Improving cancer immunotherapy through nanotechnology.

The 2018 Nobel Prize in Physiology or Medicine was awarded to pioneers in the field of cancer immunotherapy, as the utility of leveraging a patient's coordinated and adaptive immune system to fight the patient's unique tumour has now been validated robustly in the clinic. Still, the proportion of patients who respond to immunotherapy remains modest (~15% objective response rate across indications), as tumours have multiple means of immune evasion. The immune system is spatiotemporally controlled, so therapies that influence the immune system should be spatiotemporally controlled as well, in order to maximize the therapeutic index. Nanoparticles and biomaterials enable one to program the location, pharmacokinetics and co-delivery of immunomodulatory compounds, eliciting responses that cannot be achieved upon administration of such compounds in solution. The convergence of cancer immunotherapy, nanotechnology, bioengineering and drug delivery is opportune, as each of these fields has matured independently to the point that it can now be used to complement the others substantively and rationally, rather than modestly and empirically. As a result, unmet needs increasingly can be addressed with deductive intention. This Review explores how nanotechnology and related approaches are being applied to augmenting both endogenous leukocytes and adoptively transferred ones by informing specificity, influencing localization and improving function.

Combination therapy targeting both innate and adaptive immunity improves survival in a pre-clinical model of ovarian cancer.

BACKGROUND: Despite major advancements in immunotherapy among a number of solid tumors, response rates among ovarian cancer patients remain modest. Standard treatment for ovarian cancer is still surgery followed by taxane- and platinum-based chemotherapy. Thus, there is an urgent need to develop novel treatment options for clinical translation. METHODS: Our approach was to analyze the effects of standard chemotherapy in the tumor microenvironment of mice harboring orthotopic, syngeneic ID8-Vegf-Defb29 ovarian tumors in order to mechanistically determine a complementary immunotherapy combination. Specifically, we interrogated the molecular and cellular consequences of chemotherapy by analyzing gene expression and flow cytometry data. RESULTS: These data show that there is an immunosuppressive shift in the myeloid compartment, with increased expression of IL-10 and ARG1, but no activation of CD3+ T cells shortly after chemotherapy treatment. We therefore selected immunotherapies that target both the innate and adaptive arms of the immune system. Survival studies revealed that standard chemotherapy was complemented most effectively by a combination of anti-IL-10, 2'3'-cGAMP, and anti-PD-L1. Immunotherapy dramatically decreased the immunosuppressive myeloid population while chemotherapy effectively activated dendritic cells. Together, combination treatment increased the number of activated T and dendritic cells as well as expression of cytotoxic factors. It was also determined that the immunotherapy had to be administered concurrently with the chemotherapy to reverse the acute immunosuppression caused by chemotherapy. Mechanistic studies revealed that antitumor immunity in this context was driven by CD4+ T cells, which acquired a highly activated phenotype. Our data suggest that these CD4+ T cells can kill cancer cells directly via granzyme B-mediated cytotoxicity. Finally, we showed that this combination therapy is also effective at delaying tumor growth substantially in an aggressive model of lung cancer, which is also treated clinically with taxane- and platinum-based chemotherapy. CONCLUSIONS: This work highlights the importance of CD4+ T cells in tumor immunology. Furthermore, the data support the initiation of clinical trials in ovarian cancer that target both innate and adaptive immunity, with a focus on optimizing dosing schedules.

CRISPR-Mediated Editing of the B Cell Receptor in Primary Human B Cells.

Vaccination approaches have generally focused on the antigen rather than the resultant antibodies generated, which differ greatly in quality and function between individuals. The ability to replace the variable regions of the native B cell receptor (BCR) heavy and light chain loci with defined recombined sequences of a preferred monoclonal antibody could enable curative adoptive cell transfer. We report CRISPR-mediated homologous recombination (HR) into the BCR of primary human B cells. Ribonucleoprotein delivery enabled editing at the model CXCR4 locus, as demonstrated by T7E1 assay, flow cytometry, and TIDE analysis. Insertion via HR was confirmed by sequencing, cross-boundary PCR, and restriction digest. Optimized conditions were used to achieve HR at the BCR variable heavy and light chains. Insertion was confirmed at the DNA level, and transgene expression from the native BCR promoters was observed. Reprogramming the specificity of antibodies in the genomes of B cells could have clinical importance.

Neutrophil extracellular traps produced during inflammation awaken dormant cancer cells in mice.

Cancer cells from a primary tumor can disseminate to other tissues, remaining dormant and clinically undetectable for many years. Little is known about the cues that cause these dormant cells to awaken, resume proliferating, and develop into metastases. Studying mouse models, we found that sustained lung inflammation caused by tobacco smoke exposure or nasal instillation of lipopolysaccharide converted disseminated, dormant cancer cells to aggressively growing metastases. Sustained inflammation induced the formation of neutrophil extracellular traps (NETs), and these were required for awakening dormant cancer. Mechanistic analysis revealed that two NET-associated proteases, neutrophil elastase and matrix metalloproteinase 9, sequentially cleaved laminin. The proteolytically remodeled laminin induced proliferation of dormant cancer cells by activating integrin α3ß1 signaling. Antibodies against NET-remodeled laminin prevented awakening of dormant cells. Therapies aimed at preventing dormant cell awakening could potentially prolong the survival of cancer patients.

Extended release of perioperative immunotherapy prevents tumor recurrence and eliminates metastases.

Cancer immunotherapy can confer durable benefit, but the percentage of patients who respond to this approach remains modest. The ability to concentrate immunostimulatory compounds at the site of disease can overcome local immune tolerance and reduce systemic toxicity. Surgical resection of tumors may improve the efficacy of immunotherapy by removing the concentrated immunosuppressive microenvironment; however, it also removes tumor-specific leukocytes as well as tumor antigens that may be important to establishing antitumor immunity. Moreover, surgery produces a transient immunosuppressive state associated with wound healing that has been correlated with increased metastasis. Using multiple models of spontaneous metastasis, we show that extended release of agonists of innate immunity-including agonists of Toll-like receptor 7/8 (TLR7/8) or stimulator of interferon genes (STING)-from a biodegradable hydrogel placed in the tumor resection site cured a much higher percentage of animals than systemic or local administration of the same therapy in solution. Depletion and neutralization experiments confirmed that the observed prevention of local tumor recurrence and eradication of existing metastases require both the innate and adaptive arms of the immune system. The localized therapy increased the numbers of activated natural killer (NK) cells, dendritic cells, and T cells and induced production of large amounts of type I interferons, thereby converting an immunosuppressive post-resection microenvironment into an immunostimulatory one. The results suggest that the perioperative setting may prove to be a useful context for immunotherapy, particularly when the release of the therapy is extended locally.

Recurrent ubiquitin B silencing in gynecological cancers establishes dependence on ubiquitin C.

Transcriptional repression of ubiquitin B (UBB) is a cancer-subtype-specific alteration that occurs in a substantial population of patients with cancers of the female reproductive tract. UBB is 1 of 2 genes encoding for ubiquitin as a polyprotein consisting of multiple copies of ubiquitin monomers. Silencing of UBB reduces cellular UBB levels and results in an exquisite dependence on ubiquitin C (UBC), the second polyubiquitin gene. UBB is repressed in approximately 30% of high-grade serous ovarian cancer (HGSOC) patients and is a recurrent lesion in uterine carcinosarcoma and endometrial carcinoma. We identified ovarian tumor cell lines that retain UBB in a repressed state, used these cell lines to establish orthotopic ovarian tumors, and found that inducible expression of a UBC-targeting shRNA led to tumor regression, and substantial long-term survival benefit. Thus, we describe a recurrent cancer-specific lesion at the level of ubiquitin production. Moreover, these observations reveal the prognostic value of UBB repression and establish UBC as a promising therapeutic target for ovarian cancer patients with recurrent UBB silencing.

T cell-targeting nanoparticles focus delivery of immunotherapy to improve antitumor immunity.

Targeted delivery of compounds to particular cell subsets can enhance therapeutic index by concentrating their action on the cells of interest. Because attempts to target tumors directly have yielded limited benefit, we instead target endogenous immune cell subsets in the circulation that can migrate actively into tumors. We describe antibody-targeted nanoparticles that bind to CD8+ T cells in the blood, lymphoid tissues, and tumors of mice. PD-1+ T cells are successfully targeted in the circulation and tumor. The delivery of an inhibitor of TGFß signaling to PD-1-expressing cells extends the survival of tumor-bearing mice, whereas free drugs have no effect at such doses. This modular platform also enables PD-1-targeted delivery of a TLR7/8 agonist to the tumor microenvironment, increasing the proportion of tumor-infiltrating CD8+ T cells and sensitizing tumors to subsequent anti-PD-1. Targeted delivery of immunotherapy to defined subsets of endogenous leukocytes may be superior to administration of free drugs.

DNA Damage and Repair Biomarkers of Immunotherapy Response.

DNA-damaging agents are widely used in clinical oncology and exploit deficiencies in tumor DNA repair. Given the expanding role of immune checkpoint blockade as a therapeutic strategy, the interaction of tumor DNA damage with the immune system has recently come into focus, and it is now clear that the tumor DNA repair landscape has an important role in driving response to immune checkpoint blockade. Here, we summarize the mechanisms by which DNA damage and genomic instability have been found to shape the antitumor immune response and describe clinical efforts to use DNA repair biomarkers to guide use of immune-directed therapies.Significance: Only a subset of patients respond to immune checkpoint blockade, and reliable predictive biomarkers of response are needed to guide therapy decisions. DNA repair deficiency is common among tumors, and emerging experimental and clinical evidence suggests that features of genomic instability are associated with response to immune-directed therapies. Cancer Discov; 7(7); 675-93. ©2017 AACR.

Surface modulation of polymeric nanocarriers enhances the stability and delivery of proteins and small molecules.

AIM: We aimed to enhance the stability and therapeutic efficiency of protein-based therapeutic formulations. MATERIALS & METHODS: Proteins were immobilized on the surface of nanoparticles (NPs) to improve both protein stability and protein function, especially enzymatic activity. The modularity of the platform was demonstrated by coating proteins of varied molecular weights and functionalities on the surface of poly(lactic-co-glycolic acid)-based NPs. RESULTS: Coating proteins to the particle surface greatly enhanced the stability of the NPs, preventing particle aggregation and improving enzymatic potency, including in vivo. Specifically, coating of collagenase I to the particle surface greatly improved the ability of the enzyme to degrade tumor collagen relative to free enzyme, thereby increasing the penetration of adjuvant chemotherapy (doxorubicin). Additionally, the protein coating reduced the rate of doxorubicin release, enabling sustained release of the small-molecule payload. CONCLUSION: The straightforward procedure described herein permits the formulation of modular NPs that can combine and sustain the benefits of small molecules and biologics.

Cancer cells induce metastasis-supporting neutrophil extracellular DNA traps.

Neutrophils, the most abundant type of leukocytes in blood, can form neutrophil extracellular traps (NETs). These are pathogen-trapping structures generated by expulsion of the neutrophil's DNA with associated proteolytic enzymes. NETs produced by infection can promote cancer metastasis. We show that metastatic breast cancer cells can induce neutrophils to form metastasis-supporting NETs in the absence of infection. Using intravital imaging, we observed NET-like structures around metastatic 4T1 cancer cells that had reached the lungs of mice. We also found NETs in clinical samples of triple-negative human breast cancer. The formation of NETs stimulated the invasion and migration of breast cancer cells in vitro. Inhibiting NET formation or digesting NETs with deoxyribonuclease I (DNase I) blocked these processes. Treatment with NET-digesting, DNase I-coated nanoparticles markedly reduced lung metastases in mice. Our data suggest that induction of NETs by cancer cells is a previously unidentified metastasis-promoting tumor-host interaction and a potential therapeutic target.

Improving cancer immunotherapy with DNA methyltransferase inhibitors.

Immunotherapy confers durable clinical benefit to melanoma, lung, and kidney cancer patients. Challengingly, most other solid tumors, including ovarian carcinoma, are not particularly responsive to immunotherapy, so combination with a complementary therapy may be beneficial. Recent findings suggest that epigenetic modifying drugs can prime antitumor immunity by increasing expression of tumor-associated antigens, chemokines, and activating ligands by cancer cells as well as cytokines by immune cells. This review, drawing from both preclinical and clinical data, describes some of the mechanisms of action that enable DNA methyltransferase inhibitors to facilitate the establishment of antitumor immunity.

The PARP1 inhibitor BMN 673 exhibits immunoregulatory effects in a Brca1(-/-) murine model of ovarian cancer.

Familial breast and ovarian cancer are often caused by inherited mutations of BRCA1. While current prognoses for such patients are rather poor, inhibition of poly-ADP ribose polymerase 1 (PARP1) induces synthetic lethality in cells that are defective in homologous recombination. BMN 673 is a potent PARP1 inhibitor that is being clinically evaluated for treatment of BRCA-mutant cancers. Using the Brca1-deficient murine epithelial ovarian cancer cell line BR5FVB1-Akt, we investigated whether the antitumor effects of BMN 673 extend beyond its known pro-apoptotic function. Administration of modest amounts of BMN 673 greatly improved the survival of mice bearing subcutaneous or intraperitoneal tumors. We thus hypothesized that BMN 673 may influence the composition and function of immune cells in the tumor microenvironment. Indeed, BMN 673 significantly increases the number of peritoneal CD8(+) T cells and NK cells as well as their production of IFN-γ and TNF-α. These data suggest that the cell stress caused by BMN 673 induces not only cancer cell-intrinsic apoptosis but also cancer cell-extrinsic antitumor immune effects in a syngeneic murine model of ovarian cancer. BMN 673 may therefore serve as a promising adjuvant therapy to immunotherapy to achieve durable responses among patients whose tumors harbor defects in homologous recombination.

Decitabine Enhances Lymphocyte Migration and Function and Synergizes with CTLA-4 Blockade in a Murine Ovarian Cancer Model.

The lack of second-line treatment for relapsed ovarian cancer necessitates the development of improved combination therapies. Targeted therapy and immunotherapy each confer clinical benefit, albeit limited as monotherapies. Ovarian cancer is not particularly responsive to immune checkpoint blockade, so combination with a complementary therapy may be beneficial. Recent studies have revealed that a DNA methyl transferase inhibitor, azacytidine, alters expression of immunoregulatory genes in ovarian cancer. In this study, the antitumor effects of a related DNA methyl transferase inhibitor, decitabine (DAC), were demonstrated in a syngeneic murine ovarian cancer model. Low-dose DAC treatment increases the expression of chemokines that recruit NK cells and CD8(+) T cells, promotes their production of IFNγ and TNFα, and extends the survival of mice bearing subcutaneous or orthotopic tumors. While neither DAC nor immune checkpoint blockade confers durable responses as a monotherapy in this model, the efficacy of anti-CTLA-4 was potentiated by combination with DAC. This combination promotes differentiation of naïve T cells into effector T cells and prolongs cytotoxic lymphocyte responses as well as mouse survival. These results suggest that this combination therapy may be worthy of further consideration for improved treatment of drug-resistant ovarian cancer.

Epithelial PD-L2 Expression Marks Barrett's Esophagus and Esophageal Adenocarcinoma.

Esophageal adenocarcinoma is an increasingly common disease with a dismal 5-year survival rate of 10% to 15%. In the first systematic evaluation of the PD-1 pathway in esophageal adenocarcinoma, we identify expression of PD-L2 in cancer cells in 51.7% of esophageal adenocarcinomas. Epithelial PD-L1 was expressed on only 2% of cases, although PD-L1(+) immune cells were observed in 18% of esophageal adenocarcinomas. We also evaluated expression in the precursor lesion of esophageal adenocarcinoma, Barrett's esophagus, which emerges following gastric reflux-induced esophageal inflammation, and found PD-L2 expression in Barrett's esophagus but not in non-Barrett's esophagus esophagitis. Because the progression from squamous esophagitis to Barrett's esophagus is accompanied by a transition from a TH1 to TH2 immune response, we hypothesized that the TH2 cytokines IL4/IL13 could contribute to PD-L2 induction. We confirmed that these cytokines can augment PD-L2 expression in esophageal adenocarcinoma cell lines. These results suggest that the inflammatory environment in Barrett's esophagus and esophageal adenocarcinoma may contribute to the expression of PD-L2. Furthermore, the potential for PD-1 receptor blockade to be effective in esophageal adenocarcinomas with epithelial PD-L2 or immune cell PD-L1 expression should be evaluated in clinical trials.

Cosilencing of PKM-2 and MDR-1 Sensitizes Multidrug-Resistant Ovarian Cancer Cells to Paclitaxel in a Murine Model of Ovarian Cancer.

Tumor multidrug resistance (MDR) is a serious clinical challenge that significantly limits the effectiveness of cytotoxic chemotherapy. As such, complementary therapeutic strategies are being explored to prevent relapse. The altered metabolic state of cancer cells, which perform aerobic glycolysis, represents an interesting target that can enable discrimination between healthy cells and cancer cells. We hypothesized that cosilencing of genes responsible for aerobic glycolysis and for MDR would have synergistic antitumor effect. In this study, siRNA duplexes against pyruvate kinase M2 and multidrug resistance gene-1 were encapsulated in hyaluronic acid-based self-assembling nanoparticles. The particles were characterized for morphology, size, charge, encapsulation efficiency, and transfection efficiency. In vivo studies included biodistribution assessment, gene knockdown confirmation, therapeutic efficacy, and safety analysis. The benefit of active targeting of cancer cells was confirmed by modifying the particles' surface with a peptide targeted to epidermal growth factor receptor, which is overexpressed on the membranes of the SKOV-3 cancer cells. To augment the studies involving transplantation of a paclitaxel-resistant cell line, an in vivo paclitaxel resistance model was developed by injecting repeated doses of paclitaxel following tumor inoculation. The nanoparticles accumulated significantly in the tumors, hindering tumor volume doubling time (P < 0.05) upon combination therapy in both the wild-type (2-fold) and resistant (8-fold) xenograft models. Although previous studies indicated that silencing of MDR-1 alone sensitized MDR ovarian cancer to paclitaxel only modestly, these data suggest that concurrent silencing of PKM-2 improves the efficacy of paclitaxel against MDR ovarian cancer.

Immunoengineering: how nanotechnology can enhance cancer immunotherapy.

Although cancer immunotherapy can lead to durable outcomes, the percentage of patients who respond to this disruptive approach remains modest to date. Encouragingly, nanotechnology can enhance the efficacy of immunostimulatory small molecules and biologics by altering their co-localization, biodistribution, and release kinetics.

Targeting myeloid cells using nanoparticles to improve cancer immunotherapy.

While nanoparticles have traditionally been used to deliver cytotoxic drugs directly to tumors to induce cancer cell death, emerging data suggest that nanoparticles are likely to generate a larger impact on oncology through the delivery of agents that can stimulate antitumor immunity. Tumor-targeted nanocarriers have generally been used to localize chemotherapeutics to tumors and thus decrease off-target toxicity while enhancing efficacy. Challengingly, tumor heterogeneity and evolution render tumor-intrinsic approaches likely to succumb to relapse. The immune system offers exquisite specificity, cytocidal potency, and long-term activity that leverage an adaptive memory response. For this reason, the ability to manipulate immune cell specificity and function would be desirable, and nanoparticles represent an exciting means by which to perform such manipulation. Dendritic cells and tumor-associated macrophages are cells of the myeloid lineage that function as natural phagocytes, so they naturally take up nanoparticles. Dendritic cells direct the specificity and potency of cellular immune responses that can be targeted for cancer vaccines. Herein, we discuss the specific criteria needed for efficient vaccine design, including but not limited to the route of administration, size, morphology, surface charge, targeting ligands, and nanoparticle composition. In contrast, tumor-associated macrophages are critical mediators of immunosuppression whose trans-migratory abilities can be exploited to localize therapeutics to the tumor core and which can be directly targeted for elimination or for repolarization to a tumor suppressive phenotype. It is likely that a combination of targeting dendritic cells to stimulate antitumor immunity and tumor-associated macrophages to reduce immune suppression will impart significant benefits and result in durable antitumor responses.

Dual-layer surface coating of PLGA-based nanoparticles provides slow-release drug delivery to achieve metronomic therapy in a paclitaxel-resistant murine ovarian cancer model.

Development of drug resistance is a central challenge to the treatment of ovarian cancer. Metronomic chemotherapy decreases the extent of drug-free periods, thereby hindering development of drug resistance. Intraperitoneal chemotherapy allows for treatment of tumors confined within the peritoneum, but achieving sustained tumor-localized chemotherapy remains difficult. We hypothesized that modulating the surface properties of poly(lactic-co-glycolic acid) (PLGA)-based nanoparticles could enhance their drug retention ability and extend their release profile, thereby enabling metronomic, localized chemotherapy in vivo. Paclitaxel was encapsulated in particles coated with a layer of polydopamine and a subsequent layer of poly(ethylene glycol) (PEG). These particles achieved a 3.8-fold higher loading content compared to that of nanoparticles formulated from linear PLGA-PEG copolymers. In vitro release kinetic studies and in vivo drug distribution profiles demonstrate sustained release of paclitaxel. Although free drug conferred no survival advantage, low-dose intraperitoneal administration of paclitaxel-laden surface-coated nanoparticles to drug-resistant ovarian tumor-bearing mice resulted in significant survival benefits in the absence of any apparent systemic toxicity.

Silencing HoxA1 by intraductal injection of siRNA lipidoid nanoparticles prevents mammary tumor progression in mice.

With advances in screening, the incidence of detection of premalignant breast lesions has increased in recent decades; however, treatment options remain limited to surveillance or surgical removal by lumpectomy or mastectomy. We hypothesized that disease progression could be blocked by RNA interference (RNAi) therapy and set out to develop a targeted therapeutic delivery strategy. Using computational gene network modeling, we identified HoxA1 as a putative driver of early mammary cancer progression in transgenic C3(1)-SV40TAg mice. Silencing this gene in cultured mouse or human mammary tumor spheroids resulted in increased acinar lumen formation, reduced tumor cell proliferation, and restoration of normal epithelial polarization. When the HoxA1 gene was silenced in vivo via intraductal delivery of nanoparticle-formulated small interfering RNA (siRNA) through the nipple of transgenic mice with early-stage disease, mammary epithelial cell proliferation rates were suppressed, loss of estrogen and progesterone receptor expression was prevented, and tumor incidence was reduced by 75%. This approach that leverages new advances in systems biology and nanotechnology offers a novel noninvasive strategy to block breast cancer progression through targeted silencing of critical genes directly within the mammary epithelium.

Transcription factor/microRNA axis blocks melanoma invasion program by miR-211 targeting NUAK1.

Melanoma is one of the deadliest human cancers, responsible for approximately 80% of skin cancer mortalities. The aggressiveness of melanoma is due to its capacity to proliferate and rapidly invade surrounding tissues, leading to metastases. A recent model suggests melanoma progresses by reversibly switching between proliferation and invasion transcriptional signatures. Recent studies show that cancer cells are more sensitive to microRNA (miRNA) perturbation than are non-cancer cells; however, the roles of miRNAs in melanoma plasticity remain unexplored. Here, we use the gene expression profiles of melanoma and normal melanocytes to characterize the transcription factor-miRNA relationship that modulates the proliferative and invasive programs of melanoma. We identified two sets of miRNAs that likely regulate these programs. Interestingly, one of the miRNAs involved in melanoma invasion is miR-211, a known target of the master regulator microphthalmia-associated transcription factor (MITF). We demonstrate that miR-211 contributes to melanoma adhesion by directly targeting a gene, NUAK1. Inhibition of miR-211 increases NUAK1 expression and decreases melanoma adhesion, whereas upregulation of miR-211 restores adhesion through NUAK1 repression. This study defines the MITF/miR-211 axis that inhibits the invasive program by blocking adhesion. Furthermore, we have identified NUAK1 as a potential target for the treatment of metastatic melanoma.