"Evaluating trends in pharmacist e-prescribing: 2019-2022.

BACKGROUND: Pharmacists have some prescriptive authority in all fifty states through dependent and independent prescribing. Data describing the volume and characteristics of pharmacist prescribing are not widely available, and these insights are critical to gauge the impact of regulations supporting pharmacist prescriptive authority. OBJECTIVE: To identify trends in pharmacist prescribing and compare them to primary care provider (PCP) prescribing trends by analyzing e-prescriptions initiated from electronic health records systems (EHRs) from 2019 through 2022. METHODS: This cross-sectional study used e-prescriptions from a national health information network to identify e-prescriptions ordered by pharmacists and PCPs from January 7, 2019, to January 1, 2023. E-prescriptions ordered by pharmacists and PCPs were analyzed to identify annual volume by prescriber type and most prescribed therapeutic classes. States with the highest volume of e-prescriptions ordered by pharmacists were identified. RESULTS: The number of e-prescriptions prescribed by a pharmacist increased 47% from 2019 (n=814,726) to 2022 (n=1,199,601). The number of pharmacists prescribing in 2019 was 1650, and this increased by 122% to 3664 in 2022. The number of e-prescriptions prescribed by PCPs increased by 4% from 2019 (n=927,890,123) to 2022 (n=965,803,376) while the number of PCPs prescribing increased by 8% from 2019 (n=364,995) to 2022 (n=394,753). CONCLUSION: Pharmacist e-prescribing increased across the four years studied while PCP e-prescribing modestly increased. Factors like access to technology, such as electronic health records, state regulations, and reimbursement impact a pharmacist's ability to prescribe.

Characterization and Modeling of Reversible Antibody Self-Association Provide Insights into Behavior, Prediction, and Correction.

Reversible antibody self-association, while having major developability and therapeutic implications, is not fully understood or readily predictable and correctable. For a strongly self-associating humanized mAb variant, resulting in unacceptable viscosity, the monovalent affinity of self-interaction was measured in the low µM range, typical of many specific and biologically relevant protein-protein interactions. A face-to-face interaction model extending across both the heavy-chain (HC) and light-chain (LC) Complementary Determining Regions (CDRs) was apparent from biochemical and mutagenesis approaches as well as computational modeling. Light scattering experiments involving individual mAb, Fc, Fab, and Fab'2 domains revealed that Fabs self-interact to form dimers, while bivalent mAb/Fab'2 forms lead to significant oligomerization. Site-directed mutagenesis of aromatic residues identified by homology model patch analysis and self-docking dramatically affected self-association, demonstrating the utility of these predictive approaches, while revealing a highly specific and tunable nature of self-binding modulated by single point mutations. Mutagenesis at these same key HC/LC CDR positions that affect self-interaction also typically abolished target binding with notable exceptions, clearly demonstrating the difficulties yet possibility of correcting self-association through engineering. Clear correlations were also observed between different methods used to assess self-interaction, such as Dynamic Light Scattering (DLS) and Affinity-Capture Self-Interaction Nanoparticle Spectroscopy (AC-SINS). Our findings advance our understanding of therapeutic protein and antibody self-association and offer insights into its prediction, evaluation and corrective mitigation to aid therapeutic development.

Cryo-EM structures of inhibitory antibodies complexed with arginase 1 provide insight into mechanism of action.

Human Arginase 1 (hArg1) is a metalloenzyme that catalyzes the hydrolysis of L-arginine to L-ornithine and urea, and modulates T-cell-mediated immune response. Arginase-targeted therapies have been pursued across several disease areas including immunology, oncology, nervous system dysfunction, and cardiovascular dysfunction and diseases. Currently, all published hArg1 inhibitors are small molecules usually less than 350 Da in size. Here we report the cryo-electron microscopy structures of potent and inhibitory anti-hArg antibodies bound to hArg1 which form distinct macromolecular complexes that are greater than 650 kDa. With local resolutions of 3.5 Å or better we unambiguously mapped epitopes and paratopes for all five antibodies and determined that the antibodies act through orthosteric and allosteric mechanisms. These hArg1:antibody complexes present an alternative mechanism to inhibit hArg1 activity and highlight the ability to utilize antibodies as probes in the discovery and development of peptide and small molecule inhibitors for enzymes in general.

Crystal Structure and Characterization of Human Heavy-Chain Only Antibodies Reveals a Novel, Stable Dimeric Structure Similar to Monoclonal Antibodies.

We report the novel crystal structure and characterization of symmetrical, homodimeric humanized heavy-chain-only antibodies or dimers (HC2s). HC2s were found to be significantly coexpressed and secreted along with mAbs from transient CHO HC/LC cotransfection, resulting in an unacceptable mAb developability attribute. Expression of full-length HC2s in the absence of LC followed by purification resulted in HC2s with high purity and thermal stability similar to conventional mAbs. The VH and CH1 portion of the heavy chain (or Fd) was also efficiently expressed and yielded a stable, covalent, and reducible dimer (Fd2). Mutagenesis of all heavy chain cysteines involved in disulfide bond formation revealed that Fd2 intermolecular disulfide formation was similar to Fabs and elucidated requirements for Fd2 folding and expression. For one HC2, we solved the crystal structure of the Fd2 domain to 2.9 Å, revealing a highly symmetrical homodimer that is structurally similar to Fabs and is mediated by conserved (CH1) and variable (VH) contacts with all CDRs positioned outward for target binding. Interfacial dimer contacts revealed by the crystal structure were mutated for two HC2s and were found to dramatically affect HC2 formation while maintaining mAb bioactivity, offering a potential means to modulate novel HC2 formation through engineering. These findings indicate that human heavy-chain dimers can be secreted efficiently in the absence of light chains, may show good physicochemical properties and stability, are structurally similar to Fabs, offer insights into their mechanism of formation, and may be amenable as a novel therapeutic modality.

Predicting Antibody Developability Profiles Through Early Stage Discovery Screening.

Monoclonal antibodies play an increasingly important role for the development of new drugs across multiple therapy areas. The term 'developability' encompasses the feasibility of molecules to successfully progress from discovery to development via evaluation of their physicochemical properties. These properties include the tendency for self-interaction and aggregation, thermal stability, colloidal stability, and optimization of their properties through sequence engineering. Selection of the best antibody molecule based on biological function, efficacy, safety, and developability allows for a streamlined and successful CMC phase. An efficient and practical high-throughput developability workflow (100 s-1,000 s of molecules) implemented during early antibody generation and screening is crucial to select the best lead candidates. This involves careful assessment of critical developability parameters, combined with binding affinity and biological properties evaluation using small amounts of purified material (<1 mg), as well as an efficient data management and database system. Herein, a panel of 152 various human or humanized monoclonal antibodies was analyzed in biophysical property assays. Correlations between assays for different sets of properties were established. We demonstrated in two case studies that physicochemical properties and key assay endpoints correlate with key downstream process parameters. The workflow allows the elimination of antibodies with suboptimal properties and a rank ordering of molecules for further evaluation early in the candidate selection process. This enables any further engineering for problematic sequence attributes without affecting program timelines.

Biophysical and Immunological Characterization and In Vivo Pharmacokinetics and Toxicology in Nonhuman Primates of the Anti-PD-1 Antibody Pembrolizumab.

The programmed cell death 1 (PD-1) pathway represents a major immune checkpoint, which may be engaged by cells in the tumor microenvironment to overcome active T-cell immune surveillance. Pembrolizumab (Keytruda®, MK-3475) is a potent and highly selective humanized mAb of the IgG4/kappa isotype designed to directly block the interaction between PD-1 and its ligands, PD-L1 and PD-L2. This blockade enhances the functional activity of T cells to facilitate tumor regression and ultimately immune rejection. Pembrolizumab binds to human and cynomolgus monkey PD-1 with picomolar affinity and blocks the binding of human and cynomolgus monkey PD-1 to PD-L1 and PD-L2 with comparable potency. Pembrolizumab binds both the C'D and FG loops of PD-1. Pembrolizumab overcomes human and cynomolgus monkey PD-L1-mediated immune suppression in T-cell cultures by enhancing IL2 production following staphylococcal enterotoxin B stimulation of healthy donor and cancer patient cells, and IFNγ production in human primary tumor histoculture. Ex vivo and in vitro studies with human and primate T cells show that pembrolizumab enhances antigen-specific T-cell IFNγ and IL2 production. Pembrolizumab does not mediate FcR or complement-driven effector function against PD-1-expressing cells. Pembrolizumab displays dose-dependent clearance and half-life in cynomolgus monkey pharmacokinetic and toxicokinetic studies typical for human IgG4 antibodies. In nonhuman primate toxicology studies, no findings of toxicologic significance were observed. The preclinical data for pembrolizumab are consistent with the clinical anticancer activity and safety that has been demonstrated in human clinical trials.

Flexibility of BIV TAR-Tat: models of peptide binding.

A new approach in determining local residue flexibility from base-amino acid contact frequencies is applied to the twelve million lattice chains modeling BIV Tat peptide binding to TAR RNA fragment. Many of the resulting key features in flexibility correspond to RMSD calculations derived from a set of five NMR derived structures (X. Ye, R. A. Kumar, and D. J. Patel, Protein Data Bank: Database of three-dimensional structures determined from NMR (1996)) and binding studies of mutants (L. Chen and A. D. Frankel, Proc. Natl. Acad. Sci. USA 92, 5077-5081 (1995)). The lattice and RMSD calculations facilitate the identification of peptide hinge regions that can best utilize the introduction of Gly or other flexible residues. This approach for identifying potential sites amenable to substitution of more flexible residues to enhance peptide binding to RNA targets could be a useful design tool.