SUMMARY Bacillus thuringiensis produces crystalline protein inclusions with insecticidal or nematocidal properties. These crystal (Cry) proteins determine a particular strain's toxicity profile. Transgenic crops expressing one or more recombinant Cry toxins have become agriculturally important. Individual Cry toxins are usually toxic to only a few species within an order, and receptors on midgut epithelial cells have been shown to be critical determinants of Cry specificity. The best characterized of these receptors have been identified for lepidopterans, and two major receptor classes have emerged: the aminopeptidase N (APN) receptors and the cadherin-like receptors. Currently, 38 different APNs have been reported for 12 different lepidopterans. Each APN belongs to one of five groups that have unique structural features and Cry-binding properties. While 17 different APNs have been reported to bind to Cry toxins, only 2 have been shown to mediate toxin susceptibly in vivo. In contrast, several cadherin-like proteins bind to Cry toxins and confer toxin susceptibility in vitro, and disruption of the cadherin gene has been associated with toxin resistance. Nonetheless, only a small subset of the lepidopteran-specific Cry toxins has been shown to interact with cadherin-like proteins. This review analyzes the interactions between Cry toxins and their receptors, focusing on the identification and validation of receptors, the molecular basis for receptor recognition, the role of the receptor in resistant insects, and proposed models to explain the sequence of events at the cell surface by which receptor binding leads to cell death.

eEF2K (eukaryotic elongation factor 2 kinase) is a Ca2+/CaM (calmodulin)-dependent protein kinase which regulates the translation elongation machinery. eEF2K belongs to the small group of so-called ‘α-kinases’ which are distinct from the main eukaryotic protein kinase superfamily. In addition to the α-kinase catalytic domain, other domains have been identified in eEF2K: a CaM-binding region, N-terminal to the kinase domain; a C-terminal region containing several predicted α-helices (resembling SEL1 domains); and a probably rather unstructured ‘linker’ region connecting them. In the present paper, we demonstrate: (i) that several highly conserved residues, implicated in binding ATP or metal ions, are critical for eEF2K activity; (ii) that Ca2+/CaM enhance the ability of eEF2K to bind to ATP, providing the first insight into the allosteric control of eEF2K; (iii) that the CaM-binding/α-kinase domain of eEF2K itself possesses autokinase activity, but is unable to phosphorylate substrates in trans; (iv) that phosphorylation of these substrates requires the SEL1-like domains of eEF2K; and (v) that highly conserved residues in the C-terminal tip of eEF2K are essential for the phosphorylation of eEF2, but not a peptide substrate. On the basis of these findings, we propose a model for the functional organization and control of eEF2K.

f Acidification of the extracellular and/or intracellular environment is involved in many aspects of cell physiology and pathology. Eukaryotic elongation factor 2 kinase (eEF2K) is a Ca 2؉ /calmodulin-dependent kinase that regulates translation elongation by phosphorylating and inhibiting eEF2. Here we show that extracellular acidosis elicits activation of eEF2K in vivo, leading to enhanced phosphorylation of eEF2. We identify five histidine residues in eEF2K that are crucial for the activation of eEF2K during acidosis. Three of them (H80, H87, and H94) are in its calmodulin-binding site, and their protonation appears to enhance the ability of calmodulin to activate eEF2K. The other two histidines (H227 and H230) lie in the catalytic domain of eEF2K. We also identify His108 in calmodulin as essential for activation of eEF2K. Acidification of cancer cell microenvironments is a hallmark of malignant solid tumors. Knocking down eEF2K in cancer cells attenuated the decrease in global protein synthesis when cells were cultured at acidic pH. Importantly, activation of eEF2K is linked to cancer cell survival under acidic conditions. Inhibition of eEF2K promotes cancer cell death under acidosis.

Cry proteins are a large family of crystalline toxins produced by Bacillus thuringiensis. Individually, the family members are highly specific, but collectively, they target a diverse range of insects and nematodes. Domain II of the toxins is important for target specificity, and three loops at its apex have been studied extensively. There is considerable interest in determining whether modifications in this region may lead to toxins with novel specificity or potency. In this work, we studied the effect of loop substitution on toxin stability and specificity. For this purpose, sequences derived from antibody complementarity-determining regions (CDR) were used to replace native domain II apical loops to create "Crybodies." Each apical loop was substituted either individually or in combination with a library of third heavy-chain CDR (CDR-H3) sequences to create seven distinct Crybody types. An analysis of variants from each library indicated that the Cry1Aa framework can tolerate considerable sequence diversity at all loop positions but that some sequence combinations negatively affect structural stability and protease sensitivity. CDR-H3 substitution showed that loop position was an important determinant of insect toxicity: loop 2 was essential for activity, whereas the effects of substitutions at loop 1 and loop 3 were sequence dependent. Unexpectedly, differences in toxicity did not correlate with binding to cadherins-a major class of toxin receptors-since all Crybodies retained binding specificity. Collectively, these results serve to better define the role of the domain II apical loops as determinants of specificity and establish guidelines for their modification.Bacillus thuringiensis encompasses a group of aerobic, grampositive spore-forming bacteria that produce large crystalline inclusions consisting of entomocidal protein toxins (67). Cry toxins are a major constituent of these inclusions and the screening of thousands of B. thuringiensis strains has revealed that these proteins comprise a large family of related toxins with diverse specificities. The majority are toxic toward lepidopterans (butterflies and moths), dipterans (flies and mosquitoes), and coleopterans (beetles) (67), but some Cry toxins have been reported to kill hymenopterans (wasps and bees) (27) and nematodes (52, 78). Comparative sequence analyses have revealed five blocks of conserved amino acids found in most toxins (31) and that toxin genes typically encode proteins of 130 to 140 kDa or approximately 70 kDa. Phylogenetic analyses have shown a correlation between sequence similarity and insect order specificity (9, 17).Cry toxin protein inclusions must be activated to be functional (67). In lepidopterans, this multistep process starts when crystals are ingested by susceptible larvae. The high pH of the midgut promotes crystal solubilization and the release of protoxins. This exposes protoxin cleavage sites that are cut by host proteases to release the activated core. The toxin then binds to receptors on the surface of the midgut epith...

Cell therapy using T cell receptors (TCRs) and chimeric antigen receptors (CARs) represents a new wave of immunotherapies garnering considerable attention and investment. Further progress in this area of medicine depends in part on improving the functional capabilities of the engineered components, while maintaining the overall size of recombinant constructs to ensure their compatibility with existing gene delivery vehicles. We describe a single-variable-domain TCR (svd TCR) that utilizes only the variable domain of the β chain (Vβ). This Vβ module not only works in TCR and CAR formats, but also can be used to create single-chain bispecific CARs and TCRs. Comparison of individual ligand-binding Vβ domains in different formats suggests that the lone Vβ sequence controls the sensitivity and a major part of the specificity of the CAR or TCR construct, regardless of signaling format, in Jurkat and primary T cells.

Supplemental Digital Content is available in the text.

This is a PDF file of an article that has undergone enhancements after acceptance, such as the addition of a cover page and metadata, and formatting for readability, but it is not yet the definitive version of record. This version will undergo additional copyediting, typesetting and review before it is published in its final form, but we are providing this version to give early visibility of the article. Please note that, during the production process, errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

While Interleukin-2 (IL-2) has produced remarkable clinical efficacy in a fraction of cancer patients, its clinical use is limited by its narrow therapeutic index due to systemic and pleotropic activation of both inflammatory and suppressive lymphocytes. Numerous approaches to improve the specificity and activity profile of IL-2 are being evaluated. Using Tentarix’s propriety Tentacles™ platform, which is based on fully human stabilized antibody VH domains, we generated TNRX-257, a novel multispecific biologic that effectively blocks LAG3 while simultaneously delivering IL2Rγ/β agonism to LAG3+ cells in a highly conditional manner. LAG3 expression is restricted to antigen-experienced and tumor-reactive immune cells with little expression on peripheral PBMC or immune cells in normal tissues. TNRX-257 was designed to combine LAG3 inhibition with an IL2R agonist moiety to activate and expand LAG3+ tumor-reactive T cells in tumor microenvironment while enhancing their effector function and anti-tumor immunity with minimal systemic toxicity. TNRX-257 blocks the interaction of LAG3 with MHC-II and enhances TCR signaling with similar potency as Relatlimab. TNRX-257 also selectively induces pSTAT5 on LAG3+ immune cells with little activity on LAG3- immune cells, including resting human PBMCs. Moreover, the lower level of pSTAT5 (Emax) induced by TNRX-257 than IL-2 indicates TNRX-257 mediates its activity as a partial agonist, a phenotype that preserves stemness. TNRX-257 induces higher and preferential activation of CD8+ T cells over CD4+ T cells due to higher expression of LAG3 on CD8+ T cells than CD4+ T cells, particularly within the tumor microenvironment. Unlike an undirected IL-15 agonist that induced total CD8+ T cell proliferation, TNRX-257 selectively induced proliferation of LAG3+ CD8+ T cells in a human melanoma TIL stimulation assay without expansion of Tregs. TNRX-257 treatment also preserved the stem-like CD8+ population compared to the undirected IL-15 agonist. TNRX-257 showed in vitro and in vivo molecular stability, as well as good pharmacokinetic (PK) and pharmacodynamic (PD) properties. TNRX-257 induced robust anti-tumor efficacy when tested in early and established tumor models using melanoma A375-CMV-pp65 or colorectal cancer HT-29 cells in humanized mice. In contrast, the corresponding untargeted IL2Rγ/β Tentacle had no efficacy. Together, these data show that TNRX-257 has drug-like properties and elicits strong anti-tumor efficacy, supporting its clinical development. Citation Format: Rajesh K. Sharma, Jianying Dong, Natasha Del Cid, Yasamine Ghorbanian, Christina Carnevale, Christen Buetz, Matthew Lundberg, Glenn Capodagli, Jayd Hannah, Gavin Hong, Pricilla Walters, Arlene Sereno, Falene Chai, Abby Lin, James Furney, Wendy Zhang, Craig Pigott, Paul Kang, Michael Gallo, Margaret Karow, Stephen Demarest. TNRX-257, a novel multifunctional biologic effectively blocks LAG3 and conditionally delivers IL2Rg/b agonism to LAG3+ cells for robust anti-tumor immunity [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 2956.