The nucleus of the solitary tract (NST) processes gustatory and related somatosensory information rostrally and general viscerosensory information caudally. To compare its connections with those of other rodents, this study in the C57BL/6J mouse provides a subnuclear cytoarchitectonic parcellation (Nissl stain) of the NST into rostral, intermediate, and caudal divisions. Subnuclei are further characterized by NADPH staining and P2X2 immunoreactivity (IR). Cholera toxin subunit B (CTb) labeling revealed those NST subnuclei receiving chorda tympani nerve (CT) afferents, those connecting with the parabrachial nucleus (PBN) and reticular formation (RF), and those interconnecting NST subnuclei. CT terminals are densest in the rostral central (RC) and medial (M) subnuclei; less dense in the rostral lateral (RL) subnucleus; and sparse in the ventral (V), ventral lateral (VL), and central lateral (CL) subnuclei. CTb injection into the PBN retrogradely labels cells in the aforementioned subnuclei; RC and M providing the largest source of PBN projection neurons. Pontine efferent axons terminate mainly in V and rostral medial (RM) subnuclei. CTb injection into the medullary RF labels cells and axonal endings predominantly in V at rostral and intermediate NST levels. Small CTb injections within the NST label extensive projections from the rostral division to caudal subnuclei. Projections from the caudal division primarily interconnect subnuclei confined to the caudal division of the NST; they also connect with the area postrema. P2X2-IR identifies probable vagal nerve terminals in the central (Ce) subnucleus in the intermediate/caudal NST. Ce also shows intense NADPH staining and does not project to the PBN. J. Comp. Neurol. 522:1565–1596, 2014.

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Nerve fibers that surround and innervate the taste bud were visualized with inherent fluorescence using Brainbow transgenic mice that were generated by mating the founder line L with nestin-cre mice. Multicolor fluorescence revealed perigemmal fibers as branched within the non-taste epithelium and ending in clusters of multiple rounded swellings surrounding the taste pore. Brainbow-labeling also revealed the morphology and branching pattern of single intragemmal fibers. These taste bud fibers frequently innervated both the peripheral bud, where immature gemmal cells are located, and the central bud, where mature, differentiated cells are located. The fibers typically bore preterminal and terminal swellings, growth cones with filopodia, swellings, and rounded retraction bulbs. These results establish an anatomical substrate for taste nerve fibers to contact and remodel among receptor cells at all stages of their differentiation, an interpretation that was supported by staining with GAP-43, a marker for growing fibers and growth cones.

Introduction: Epidermal growth receptor (EGFR) is the most expressed membrane oncogenic protein in human cancers. KRAS and BRAF mutations are significant drivers of resistance to EGFR-targeted therapies. Unlike other treatments, EGFR-targeting, CD3 bispecific T cell engagers (TCEs) can potentially retain activity against tumors bearing resistance mutations. However, cytokine release syndrome (CRS), on-target off-tumor toxicities, and poor pharmacokinetics (PK) properties present significant clinical limitations for these potent immunomodulators. To overcome these challenges, Janux has developed JANX008, an EGFR- and CD3-targeted tumor-activated T cell engager (TRACTr). JANX008 is a humanized tri-specific protein that contains EGFR- and CD3-binding domains, an albumin binding domain to extend circulating half-life, and two different peptide masks fused to the molecule through tumor protease cleavable linkers. One peptide mask inhibits EGFR engagement on target cells, and the other inhibits CD3 engagement on T cells. Once the cleavage sequences undergo proteolysis by tumor proteases, the EGFR and CD3 masks are released, and the resulting active molecule can bind EGFR and CD3 on target cells. Methods: Peptide masks against EGFR- and CD3-binding domains were identified via phage display. The efficiency of the masks was evaluated using human EGFR and CD3 ELISAs. JANX008-induced cleavage-dependent T cell killing was evaluated in human PBMC/tumor cell co-culture assays. Anti-tumor efficacy of JANX008 was tested in multiple preclinical models, including EGFR antibody-resistant KRAS- and PIK3CA-mutant mouse colon cancer model (HCT116) and a fully human primary colorectal cancer (CRC) tumoroid system. The pharmacokinetic and safety profile of JANX008 was evaluated in non-human primate studies. Results: JANX008 target engagement was cleavage-dependent where masking reduced EGFR and CD3 binding by >300x and >1,000x, respectively. JANX008 exhibited potent cleavage- and dose-dependent activity in multiple preclinical models, including EGFR antibody-resistant tumor and T cell co-culture assays, humanized mouse CRC model, and a human primary CRC tumoroids with an intact tumor microenvironment. JANX008 showed a significantly enhanced safety profile in NHPs compared to non-masked EGFR-TCE, including decreased CRS-associated cytokines and healthy tissue toxicities at high exposures. Clinical chemistry, hematology, and pathology measurements all supported No-Observed-Adverse-Effect-Level ≥ 0.6 mg/kg/dose. Finally, the cleavable albumin-binding domain extended the half-life of JANX008 to ~94h, relative to the ~1.3h half-life of non-masked TCE, supporting its weekly clinical dosing. Conclusions: Preclinical data demonstrate key characteristics of JANX008, including cleavage-dependent activity, half-life extended PK, the potential for superior safety, and manufacturability properties that could mitigate significant limitations of TCEs and support JANX008 clinical development. Citation Format: Thomas R DiRaimondo, Natalija Budimir, Lina Ma, Simon Shenhav, Vanessa Cicchini, Hu Wu, Renee Jocic, Fabrece Roup, Calvin Campbell, Carolina Caffaro, Hans Aerni, Ugur Eskiocak, Wayne Godfrey, Charles Winter, Marc Nasoff, Neil Gibson, David Campbell, shahram Salek-Ardakani. Preclinical activity and safety profile or JANX008, a novel EGFR-targeting tumor-activated T cell engager for treatment of solid tumors [abstract]. In: Proceedings of the AACR Special Conference: Tumor Immunology and Immunotherapy; 2022 Oct 21-24; Boston, MA. Philadelphia (PA): AACR; Cancer Immunol Res 2022;10(12 Suppl):Abstract nr B04.

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Natural Killer Group 2, member D (NKG2D) is an activating receptor that is expressed on the key immune cells involved in the cytotoxic arm of the immune system. Ligands of NKG2D, such as MICA and MICB, are not normally expressed but are upregulated in response to stress, such as oxygen or nutrient deprivation, viral infection or oncogenic transformation. One mechanism that tumor cells, that express MICA and MICB, have adopted to evade the cytotoxic potential of NK and T cells, that express the NKG2D receptor, is to cleave MICA/B from the cell surface. Proteolytic cleavage of MICA/B occurs within the α-3 domain of MICA/B. In order to evaluate the therapeutic potential of blocking cleavage of MICA/B we have generated multiple antibodies that bind to the α-3 domain of MICA/B. The lead antibody, PDI-01, was found to bind to multiple recombinant human MICA alleles and human MICB and to recombinant MIC proteins derived from non-human primate sequences with low nM affinity. PDI-01 bound to target antigen on the surface of multiple cancer cell lines including HCT-116 colon carcinoma, HCC1534 lung carcinoma and PLC/PRF/5 liver cancer cell lines. Each of those tumor cell lines were positive for MICA and MICB expression. Low expression of other NKG2D ligands, known as UL-16 binding proteins 1-6 (ULBP 1-6), has been shown in those cell lines. PDI-01 was found to prevent cleavage of MICA from the surface of HCT-116, HCC1534 and PLC/PRF/5 cells via ELISA quantification of soluble MICA levels. Exposure of HCT-116, HCC1534 and PLC/PRF/5 tumor cell lines to PDI-01 increased surface MICA levels as measured by flow cytometry analysis. An increase in immune cell mediated killing of HCT-116, HCC1534 and PLC/PRF/5 cells was observed when tumor cells were incubated with NK-92 cells (NKG2D positive) in the presence of PDI-01. PDI-01 also enhanced antibody-dependent cell-mediated cytotoxicity (ADCC) against these three tumor cell lines. Co-culture of PBMCs, or purified NK cells, with PLC/PRF/5 cells in the presence of PDI-01 led to an increase in TNF-α, IFN-γ, CCL4, CCL5 and IL-6. PDI-01 is being advanced into clinical development and the Phase 1 program is scheduled to be initiated in the 2Q, 2020. Citation Format: Safak Yalcin, Vanessa Cicchini, Xin Du, Xin Wu, Tom Kaever, Neil Gibson. Inhibition of proteolytic cleavage of NKG2D ligands increases immune cell-mediated cell killing of tumor cells in vitro [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 6707.

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