The physiological interactome of TCR-like antibody therapeutics in human tissues.

Selective binding of TCR-like antibodies that target a single tumour-specific peptide antigen presented by human leukocyte antigens (HLA) is the absolute prerequisite for their therapeutic suitability and patient safety. To date, selectivity assessment has been limited to peptide library screening and predictive modeling. We developed an experimental platform to de novo identify interactomes of TCR-like antibodies directly in human tissues using mass spectrometry. As proof of concept, we confirm the target epitope of a MAGE-A4-specific TCR-like antibody. We further determine cross-reactive peptide sequences for ESK1, a TCR-like antibody with known off-target activity, in human liver tissue. We confirm off-target-induced T cell activation and ESK1-mediated liver spheroid killing. Off-target sequences feature an amino acid motif that allows a structural groove-coordination mimicking that of the target peptide, therefore allowing the interaction with the engager molecule. We conclude that our strategy offers an accurate, scalable route for evaluating the non-clinical safety profile of TCR-like antibody therapeutics prior to first-in-human clinical application.

Application of a gut-liver-on-a-chip device and mechanistic modelling to the quantitative in vitro pharmacokinetic study of mycophenolate mofetil.

Microphysiological systems (MPS) consisting of multiple linked organ-on-a-chip (OoC) components are highly promising tools with potential to provide more relevant in vitro to in vivo translation of drug disposition, efficacy and toxicity. A gut-liver OoC system was employed with Caco2 cells in co-culture with HT29 cells in the intestinal compartment and single donor primary hepatocytes in the hepatic compartment for the investigation of intestinal permeability, metabolism (intestinal and hepatic) and potential interplay of those processes. The prodrug mycophenolate mofetil was tested for quantitative evaluation of the gut-liver OoC due to the contribution of both gut and liver in its metabolism. Conversion of mycophenolate mofetil to active drug mycophenolic acid and further metabolism to a glucuronide metabolite was assessed over time in the gut apical, gut basolateral and liver compartments. Mechanistic modelling of experimental data was performed to estimate clearance and permeability parameters for the prodrug, active drug and glucuronide metabolite. Integration of gut-liver OoC data with in silico modelling allowed investigation of the complex combination of intestinal and hepatic processes, which is not possible with standard single tissue in vitro systems. A comprehensive evaluation of the mechanistic model, including structural model and parameter identifiability and global sensitivity analysis, enabled a robust experimental design and estimation of in vitro pharmacokinetic parameters. We propose that similar methodologies may be applied to other multi-organ microphysiological systems used for drug metabolism studies or wherever quantitative knowledge of changing drug concentration with time enables better understanding of biological effect.

Targeting intracellular WT1 in AML with a novel RMF-peptide-MHC-specific T-cell bispecific antibody.

Antibody-based immunotherapy is a promising strategy for targeting chemoresistant leukemic cells. However, classical antibody-based approaches are restricted to targeting lineage-specific cell surface antigens. By targeting intracellular antigens, a large number of other leukemia-associated targets would become accessible. In this study, we evaluated a novel T-cell bispecific (TCB) antibody, generated by using CrossMAb and knob-into-holes technology, containing a bivalent T-cell receptor-like binding domain that recognizes the RMFPNAPYL peptide derived from the intracellular tumor antigen Wilms tumor protein (WT1) in the context of HLA-A*02. Binding to CD3ε recruits T cells irrespective of their T-cell receptor specificity. WT1-TCB elicited antibody-mediated T-cell cytotoxicity against AML cell lines in a WT1- and HLA-restricted manner. Specific lysis of primary acute myeloid leukemia (AML) cells was mediated in ex vivo long-term cocultures by using allogeneic (mean ± standard error of the mean [SEM] specific lysis, 67 ± 6% after 13-14 days; n = 18) or autologous, patient-derived T cells (mean ± SEM specific lysis, 54 ± 12% after 11-14 days; n = 8). WT1-TCB-treated T cells exhibited higher cytotoxicity against primary AML cells than an HLA-A*02 RMF-specific T-cell clone. Combining WT1-TCB with the immunomodulatory drug lenalidomide further enhanced antibody-mediated T-cell cytotoxicity against primary AML cells (mean ± SEM specific lysis on days 3-4, 45.4 ± 9.0% vs 70.8 ± 8.3%; P = .015; n = 9-10). In vivo, WT1-TCB-treated humanized mice bearing SKM-1 tumors exhibited a significant and dose-dependent reduction in tumor growth. In summary, we show that WT1-TCB facilitates potent in vitro, ex vivo, and in vivo killing of AML cell lines and primary AML cells; these results led to the initiation of a phase 1 trial in patients with relapsed/refractory AML (#NCT04580121).

Mechanical Stretching of Fibronectin Fibers Upregulates Binding of Interleukin-7.

Since evidence is rising that extracellular matrix (ECM) fibers might serve as reservoirs for growth factors and cytokines, we investigated the interaction between fibronectin (FN) and interleukin-7 (IL-7), a cytokine of immunological significance and a target of several immunotherapies. By employing a FN fiber stretch assay and Förster resonance energy transfer (FRET) confocal microscopy, we found that stretching of FN fibers increased IL-7 binding. We localized the FN binding site on the CD loop of IL-7, since a synthetic CD loop peptide also bound stronger to stretched than to relaxed FN fibers. On the basis of a structural model, we propose that the CD loop can bind to FN, while IL-7 is bound to its cognate cell surface receptors. Sequence alignment with bacterial adhesins, which also bind the FN N-terminus, suggests that a conserved motif on the CD loop (110TKSLEEN116 and the truncated 112SLEE115 in human and mouse IL-7, respectively) might bind to the second FN type I module (FnI2) and that additional epitopes enhance the stretch-upregulated binding. FN fiber stretching might thus serve as a mechano-regulated mechanism to locally concentrate IL-7 in an ECM-bound state, thereby upregulating the potency of IL-7 signaling. A feedback model mechanism is proposed that could explain the well-known, but poorly understood, function of IL-7 in ECM homeostasis. Understanding how local IL-7 availability and signaling might be modulated by the tensional state of the ECM niche, which is adjusted by residing stroma cells, is highly relevant for basic science but also for advancing IL-7 based immunotherapies.