The physiological asymmetry between daughters of a mother bacterium is produced by the inheritance of either old poles, carrying non-genetic damage, or newly synthesized poles. However, as bacteria display long-term growth stability leading to physiological immortality, there is controversy on whether asymmetry corresponds to aging. Here we show that deterministic age structure landscapes emerge from physiologically immortal bacterial lineages. Through single-cell microscopy and microfluidic techniques, we demonstrate that aging and rejuvenating bacterial lineages reach two distinct states of growth equilibria. These equilibria display stabilizing properties, which we quantified according to the compensatory trajectories of continuous lineages throughout generations. Finally, we show that the physiological asymmetry between aging and rejuvenating lineages produces complex age structure landscapes, resulting in a deterministic phenotypic heterogeneity that is neither an artifact of starvation nor a product of extrinsic damage. These findings indicate that physiological immortality and cellular aging can both be manifested in single celled organisms.

Bacteria have the ability to produce minicells, or small spherical versions of themselves that lack chromosomal DNA and are unable to replicate. A minicell can constitute as much as 20% of the cell’s volume. Although molecular biology and biotechnology have used minicells as laboratory tools for several decades, it is still puzzling that bacteria should produce such costly but potentially nonfunctional structures. Here, we show that bacteria gain a benefit by producing minicells and using them as a mechanism to eliminate damaged or oxidated proteins. The elimination allows the bacteria to tolerate higher levels of stress, such as increasing levels of streptomycin. If this mechanism extends from streptomycin to other antibiotics, minicell production could be an overlooked pathway that bacteria are using to resist antimicrobials.

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.

BackgroundSAR444245 is a non-alpha IL-2 Synthorin TM molecule designed with a site-specific non-natural amino acid serving as a bioconjugation site for a single PEG. The non-natural amino acid is positioned to enable the PEG bioconjugation to obscure block binding to the IL-2 alpha receptor, while retaining near-native affinity with the intermediate affinity βγ IL-2 receptor. The non-alpha features of SAR444245 minimize activation of immune suppressive regulatory CD4+ T cells, while retaining activity on CD8+ T cells and NK cells expressing the IL-2 βγ receptors. NK cells exert anti-tumor activity through antibody dependent cellular cytotoxicity (ADCC) of IgG antibodies as well as antibody independent mechanisms.MethodsHere, we utilized a panel of human primary PBMC based immunoassays and transcriptomic analysis to evaluate whether SAR444245 may improve ADCC function of IgG1 anti-tumor target antibodies.ResultsWe characterized the ability of SAR444245 to enhance the cytolytic function of NK cells towards the prototypic NK target cell K562 as well as to modulate NK cell ADCC in combination with EGFR or CD20-targeting antibodies. In vitro assays demonstrated that SAR444245 can activate NK cells, promote NK cell proliferation and improve cytotoxicity of NK cells against K562 cells and across a panel of human EGFR and CD20 positive cell lines. In PBMC based ADCC assays with 1ug/ml of antibody, SAR444245 improved ADCC function maximally by 9-fold for an anti-EGFR antibody and at 5-fold for an anti-CD20 antibody. SAR444245 exhibited dose-dependent enhancement of NK cell ADCC function. Notably, this activity was observed in cell lines expressing varying levels of EGFR and CD20. SAR444245 treatment was associated with dose dependent increases in NK cell degranulation and IFN-γ production. Transcriptomic profiling revealed that SAR444245 had broad effects on NK cell biology leading to changes in inhibitory and activating receptors.ConclusionsIn summary, these results indicate that SAR444245 can enhance the cytolytic activity of NK cells and enhance the ADCC effect of tumor-directed antibodies by activating NK cells.