Attacking Tumors From All Sides: Personalized Multiplex Vaccines to Tackle Intratumor Heterogeneity

Tumor vaccines are an important asset in the field of cancer immunotherapy. Whether prophylactic or therapeutic, these vaccines aim to enhance the T cell-mediated anti-tumor immune response that is orchestrated by dendritic cells. Although promising preclinical and early-stage clinical results have been obtained, large-scale clinical implementation of cancer vaccination is stagnating due to poor clinical response. The challenges of clinical efficacy of tumor vaccines can be mainly attributed to tumor induced immunosuppression and poor immunogenicity of the chosen tumor antigens. Recently, intratumor heterogeneity and the relation with tumor-specific neoantigen clonality were put in the equation.In this perspective we provide an overview of recent studies showing how personalized tumor vaccines containing multiple neoantigens can broaden and enhance the anti-tumor immune response. Furthermore, we summarize advances in the understanding of the intratumor mutational landscape containing different tumor cell subclones and the temporal and spatial diversity of neoantigen presentation and burden, and the relation between these factors with respect to tumor immunogenicity. Together, the presented knowledge calls for the investment in the characterization of neoantigens in the context of intratumor heterogeneity to improve clinical efficacy of personalized tumor vaccines

Biomaterial-Based Activation and Expansion of Tumor-Specific T Cells

Traditional tumor vaccination approaches mostly focus on activating dendritic cells (DCs) by providing them with a source of tumor antigens and/or adjuvants, which in turn activate tumor-reactive T cells. Novel biomaterial-based cancer immunotherapeutic strategies focus on directly activating and stimulating T cells through molecular cues presented on synthetic constructs with the aim of improving T cell survival, more precisely steer T cell activation and direct T cell differentiation. Synthetic artificial antigen presenting cells (aAPCs) decorated with T cell-activating ligands are being developed to induce robust tumor-specific T cell responses, essentially bypassing DCs. In this perspective, we approach these promising new technologies from an immunological angle, first by identifying the CD4+ and CD8+ T cell subtypes that are imperative for robust anti-cancer immunity and subsequently discussing the molecular cues needed to induce these cells types. We will elaborate on how biomaterials can be applied to stimulate T cells in vitro and in vivo to improve their survival, activation and function. Scaffold-based methods can also be used as delivery vehicles for adoptive transfer of T cells, including tumor-infiltrating lymphocytes (TILs) and chimeric antigen receptor expressing (CAR) T cells, while simultaneously stimulating these cells. Finally, we provide suggestions on how these insights could advance the field of biomaterial-based activation and expansion of tumor-specific T cells in the future

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

Cathepsin B abundance, activity and microglial localisation in Alzheimer's disease-Down syndrome and early onset Alzheimer's disease; the role of elevated cystatin B

Cathepsin B is a cysteine protease that is implicated in multiple aspects of Alzheimer's disease pathogenesis. The endogenous inhibitor of this enzyme, cystatin B (CSTB) is encoded on chromosome 21. Thus, individuals who have Down syndrome, a genetic condition caused by having an additional copy of chromosome 21, have an extra copy of an endogenous inhibitor of the enzyme. Individuals who have Down syndrome are also at significantly increased risk of developing early-onset Alzheimer's disease (EOAD). The impact of the additional copy of CSTB on Alzheimer's disease development in people who have Down syndrome is not well understood. Here we compared the biology of cathepsin B and CSTB in individuals who had Down syndrome and Alzheimer's disease, with disomic individuals who had Alzheimer's disease or were ageing healthily. We find that the activity of cathepsin B enzyme is decreased in the brain of people who had Down syndrome and Alzheimer's disease compared with disomic individuals who had Alzheimer's disease. This change occurs independently of an alteration in the abundance of the mature enzyme or the number of cathepsin B+ cells. We find that the abundance of CSTB is significantly increased in the brains of individuals who have Down syndrome and Alzheimer's disease compared to disomic individuals both with and without Alzheimer's disease. In mouse and human cellular preclinical models of Down syndrome, three-copies of CSTB increases CSTB protein abundance but this is not sufficient to modulate cathepsin B activity. EOAD and Alzheimer's disease-Down syndrome share many overlapping mechanisms but differences in disease occur in individuals who have trisomy 21. Understanding this biology will ensure that people who have Down syndrome access the most appropriate Alzheimer's disease therapeutics and moreover will provide unique insight into disease pathogenesis more broadly

A fluorogenic probe for granzyme B enables in-biopsy evaluation and screening of response to anticancer immunotherapies

Immunotherapy promotes the attack of cancer cells by the immune system; however, it is difficult to detect early responses before changes in tumor size occur. Here, we report the rational design of a fluorogenic peptide able to detect picomolar concentrations of active granzyme B as a biomarker of immune-mediated anticancer action. Through a series of chemical iterations and molecular dynamics simulations, we synthesize a library of FRET peptides and identify probe H5 with an optimal fit into granzyme B. We demonstrate that probe H5 enables the real-time detection of T cell-mediated anticancer activity in mouse tumors and in tumors from lung cancer patients. Furthermore, we show image-based phenotypic screens, which reveal that the AKT kinase inhibitor AZD5363 shows immune-mediated anticancer activity. The reactivity of probe H5 may enable the monitoring of early responses to anticancer treatments using tissue biopsies

The Phosphoinositide Kinase PIKfyve Promotes Cathepsin-S-Mediated Major Histocompatibility Complex Class II Antigen Presentation

Contains fulltext : 201259.pdf (publisher's version ) (Open Access

Non-Invasive Imaging of Cysteine Cathepsin Activity in Solid Tumors Using a 64Cu-Labeled Activity-Based Probe

The papain family of cysteine cathepsins are actively involved in multiple stages of tumorigenesis. Because elevated cathepsin activity can be found in many types of human cancers, they are promising biomarkers that can be used to target radiological contrast agents for tumor detection. However, currently there are no radiological imaging agents available for these important molecular targets. We report here the development of positron emission tomography (PET) radionuclide-labeled probes that target the cysteine cathepsins by formation of an enzyme activity-dependent bond with the active site cysteine. These probes contain an acyloxymethyl ketone (AOMK) functional group that irreversibly labels the active site cysteine of papain family proteases attached to a 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA) tag for labeling with 64Cu for PET imaging studies. We performed biodistribution and microPET imaging studies in nude mice bearing subcutaneous tumors expressing various levels of cysteine cathepsin activity and found that the extent of probe uptake by tumors correlated with overall protease activity as measured by biochemical methods. Furthermore, probe signals could be reduced by pre-treatment with a general cathepsin inhibitor. We also found that inclusion of a Cy5 tag on the probe increased tumor uptake relative to probes lacking this fluorogenic dye. Overall, these results demonstrate that small molecule activity-based probes carrying radio-tracers can be used to image protease activity in living subjects

Inhibitory Prodrug Approach for Selective Elimination of Immunosuppressive M2 Macrophages

Tumor associated macrophages (TAMs) support tumor 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 in response to inhibition of pro-tumor cysteine cathepsin activity. Such inhibitory prodrug (IPD) establishes a self-regulated delivery 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 this self-regulation concept with a fluorogenic IPD model. We have applied our IPD strategy to two cytotoxic agents, doxorubicin and monomethyl auristatin E, which could be efficiently released from the IPD scaffold to induce concentration dependent toxicity in RAW macrophages. Lastly, by taking advantage of the increased cathepsin activity in TAM-like M2 polarized bone marrow derived 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 tumor microenvironment

Autophagy regulates long‐term cross‐presentation by murine dendritic cells

Autophagy has been reported to be involved in supporting antigen cross‐presentation by dendritic cells (DCs). We have shown that DCs have the ability to store antigen for a prolonged time in endo‐lysosomal compartments and thereby sustain MHCI antigen cross‐presentation to CD8+ T cells. In the current study, we investigated the role of autophagy in long‐term antigen presentation. We show that the autophagy machinery has a negative impact on storage of antigen in DCs. Atg5–/– DCs which are deficient in autophagy or DCs treated with common autophagy inhibitors showed enhanced antigen storage and antigen cross‐presentation. This augmented antigen cross‐presentation effect is independent of altered proteasome enzyme activity or MHCI surface expression on DCs. We visualized that the storage compartments are in close proximity to LC3 positive autophagosomes. Our results indicate that autophagosomes disrupt antigen storage in DCs and thereby regulate long‐term MHCI cross‐presentation