construct (TriTAC) consisting of three binding domains: a N-terminal single chain Fv (scFv) that binds to CD3ϵ of the T cell receptor (TCR), a middle single domain antibody (sdAb) that binds to human serum albumin (HSA) to extend the half-life, and a C-terminal sdAb that binds to human DLL3. HPN328 is designed to simultaneously engage DLL3 on a target SCLC cell and CD3 on a T cell resulting in T cell activation, proliferation, and the eventual lysis of the target lung cancer cell. HPN328 is a highly stable single polypeptide of ~ 53 kDa expressed in CHO cells. It binds to human and cynomolgus monkey DLL3, albumin, and CD3ϵ with similar affinities. Flow cytometry analysis of T cells from various normal donors and a panel of DLL3 positive and DLL3 negative tumor cell lines confirmed binding of HPN328 to its native targets expressed on the cell surface. HPN328 induces potent killing of DLL3 expressing SCLC cell lines in vitro. In co-cultures of T cells from normal human donors, target tumor cells, and HSA, HPN328 mediated dose-dependent and DLL3-dependent cytotoxicity. Concomitant with target tumor cell killing, HPN328 also mediated dose-dependent upregulation of CD25 and CD69 on T cells in the TDCC co-cultures when DLL3 positive tumor cells were present. Consistent with the mechanism of action of CD3-based T cell engaging molecules, T cell derived cytokines, including TNFα, IL-2 and IFNγ, were detected. Nonclinical in vivo properties of HPN328 were evaluated in an NCI-H82 SCLC established tumor model. When administered to mice bearing human SCLC xenografts and human T cells, HPN328 eradicated subcutaneous tumors. In a single dose pilot toxicity study in cynomolgus monkeys, HPN328 was well tolerated at 1 and 10 mg/kg. Pharmacodynamic changes such as transient cytokine elevation mainly at the high dose were observed, consistent with the expected mechanism of action of T cell engagers. There were no clinically significant or adverse test article-related changes in hematology or clinical chemistry, and no apparent adverse findings at terminal and recovery necropsy. HPN328 exhibited linear PK properties in the given dose range with a serum half-life of 64 to 85 hours. Pharmacokinetic analysis supports weekly administration of HPN328 in humans. Preclinical and nonclinical characterization suggests that HPN328 is a highly efficacious, safe, and novel therapeutic candidate. A first-in-human phase 1 clinical trial is planned to evaluate HPN328 in SCLC. *1. Saunders, L et al. (2015) Sci Transl Med. 7(302): 302ra136. *2. Navarro, A et al. (2017) Transl Lung Cancer Res. 6(1): S78–S83. Citation Format: Wade H Aaron, Richard Austin, Manasi Barath, Evan Callihan, Michael Cremin, Thomas Evans, Maria Gamez, Vaishnavi Ganti, Golzar Hemmati, Kathryn Kwant, Che-Leung Law, Bryan Lemon, Llewelyn Lao, Mary Ellen Molloy, Jessica O’Rear, Laura Sun, Holger Wesche, Stephen Yu, Timothy Yu. HPN328: An anti-DLL3 T cell engager for treatment of small cell lung cancer [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference on Molecular Targets and Cancer Therapeutics; 2019 Oct 26-30; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2019;18(12 Suppl):Abstract nr C033. doi:10.1158/1535-7163.TARG-19-C033

It is becoming increasingly appreciated that the nervous and immune systems communicate bidirectionally to regulate immunological outcomes in a variety of organs including the lung. Activation of neuronal signaling can be induced by inflammation, tissue damage, or pathogens to evoke or reduce immune cell activation in what has been termed a neuro-immune reflex. In the periphery, these reflexes include the cholinergic anti-inflammatory pathway, sympathetic reflex, and sensory nociceptor-immune cell pathways. Continual advances in neuroimmunology in peripheral organ systems have fueled small-scale clinical trials that have yielded encouraging results for a range of immunopathologies such as rheumatoid arthritis. Despite these successes, several limitations should give clinical investigators pause in the application of neural stimulation as a therapeutic for lung inflammation, especially if inflammation arises from a novel pathogen. In this review, the general mechanisms of each reflex, the evidence for these circuits in the control of lung inflammation, and the key knowledge gaps in our understanding of these neuroimmune circuits will be discussed. These limitations can be overcome not only through better understanding of neuroanatomy but also through a systematic evaluation of stimulation parameters using immune activation in lung tissues as primary readouts. Our rapidly evolving understanding of the nervous and immune systems highlights the importance of bidirectional communication between these cells in health and disease. This integrative approach has tremendous potential in the development of targeted therapeutics, if specific challenges can be overcome.

BackgroundEpithelial cell adhesion molecule (EpCAM) is highly expressed in many solid tumors. However, therapeutics targeting EpCAM have had limited clinical utility or failed in clinical development likely due to the expression of EpCAM in normal tissues. For example, clinical testing of solitomab, an EpCAM-targeting T cell engager, resulted in severe dose-limiting toxicities, including elevated liver transaminases, hyperbilirubinemia, and diarrhea. Designing an EpCAM-targeting T cell engager that is only active in the tumor would expand its therapeutic window and improve its safety profile.MethodsUsing a T cell engager prodrug platform named ProTriTAC that couples therapeutic half-life extension with functional masking, we have engineered HPN601, a protease-activated EpCAM-targeting T cell engager. HPN601 is a single polypeptide with three binding domains: anti-albumin for half-life extension, anti-CD3e for T cell engagement, and anti-EpCAM for tumor cell engagement. The anti-albumin domain contains a masking moiety and a protease-cleavable linker and keeps the molecule inert outside the tumor microenvironment. Activation by tumor-associated proteases removes the anti-albumin domain along with the masking moiety to reveal a potently active drug inside the tumor. This active drug has minimal activity outside of tumor because, without an albumin binding domain, it is rapidly cleared in circulation.ResultsA humanized rodent tumor model was used to simultaneously measure anti-tumor efficacy and clinically relevant toxicity endpoints. In this model, a surrogate molecule of HPN601 was safely administered at a dose ten-fold higher than the minimal efficacious dose required for durable tumor regression. Higher doses produced toxicities including elevated ALT/AST and bilirubin, body weight loss, and evidence of tissue damage by histopathology. In contrast, a constitutively active EpCAM-targeting T cell engager could only be dosed safely up to its minimal efficacious dose. The improved safety profile of HPN601 is further supported by a toxicokinetic study in non-human primates: compared to a constitutively active EpCAM-targeting T cell engager, HPN601 had significantly attenuated cytokine production, including IFN-g, IL-2, IL-6, and IL-10.ConclusionsHPN601 is a conditionally active EpCAM-targeting T cell engager with a ten-fold improved therapeutic window compared to a constitutively active EpCAM-targeting T cell engager. An EpCAM-specific T cell engager with an improved safety profile could address unmet needs in many solid tumors and demonstrate the feasibility of using conditionally active T cell engagers to target more solid tumor antigens.Ethics ApprovalThe study was reviewed and approved by Harpoon’s Institutional Animal Care and Use Committee.

Mucosal immunity is critical to host protection from enteric pathogens and must be carefully controlled to prevent immunopathology. Regulation of immune responses can occur through a diverse range of mechanisms including bi-directional communication with the neurons. Among which include specialized sensory neurons that detect noxious stimuli due to the expression of transient receptor potential vanilloid receptor 1 (TRPV1) ion channel and have a significant role in the coordination of host-protective responses to enteric bacterial pathogens. Here we have used the mouse-adapted attaching and effacing pathogen Citrobacter rodentium to assess the specific role of the TRPV1 channel in coordinating the host response. TRPV1 knockout (TRPV1-/-) mice had a significantly higher C. rodentium burden in the distal colon and fecal pellets compared to wild-type (WT) mice. Increased bacterial burden was correlated with significantly increased colonic crypt hyperplasia and proliferating intestinal epithelial cells in TRPV1-/- mice compared to WT. Despite the increased C. rodentium burden and histopathology, the recruitment of colonic T cells producing IFNγ, IL-17, or IL-22 was similar between TRPV1-/- and WT mice. In evaluating the innate immune response, we identified that colonic neutrophil recruitment in C. rodentium infected TRPV1-/- mice was significantly reduced compared to WT mice; however, this was independent of neutrophil development and maturation within the bone marrow compartment. TRPV1-/- mice were found to have significantly decreased expression of the neutrophil-specific chemokine Cxcl6 and the adhesion molecules Icam1 in the distal colon compared to WT mice. Corroborating these findings, a significant reduction in ICAM-1 and VCAM-1, but not MAdCAM-1 protein on the surface of colonic blood endothelial cells from C. rodentium infected TRPV1-/- mice compared to WT was observed. These findings demonstrate the critical role of TRPV1 in regulating the host protective responses to enteric bacterial pathogens, and mucosal immune responses.

Background: Vagus nerve stimulation (VNS) has recently been introduced as a nonpharmacological therapy for disease characterized by an exacerbated immune response, with clinical trials demonstrating its efficacy for autoimmunity and sepsis by reducing inflammatory proteins and improving disease scores. To date, much of these anti-inflammatory effects have been attributed to vagal efferent (motor) signaling in a mechanism referred to as the cholinergic anti-inflammatory pathway (CAIP). Recent studies from our lab, as well as others, have begun to highlight alternative neuro-immune regulation by the vagus nerve, independent from the CAIP. The scope of these neuro-immune interactions, including target tissues and cell types, however, is still unclear. Although the lung is densely innervated, it is unknown how VNS can reduce acute TLR7 agonist-induced lung inflammation. Hypothesis: We sought to determine whether VNS was effective at reducing acute lung inflammation induced by viral mimetic and TLR7/8 agonist Resiquimod (R848). Methods: The right cervical vagus nerves of C57BL/6 mice were stimulated electrically (5V, 5Hz) for 20 minutes. A 0.25mg/kg dose of R848 was instilled 10 minutes after beginning VNS. 1 hour post-R848 challenge, relevant tissues were prepared for qPCR, flow cytometry, or ELISA. Results: Stimulation of the right vagus nerve was able to significantly decrease R848-induced mRNA expression of pro-inflammatory cytokines TNFα and IFNβ, as well as chemokines CCL4 and CXCL1 within the lung. This decrease occurred independently of the cells responsible for the CAIP (CD4+ ChAT+ T-cells), indicating a novel neuro-immune mechanism with a new target organ. Flow cytometry revealed that alveolar macrophages and neutrophils were the primary immune cell populations responding to R848 and subsequently modified by VNS, with significant reductions in intracellular staining for TNFα. Epinephrine and norepinephrine were significantly elevated within the serum during the first 5 minutes of VNS, but fluid from bronchoalveolar lavage revealed significant increases of only epinephrine within the lung. Subsequently, removal of the adrenal glands, the primary source of epinephrine, eliminated the anti-inflammatory effect of VNS. Elevation of epinephrine suggests an immunomodulatory role for the β2AR, with further studies using a highly selective antagonist ICI 118 551 or β2AR KO mice eliminating VNS-induced protection. Finally, using optogenetics we were able to identify vagal afferent neurons as the fibers responsible for modulating immune cell activation within the lung. Conclusion: These studies highlight a novel neuro-immune mechanism within the lung capable of regulating viral mimetic-induced inflammation. Facilitated by vagal afferent neurons, we show that VNS elicits adrenal gland-derived epinephrine release that acts through the β2AR to reduce macrophage activation and neutrophil recruitment in the lung. Chan-Zuckerberg Initiative (2020-217656); Graduate Student Support Program (UC Davis: School of Veterinary Medicine); NIH T32 GM (GM099608, GM144303) This is the full abstract presented at the American Physiology Summit 2023 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.