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Innovative cell-based therapies are important new weapons in the fight against difficult-to-treat cancers. One promising strategy involves cell therapies equipped with multiple receptors to integrate signals from more than one antigen. We developed a specific embodiment of this approach called Tmod, a two-receptor system that combines activating and inhibitory inputs to distinguish between tumor and normal cells. The selectivity of Tmod is enforced by the inhibitory receptor (blocker) that recognizes an antigen, such as an HLA allele, whose expression is absent from tumors because of loss of heterozygosity. Although unwanted cross-reactivity of the blocker likely reduces efficacy rather than safety, it is important to verify the blocker's specificity. We have tested an A∗02-directed blocker derived from the PA2.1 mouse antibody as a safety mechanism paired with a mesothelin-specific activating CAR in our Tmod construct. We solved the crystal structure of humanized PA2.1 Fab in complex with HLA-A∗02 to determine its binding epitope, which was used to bioinformatically select specific class I HLA alleles to test the blocker's functional specificity in vitro . We found that this A∗02-directed blocker is highly specific for its cognate antigen, with only one cross-reactive allele (A∗69) capable of triggering comparable function., Competing Interests: A.E.H., A.K., J.-Y.M., A.W., T.P.R., R.B., M.S.N., S.S., C.J., R.E., C.G., and D.T.-W. are current or former employees and shareholders of A2 Biotherapeutics, Inc., (© 2022 A2 Biotherapeutics, Inc.)

Neoantigens are among the most intriguing potential immuno-oncology targets because, unlike many cancer targets that are expressed on normal tissues, they are by definition restricted to cancer cells. Medicines directed at common neoantigens such as mutant KRAS are especially interesting because they may offer the convenience and cost of an off-the-shelf therapy. However, all common KRAS mutations produce proteins that differ from the wild type at a single amino acid, creating challenges for molecular discrimination. We have undertaken an effort to optimize single-chain variable fragments (scFv) against peptide/major histocompatibility antigen complexes composed of HLA-A*11 and either G12V- or G12D-mutant KRAS peptides. These scFvs could in principle be used in chimeric antigen receptor (CAR) T-cell therapies for selected patients whose tumors bear either of these mutations. Here we show that optimization of such CARs involves a trade-off between potency and selectivity. We further show that targeting this family without high selectivity engenders risks of cross-reactivity against other members of the G-protein family to which KRAS belongs., Significance: We report an effort to generate high potency, selective CARs directed at mutant KRAS peptides. Although the heavily optimized CARs maintain high selectivity against wild-type KRAS, they lose selectivity against other KRAS-related peptides derived from human proteins. To our knowledge, this work is the first to examine the trade-off between potency and selectivity with regard to KRAS pMHC-directed CARs, illustrating the challenge to achieve both sufficient potency and high selectivity., Competing Interests: T. Tokatlian reports personal fees from A2 Biotherapeutics during the conduct of the study; personal fees from A2 Biotherapeutics outside the submitted work; in addition, T. Tokatlian has a patent to Polypeptides targeting KRAS and methods of use thereof pending. G.E. Asuelime reports personal fees from A2 Biotherapeutics during the conduct of the study; other from A2 Biotherapeutics outside the submitted work; in addition, G.E. Asuelime has a patent to Polypeptides targeting KRAS and methods of use thereof pending. M.S. Naradikian reports personal fees from A2 Biotherapeutics during the conduct of the study; personal fees from A2 Biotherapeutics outside the submitted work; in addition, M.S. Naradikian has a patent to Polypeptides targeting KRAS and methods of use thereof pending. J.-Y. Mock reports personal fees from A2 Biotherapeutics during the conduct of the study; personal fees from A2 Biotherapeutics outside the submitted work; in addition, J. Mock has a patent to Polypeptides targeting KRAS and methods of use thereof pending. M.E. Daris reports personal fees from A2 Biotherapeutics during the conduct of the study; personal fees from A2 Biotherapeutics outside the submitted work; in addition, M.E. Daris has a patent to Polypeptides targeting KRAS and methods of use thereof pending. A.D. Martin reports personal fees from A2 Biotherapeutics during the conduct of the study; personal fees from A2 Biotherapeutics outside the submitted work; in addition, A.D. Martin has a patent to Polypeptides targeting KRAS and methods of use thereof pending. D. Toledo Warshaviak reports personal fees from a. A2 Biotherapeutics during the conduct of the study; personal fees from A2 Biotherapeutics outside the submitted work; in addition, D. Toledo Warshaviak has a patent to Polypeptides targeting KRAS and methods of use thereof pending. A. Kamb reports personal fees from A2 Biotherapeutics during the conduct of the study; personal fees from A2 Biotherapeutics outside the submitted work; in addition, A. Kamb has a patent to Polypeptides targeting KRAS and methods of use thereof pending. A.E. Hamburger reports personal fees from A2 Biotherapeutics during the conduct of the study; personal fees from A2 Biotherapeutics outside the submitted work; in addition, A.E. Hamburger has a patent to Polypeptides targeting KRAS and methods of use thereof pending., (© 2022 The Authors; Published by the American Association for Cancer Research.)

Background: Mesothelin (MSLN) is a classic tumor-associated antigen that is expressed in lung cancer and many other solid tumors. However, MSLN is also expressed in normal mesothelium which creates a significant risk of serious inflammation for MSLN-directed therapeutics. We have developed a dual-receptor (Tmod™) system that exploits the difference between tumor and normal tissue in a subset of patients with defined heterozygous gene loss (LOH) in their tumors., Methods: T cells engineered with the MSLN CAR Tmod construct described here contain (1) a novel MSLN-activated CAR and (2) an HLA-A*02-gated inhibitory receptor (blocker). A*02 binding is intended to override T-cell cytotoxicity, even in the presence of MSLN. The Tmod system is designed to treat heterozygous HLA class I patients, selected for HLA LOH. When A*02 is absent from tumors selected for LOH, the MSLN Tmod cells are predicted to mediate potent killing of the MSLN(+)A*02(-) malignant cells., Results: The sensitivity of the MSLN Tmod cells is comparable with a benchmark MSLN CAR-T that was active but toxic in the clinic. Unlike MSLN CAR-T cells, the Tmod system robustly protects surrogate "normal" cells even in mixed-cell populations in vitro and in a xenograft model. The MSLN CAR can also be paired with other HLA class I blockers, supporting extension of the approach to patients beyond A*02 heterozygotes., Conclusions: The Tmod mechanism exemplified by the MSLN CAR Tmod construct provides an alternative route to leverage solid-tumor antigens such as MSLN in safer, more effective ways than previously possible., Competing Interests: Competing interests: All authors are current or former employees and shareholders of A2 Biotherapeutics, Inc., (© Author(s) (or their employer(s)) 2021. Re-use permitted under CC BY-NC. No commercial re-use. See rights and permissions. Published by BMJ.)

Safety pharmacology screening against a wide range of unintended vital targets using in vitro assays is crucial to understand off-target interactions with drug candidates. With the increasing demand for in vitro assays, ligand- and structure-based virtual screening approaches have been evaluated for potential utilization in safety profiling. Although ligand based approaches have been actively applied in retrospective analysis or prospectively within well-defined chemical space during the early discovery stage (i.e., HTS screening and lead optimization), virtual screening is rarely implemented in later stage of drug discovery (i.e., safety). Here we present a case study to evaluate ligand-based 3D QSAR models built based on in vitro antagonistic activity data against adenosine receptor 2A (A2A). The resulting models, obtained from 268 chemically diverse compounds, were used to test a set of 1,897 chemically distinct drugs, simulating the real-world challenge of safety screening when presented with novel chemistry and a limited training set. Due to the unique requirements of safety screening versus discovery screening, the limitations of 3D QSAR methods (i.e., chemotypes, dependence on large training set, and prone to false positives) are less critical than early discovery screen. We demonstrated that 3D QSAR modeling can be effectively applied in safety assessment prior to in vitro assays, even with chemotypes that are drastically different from training compounds. It is also worth noting that our model is able to adequately make the mechanistic distinction between agonists and antagonists, which is important to inform subsequent in vivo studies. Overall, we present an in-depth analysis of the appropriate utilization and interpretation of pharmacophore-based 3D QSAR models for safety screening., Competing Interests: The authors declare the following competing interests: Amgen Inc. provided support in the form of salaries for authors FF, DTW, HDH, RTD; and at the initial planning stage of the presented study, DTW was an employee for Schrodinger Inc. This does not alter our adherence to PLOS ONE policies on sharing data and materials. There are no patents, products in development or marketed products associated with this research to declare.

We present the Consensus Induced Fit Docking (cIFD) approach for adapting a protein binding site to accommodate multiple diverse ligands for virtual screening. This novel approach results in a single binding site structure that can bind diverse chemotypes and is thus highly useful for efficient structure-based virtual screening. We first describe the cIFD method and its validation on three targets that were previously shown to be challenging for docking programs (COX-2, estrogen receptor, and HIV reverse transcriptase). We then demonstrate the application of cIFD to the challenging discovery of irreversible Crm1 inhibitors. We report the identification of 33 novel Crm1 inhibitors, which resulted from the testing of 402 purchased compounds selected from a screening set containing 261,680 compounds. This corresponds to a hit rate of 8.2 %. The novel Crm1 inhibitors reveal diverse chemical structures, validating the utility of the cIFD method in a real-world drug discovery project. This approach offers a pragmatic way to implicitly account for protein flexibility without the additional computational costs of ensemble docking or including full protein flexibility during virtual screening.