HLA class II upregulation during viral infection leads to HLA-DP-directed graft-versus-host disease after CD4+ donor lymphocyte infusion.

CD8+ T cell-depleted (TCD) donor lymphocyte infusion (DLI) after TCD allogeneic hematopoietic stem cell transplantation (alloSCT) has been associated with a reduced risk of graft-versus-host disease (GVHD) while preserving conversion to donor hematopoiesis and antitumor immunity, providing a rationale for exploring CD4+ T cell-based immunotherapy for hematologic malignancies. Here, we analyzed the clinical course and specificity of T cell immune responses in 2 patients with acute myeloid leukemia (AML) who converted to full-donor chimerism but developed severe acute GVHD after prophylactic CD4+ DLI after 10/10-HLA-matched, but HLA-DPB1-mismatched TCD-alloSCT. Clonal analysis of activated T cells isolated during GVHD demonstrated allo-reactivity exerted by CD4+ T cells directed against patient-mismatched HLA-DPB1 molecules on hematopoietic cells and skin-derived fibroblasts only when cultured under inflammatory conditions. At the time of CD4+ DLI, both patients contained residual patient-derived T cells, including cytomegalovirus (CMV)-specific T cells as a result of CMV reactivations. Once activated by CMV antigens, these CMV-specific T cells could stimulate HLA-DPB1-specific CD4+ T cells, which in turn could target nonhematopoietic tissues in GVHD. In conclusion, our data demonstrate that GVHD after HLA-DPB1-mismatched CD4+ DLI can be mediated by allo-reactive HLA-DPB1-directed CD4+ T cells and that ongoing viral infections inducing HLA class II expression on nonhematopoietic cells may increase the likelihood of GVHD development. This trial is registered at http://www.controlled-trials.com/ISRCTN51398568/LUMC as #51398568.

Donor T cells administered over HLA class II barriers mediate antitumor immunity without broad off-target toxicity in a NOD/scid mouse model of acute leukemia.

Alloreactive (allo)-HLA-directed T cell responses after HLA-mismatched allogeneic hematopoietic stem cell transplantation and donor lymphocyte infusion are typically considered detrimental responses mediating graft-versus-host disease (GVHD). Allo-HLA-reactive T cells with beneficial and selective graft-versus-leukemia (GVL) reactivity, however, can also be identified within an HLA-mismatched context. We investigated whether allo-HLA class II-directed T cells with beneficial GVL reactivity induced in NOD/scid mice engrafted with human chronic myelogenous leukemia in lymphoid blast crisis after treatment with donor lymphocyte infusion - mediated detrimental xenogeneic GVHD as a result of broad off-target cross-reactivity. The results demonstrate that beneficial GVL reactivity and xenogeneic GVHD are mediated by separate T cells. GVL reactivity was mediated by human T cells recognizing allo-HLA class II molecules, whereas xenoreactivity was exerted by human T cells recognizing H-2 molecules. Taken together, our data indicate a limited risk for detrimental off-target effects by allo-HLA class II-directed T cells and thereby provide a basis for the development of strategies for selecting allo-HLA-restricted T cells with selective GVL reactivity for adoptive transfer after HLA-mismatched allogeneic hematopoietic stem cell transplantation.

Design of Pt-Sn-Zn Nanomaterials for Successful Methanol Electrooxidation Reaction.

This work highlights the potential for the synthesis of new PtSnZn catalysts with enhanced efficiency and durability for methanol oxidation reaction (MOR) in low-temperature fuel cells. In this research, PtZn and PtSnZn nanoparticles deposited on high surface area Vulcan XC-72R Carbon support were created by a microwave-assisted polyol method. The electrochemical performances of synthesized catalysts were analyzed by cyclic voltammetry and by the electrooxidation of adsorbed CO and the chronoamperometric method. The physicochemical properties of obtained catalysts were characterized by transmission electron microscopy (TEM), thermogravimetric (TGA) analysis, energy dispersive spectroscopy (EDS) and by X-ray diffraction (XRD). The obtained findings showed the successful synthesis of platinum-based catalysts. It was established that PtSnZn/C and PtZn/C catalysts have high electrocatalytic performance in methanol oxidation reactions. Catalysts stability tests were obtained by chronoamperometry. Stability tests also confirmed decreased poisoning and indicated improved stability and better tolerance to CO-like intermediate species. According to activity and stability measurements, the PtSnZn/C catalyst possesses the best electrochemical properties for the methanol oxidation reaction. The observed great electrocatalytic activity in the methanol oxidation reaction of synthesized catalysts can be attributed to the beneficial effects of microwave synthesis and the well-balanced addition of alloying metals in PtSnZn/C catalysts.

Relationships between structure and activity of carbon as a multifunctional support for electrocatalysts.

We report on new insights into the relationships between structure and activity of glassy carbon (GC), as a model material for electrocatalyst support, during its anodization in acid solution. Our investigation strongly confirms the role of CFGs in promotion of Pt activity by the "spill-over" effect related to CO(ads) for methanol electrooxidation (MEO) on a carbon-supported Pt catalyst. Combined analysis of voltammetric and impedance behaviour as well as changes in GC surface morphology induced by intensification of anodizing conditions reveal an intrinsic influence of the carbon functionalization and the structure of a graphene oxide (GO) layer on the electrical and electrocatalytic properties of activated GC. Although GO continuously grows during anodization, it structurally changes from being a graphite inter-layer within graphite ribbons toward a continuous GO surface layer that deteriorates the native structure of GC. As a consequence of the increased distance between GO-spaced graphite layers, the GC conductivity decreases until the case of profound GO exfoliation under drastic anodizing conditions. This exposes the native, yet abundantly functionalized, GC texture. While GC capacitance continuously increases with intensification of anodizing conditions, the surface nano-roughness and GO resistance reach the highest values at modest anodizing conditions, and then decrease upon drastic anodization due to the onset of GO exfoliation. We found for the first time that the activity of a GC-supported Pt catalyst in MEO, as one of the promising half-reactions in polymer electrolyte fuel cells, strictly follows the changes in GC nano-roughness and GO-induced GC resistance. The highest GC/Pt MEO activity is reached when optimal distance between graphite layers and optimal degree of GC functionalization bring the highest amount of CFGs into intimate contact with the Pt surface. This confirms the promoting role of CFGs in MEO catalysis.

Bacterial nanocellulose as green support of platinum nanoparticles for effective methanol oxidation.

Bacterial nanocellulose, BNC, has emerged as a new class of nanomaterials recognized as renewable, biodegradable, biocompatible and material for versatile applications. BNC also proved as a perfect support matrix for metallic nanoparticle synthesis and appeared as suitable alternative for widely used carbon based materials. Following the idea to replace commonly used carbon based materials for platinum supports with the green and sustainable one, BNC appeared as an excellent candidate. Herein, microwave assisted synthesis has been reported for the first time for platinum nanoparticles supported on BNC as green material. Bacterial nanocelullose-platinum catalyst, Pt/BNC, was investigated by Fourier transform infrared spectroscopy (FTIR), differential scanning calorimetry (DSC), atomic force microscopy (AFM), X-ray diffractometry (XRD) and transmission-electron microscopy (TEM) analysis. The obtained results confirmed successful synthesis of new Pt-based catalyst. It was found that Pt/BNC catalyst has high electrocatalytic performance in methanol oxidation reaction. Green/sustainable catalytic system is highly desirable and provided by the elegant microwave assisted synthesis of Pt/BNC will pave the way for a larger scale application and expedite the market penetration of such fuel cells.

Upcycling Biodegradable PVA/Starch Film to a Bacterial Biopigment and Biopolymer.

Meeting the challenge of circularity for plastics requires amenability to repurposing post-use, as equivalent or upcycled products. In a compelling advancement, complete circularity for a biodegradable polyvinyl alcohol/thermoplastic starch (PVA/TPS) food packaging film was demonstrated by bioconversion to high-market-value biopigments and polyhydroxybutyrate (PHB) polyesters. The PVA/TPS film mechanical properties (tensile strength (σu), 22.2 ± 4.3 MPa; strain at break (εu), 325 ± 73%; and Young's modulus (E), 53-250 MPa) compared closely with low-density polyethylene (LDPE) grades used for food packaging. Strong solubility of the PVA/TPS film in water was a pertinent feature, facilitating suitability as a carbon source for bioprocessing and microbial degradation. Biodegradability of the film with greater than 50% weight loss occurred within 30 days of incubation at 37 °C in a model compost. Up to 22% of the PVA/TPS film substrate conversion to biomass was achieved using three bacterial strains, Ralstonia eutropha H16 (Cupriavidus necator ATCC 17699), Streptomyces sp. JS520, and Bacillus subtilis ATCC6633. For the first time, production of the valuable biopigment (undecylprodigiosin) by Streptomyces sp. JS520 of 5.3 mg/mL and the production of PHB biopolymer at 7.8% of cell dry weight by Ralstonia eutropha H16 from this substrate were reported. This low-energy, low-carbon post-use PVA/TPS film upcycling model approach to plastic circularity demonstrates marked progress in the quest for sustainable and circular plastic solutions.

TCR-engineered T cells targeting E7 for patients with metastatic HPV-associated epithelial cancers.

Genetically engineered T cell therapy can induce remarkable tumor responses in hematologic malignancies. However, it is not known if this type of therapy can be applied effectively to epithelial cancers, which account for 80-90% of human malignancies. We have conducted a first-in-human, phase 1 clinical trial of T cells engineered with a T cell receptor targeting HPV-16 E7 for the treatment of metastatic human papilloma virus-associated epithelial cancers (NCT02858310). The primary endpoint was maximum tolerated dose. Cell dose was not limited by toxicity with a maximum dose of 1 × 1011 engineered T cells administered. Tumor responses following treatment were evaluated using RECIST (Response Evaluation Criteria in Solid Tumors) guidelines. Robust tumor regression was observed with objective clinical responses in 6 of 12 patients, including 4 of 8 patients with anti-PD-1 refractory disease. Responses included extensive regression of bulky tumors and complete regression of most tumors in some patients. Genomic studies, which included intra-patient tumors with dichotomous treatment responses, revealed resistance mechanisms from defects in critical components of the antigen presentation and interferon response pathways. These findings demonstrate that engineered T cells can mediate regression of common carcinomas, and they reveal immune editing as a constraint on the curative potential of cellular therapy and possibly other immunotherapies in advanced epithelial cancer.

On optimal and near-optimal shapes of external shading of windows in apartment buildings.

We studied previously optimal shape of external shading of windows in a cellular office with an outer edge modeled by a non-uniform rational basis spline (NURBS) curve whose control points were placed uniformly around western fin, overhang and eastern fin of the window, and whose depths were allowed to vary independently. We observed there that for each climate considered in the study there exists a shading shape close to the optimal one, but with a substantially simpler structure of control points for the NURBS curve. This simpler structure was reflected in partitioning control points into six groups such that all control points in the same group have equal depths, with groups corresponding to lower part of the western fin, upper part of the western fin, joint of the western fin and the overhang, internal part of the overhang, joint of the overhang and the eastern fin and the remaining part of the eastern fin. Here we confirm that shadings with control point structure restricted in such way can perform as well as shadings with unrestricted control points by optimising shape of external shading of windows in an apartment room for both restricted and unrestricted control point structure for the same range of climates, and showing that differences in heating and cooling demands between Pareto optimal shadings in both cases are negligibly small. This grouping of control points thus gives a simple and natural division of shading into a small number of basic constituents that have most impact on its heating and cooling demands. We further consider the convex hull of the Pareto front for shadings with restricted control points, as it contains shadings that minimise equivalent source energy in terms of the ratio of efficiencies and source energy conversion factors for district heating and cooling. We show that, in cases when depths of control point groups in convex hull shadings do not experience sudden changes between their extremal values, these depths can be fitted reasonably well by a sigmoid function that results in functional shadings that satisfactorily approximate heating and cooling demands of shadings in the Pareto front.

Tumor-infiltrating human CD4+ regulatory T cells display a distinct TCR repertoire and exhibit tumor and neoantigen reactivity.

CD4+ regulatory T (Treg) cells have an essential function in maintaining self-tolerance; however, they may also play a detrimental role in antitumor immune responses. The presence of elevated frequencies of Treg cells in tumors correlates with disease progression and poor survival in patients with cancer. The antigen specificity of Treg cells that have expanded in the tumor microenvironment is poorly understood; answering this question may provide important insights for immunotherapeutic approaches. To address this, we used a novel combinatorial approach to characterizing the T cell receptor (TCR) profiles of intratumoral Treg cells from patients with metastatic melanoma, gastrointestinal, and ovarian cancers and elucidated their antigen specificities. The TCR repertoires of tumor-resident Treg cells were diverse yet displayed significant overlap with circulating Treg cells but not with conventional T cells in tumor or blood. TCRs isolated from Treg cells displayed specific reactivity against autologous tumors and mutated neoantigens, suggesting that intratumoral Treg cells act in a tumor antigen-selective manner leading to their activation and clonal expansion in the tumor microenvironment. Tumor antigen-specific Treg-derived TCRs resided in the tumor and in the circulation, suggesting that both Treg cell compartments may serve as a source for tumor-specific TCRs. These findings provide insights into the TCR specificity of tumor-infiltrating human Treg cells that may have potential implications for cancer immunotherapy.

Cancer targeting by TCR gene-engineered T cells directed against Kita-Kyushu Lung Cancer Antigen-1.

T cell receptor (TCR) gene-engineered T cells have shown promise in the treatment of melanoma and synovial cell sarcoma, but their application to epithelial cancers has been limited. The identification of novel therapeutic TCRs for the targeting of these tumors is important for the development of new treatments. Here, we describe the preclinical characterization of a TCR directed against Kita-Kyushu Lung Cancer Antigen-1 (KK-LC-1, encoded by CT83), a cancer germline antigen with frequent expression in human epithelial malignancies including gastric cancer, breast cancer, and lung cancer. Gene-engineered T cells expressing the KK-LC-1 TCR (KK-LC-1 TCR-Ts) demonstrated recognition of CT83+ tumor lines in vitro and mediated regression of established CT83+ xenograft tumors in immunodeficient mouse models. Cross-reactivity studies based on experimental determination of the recognition motifs for the target epitope did not demonstrate cross-reactivity against other human proteins. CT83 gene expression studies in 51 non-neural tissues and 24 neural tissues showed expression restricted exclusively to germ cells. CT83 was however expressed by a range of epithelial cancers, with the highest expression noted in gastric cancer. Collectively, these findings support the further investigation and clinical testing of KK-LC-1 TCR-Ts for gastric cancer and possibly other malignancies.

T-Cell Receptor Gene Therapy for Human Papillomavirus-Associated Epithelial Cancers: A First-in-Human, Phase I/II Study.

PURPOSE: Genetically engineered T-cell therapy is an emerging treatment of hematologic cancers with potential utility in epithelial cancers. We investigated T-cell therapy for the treatment of metastatic human papillomavirus (HPV)-associated epithelial cancers. METHODS: This phase I/II, single-center trial enrolled patients with metastatic HPV16-positive cancer from any primary tumor site who had received prior platinum-based therapy. Treatment consisted of autologous genetically engineered T cells expressing a T-cell receptor directed against HPV16 E6 (E6 T-cell receptor T cells), a conditioning regimen, and systemic aldesleukin. RESULTS: Twelve patients were treated in the study. No dose-limiting toxicities were observed in the phase I portion. Two patients, both in the highest-dose cohort, experienced objective tumor responses. A patient with three lung metastases experienced complete regression of one tumor and partial regression of two tumors, which were subsequently resected; she has no evidence of disease 3 years after treatment. All patients demonstrated high levels of peripheral blood engraftment with E6 T-cell receptor T cells 1 month after treatment (median, 30%; range, 4% to 53%). One patient's resistant tumor demonstrated a frameshift deletion in interferon gamma receptor 1, which mediates response to interferon gamma, an essential molecule for T-cell-mediated antitumor activity. Another patient's resistant tumor demonstrated loss of HLA-A*02:01, the antigen presentation molecule required for this therapy. A tumor from a patient who responded to treatment did not demonstrate genetic defects in interferon gamma response or antigen presentation. CONCLUSION: Engineered T cells can induce regression of epithelial cancer. Tumor resistance was observed in the context of T-cell programmed death-1 expression and defects in interferon gamma and antigen presentation pathway components. These findings have important implications for development of cellular therapy in epithelial cancers.

A Phase II Study of Tumor-infiltrating Lymphocyte Therapy for Human Papillomavirus-associated Epithelial Cancers.

PURPOSE: Cellular therapy is an emerging cancer treatment modality, but its application to epithelial cancers has been limited. This clinical trial evaluated tumor-infiltrating lymphocyte (TIL) therapy for the treatment of patients with metastatic human papillomavirus (HPV)-associated carcinomas. PATIENTS AND METHODS: The trial was a phase II design with two cohorts, cervical cancers and noncervical cancers. Cell infusion was preceded by a lymphocyte-depleting conditioning regimen and followed by systemic high-dose aldesleukin. RESULTS: Objective tumor responses occurred in 5 of 18 (28%) patients in the cervical cancer cohort and 2 of 11 (18%) patients in the noncervical cancer cohort. Two of the responses in cervical cancer were complete and are ongoing 67 and 53 months after treatment. Responses in the noncervical cancer cohort were in anal cancer and oropharyngeal cancer. The HPV reactivity of the infused T cells correlated with clinical response. Peripheral blood repopulation with HPV-reactive T cells also correlated with clinical response. CONCLUSIONS: These findings support the concept that cellular therapy can mediate the regression of epithelial cancers, and they suggest the importance of predictive biomarkers and novel treatment platforms for more effective therapies.

Optimisation of curvilinear external shading of windows in cellular offices.

Shading of windows influences building cooling and heating loads through control of solar heat gains, and lighting load through access to available daylight. Shading shape thus presents an important factor both in building energy analysis and building aesthetics. Curvilinearity of solar paths suggests that the optimal shading shape may be curvilinear as well, and our aim here is to test this expectation. To accommodate curvilinearity of shading shape, outer edges of shading, which consists of overhang, western and eastern fins, are modeled as non-uniform rational basis spline (NURBS) curves, a widely accepted representation standard for curves in design industry. As a case study, a cellular office is considered in the Pacific Northwest National Laboratory (PNNL) office building model, with its overhang lined up by seven control points, and the fins lined up by five control points each, with two ending control points joint for the overhang and the fins. With control points allowed to take on nine different alternative depths, genetic optimisation is employed for 16 representative USA climates with respect to total equivalent source energy for heating, cooling and lighting loads. The main finding is that in a very close proximity to optimal shadings found by genetic optimisation there exist shadings with much simpler control point structure, obtained by identifying depths of successive control points, that have nearly rectangular overhangs. Since the difference between these simpler shadings and the optimal ones is less than 0.24%, this partially rejects the expectation that the optimal shading shape should be curvilinear. Structure of these near-optimal shadings also suggests a new way to partition shadings into independent regions: the lower and the upper parts of the western fin, joints of the overhang with the western and the eastern fin, the interior part of the overhang and the rest of the eastern fin.

Engineered T cells targeting E7 mediate regression of human papillomavirus cancers in a murine model.

T cell receptor (TCR) T cell therapy is a promising cancer treatment modality. However, its successful development for epithelial cancers may depend on the identification of high-avidity TCRs directed against tumor-restricted target antigens. The human papillomavirus (HPV) E7 antigen is an attractive therapeutic target that is constitutively expressed by HPV+ cancers but not by healthy tissues. It is unknown if genetically engineered TCR T cells that target E7 can mediate regression of HPV+ cancers. We identified an HPV-16 E7-specific, HLA-A*02:01-restricted TCR from a uterine cervix biopsy from a woman with cervical intraepithelial neoplasia. This TCR demonstrated high functional avidity, with CD8 coreceptor-independent tumor targeting. Human T cells transduced to express the TCR specifically recognized and killed HPV-16+ cervical and oropharyngeal cancer cell lines and mediated regression of established HPV-16+ human cervical cancer tumors in a mouse model. These findings support the therapeutic potential of this approach and established the basis for an E7 TCR gene therapy clinical trial in patients with metastatic HPV+ cancers (NCT02858310).

Human Papillomavirus T-Cell Cross-reactivity in Cervical Cancer: Implications for Immunotherapy Clinical Trial Design.

Importance: Clinical trials are testing vaccines that target human papillomavirus 16 (HPV-16) oncoproteins for the treatment of cervical cancer regardless of the HPV type of the tumor. For patients with HPV-18-positive cancers, this strategy relies on cross-reactivity of HPV-16-reactive T cells against the HPV-18 oncoproteins. Objectives: To determine the prevalence of HPV-16 and HPV-18 metastatic cervical cancers in women enrolling in clinical trials at a US medical center and to assess whether HPV oncoprotein-targeting tumor-infiltrating lymphocytes (TILs) and T-cell receptors (TCRs) possess HPV-16/HPV-18 oncoprotein cross-reactivity. Design, Setting, and Participants: This study was conducted at the National Institutes of Health Clinical Center, a tertiary care research hospital in the United States. The HPV type of the tumors from 65 consecutive patients with cervical cancer who were evaluated for participation in clinical trials was determined by retrospective medical record review. Immunological assays testing HPV cross-reactivity were conducted on all available archived samples of oncoprotein-reactive TILs from HPV-positive tumors (n = 16) and on a library of previously identified TCRs (n = 10). Interventions: The HPV genotype of each patient's tumor was determined. The cross-reactivity of archived TILs and a library of TCRs was assessed. Main Outcomes and Measures: The main outcomes were the prevalence of each HPV genotype and the frequency of TILs or TCRs with HPV oncoprotein-T-cell cross-reactivity. Cross-reactivity was assessed by enzyme-linked immunospot assays and interferon-γ production assays. Results: The median (range) age of 65 referred patients was 44 (24-64) years. Ethnicity was recorded for 39 of 65 patients; 35 (89.7%) were white, 3 (7.7%) were Asian, and 1 (2.6%) was American Indian/Alaskan Native. Histologic tumor subtype was recorded for 41 of 65 patients; 25 (61.0%) were squamous cell carcinomas, 12 (29.3%) were adenocarcinomas, 2 (4.9%) were adenosquamous cell carcinomas, and 2 (4.9%) were neuroendocrine tumors. Thirty-nine of 65 patients (60.0%) had HPV-16-positive tumors and 21 patients (32.3%) had HPV-18-positive tumors. In the analysis of cross-reactivity, 1 of 16 oncoprotein-reactive archived TILs (9 from cervical cancers and 7 from other cancers) displayed HPV-16/HPV-18 cross-reactivity. None of the 10 oncoprotein-reactive TCRs displayed HPV-16/HPV-18 cross-reactivity. Conclusions and Relevance: Cervical cancers that tested positive for HPV-18 were common in this study and may be common in other US clinical trial populations. Results showed that HPV-16/HPV-18 intergenotype T-cell cross-reactivity of T cells from HPV-16-positive and HPV-18-positive cancers was uncommon. These findings support clinical trial designs in which the HPV type targeted by a therapeutic vaccine is matched with the HPV type of a cancer and suggest a change is necessary in the design of active clinical trials.

Circulating Cell-free DNA for Metastatic Cervical Cancer Detection, Genotyping, and Monitoring.

Purpose: Circulating cell-free (ccf) human papillomavirus (HPV) DNA may serve as a unique tumor marker for HPV-associated malignancies, including cervical cancer. We developed a method to genotype and quantify circulating HPV DNA in patients with HPV16- or HPV18-positive metastatic cervical cancer for potential disease monitoring and treatment-related decision making.Experimental Design: In this retrospective study, HPV ccfDNA was measured in serum samples from 19 metastatic cervical cancer patients by duplex digital droplet PCR (ddPCR). Nine patients had received tumor-infiltrating lymphocyte (TIL) immunotherapy. ccfDNA data were aligned with the tumor HPV genotype, drug treatment, and clinical outcome.Results: In blinded tests, HPV ccfDNA was detected in 19 of 19 (100%) patients with HPV-positive metastatic cervical cancer but not in any of the 45 healthy blood donors. The HPV genotype harbored in the patients' tumors was correctly identified in 87 of 87 (100%) sequential patient serum samples from 9 patients who received TIL immunotherapy. In three patients who experienced objective cancer regression after TIL treatment, a transient HPV ccfDNA peak was detected 2-3 days after TIL infusion. Furthermore, persistent clearance of HPV ccfDNA was only observed in two patients who experienced complete response (CR) after TIL immunotherapy.Conclusions: HPV ccfDNA represents a promising tumor marker for noninvasive HPV genotyping and may be used in selecting patients for HPV type-specific T-cell-based immunotherapies. It may also have value in detecting antitumor activity of therapeutic agents and in the long-term follow-up of cervical cancer patients in remission. Clin Cancer Res; 23(22); 6856-62. ©2017 AACR.

Landscape of immunogenic tumor antigens in successful immunotherapy of virally induced epithelial cancer.

Immunotherapy has clinical activity in certain virally associated cancers. However, the tumor antigens targeted in successful treatments remain poorly defined. We used a personalized immunogenomic approach to elucidate the global landscape of antitumor T cell responses in complete regression of human papillomavirus-associated metastatic cervical cancer after tumor-infiltrating adoptive T cell therapy. Remarkably, immunodominant T cell reactivities were directed against mutated neoantigens or a cancer germline antigen, rather than canonical viral antigens. T cells targeting viral tumor antigens did not display preferential in vivo expansion. Both viral and nonviral tumor antigen-specific T cells resided predominantly in the programmed cell death 1 (PD-1)-expressing T cell compartment, which suggests that PD-1 blockade may unleash diverse antitumor T cell reactivities. These findings suggest a new paradigm of targeting nonviral antigens in immunotherapy of virally associated cancers.

Targeting liposomes with protein drugs to the blood-brain barrier in vitro.

In this study, we aim to target pegylated liposomes loaded with horseradish peroxidase (HRP) and tagged with transferrin (Tf) to the BBB in vitro. Liposomes were prepared with the post-insertion technique: micelles of polyethylene glycol (PEG) and PEG-Tf were inserted into pre-formed liposomes containing HRP. Tf was measured indirectly by measuring iron via atomic absorption spectroscopy. All liposomes were around 100 nm in diameter, contained 5-13 microg HRP per mumol phospholipid and 63-74 Tf molecules per liposome (lipo Tf) or no Tf (lipo C). Brain capillary endothelial cells (BCEC) were incubated with liposomes at 4 degrees C (to determine binding) or at 37 degrees C (to determine association, i.e. binding+endocytosis) and the HRP activity, rather than the HRP amount was determined in cell lysates. Association of lipo Tf was two- to three-fold higher than association of lipo C. Surprisingly, the binding of lipo Tf at 4 degrees C was four-fold higher than the association of at 37 degrees C. Most likely this high binding and low endocytosis is explained by intracellular degradation of endocytosed HRP. In conclusion, we have shown targeting of liposomes loaded with protein or peptide drugs to the BCEC and more specifically to the lysosomes. This is an advantage for the treatment of lysosomal storage disease. However, drug targeting to other intracellular targets also results in intracellular degradation of the drug. Our experiments suggest that liposomes release some of their content within the BBB, making targeting of liposomes to the TfR on BCEC an attractive approach for brain drug delivery.

Targeting of HPV-16+ Epithelial Cancer Cells by TCR Gene Engineered T Cells Directed against E6.

PURPOSE: The E6 and E7 oncoproteins of HPV-associated epithelial cancers are in principle ideal immunotherapeutic targets, but evidence that T cells specific for these antigens can recognize and kill HPV(+) tumor cells is limited. We sought to determine whether TCR gene engineered T cells directed against an HPV oncoprotein can successfully target HPV(+) tumor cells. EXPERIMENTAL DESIGN: T-cell responses against the HPV-16 oncoproteins were investigated in a patient with an ongoing 22-month disease-free interval after her second resection of distant metastatic anal cancer. T cells genetically engineered to express an oncoprotein-specific TCR from this patient's tumor-infiltrating T cells were tested for specific reactivity against HPV(+) epithelial tumor cells. RESULTS: We identified, from an excised metastatic anal cancer tumor, T cells that recognized an HLA-A*02:01-restricted epitope of HPV-16 E6. The frequency of the dominant T-cell clonotype from these cells was approximately 400-fold greater in the patient's tumor than in her peripheral blood. T cells genetically engineered to express the TCR from this clonotype displayed high avidity for an HLA-A*02:01-restricted epitope of HPV-16, and they showed specific recognition and killing of HPV-16(+) cervical, and head and neck cancer cell lines. CONCLUSIONS: These findings demonstrate that HPV-16(+) tumors can be targeted by E6-specific TCR gene engineered T cells, and they provide the foundation for a novel cellular therapy directed against HPV-16(+) malignancies, including cervical, oropharyngeal, anal, vulvar, vaginal, and penile cancers.

Complete regression of metastatic cervical cancer after treatment with human papillomavirus-targeted tumor-infiltrating T cells.

PURPOSE: Metastatic cervical cancer is a prototypical chemotherapy-refractory epithelial malignancy for which better treatments are needed. Adoptive T-cell therapy (ACT) is emerging as a promising cancer treatment, but its study in epithelial malignancies has been limited. This study was conducted to determine if ACT could mediate regression of metastatic cervical cancer. PATIENTS AND METHODS: Patients enrolled onto this protocol were diagnosed with metastatic cervical cancer and had previously received platinum-based chemotherapy or chemoradiotherapy. Patients were treated with a single infusion of tumor-infiltrating T cells selected when possible for human papillomavirus (HPV) E6 and E7 reactivity (HPV-TILs). Cell infusion was preceded by lymphocyte-depleting chemotherapy and was followed by administration of aldesleukin. RESULTS: Three of nine patients experienced objective tumor responses (two complete responses and one partial response). The two complete responses were ongoing 22 and 15 months after treatment, respectively. One partial response was 3 months in duration. The HPV reactivity of T cells in the infusion product (as measured by interferon gamma production, enzyme-linked immunospot, and CD137 upregulation assays) correlated positively with clinical response (P = .0238 for all three assays). In addition, the frequency of HPV-reactive T cells in peripheral blood 1 month after treatment was positively associated with clinical response (P = .0238). CONCLUSION: Durable, complete regression of metastatic cervical cancer can occur after a single infusion of HPV-TILs. Exploratory studies suggest a correlation between HPV reactivity of the infusion product and clinical response. Continued investigation of this therapy is warranted.