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Tumor-associated peptide-human leukocyte antigen complexes (pHLAs) represent the largest pool of cell surface-expressed cancer-specific epitopes, making them attractive targets for cancer therapies. Soluble bispecific molecules that incorporate an anti-CD3 effector function are being developed to redirect T cells against these targets using 2 different approaches. The first achieves pHLA recognition via affinity-enhanced versions of natural TCRs (e.g., immune-mobilizing monoclonal T cell receptors against cancer [ImmTAC] molecules), whereas the second harnesses an antibody-based format (TCR-mimic antibodies). For both classes of reagent, target specificity is vital, considering the vast universe of potential pHLA molecules that can be presented on healthy cells. Here, we made use of structural, biochemical, and computational approaches to investigate the molecular rules underpinning the reactivity patterns of pHLA-targeting bispecifics. We demonstrate that affinity-enhanced TCRs engage pHLA using a comparatively broad and balanced energetic footprint, with interactions distributed over several HLA and peptide side chains. As ImmTAC molecules, these TCRs also retained a greater degree of pHLA selectivity, with less off-target activity in cellular assays. Conversely, TCR-mimic antibodies tended to exhibit binding modes focused more toward hot spots on the HLA surface and exhibited a greater degree of crossreactivity. Our findings extend our understanding of the basic principles that underpin pHLA selectivity and exemplify a number of molecular approaches that can be used to probe the specificity of pHLA-targeting molecules, aiding the development of future reagents., Introduction The ability to selectively target tumor-specific antigens holds great promise for the development of specific cancer treatments, but their identification remains a key challenge. Peptide fragments presented on the [...]

T-cell-engaging bispecifics have great clinical potential for the treatment of cancer and infectious diseases. The binding affinity and kinetics of a bispecific molecule for both target and T-cell CD3 have substantial effects on potency and specificity, but the rules governing these relationships are not fully understood. Using immune mobilizing monoclonal TCRs against cancer (ImmTAC) molecules as a model, we explored the impact of altering affinity for target and CD3 on the potency and specificity of the redirected T-cell response. This class of bispecifics binds specific target peptides presented by human leukocyte antigen on the cell surface via an affinity-enhanced T-cell receptor and can redirect T-cell activation with an anti-CD3 effector moiety. The data reveal that combining a strong affinity TCR with an intermediate affinity anti-CD3 results in optimal T-cell activation, while strong affinity of both targeting and effector domains significantly reduces maximum cytokine release. Moreover, by optimizing the affinity of both parts of the molecule, it is possible to improve the selectivity. These results could be effectively modelled based on kinetic proofreading with limited signalling. This model explained the experimental observation that strong binding at both ends of the molecules leads to reduced activity, through very stable target-bispecific-effector complexes leading to CD3 entering a non-signalling dark state. These findings have important implications for the design of anti-CD3-based bispecifics with optimal biophysical parameters for both activity and specificity. These studies demonstrate how to optimize the fundamental process of T-cell activation, both in terms of affinity and binding kinetics to target cell and T-cell epitopes. Current theories of T-cell biology are used to build a mathematical model that explains most features of the data. How the kinetics of target and cluster of differentiation 3 binding regulate T-cell activation in the context of the clinically validated immune mobilizing monoclonal TCRs against cancer platform, which directly targets peptide-human leukocyte antigen complexes. [ABSTRACT FROM AUTHOR]

Shallot (Allium cepa L. Aggregatum group) is an important Allium plant commonly grown in tropical countries, including Indonesia, and it is used as spice for daily cooking, medicine, cosmetics and dietary supplements. Since shallot is predominantly propagated vegetatively by bulbs, it augments pathogen accumulation which influences low seed bulb management as well as productivity. Hence, utilisation of seeds/True Seed Shallot (TSS) emerges an alternative propagation material. However, no information is available on the growth stages of shallot plants from TSS. The phenological growth stages of tropical shallot planted in lowland areas using seeds in accordance with the Biologische Bundesanstalt, Bundessortenamt nd CHemische Industrie (BBCH) scale are first described here. Five introduced TSS cultivars, that is, Tuk Tuk, Lokananta, Sanren, Maserati, Tropix and four landraces TSS cultivars, that is, Bima, Biru Lancor, Pancasona and Trisula, were used as planting materials to develop an extended numerical BBCH scale for the identification of different phenological growth stages of shallot from seed. The study was conducted in lowland areas under tropical conditions of Indonesia. Based on BBCH scale, phenology of tropical shallot from seed in lowland area exhibited five of the 10 principal stages (0–9): germination (stage 0), leaf development (stage 1), formation of lateral shoot/tiller of pseudostem (stage 2), development of bulb (harvestable vegetative part) (stage 4), and senescence (stage 9). The codification of the different growth stages is essential in determination of the correct timing for better crop management, particularly for transplanting, fertilisation, control of weeds, disease and pest, plant growth regulators application and harvest time. As an implication, these BBCH scales could be used in plant breeding activities to shorten the TSS life cycle. [ABSTRACT FROM AUTHOR]

Neoantigens derived from somatic mutations are specific to cancer cells and are ideal targets for cancer immunotherapy. KRAS is the most frequently mutated oncogene and drives the pathogenesis of several cancers. Here we show the identification and development of an affinity-enhanced T cell receptor (TCR) that recognizes a peptide derived from the most common KRAS mutant, KRASG12D, presented in the context of HLA-A*11:01. The affinity of the engineered TCR is increased by over one million-fold yet fully able to distinguish KRASG12D over KRASWT. While crystal structures reveal few discernible differences in TCR interactions with KRASWT versus KRASG12D, thermodynamic analysis and molecular dynamics simulations reveal that TCR specificity is driven by differences in indirect electrostatic interactions. The affinity enhanced TCR, fused to a humanized anti-CD3 scFv, enables selective killing of cancer cells expressing KRASG12D. Our work thus reveals a molecular mechanism that drives TCR selectivity and describes a soluble bispecific molecule with therapeutic potential against cancers harboring a common shared neoantigen. Cancers often harbor mutations in genes encoding important regulatory proteins, but therapeutic targeting of these molecules proves difficult due to their high structural similarity to their non-mutated counterpart. Here authors show the engineering of T cell engaging bispecific protein able to selectively target cancer cells with a high-frequency mutation in the KRAS oncogene. [ABSTRACT FROM AUTHOR]