Antibody-peptide conjugates deliver covalent inhibitors blocking oncogenic cathepsins.

Cysteine cathepsins are a family of proteases that are relevant therapeutic targets for the treatment of different cancers and other diseases. However, no clinically approved drugs for these proteins exist, as their systemic inhibition can induce deleterious side effects. To address this problem, we developed a modular antibody-based platform for targeted drug delivery by conjugating non-natural peptide inhibitors (NNPIs) to antibodies. NNPIs were functionalized with reactive warheads for covalent inhibition, optimized with deep saturation mutagenesis and conjugated to antibodies to enable cell-type-specific delivery. Our antibody-peptide inhibitor conjugates specifically blocked the activity of cathepsins in different cancer cells, as well as osteoclasts, and showed therapeutic efficacy in vitro and in vivo. Overall, our approach allows for the rapid design of selective cathepsin inhibitors and can be generalized to inhibit a broad class of proteases in cancer and other diseases.

Controlling Antigen Fate in Therapeutic Cancer Vaccines by Targeting Dendritic Cell Receptors.

Antigen-presenting cells (APCs) orchestrate immune responses and are therefore of interest for the targeted delivery of therapeutic vaccines. Dendritic cells (DCs) are professional APCs that excel in presentation of exogenous antigens toward CD4+ T helper cells, as well as cytotoxic CD8+ T cells. DCs are highly heterogeneous and can be divided into subpopulations that differ in abundance, function, and phenotype, such as differential expression of endocytic receptor molecules. It is firmly established that targeting antigens to DC receptors enhances the efficacy of therapeutic vaccines. While most studies emphasize the importance of targeting a specific DC subset, we argue that the differential intracellular routing downstream of the targeted receptors within the DC subset should also be considered. Here, we review the mouse and human receptors studied as target for therapeutic vaccines, focusing on antibody and ligand conjugates and how their targeting affects antigen presentation. We aim to delineate how targeting distinct receptors affects antigen presentation and vaccine efficacy, which will guide target selection for future therapeutic vaccine development.

Inhibitory prodrug mechanism for cysteine cathepsin-targeted self-controlled drug release.

Tumour-associated macrophages (TAMs) support tumour development and have emerged as important regulators of therapeutic response to cytostatic agents. To target TAMs, we have developed a novel drug delivery approach which induces drug release as it inhibits cysteine cathepsin activity. This inhibitory prodrug (IPD) approach establishes a self-regulated system where drug release stops after all cysteine cathepsins are inhibited. This could improve the therapeutic window for drugs with severe side effects. We demonstrate and characterise this self-regulation concept with a fluorogenic IPD model. Next, we applied this IPD strategy to deliver cytotoxic drugs, as doxorubicin and monomethyl auristatin E, which are efficiently released and dose-dependently eliminate RAW264.7 macrophages. Lastly, by exploiting the increased cathepsin activity in TAM-like M2-polarised primary macrophages, we show that IPD-Dox selectively eliminates M2 over M1 macrophages. This demonstrates the potential of our IPD strategy for selective drug delivery and modulation of the tumour microenvironment.

Enzyme-Activatable Chemokine Conjugates for In Vivo Targeting of Tumor-Associated Macrophages.

Increased levels of tumor-associated macrophages (TAMs) are indicators of poor prognosis in most cancers. Although antibodies and small molecules blocking the recruitment of macrophages to tumors are under evaluation as anticancer therapies, these strategies are not specific for macrophage subpopulations. Herein we report the first enzyme-activatable chemokine conjugates for effective targeting of defined macrophage subsets in live tumors. Our constructs exploit the high expression of chemokine receptors (e.g., CCR2) and the activity of cysteine cathepsins in TAMs to target these cells selectively over other macrophages and immune cells (e.g., neutrophils, T cells, B cells). Furthermore, we demonstrate that cathepsin-activatable chemokines are compatible with both fluorescent and therapeutic cargos, opening new avenues in the design of targeted theranostic probes for immune cells in the tumor microenvironment.

Enzyme-Activatable Chemokine Conjugates for In Vivo Targeting of Tumor-Associated Macrophages.

Increased levels of tumor-associated macrophages (TAMs) are indicators of poor prognosis in most cancers. Although antibodies and small molecules blocking the recruitment of macrophages to tumors are under evaluation as anticancer therapies, these strategies are not specific for macrophage subpopulations. Herein we report the first enzyme-activatable chemokine conjugates for effective targeting of defined macrophage subsets in live tumors. Our constructs exploit the high expression of chemokine receptors (e.g., CCR2) and the activity of cysteine cathepsins in TAMs to target these cells selectively over other macrophages and immune cells (e.g., neutrophils, T cells, B cells). Furthermore, we demonstrate that cathepsin-activatable chemokines are compatible with both fluorescent and therapeutic cargos, opening new avenues in the design of targeted theranostic probes for immune cells in the tumor microenvironment.

Dual Site-Specific Chemoenzymatic Antibody Fragment Conjugation Using CRISPR-Based Hybridoma Engineering.

Functionalized antibodies and antibody fragments have found applications in the fields of biomedical imaging, theranostics, and antibody-drug conjugates (ADC). In addition, therapeutic and theranostic approaches benefit from the possibility to deliver more than one type of cargo to target cells, further challenging stochastic labeling strategies. Thus, bioconjugation methods to reproducibly obtain defined homogeneous conjugates bearing multiple different cargo molecules, without compromising target affinity, are in demand. Here, we describe a straightforward CRISPR/Cas9-based strategy to rapidly engineer hybridoma cells to secrete Fab' fragments bearing two distinct site-specific labeling motifs, which can be separately modified by two different sortase A mutants. We show that sequential genetic editing of the heavy chain (HC) and light chain (LC) loci enables the generation of a stable cell line that secretes a dual tagged Fab' molecule (DTFab'), which can be easily isolated. To demonstrate feasibility, we functionalized the DTFab' with two distinct cargos in a site-specific manner. This technology platform will be valuable in the development of multimodal imaging agents, theranostics, and next-generation ADCs.

Application of a Highly Selective Cathepsin S Two-step Activity-Based Probe in Multicolor Bio-Orthogonal Correlative Light-Electron Microscopy.

Cathepsin S is a lysosomal cysteine protease highly expressed in immune cells such as dendritic cells, B cells and macrophages. Its functions include extracellular matrix breakdown and cleavage of cell adhesion molecules to facilitate immune cell motility, as well as cleavage of the invariant chain during maturation of major histocompatibility complex II. The identification of these diverse specific functions has brought the challenge of delineating cathepsin S activity with great spatial precision, relative to related enzymes and substrates. Here, the development of a potent and highly selective two-step activity-based probe for cathepsin S and the application in multicolor bio-orthogonal correlative light-electron microscopy is presented. LHVS, which has been reported as a selective inhibitor of cathepsin S with nanomolar potency, formed the basis for our probe design. However, in competitive activity-based protein profiling experiments LHVS showed significant cross-reactivity toward Cat L. Introduction of an azide group in the P2 position expanded the selectivity window for cathepsin S, but rendered the probe undetectable, as demonstrated in bio-orthogonal competitive activity-based protein profiling. Incorporation of an additional azide handle for click chemistry on the solvent-exposed P1 position allowed for selective labeling of cathepsin S. This highlights the influence of click handle positioning on probe efficacy. This probe was utilized in multicolor bio-orthogonal confocal and correlative light-electron microscopy to investigate the localization of cathepsin S activity at an ultrastructural level in bone marrow-derived dendritic cells. The tools developed in this study will aid the characterization of the variety of functions of cathepsin S throughout biology.

Molecular Repolarisation of Tumour-Associated Macrophages.

The tumour microenvironment (TME) is composed of extracellular matrix and non-mutated cells supporting tumour growth and development. Tumour-associated macrophages (TAMs) are among the most abundant immune cells in the TME and are responsible for the onset of a smouldering inflammation. TAMs play a pivotal role in oncogenic processes as tumour proliferation, angiogenesis and metastasis, and they provide a barrier against the cytotoxic effector function of T lymphocytes and natural killer (NK) cells. However, TAMs are highly plastic cells that can adopt either pro- or anti-inflammatory roles in response to environmental cues. Consequently, TAMs represent an attractive target to recalibrate immune responses in the TME. Initial TAM-targeted strategies, such as macrophage depletion or disruption of TAM recruitment, have shown beneficial effects in preclinical models and clinical trials. Alternatively, reprogramming TAMs towards a proinflammatory and tumouricidal phenotype has become an attractive strategy in immunotherapy. This work summarises the molecular wheelwork of macrophage biology and presents an overview of molecular strategies to repolarise TAMs in immunotherapy.