Urinary detection of early responses to checkpoint blockade and of resistance to it via protease-cleaved antibody-conjugated sensors.

Immune checkpoint blockade (ICB) therapy does not benefit the majority of treated patients, and those who respond to the therapy can become resistant to it. Here we report the design and performance of systemically administered protease activity sensors conjugated to anti-programmed cell death protein 1 (αPD1) antibodies for the monitoring of antitumour responses to ICB therapy. The sensors consist of a library of mass-barcoded protease substrates that, when cleaved by tumour proteases and immune proteases, are released into urine, where they can be detected by mass spectrometry. By using syngeneic mouse models of colorectal cancer, we show that random forest classifiers trained on mass spectrometry signatures from a library of αPD1-conjugated mass-barcoded activity sensors for differentially expressed tumour proteases and immune proteases can be used to detect early antitumour responses and discriminate resistance to ICB therapy driven by loss-of-function mutations in either the B2m or Jak1 genes. Biomarkers of protease activity may facilitate the assessment of early responses to ICB therapy and the classification of refractory tumours based on resistance mechanisms.

Interfacing Biomaterials with Synthetic T Cell Immunity.

The clinical success of cancer immunotherapy is providing exciting opportunities for the development of new methods to detect and treat cancer more effectively. A new generation of biomaterials is being developed to interface with molecular and cellular features of immunity and ultimately shape or control anti-tumor responses. Recent advances that are supporting the advancement of engineered T cells are focused here. This class of cancer therapy has the potential to cure disease in subsets of patients, yet there remain challenges such as the need to improve response rates and safety while lowering costs to expand their use. To provide a focused overview, recent strategies in three areas of biomaterials research are highlighted: low-cost cell manufacturing to broaden patient access, noninvasive diagnostics for predictive monitoring of immune responses, and strategies for in vivo control that enhance anti-tumor immunity. These research efforts shed light on some of the challenges associated with T cell immunotherapy and how engineered biomaterials that interface with synthetic immunity are gaining traction to solve these challenges.

Non-invasive early detection of acute transplant rejection via nanosensors of granzyme B activity.

The early detection of the onset of transplant rejection is critical for the long-term survival of patients. The diagnostic gold standard for detecting transplant rejection involves a core biopsy, which is invasive, has limited predictive power and carries a morbidity risk. Here, we show that nanoparticles conjugated with a peptide substrate specific for the serine protease granzyme B, which is produced by recipient T cells during the onset of acute cellular rejection, can serve as a non-invasive biomarker of early rejection. When administered systemically in mouse models of skin graft rejection, these nanosensors preferentially accumulate in allograft tissue, where they are cleaved by granzyme B, releasing a fluorescent reporter that filters into the recipient's urine. Urinalysis then discriminates the onset of rejection with high sensitivity and specificity before features of rejection are apparent in grafted tissues. Moreover, in mice treated with subtherapeutic levels of immunosuppressive drugs, the reporter signals in urine can be detected before graft failure. This method may enable routine monitoring of allograft status without the need for biopsies.

Nanosensors to Detect Protease Activity In Vivo for Noninvasive Diagnostics.

Proteases are multi-functional enzymes that specialize in the hydrolysis of peptide-bonds and control broad biological processes including homeostasis and allostasis. Moreover, dysregulated protease activity drives pathogenesis and is a functional biomarker of diseases such as cancer; therefore, the ability to detect protease activity in vivo may provide clinically relevant information for biomedical diagnostics. The goal of this protocol is to create nanosensors that probe for protease activity in vivo by producing a quantifiable signal in urine. These protease nanosensors consist of two components: a nanoparticle and substrate. The nanoparticle functions to increase circulation half-life and substrate delivery to target disease sites. The substrate is a short peptide sequence (6-8 AA), which is designed to be specific to a target protease or group of proteases. The substrate is conjugated to the surface of the nanoparticle and is terminated by a reporter, such as a fluorescent marker, for detection. As dysregulated proteases cleave the peptide substrate, the reporter is filtered into urine for quantification as a biomarker of protease activity. Herein we describe construction of a nanosensor for matrix metalloproteinase 9 (MMP9), which is associated with tumor progression and metastasis, for detection of colorectal cancer in a mouse model.