The L-enantiomer is the predominant type of amino acid in all living systems. However, D-amino acids, once thought to be “unnatural”, have been found to be indigenous even in mammalian systems and increasingly appear to be functioning in essential biological and neurological roles. Both D- and L-amino acid levels in the hippocampus, cortex, and blood samples from NIH Swiss mice are reported. Perfused brain tissues were analyzed for the first time, thereby eliminating artifacts due to endogenous blood, and decreased the mouse-to-mouse variability in amino acid levels. Total amino acid levels (L-plus D-enantiomers) in brain tissue are up to 10 times higher than in blood. However, all measured D-amino acid levels in brain tissue are typically ~10 to 2000 times higher than blood levels. There was a 13% reduction in almost all measured D-amino acid levels in the cortex compared to those in the hippocampus. There is an approximate inverse relationship between the prevalence of an amino acid and the percentage of its D-enantiomeric form. Interestingly, glutamic acid, unlike all other amino acids, had no quantifiable level of its D-antipode. The bioneurological reason for the unique and conspicuous absence/removal of this D-amino acid is yet unknown. However, results suggest that D-glutamate metabolism is likely a unidirectional process and not a cycle, as per the L-glutamate/glutamine cycle. The results suggest that there might be unreported D-amino acid racemases in mammalian brains. The regulation and function of specific other D-amino acids are discussed.

New cyclofructan-6 (CF6)-based chiral stationary phases (CSPs) bind barium cations. As a result, the barium-complexed CSPs exhibit enantioselectivity toward 16 chiral phosphoric and sulfonic acids in the polar organic mode (e.g., methanol or ethanol mobile phase containing a barium salt additive). Retention is predominantly governed by a strong ionic interaction between the analyte and the complexed barium cation as well as hydrogen bonding with the cyclofructan macrocycle. The log k versus log [X], where [X] = the concentration of the barium counteranion, plots for LARIHC-CF6-P were linear with negative slopes demonstrating typical anion exchange behavior. The nature of the barium counteranion also was investigated (acetate, methanesulfonate, trifluoroacetate, and perchlorate), and the apparent elution strength was found to be acetate > methanesulfonate > trifluoroacetate > perchlorate. A theory based upon a double layer model was proposed wherein kosmotropic anions are selectively adsorbed to the cyclofructan macrocycle and attenuate the effect of the barium cation. van't Hoff studies for two analytes were conducted on the LARIHC-CF6-P for three of the barium salts (acetate, trifluoroacetate, and perchlorate), and the thermodynamic parameters governing retention and enantioselectivity are discussed. Interestingly, for the entropically driven separations, enantiomeric selectivity can increase at higher temperatures, even with decreasing retention.

Background: Though antibodies for cancer treatment have achieved clinical and commercial success over the past few decades, a large portion are limited by suboptimal efficacy and on-target off-tumor toxicity due to ubiquitous expression of their targets. A plethora of molecular engineering approaches have been introduced to restrict the activity of antibodies solely to tumor instead of healthy tissue with the goal of improving the therapeutic index, such as masking the binding sites of antibodies with inhibitory domains. However, sophisticated modification is usually required while the outcomes are often mediocre. Previously we developed an ultra-pH sensitive nanoparticle platform named ON-BOARD. The strength of this technology stems from its ability to preferentially release the payload specifically in the acidic tumor microenvironment while staying intact in normal tissue. The safety and feasibility of using such a platform have been demonstrated by successful delivery of fluorophore to tumors for imaging of multiple tumor types in Phase I and II clinical trials with Pegsitacianine (formerly “ONM-100”). Pegsitacianine has been shown to be generally well-tolerated with an infusion-related reaction as the most common adverse event in the clinical trials conducted to date. Based on the clinical results, we present the ON-BOARD platform herein as a potential universal and effective tool for tumor specific activation and delivery of therapeutic antibodies without the need for sophisticated antibody chemistry or engineering. Methods: Biosimilar monoclonal antibodies of atezolizumab, cetuximab, pembrolizumab, trastuzumab, and ipilimumab with the same variable region sequences as the original pharmaceutical drugs were used to demonstrate encapsulation by the ON-BOARD platform and pH-dependent activation. Encapsulated antibodies were purified using SEC and the encapsulation efficiencies were quantified by HPLC. Particle size and uniformity were studied by DLS. The formulations were accessed for bioactivity in vitro under neutral pH or acidified conditions using appropriate cell-based reporter assays. Results: ON-BOARD nanoparticles successfully encapsulated the antibodies mentioned above without additional modification of the original antibody. Encapsulation efficiency ranged from 50-100%. The formulations were characterized as uniformly distributed particles < 100nm in size with good stability. In vitro assessment by cell-based reporter assays demonstrated > 100-fold activation window between the acid-activated and intact formulations. The pH-dependent activation was further confirmed by affinity and binding assay. Conclusions: The ON-BOARD pH-sensitive nanoparticle platform demonstrated potential as an effective and universal tool for tumor specific activation and delivery of antibody-based therapeutics. Citation Format: Gaurav Bharadwaj, Qingtai Su, Stephen Gutowski, Curran Parpia, Ashley Campbell, Jason Miller, Tian Zhao. Encapsulating therapeutic antibodies for tumor specific activation and delivery using a clinically validated pH-sensitive nanoparticle platform [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 1734.