Hallmarks of CD8+ T cell dysfunction are established within hours of tumor antigen encounter before cell division.

Tumor-specific CD8+ T cells (TST) in patients with cancer are dysfunctional and unable to halt cancer progression. TST dysfunction, also known as exhaustion, is thought to be driven by chronic T cell antigen receptor (TCR) stimulation over days to weeks. However, we know little about the interplay between CD8+ T cell function, cell division and epigenetic remodeling within hours of activation. Here, we assessed early CD8+ T cell differentiation, cell division, chromatin accessibility and transcription in tumor-bearing mice and acutely infected mice. Surprisingly, despite robust activation and proliferation, TST had near complete effector function impairment even before undergoing cell division and had acquired hallmark chromatin accessibility features previously associated with later dysfunction/exhaustion. Moreover, continued tumor/antigen exposure drove progressive epigenetic remodeling, 'imprinting' the dysfunctional state. Our study reveals the rapid divergence of T cell fate choice before cell division in the context of tumors versus infection.

Pre-Clinical Investigation of Keratose as an Excipient of Drug Coated Balloons.

BACKGROUND: Drug-coated balloons (DCBs), which deliver anti-proliferative drugs with the aid of excipients, have emerged as a new endovascular therapy for the treatment of peripheral arterial disease. In this study, we evaluated the use of keratose (KOS) as a novel DCB-coating excipient to deliver and retain paclitaxel. METHODS: A custom coating method was developed to deposit KOS and paclitaxel on uncoated angioplasty balloons. The retention of the KOS-paclitaxel coating, in comparison to a commercially available DCB, was evaluated using a novel vascular-motion simulating ex vivo flow model at 1 h and 3 days. Additionally, the locoregional biological response of the KOS-paclitaxel coating was evaluated in a rabbit ilio-femoral injury model at 14 days. RESULTS: The KOS coating exhibited greater retention of the paclitaxel at 3 days under pulsatile conditions with vascular motion as compared to the commercially available DCB (14.89 ± 4.12 ng/mg vs. 0.60 ± 0.26 ng/mg, p = 0.018). Histological analysis of the KOS-paclitaxel-treated arteries demonstrated a significant reduction in neointimal thickness as compared to the uncoated balloons, KOS-only balloon and paclitaxel-only balloon. CONCLUSIONS: The ability to enhance drug delivery and retention in targeted arterial segments can ultimately improve clinical peripheral endovascular outcomes.

Vaccination generates functional progenitor tumor-specific CD8 T cells and long-term tumor control.

Background: Immune checkpoint blockade (ICB) therapies are an important treatment for patients with advanced cancers; however only a subset of patients with certain types of cancer achieves durable remissions. Cancer vaccines are an attractive strategy to boost patient immune responses, but less is known about whether and how immunization can induce long-term tumor immune reprogramming and arrest cancer progression. We developed a clinically-relevant genetic cancer mouse model in which hepatocytes sporadically undergo oncogenic transformation. We compared how tumor-specific CD8 T cells (TST) differentiate in mice with early sporadic lesions as compared to late lesions and tested how immunotherapeutic strategies, including vaccination and ICB, reprogram TST and impact liver cancer progression. Methods: Mice with a germline floxed allele of the SV40 large T antigen (TAG) undergo spontaneous recombination and activation of the TAG oncogene, leading to rare early pre-cancerous lesions that inevitably progress to established liver cancer. We assessed the immunophenotype and function of TAG-specific CD8 T cells in mice with early and late liver lesions. We vaccinated mice, either alone or in combination with ICB, to test whether these immunotherapeutic interventions could stop liver cancer progression. Results: In mice with early lesions, a subset of TST were PD1 + TCF1 + TOX - and could produce IFNγ, while TST present in mice with late liver cancers were PD1 + TCF1 lo/- TOX + and unable to make effector cytokines. Strikingly, vaccination with attenuated TAG epitope-expressing Listeria monocytogenes (LM TAG ) blocked liver cancer development and led to a population of TST that were TCF1 + TOX - TST and polyfunctional cytokine producers. In contrast, ICB administration did not slow cancer progression or improve LM TAG vaccine efficacy. Conclusion: Vaccination, but not ICB, generated a population of progenitor TST and halted cancer progression in a clinically relevant model of sporadic liver cancer. In patients with early cancers or at high-risk of cancer recurrence, immunization may be the most effective strategy. What is already known on this topic: Immunotherapy, including immune checkpoint blockade and cancer vaccines, fails to induce long-term remissions in most patients with cancer. What this study adds: Hosts with early lesions but not hosts with advanced cancer retain a progenitor TCF1+ TST population. This population can be reprogrammed and therapeutically exploited by vaccination, but not ICB, to block tumor progression. How this study might affect research practice or policy: For people at high-risk of cancer progression, vaccination administered when a responsive progenitor TST population is present may be the optimal immunotherapy to induce long-lasting progression-free survival.

Tumor-Reactive CD8+ T Cells Enter a TCF1+PD-1- Dysfunctional State.

T cells recognize several types of antigens in tumors, including aberrantly expressed, nonmutated proteins, which are therefore shared with normal tissue and referred to as self/shared-antigens (SSA), and mutated proteins or oncogenic viral proteins, which are referred to as tumor-specific antigens (TSA). Immunotherapies such as immune checkpoint blockade (ICB) can activate T-cell responses against TSA, leading to tumor control, and also against SSA, causing immune-related adverse events (irAE). To improve anti-TSA immunity while limiting anti-SSA autoreactivity, we need to understand how tumor-specific CD8+ T cells (TST) and SSA-specific CD8+ T (SST) cells differentiate in response to cognate antigens during tumorigenesis. Therefore, we developed a genetic cancer mouse model in which we can track TST and SST differentiation longitudinally as liver cancers develop. We found that both TST and SST lost effector function over time, but while TST persisted long term and had a dysfunctional/exhausted phenotype (including expression of PD1, CD39, and TOX), SST exited cell cycle prematurely and disappeared from liver lesions. However, SST persisted in spleens in a dysfunctional TCF1+PD-1- state: unable to produce effector cytokines or proliferate in response to ICB targeting PD-1 or PD-L1. Thus, our studies identify a dysfunctional T-cell state occupied by T cells reactive to SSA: a TCF1+PD-1- state lacking in effector function, demonstrating that the type/specificity of tumor antigen may determine tumor-reactive T-cell differentiation.

Precision delivery of liquid therapy into the arterial wall for the treatment of peripheral arterial disease.

Perfusion catheters have recently emerged as a novel approach to deliver liquid anti-proliferative agents into flow obstructed arterial segments. The purpose of this study was to determine the impact of luminal delivery pressure on liquid drug penetration into the vessel wall. An ex vivo model using harvested porcine carotid arteries and a two-dimensional computational model were utilized to determine the impact of delivery pressure of liquid therapy into the arterial wall. A pig peripheral injury model determined the impact of intra-luminal delivery pressure on drug retention. Ex vivo results demonstrated that depth of fluid penetration varies from 6.93 ± 1.90% at 0 atm to 27.75 ± 6.61% penetration of the medial layer at 0.4 atm. Computational results had similar outcomes, as penetration varied between 4.4% and 22.84%. The in vivo results demonstrated significant increase in drug delivery to the arterial tissue at 0.4 atm versus 0.1 atm at 1 h (23.43 ± 13.59 ng/mg vs. 2.49 ± 1.81 ng/mg, p = 0.026) and 7 days (0.50 ± 0.39 ng/mg vs. 0.018 ± 0.023 ng/mg, p = 0.0496). The result of this study provides an innovative strategic and technical approach to enable targeted liquid therapy.

Delivery of Cell-Specific Aptamers to the Arterial Wall with an Occlusion Perfusion Catheter.

Current strategies to prevent restenosis following endovascular treatment include the local delivery of anti-proliferative agents to inhibit vascular smooth muscle cell (VSMC) proliferation and migration. These agents, not specific to VSMCs, are deposited on the luminal surface and therefore target endothelial cells and delay vascular healing. Cell-targeted therapies, (e.g., RNA aptamers), can potentially overcome these safety concerns by specifically binding to VSMC and inhibiting proliferation and migration. The purpose of this study was to therefore demonstrate the ability of a perfusion catheter to deliver cell-specific RNA aptamer inhibitors directly to the vessel wall. RNA aptamers specific to VSMCs were developed using an in vitro cell-based systematic evolution of ligand by exponential enrichment selection process. Two aptamers (Apt01 and Apt14) were evaluated ex vivo using harvested pig arteries in a pulsatile flow bioreactor. Local drug delivery of the aptamers into the medial wall was accomplished using a novel perfusion catheter. We demonstrated the feasibility to deliver aptamer-based drugs directly to the medial layer of an artery using a perfusion catheter. Such cell-specific targeted therapeutic drugs provide a potentially safer and more effective treatment option for patients with vascular disease.

In vitro and in vivo Assessment of Keratose as a Novel Excipient of Paclitaxel Coated Balloons.

Purpose: Drug coated balloons (DCB) are continually improving due to advances in coating techniques and more effective excipients. Paclitaxel, the current drug choice of DCB, is a microtubule-stabilizing chemotherapeutic agent that inhibits smooth muscle cell proliferation. Excipients work to promote coating stability and facilitate paclitaxel transfer and retention at the target lesion, although current excipients lack sustained, long-term paclitaxel retention. Keratose, a naturally derived protein, has exhibited unique properties allowing for tuned release of various therapeutic agents. However, little is known regarding its ability to support delivery of anti-proliferative agents such as paclitaxel. The goal of this project was to thus demonstrate the feasibility of keratose as a DCB-coating excipient to promote the release and delivery of paclitaxel. Methods: Keratose was combined with paclitaxel in vitro and the release kinetics of paclitaxel and keratose were evaluated through high performance liquid chromatograph-mass spectroscopy (HPLC-MS) and spectrophotometry, respectively. A custom coating method was developed to deposit keratose and paclitaxel on commercially available angioplasty balloons via an air spraying method. Coatings were then visualized under scanning electron microscopy and drug load quantified by HPLC-MS. Acute arterial transfer of paclitaxel at 1 h was assessed using a novel ex vivo model and further evaluated in vivo in a porcine ilio-femoral injury model. Results: Keratose demonstrated tunable release of paclitaxel as a function of keratose concentration in vitro. DCB coated via air spraying yielded consistent drug loading of 4.0 ± 0.70 µg/mm2. Under scanning electron microscopy, the keratose-paclitaxel DCB showed uniform coverage with a consistent, textured appearance. The acute drug transfer of the keratose-paclitaxel DCB was 43.60 ± 14.8 ng/mg at 1 h ex vivo. These measurements were further confirmed in vivo as the acute 1 h arterial paclitaxel levels were 56.60 ± 66.4 ng/mg. Conclusion: The keratose-paclitaxel coated DCB exhibited paclitaxel uptake and achieved acute therapeutic arterial tissue levels, confirming the feasibility of keratose as a novel excipient for DCB.

Coating and Pharmacokinetic Evaluation of Air Spray Coated Drug Coated Balloons.

Drug coated balloons (DCB) are becoming the standard-care treatment for peripheral arterial disease (PAD). DCB use excipients to transfer and retain anti-proliferative drugs, such as paclitaxel. Excipients thus play a vital role in the design and function of DCB, however methods to coat balloons with excipients and anti-proliferative drugs remain unknown. The goal of this study was to thus develop an approach to coat and evaluate DCB for various excipients. An air sprayer method was developed to deposit paclitaxel and various excipients onto non-coated commercially available angioplasty balloons. The coating of the angioplasty balloons was evaluated for drug deposition and coating efficiency using high performance liquid chromatography tandem mass spectrometry. Drug transfer and retention of the coated angioplasty balloons into arterial segments were evaluated ex vivo using harvested pig arteries in a pulsatile flow bioreactor. The air sprayer method successfully delivered varying excipients including bovine serum albumin (BSA), urea and iohexol. The air spray method was configured to coat four angioplasty balloons simultaneously with paclitaxel and iohexol with an average paclitaxel load of 4.0 ± 0.70 µg/mm2. The intra-day (within) and inter-day (between) coating precisions, defined as relative standard deviation (RSD), was 17.2 and 15.5%, respectively. Ex vivo deployment of iohexol-paclitaxel DCB yielded an arterial paclitaxel concentration of 123.4 ± 44.68 ng/mg (n = 3) at 1 h, 126.7 ± 25.27 ng/mg (n = 3) at 1 day, and 12.9 ± 12.88 ng/mg (n = 3) at 7 days. This work provides proof-of-concept of a quick, inexpensive approach to coat commercially available angioplasty balloons with paclitaxel and various excipients.