Radiation and anti-PD-L1 synergize by stimulating a stem-like T cell population in the tumor-draining lymph node.

Radiotherapy (RT) and anti-PD-L1 synergize to enhance local and distant (abscopal) tumor control. However, clinical results in humans have been variable. With the goal of improving clinical outcomes, we investigated the underlying synergistic mechanism focusing on a CD8+ PD-1+ Tcf-1+ stem-like T cell subset in the tumor-draining lymph node (TdLN). Using murine melanoma models, we found that RT + anti-PD-L1 induces a novel differentiation program in the TdLN stem-like population which leads to their expansion and differentiation into effector cells within the tumor. Our data indicate that optimal synergy between RT + anti-PD-L1 is dependent on the TdLN stem-like T cell population as either blockade of TdLN egress or specific stem-like T cell depletion reduced tumor control. Together, these data demonstrate a multistep stimulation of stem-like T cells following combination therapy which is initiated in the TdLN and completed in the tumor.

Microengineered In Vitro Assays for Screening and Sorting Manufactured Therapeutic T Cells.

Adoptively transferred T cells constitute a major class of current and emergent cellular immunotherapies for the treatment of disease, including but not limited to cancer. Although key advancements in molecular recognition, genetic engineering, and manufacturing have dramatically enhanced their translational potential, therapeutic potency remains limited by poor homing and infiltration of transferred cells within target host tissues. In vitro microengineered homing assays with precise control over micromechanical and biological cues can address these shortcomings by enabling interrogation, screening, sorting, and optimization of therapeutic T cells based on their homing capacity. In this article, the working principles, application, and integration of microengineered homing assays for the mechanistic study of biophysical and biomolecular cues relevant to homing of therapeutic T cells are reviewed. The potential for these platforms to enable scalable enrichment and screening of next-generation manufactured T cell therapies for cancer is also discussed.

Sustained release hydrogel for durable locoregional chemoimmunotherapy for BRAF-mutated melanoma.

The wide prevalence of BRAF mutations in diagnosed melanomas drove the clinical advancement of BRAF inhibitors in combination with immune checkpoint blockade for treatment of advanced disease. However, deficits in therapeutic potencies and safety profiles motivate the development of more effective strategies that improve the combination therapy's therapeutic index. Herein, we demonstrate the benefits of a locoregional chemoimmunotherapy delivery system, a novel thermosensitive hydrogel comprised of gelatin and Pluronic® F127 components already widely used in humans in both commercial and clinical products, for the co-delivery of a small molecule BRAF inhibitor with immune checkpoint blockade antibody for the treatment of BRAF-mutated melanoma. In vivo evaluation of administration route and immune checkpoint target effects revealed intratumoral administration of antagonistic programmed cell death protein 1 antibody (aPD-1) lead to potent antitumor therapy in combination with BRAF inhibitor vemurafenib. The thermosensitive F127-g-Gelatin hydrogel that was evaluated in multiple murine models of BRAF-mutated melanoma that facilitated prolonged local drug release within the tumor (>1 week) substantially improved local immunomodulation, tumor control, rates of tumor response, and animal survival. Thermosensitive F127-g-Gelatin hydrogels thus improve upon the clinical benefits of vemurafenib and aPD-1 in a locoregional chemoimmunotherapy approach for the treatment of BRAF-mutated melanoma.

Adhesion analysis via a tumor vasculature-like microfluidic device identifies CD8+ T cells with enhanced tumor homing to improve cell therapy.

CD8+ T cell recruitment to the tumor microenvironment is critical for the success of adoptive cell therapy (ACT). Unfortunately, only a small fraction of transferred cells home to solid tumors. Adhesive ligand-receptor interactions have been implicated in CD8+ T cell homing; however, there is a lack of understanding of how CD8+ T cells interact with tumor vasculature-expressed adhesive ligands under the influence of hemodynamic flow. Here, the capacity of CD8+ T cells to home to melanomas is modeled ex vivo using an engineered microfluidic device that recapitulates the hemodynamic microenvironment of the tumor vasculature. Adoptively transferred CD8+ T cells with enhanced adhesion in flow in vitro and tumor homing in vivo improve tumor control by ACT in combination with immune checkpoint blockade. These results show that engineered microfluidic devices can model the microenvironment of the tumor vasculature to identify subsets of T cells with enhanced tumor infiltrating capabilities, a key limitation in ACT.

Targeting vasoactive intestinal peptide-mediated signaling enhances response to immune checkpoint therapy in pancreatic ductal adenocarcinoma.

A paucity of effector T cells within tumors renders pancreatic ductal adenocarcinoma (PDAC) resistant to immune checkpoint therapies. While several under-development approaches target immune-suppressive cells in the tumor microenvironment, there is less focus on improving T cell function. Here we show that inhibiting vasoactive intestinal peptide receptor (VIP-R) signaling enhances anti-tumor immunity in murine PDAC models. In silico data mining and immunohistochemistry analysis of primary tumors indicate overexpression of the neuropeptide vasoactive intestinal peptide (VIP) in human PDAC tumors. Elevated VIP levels are also present in PDAC patient plasma and supernatants of cultured PDAC cells. Furthermore, T cells up-regulate VIP receptors after activation, identifying the VIP signaling pathway as a potential target to enhance T cell function. In mouse PDAC models, VIP-R antagonist peptides synergize with anti-PD-1 antibody treatment in improving T cell recruitment into the tumors, activation of tumor-antigen-specific T cells, and inhibition of T cell exhaustion. In contrast to the limited single-agent activity of anti-PD1 antibodies or VIP-R antagonist peptides, combining both therapies eliminate tumors in up to 40% of animals. Furthermore, tumor-free mice resist tumor re-challenge, indicating anti-cancer immunological memory generation. VIP-R signaling thus represents a tumor-protective immune-modulatory pathway that is targetable in PDAC.

Opportunities for Nitric Oxide in Potentiating Cancer Immunotherapy.

Despite nearly 30 years of development and recent highlights of nitric oxide (NO) donors and NO delivery systems in anticancer therapy, the limited understanding of exogenous NO's effects on the immune system has prevented their advancement into clinical use. In particular, the effects of exogenously delivered NO differing from that of endogenous NO has obscured how the potential and functions of NO in anticancer therapy may be estimated and exploited despite the accumulating evidence of NO's cancer therapy-potentiating effects on the immune system. After introducing their fundamentals and characteristics, this review discusses the current mechanistic understanding of NO donors and delivery systems in modulating the immunogenicity of cancer cells as well as the differentiation and functions of innate and adaptive immune cells. Lastly, the potential for the complex modulatory effects of NO with the immune system to be leveraged for therapeutic applications is discussed in the context of recent advancements in the implementation of NO delivery systems for anticancer immunotherapy applications. SIGNIFICANCE STATEMENT: Despite a 30-year history and recent highlights of nitric oxide (NO) donors and delivery systems as anticancer therapeutics, their clinical translation has been limited. Increasing evidence of the complex interactions between NO and the immune system has revealed both the potential and hurdles in their clinical translation. This review summarizes the effects of exogenous NO on cancer and immune cells in vitro and elaborates these effects in the context of recent reports exploiting NO delivery systems in vivo in cancer therapy applications.

Elicitation of stem-like CD8+ T cell responses via lymph node-targeted chemoimmunotherapy evokes systemic tumor control.

BACKGROUND: Tumor-draining lymph nodes (TdLNs) are critical in the regulation of local and systemic antitumor T cell immunity and are implicated in coordinating responses to immunomodulatory therapies. METHODS: Biomaterial nanoparticles that deliver chemotherapeutic drug paclitaxel to TdLNs were leveraged to explore its effects in combination and immune checkpoint blockade (ICB) antibody immunotherapy to determine the benefit of TdLN-directed chemoimmunotherapy on tumor control. RESULTS: Accumulation of immunotherapeutic drugs in combination within TdLNs synergistically enhanced systemic T cell responses that led to improved control of local and disseminated disease and enhanced survival in multiple murine breast tumor models. CONCLUSIONS: These findings suggest a previously underappreciated role of secondary lymphoid tissues in mediating effects of chemoimmunotherapy and demonstrate the potential for nanotechnology to unleashing drug synergies via lymph node targeted delivery to elicit improved response of breast and other cancers.

Thermosensitive hydrogel releasing nitric oxide donor and anti-CTLA-4 micelles for anti-tumor immunotherapy.

Due to their autosynchronous roles in shaping the anti-tumor immune response, complex immune regulatory networks acting both locally within the tumor microenvironment as well as in its draining lymph nodes play critical roles in the cancer immunotherapy response. We describe herein a thermosensitive co-polymer hydrogel system formed from biocompatible polymers gelatin and Pluronic® F127 that are widely used in humans to enable the sustained release of a nitric oxide donor and antibody blocking immune checkpoint cytotoxic T-lymphocyte-associated protein-4 for efficient and durable anti-tumor immunotherapy. By virtue of its unique gel formation and degradation properties that sustain drug retention at the tumor tissue site for triggered release by the tumor microenvironment and formation of in situ micelles optimum in size for lymphatic uptake, this rationally designed thermosensitive hydrogel facilitates modulation of two orthogonal immune signaling networks relevant to the regulation of the anti-tumor immune response to improve local and abscopal effects of cancer immunotherapy.

Synthetic Matrix Scaffolds Engineer the In Vivo Tumor Immune Microenvironment for Immunotherapy Screening.

Immunotherapy has emerged as one of the most powerful anti-cancer therapies but is stymied by the limits of existing preclinical models with respect to disease latency and reproducibility. Additionally, the influence of differing immune microenvironments within tumors observed clinically and associated with immunotherapeutic resistance cannot be tuned to facilitate drug testing workflows without changing model system or laborious genetic approaches. To address this testing platform gap in the immune oncology drug development pipeline, the authors deploy engineered biomaterials as scaffolds to increase tumor formation rate, decrease disease latency, and diminish variability of immune infiltrates into tumors formed from murine mammary carcinoma cell lines implanted into syngeneic mice. By altering synthetic gel formulations that reshape infiltrating immune cells within the tumor, responsiveness of the same tumor model to varying classes of cancer immunotherapies, including in situ vaccination with a molecular adjuvant and immune checkpoint blockade, diverge. These results demonstrate the significant role the local immune microenvironment plays in immunotherapeutic response. These engineered tumor immune microenvironments therefore improve upon the limitations of current breast tumor models used for immune oncology drug screening to enable immunotherapeutic testing relevant to the variability in tumor immune microenvironments underlying immunotherapeutic resistance seen in human patients.

Innovations in lymph node targeting nanocarriers.

Lymph nodes are secondary lymphoid tissues in the body that facilitate the co-mingling of immune cells to enable and regulate the adaptive immune response. They are also tissues implicated in a variety of diseases, including but not limited to malignancy. The ability to access lymph nodes is thus attractive for a variety of therapeutic and diagnostic applications. As nanotechnologies are now well established for their potential in translational biomedical applications, their high relevance to applications that involve lymph nodes is highlighted. Herein, established paradigms of nanocarrier design to enable delivery to lymph nodes are discussed, considering the unique lymph node tissue structure as well as lymphatic system physiology. The influence of delivery mechanism on how nanocarrier systems distribute to different compartments and cells that reside within lymph nodes is also elaborated. Finally, current advanced nanoparticle technologies that have been developed to enable lymph node delivery are discussed.

Drug-eluting immune checkpoint blockade antibody-nanoparticle conjugate enhances locoregional and systemic combination cancer immunotherapy through T lymphocyte targeting.

Multiple small molecule immune modulators have been identified as synergistic with immune checkpoint blockade (ICB) in their effects on T lymphocytes, but are limited in their successful application to combination cancer immunotherapy due to their short in vivo retention and lack of affinity for T cells. We engineered an antibody-nanoparticle conjugate (ANC) platform consisting of 30 nm polymer nanoparticles that, due to their size and formulation, efficiently distribute after administration to lymph nodes, tissues highly enriched in lymphocytes that contribute to tumor control mediated by ICB. Displaying monoclonal antibodies against surface-expressed T cell markers, NP delivery in vivo to circulating and lymph node-resident lymphocytes was substantially enhanced, as was delivery of small molecules formulated into the NP by passive encapsulation. Using ICB monoclonal antibodies as both targeting moiety and signal-blocking therapeutic, ANCs improved the local and systemic anti-tumor effects of small molecule TGFß receptor 1 inhibitor and an adenosine 2A antagonist when administered either locoregionally or systemically into the circulation in two syngeneic, aggressive tumor models, slowing tumor growth and prolonging animal survival. As these benefits were lost in the absence of ANC targeting, co-formulation strategies enabling the targeted co-delivery of multiple immunotherapeutics to T lymphocytes have high potential to improve ICB cancer immunotherapy by concurrent inhibition of non-redundant suppressive pathways.

The Kinetics of Lymphatic Dysfunction and Leukocyte Expansion in the Draining Lymph Node during LTB4 Antagonism in a Mouse Model of Lymphedema.

The mechanisms of lymphedema development are not well understood, but emerging evidence highlights the crucial role the immune system plays in driving its progression. It is well known that lymphatic function deteriorates as lymphedema progresses; however, the connection between this progressive loss of function and the immune-driven changes that characterize the disease has not been well established. In this study, we assess changes in leukocyte populations in lymph nodes within the lymphatic drainage basin of the tissue injury site (draining lymph nodes, dLNs) using a mouse tail model of lymphedema in which a pair of draining collecting vessels are left intact. We additionally quantify lymphatic pump function using established near infrared (NIR) lymphatic imaging methods and lymph-draining nanoparticles (NPs) synthesized and employed by our team for lymphatic tissue drug delivery applications to measure lymphatic transport to and resulting NP accumulation within dLNs associated with swelling following surgery. When applied to assess the effects of the anti-inflammatory drug bestatin, which has been previously shown to be a possible treatment for lymphedema, we find lymph-draining NP accumulation within dLNs and lymphatic function to increase as lymphedema progresses, but no significant effect on leukocyte populations in dLNs or tail swelling. These results suggest that ameliorating this loss of lymphatic function is not sufficient to reverse swelling in this surgically induced disease model that better recapitulates the extent of lymphatic injury seen in human lymphedema. It also suggests that loss of lymphatic function during lymphedema may be driven by immune-mediated mechanisms coordinated in dLNs. Our work indicates that addressing both lymphatic vessel dysfunction and immune cell expansion within dLNs may be required to prevent or reverse lymphedema when partial lymphatic function is sustained.

In Situ Crosslinked Hydrogel Depot for Sustained Antibody Release Improves Immune Checkpoint Blockade Cancer Immunotherapy.

The therapeutic inhibition of immune checkpoints, including cytotoxic T lymphocyte-associated protein (CTLA)-4 and programmed cell death 1 (PD-1), through the use of function blocking antibodies can confer improved clinical outcomes by invigorating CD8+ T cell-mediated anticancer immunity. However, low rates of patient responses and the high rate of immune-related adverse events remain significant challenges to broadening the benefit of this therapeutic class, termed immune checkpoint blockade (ICB). To overcome these significant limitations, controlled delivery and release strategies offer unique advantages relevant to this therapeutic class, which is typically administered systemically (e.g., intravenously), but more recently, has been shown to be highly efficacious using locoregional routes of administration. As such, in this paper, we describe an in situ crosslinked hydrogel for the sustained release of antibodies blocking CTLA-4 and PD-1 signaling from a locoregional injection proximal to the tumor site. This formulation results in efficient and durable anticancer effects with a reduced systemic toxicity compared to the bolus delivery of free antibody using an equivalent injection route. This formulation and strategy thus represent an approach for achieving the efficient and safe delivery of antibodies for ICB cancer immunotherapy.

Tumor-draining lymph nodes are survival niches that support T cell priming against lymphatic transported tumor antigen and effects of immune checkpoint blockade in TNBC.

Triple negative breast cancer (TNBC) is a significant clinical problem to which immunotherapeutic strategies have been applied with limited success. Using the syngeneic E0771 TNBC mouse model, this work explores the potential for antitumor CD8+ T cell immunity to be primed extratumorally in lymphoid tissues and therapeutically leveraged. CD8+ T cell viability and responses within the tumor microenvironment (TME) were found to be severely impaired, effects coincident with local immunosuppression that is recapitulated in lymphoid tissues in late stage disease. Prior to onset of a locally suppressed immune microenvironment, however, CD8+ T cell priming within lymph nodes (LN) that depended on tumor lymphatic drainage remained intact. These results demonstrate tumor-draining LNs (TdLN) to be lymphoid tissue niches that support the survival and antigenic priming of CD8+ T lymphocytes against lymph-draining antigen. The therapeutic effects of and CD8+ T cells response to immune checkpoint blockade were furthermore improved when directed to LNs within the tumor-draining lymphatic basin. Therefore, TdLNs represent a unique potential tumor immunity reservoir in TNBC for which strategies may be developed to improve the effects of ICB immunotherapy.

Lymph Node Subcapsular Sinus Microenvironment-On-A-Chip Modeling Shear Flow Relevant to Lymphatic Metastasis and Immune Cell Homing.

A lymph node sinus-on-a-chip adhesion microfluidic platform that recapitulates the hydrodynamic microenvironment of the lymph node subcapsular sinus was engineered. This device was used to interrogate the effects of lymph node remodeling on cellular adhesion in fluid flow relevant to lymphatic metastasis. Wall shear stress levels analytically estimated and modeled after quiescent and diseased/inflamed lymph nodes were experimentally recapitulated using a flow-based microfluidic perfusion system to assess the effects of physiological flow fields on human metastatic cancer cell adhesion. Results suggest that both altered fluid flow profiles and presentation of adhesive ligands, which are predicted to manifest within the lymph node subcapsular sinus as a result of inflammation-induced remodeling, and the presence of lymph-borne monocytic cells may synergistically contribute to the dynamic extent of cell adhesion in flow relevant to lymph node invasion by cancer and monocytic immune cells during lymphatic metastasis.

Lymphatic immunomodulation using engineered drug delivery systems for cancer immunotherapy.

Though immunotherapy has revolutionized the treatment of cancer to improve disease outcomes, an array of challenges remain that limit wider clinical success, including low rate of response and immune-related adverse events. Targeting immunomodulatory drugs to therapeutically relevant tissues offers a way to overcome these challenges by potentially enabling enhanced therapeutic efficacy and decreased incidence of side effects. Research highlighting the importance of lymphatic tissues in the response to immunotherapy has increased interest in the application of engineered drug delivery systems (DDSs) to enable specific targeting of immunomodulators to lymphatic tissues and cells that they house. To this end, a variety of DDS platforms have been developed that enable more efficient uptake into lymphatic vessels and lymph nodes to provide targeted modulation of the immune response to cancer. This can occur either by delivery of immunotherapeutics to lymphatics tissues or by direct modulation of the lymphatic vasculature itself due to their direct involvement in tumor immune processes. This review will highlight DDS platforms that, by enabling the activities of cancer vaccines, chemotherapeutics, immune checkpoint blockade (ICB) antibodies, and anti- or pro-lymphangiogenic factors to lymphatic tissues through directed delivery and controlled release, augment cancer immunotherapy.

Blockade of immune checkpoints in lymph nodes through locoregional delivery augments cancer immunotherapy.

Systemic administration of immune checkpoint blockade (ICB) monoclonal antibodies (mAbs) can unleash antitumor functions of T cells but is associated with variable response rates and off-target toxicities. We hypothesized that antitumor efficacy of ICB is limited by the minimal accumulation of mAb within tissues where antitumor immunity is elicited and regulated, which include the tumor microenvironment (TME) and secondary lymphoid tissues. In contrast to systemic administration, intratumoral and intradermal routes of administration resulted in higher mAb accumulation within both the TME and its draining lymph nodes (LNs) or LNs alone, respectively. The use of either locoregional administration route resulted in pronounced T cell responses from the ICB therapy, which developed in the secondary lymphoid tissues and TME of treated mice. Targeted delivery of mAb to tumor-draining lymph nodes (TdLNs) alone was associated with enhanced antitumor immunity and improved therapeutic effects compared to conventional systemic ICB therapy, and these effects were sustained at reduced mAb doses and comparable to those achieved by intratumoral administration. These data suggest that locoregional routes of administration of ICB mAb can augment ICB therapy by improving immunomodulation within TdLNs.