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The antitumor immunity can be enhanced through the synchronized codelivery of antigens and immunostimulatory adjuvants to antigen-presenting cells, particularly dendritic cells (DCs), using nanovaccines (NVs). To study the influence of intracellular vaccine cargo release kinetics on the T cell activating capacities of DCs, we compared stimuli-responsive to nonresponsive polymersome NVs. To do so, we employed “AND gate” multiresponsive (MR) amphiphilic block copolymers that decompose only in response to the combination of chemical cues present in the environment of the intracellular compartments in antigen cross-presenting DCs: low pH and high reactive oxygen species (ROS) levels. After being unmasked by ROS, pH-responsive side chains are exposed and can undergo a charge shift within a relevant pH window of the intracellular compartments in antigen cross-presenting DCs. NVs containing the model antigen Ovalbumin (OVA) and the iNKT cell activating adjuvant a-Galactosylceramide (a-Galcer) were fabricated using microfluidics self-assembly. The MR NVs outperformed the nonresponsive NV in vitro, inducing enhanced classical- and cross-presentation of the OVA by DCs, effectively activating CD8+, CD4+ T cells, and iNKT cells. Interestingly, in vivo, the nonresponsive NVs outperformed the responsive vaccines. These differences in polymersome vaccine performance are likely linked to the kinetics of cargo release, highlighting the crucial chemical requirements for successful cancer nanovaccines.

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.

Adoptive T cell therapy has successfully been implemented for the treatment of cancer. Nevertheless, ex vivo expansion of T cells by artificial antigen-presenting cells (aAPCs) remains cumbersome and can compromise T cell functionality, thereby limiting their therapeutic potential. We propose a radically different approach aimed at direct expansion of T cells in vivo, thereby omitting the need for large-scale ex vivo T cell production. We engineered nanosized immunofilaments (IFs), with a soluble semiflexible polyisocyanopeptide backbone that presents peptide-loaded major histocompatibility complexes and costimulatory molecules multivalently. IFs readily activated and expanded antigen-specific T cells like natural APCs, as evidenced by transcriptomic analyses of T cells. Upon intravenous injection, IFs reach the spleen and lymph nodes and induce antigen-specific T cell responses in vivo. Moreover, IFs display strong antitumor efficacy resulting in inhibition of the formation of melanoma metastases and reduction of primary tumor growth in synergy with immune checkpoint blockade. In conclusion, nanosized IFs represent a powerful modular platform for direct activation and expansion of antigen-specific T cells in vivo, which can greatly contribute to cancer immunotherapy.

Synthetic cancer vaccines may boost anticancer immune responses by co-delivering tumor antigens and adjuvants to dendritic cells (DCs). The accessibility of cancer vaccines to DCs and thereby the delivery efficiency of antigenic material greatly depends on the vaccine platform that is used. Three-dimensional scaffolds have been developed to deliver antigens and adjuvants locally in an immunostimulatory environment to DCs to enable sustained availability. However, current systems have little control over the release profiles of the cargo that is incorporated and are often characterized by an initial high-burst release. Here, an alternative system is designed that co-delivers antigens and adjuvants to DCs through cargo-loaded nanoparticles (NPs) incorporated within biomaterial-based scaffolds. This creates a programmable system with the potential for controlled delivery of their cargo to DCs. Cargo-loaded poly(d,l-lactic-co-glycolic acid) NPs are entrapped within the polymer walls of alginate cryogels with high efficiency while retaining the favorable physical properties of cryogels, including syringe injection. DCs cultured within these NP-loaded scaffolds acquire strong antigen-specific T cell-activating capabilities. These findings demonstrate that introduction of NPs into the walls of macroporous alginate cryogels creates a fully synthetic immunostimulatory niche that stimulates DCs and evokes strong antigen-specific T cell responses.

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.

The human dendritic cell (DC) family has recently been expanded by CD1c+CD14+CD163+DCs, introduced as DC3s. DC3s are found in tumors and peripheral blood of cancer patients. Here, we report elevated frequencies of CD14+cDC2s, which restore to normal frequencies after tumor resection, in non-small cell lung cancer patients. These CD14+cDC2s phenotypically resemble DC3s and exhibit increased PD-L1, MERTK, IL-10, and IDO expression, consistent with inferior T cell activation ability compared with CD14−cDC2s. In melanoma patients undergoing CD1c+DC vaccinations, increased CD1c+CD14+DC frequencies correlate with reduced survival. We demonstrate conversion of CD5+/−CD1c+CD14−cDC2s to CD14+cDC2s by tumor-associated factors, whereas monocytes failed to express CD1c under similar conditions. Targeted proteomics identified IL-6 and M-CSF as dominant drivers, and we show that IL-6R and CSF1R inhibition prevents tumor-induced CD14+cDC2s. Together, this indicates cDC2s as direct pre-cursors of DC3-like CD1c+CD14+DCs and provides insights into the importance and modulation of CD14+DC3s in anti-tumor immune responses.

Cancer vaccines are a promising strategy to increase tumor-specific immune responses in patients who do not adequately respond to checkpoint inhibitors. Cancer vaccines that contain patient-specific tumor antigens are most effective but also necessitate the production of patient-specific vaccines. This study aims to develop a versatile cancer vaccine format in which patient-specific tumor antigens can be site-specifically conjugated by a proximity-based Sortase A (SrtA)-mediated ligation (PBSL) approach to antibodies that specifically bind to antigen-presenting cells to stimulate immune responses. DEC205 and CD169 are both receptors expressed on antigen-presenting cells that can be targeted to deliver antigens and stimulate T-cell responses. We used the CRISPR/HDR platform to produce mouse heavy chain IgG2a antibodies with DEC205 or CD169 specificity containing an SrtA recognition motif followed by a SpyTag at the C-terminus. Using a recombinant protein of SrtA linked to SpyCatcher, we applied proximity-based SrtA-mediated ligation to ligate fluorescein isothiocyanate (FITC)-labeled or antigenic peptides to the antibodies. Ligated antibodies bound to DEC205-expressing dendritic cells or CD169-expressing macrophages both in vitroand in vivo. More importantly, immunization with DEC205- or CD169-specific Abs linked to T-cell epitopes efficiently stimulated T-cell responses in vivo. To conclude, we have developed a cancer vaccine format using PBSL that enables the rapid incorporation of tumor antigens and could potentially be implemented for the synthesis of personalized cancer vaccines.

Polymer brushes are extensively used for the preparation of bioactive surfaces. They form a platform to attach functional (bio)molecules and control the physicochemical properties of the surface. These brushes are nearly exclusively prepared from flexible polymers, even though much stiffer brushes from semiflexible polymers are frequently found in nature, which exert bioactive functions that are out of reach for flexible brushes. Synthetic semiflexible polymers, however, are very rare. Here, we use polyisocyanopeptides (PICs) to prepare high-density semiflexible brushes on different substrate geometries. For bioconjugation, we developed routes with two orthogonal click reactions, based on the strain-promoted azide–alkyne cycloaddition reaction and the (photoactivated) tetrazole–ene cycloaddition reaction. We found that for high brush densities, multiple bonds between the polymer and the substrate are necessary, which was achieved in a block copolymer strategy. Whether the desired biomolecules are conjugated to the PIC polymer before or after brush formation depends on the dimensions and required densities of the biomolecules and the curvature of the substrate. In either case, we provide mild, aqueous, and highly modular reaction strategies, which make PICs a versatile addition to the toolbox for generating semiflexible bioactive polymer brush surfaces.

Cancer cells can evade immune surveillance through the expression of inhibitory ligands that bind their cognate receptors on immune effector cells. Expression of programmed death ligand 1 in tumor microenvironments is a major immune checkpoint for tumor-specific T cell responses as it binds to programmed cell death protein-1 on activated and dysfunctional T cells1. The activity of myeloid cells such as macrophages and neutrophils is likewise regulated by a balance between stimulatory and inhibitory signals. In particular, cell surface expression of the CD47 protein creates a ‘don’t eat me’ signal on tumor cells by binding to SIRPα expressed on myeloid cells2–5. Using a haploid genetic screen, we here identify glutaminyl-peptide cyclotransferase-like protein (QPCTL) as a major component of the CD47-SIRPα checkpoint. Biochemical analysis demonstrates that QPCTL is critical for pyroglutamate formation on CD47 at the SIRPα binding site shortly after biosynthesis. Genetic and pharmacological interference with QPCTL activity enhances antibody-dependent cellular phagocytosis and cellular cytotoxicity of tumor cells. Furthermore, interference with QPCTL expression leads to a major increase in neutrophil-mediated killing of tumor cells in vivo. These data identify QPCTL as a novel target to interfere with the CD47 pathway and thereby augment antibody therapy of cancer.

Irreversible covalent inhibitors can have a beneficial pharmacokinetic/pharmacodynamics profile but are still often avoided due to the risk of indiscriminate covalent reactivity and the resulting adverse effects. To overcome this potential liability, we introduced an alkyne moiety as a latent electrophile into small molecule inhibitors of cathepsin K (CatK). Alkyne-based inhibitors do not show indiscriminate thiol reactivity but potently inhibit CatK protease activity by formation of an irreversible covalent bond with the catalytic cysteine residue, confirmed by crystal structure analysis. The rate of covalent bond formation (kinact) does not correlate with electrophilicity of the alkyne moiety, indicative of a proximity-driven reactivity. Inhibition of CatK-mediated bone resorption is validated in human osteoclasts. Together, this work illustrates the potential of alkynes as latent electrophiles in small molecule inhibitors, enabling the development of irreversible covalent inhibitors with an improved safety profile.

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.

Active enzymes, such as proteases, often serve as valuable biomarkers for various disease pathologies. Therefore, methods to detect specific enzyme activities in biological samples can provide information to guide disease detection and diagnosis and to increase our understanding of the biological roles of specific enzyme targets. In this protocol, we outline methods for the topical application of fluorescently quenched activity-based probes (qABPs) to fresh-frozen tissue samples. This technique enables rapid imaging of enzyme activity at cellular resolution, and it can be combined with antibody labeling for immunodiagnosis. In this method, fresh-frozen tissue sections are fixed, incubated with the probe and imaged using fluorescence microscopy. This provides an advance over classical immunohistochemistry (IHC) in that it is rapid (4–8 h) and inexpensive, and it provides information on enzyme activity. Furthermore, it can be used with any of the growing number of fluorescent ABPs to provide data for more effective disease monitoring and diagnosis.

There is a need for new molecular-guided contrast agents to enhance surgical procedures such as tumor resection that require a high degree of precision. Cysteine cathepsins are highly up-regulated in a wide variety of cancers, both in tumor cells and in the tumor-supporting cells of the surrounding stroma. Therefore, tools that can be used to dynamically monitor their activity in vivocould be used as imaging contrast agents for intraoperative fluorescence image guided surgery (FGS). Although multiple classes of cathepsin-targeted substrate probes have been reported, most suffer from overall fast clearance from sites of protease activation, leading to reduced signal intensity and duration in vivo. Here we describe the design and synthesis of a series of near-infrared fluorogenic probes that exploit a latent cationic lysosomotropic effect (LLE) to promote cellular retention upon protease activation. These probes show tumor-specific retention, fast activation kinetics, and rapid systemic distribution. We demonstrate that they are suitable for detection of diverse cancer types including breast, colon and lung tumors. Most importantly, the agents are compatible with the existing, FDA approved, da Vinci surgical system for fluorescence guided tumor resection. Therefore, our data suggest that the probes reported here can be used with existing clinical instrumentation to detect tumors and potentially other types of inflammatory lesions to guide surgical decision making in real time.

The ubiquitin-proteasome system (UPS) is a potential pathway for therapeutic intervention for pathogens such as Plasmodium, the causative agent of malaria. However, due to the essential nature of this proteolytic pathway, proteasome inhibitors must avoid inhibition of the host enzyme complex to prevent toxic side effects. The Plasmodiumproteasome is poorly characterized, making rational design of inhibitors that induce selective parasite killing difficult. In this study, we developed a chemical probe that labels all catalytic sites of the Plasmodiumproteasome. Using this probe, we identified several subunit selective small molecule inhibitors of the parasite enzyme complex. Treatment with an inhibitor that is specific for the β5 subunit during blood stage schizogony led to a dramatic decrease in parasite replication while short-term inhibition of the β2 subunit did not affect viability. Interestingly, coinhibition of both the β2 and β5 catalytic subunits resulted in enhanced parasite killing at all stages of the blood stage life cycle and reduced parasite levels in vivoto barely detectable levels. Parasite killing was achieved with overall low host toxicity, something that has not been possible with existing proteasome inhibitors. Our results highlight differences in the subunit dependency of the parasite and human proteasome, thus providing a strategy for development of potent antimalarial drugs with overall low host toxicity.

Activity-based protein profiling (ABPP) is a functional proteomics technique for directly monitoring the expression of active enzymes in cell extracts and living cells. The technique relies on irreversible inhibitors equipped with reactive groups (warheads) that covalently attach to the active site of enzymes and fluorescent or affinity tags for imaging and purification purposes, respectively. Here, a high-throughput and robust protocol for high-resolution quantitative activity-based proteasome profiling is described. We use both panreactive and subunit-specific fluorescent activity-based probes (ABPs) to quantify the proteasome activity in living cells, in the presence or absence of the potent proteasome inhibitor bortezomib. Active proteasome subunits from cell lysates are affinity-purified via a biotinylated ABP. Purification from live cells involves a two-step ABP approach using a reagent with a cell-permeable azide-warhead and postlysis installation of biotin. By means of liquid chromatography–mass spectrometry (LC-MS)-based proteomics, we can accurately identify the enriched proteins and the active site peptides of the enzymes, and relatively quantify all the proteasome activities in one experiment. The fluorescence ABPP protocols takes 2–3 d, and approximately 8–10 d are needed to complete the entire protocol.

Proteolysis, or the processing and degradation of proteins, has emerged as one of the most widely studied processes in biology today. Long viewed as a dead end process, of importance only for the removal of obsolete peptides and proteins, proteolytic events are now associated with numerous biological events. The main proteolytic pathway in the eukaryotic cytoplasm and nucleus, responsible for the degradation of 80–90  of all cellular proteins is known as the Ubiquitin Proteasome System UPS. Proteasomes are the central proteases in this tightly controlled ATP and ubiquitindependent proteolytic pathway. Proteasomes are multicatalytic, compartmentalized proteinase complexes. Their substrates include abnormal and damaged proteins, cellcycle regulators, oncogens and tumor suppressors. Furthermore, proteasomal degradation is imperative for the generation of MHC class I antigenic peptides. The recent approval of a proteasome inhibitor as a cancer drug has boosted proteasome research. This microreview highlights the recent advances in the development of chemical tools to study proteasome activity.© WileyVCH Verlag GmbH & Co. KGaA, 69451 Weinheim, Germany, 2009

Tag for professionals: A fluorescently tagged clustered mannoside DCG04 analogue see structure is designed and synthesized using a modular approach. Uptake of the probe in professional antigen presenting cells and subsequent labeling of cathepsins proceeded in a mannosereceptor dependent manner.

Speziell für professionelle Zellen: Ein MannosidDCG04Analog mit Fluoreszenzmarkierung siehe Struktur wurde nach einem modularen Verfahren entworfen und synthetisiert. Die Aufnahme der Sonde in professionelle antigenpräsentierende Zellen und die anschließende Markierung von Cathepsinen waren vom Mannoserezeptor abhängig.

Anticancer drug delivery strategies are designed to take advantage of the differential chemical environment in solid tumors independently, or to high levels of reactive oxygen species (ROS) or to low pH, compared to healthy tissue. Here, the design and thorough characterization of two functionalizable “AND gate” multiresponsive (MR) block amphiphilic copolymers are reported, aimed to take full advantage of the coexistence of two chemical cues—ROS and low pH—present in the tumor microenvironment. The hydrophobic blocks contain masked pH‐responsive side chains, which are exposed exclusively in response to ROS. Hence, the hydrophobic polymer side chains will undergo a charge shift in a very relevant pH window present in the extracellular milieu in most solid tumors (pH 5.6–7.2) after demasking by ROS. Doxorubicin (DOX)‐loaded nanosized “AND gate” MR polymersomes (MRPs) are fabricated via microfluidic self‐assembly. Chemical characterization reveals ROS‐dependent pH sensitivity and accelerated DOX release under influence of both ROS and low pH. Treatment of tumor‐bearing mice with DOX‐loaded nonresponsive and “AND gate” MRPs dramatically decreases cardiac toxicity. The most optimal “AND gate” MRPs outperform free DOX in terms of tumor growth inhibition and survival, shedding light on chemical requirements for successful cancer nanomedicine. To take full advantage of the coexistence of two chemical cues present in tumors, functionalizable, reactive oxygen species (ROS) and pH “AND gate” multiresponsive amphiphilic block copolymers are developed for cancer drug delivery applications. With these, nanosized “AND gate” multiresponsive polymersomes loaded with doxorubicin are fabricated which outperformed free doxorubicin in terms of tumor growth inhibition and survival with decreased cardiac toxicity. ​

A variety of bioactive materials developed to expand T cells for adoptive transfer into cancer patients are currently evaluated in the clinic. In most cases, T cell activating biomolecules are attached to rigid surfaces or matrices and form a static interface between materials and the signaling receptors on the T cells. We hypothesized that a T cell activating polymer brush interface might better mimic the cell surface of a natural antigen-presenting cell, facilitating receptor movement and concomitant advantageous mechanical forces to provide enhanced T cell activating capacities. Here, as a proof of concept, we synthesized semiflexible polyisocyanopeptide (PIC) polymer-based immunobrushes equipped with T cell activating agonistic anti-CD3 (αCD3) and αCD28 antibodies placed on magnetic microbeads. We demonstrated enhanced efficiency of ex vivoexpansion of activated primary human T cells even at very low numbers of stimulating antibodies compared to rigid beads. Importantly, the immunobrush architecture appeared crucial for this improved T cell activating capacity. Immunobrushes outperform current benchmarks by producing higher numbers of T cells exhibiting a combination of beneficial phenotypic characteristics, such as reduced exhaustion marker expression, high cytokine production, and robust expression of cytotoxic hallmarks. This study indicates that semiflexible immunobrushes have great potential in making T cell-based immunotherapies more effective.

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