Using Oncolytic Virus to Retask CD19-Chimeric Antigen Receptor T Cells for Treatment of Pancreatic Cancer: Toward a Universal Chimeric Antigen Receptor T-Cell Strategy for Solid Tumor.

BACKGROUND: Chimeric antigen receptor (CAR) T cells targeting the B-cell antigen CD19 are standard therapy for relapsed or refractory B-cell lymphoma and leukemia. CAR T cell therapy in solid tumors is limited due to an immunosuppressive tumor microenvironment and a lack of tumor-restricted antigens. We recently engineered an oncolytic virus (CF33) with high solid tumor affinity and specificity to deliver a nonsignaling truncated CD19 antigen (CD19t), allowing targeting by CD19-CAR T cells. Here, we tested this combination against pancreatic cancer. STUDY DESIGN: We engineered CF33 to express a CD19t (CF33-CD19t) target. Flow cytometry and ELISA were performed to quantify CD19t expression, immune activation, and killing by virus and CD19-CAR T cells against various pancreatic tumor cells. Subcutaneous pancreatic human xenograft tumor models were treated with virus, CAR T cells, or virus+CAR T cells. RESULTS: In vitro, CF33-CD19t infection of tumor cells resulted in >90% CD19t cell-surface expression. Coculturing CD19-CAR T cells with infected cells resulted in interleukin-2 and interferon gamma secretion, upregulation of T-cell activation markers, and synergistic cell killing. Combination therapy of virus+CAR T cells caused significant tumor regression (day 13): control (n = 16, 485 ± 20 mm 3 ), virus alone (n = 20, 254 ± 23 mm 3 , p = 0.0001), CAR T cells alone (n = 18, 466 ± 25 mm 3 , p = NS), and virus+CAR T cells (n = 16, 128 ± 14 mm 3 , p < 0.0001 vs control; p = 0.0003 vs virus). CONCLUSIONS: Engineered CF33-CD19t effectively infects and expresses CD19t in pancreatic tumors, triggering cell killing and increased immunogenic response by CD19-CAR T cells. Notably, CF33-CD19t can turn cold immunologic tumors hot, enabling solid tumors to be targetable by agents designed against liquid tumor antigens.

Chimeric Antigen Receptor-T Cell and Oncolytic Viral Therapies for Gastric Cancer and Peritoneal Carcinomatosis of Gastric Origin: Path to Improving Combination Strategies.

Precision immune oncology capitalizes on identifying and targeting tumor-specific antigens to enhance anti-tumor immunity and improve the treatment outcomes of solid tumors. Gastric cancer (GC) is a molecularly heterogeneous disease where monoclonal antibodies against human epidermal growth factor receptor 2 (HER2), vascular endothelial growth factor (VEGF), and programmed cell death 1 (PD-1) combined with systemic chemotherapy have improved survival in patients with unresectable or metastatic GC. However, intratumoral molecular heterogeneity, variable molecular target expression, and loss of target expression have limited antibody use and the durability of response. Often immunogenically "cold" and diffusely spread throughout the peritoneum, GC peritoneal carcinomatosis (PC) is a particularly challenging, treatment-refractory entity for current systemic strategies. More adaptable immunotherapeutic approaches, such as oncolytic viruses (OVs) and chimeric antigen receptor (CAR) T cells, have emerged as promising GC and GCPC treatments that circumvent these challenges. In this study, we provide an up-to-date review of the pre-clinical and clinical efficacy of CAR T cell therapy for key primary antigen targets and provide a translational overview of the types, modifications, and mechanisms for OVs used against GC and GCPC. Finally, we present a novel, summary-based discussion on the potential synergistic interplay between OVs and CAR T cells to treat GCPC.

A Thin-Film Pinned-Photodiode Imager Pixel with Fully Monolithic Fabrication and beyond 1Me- Full Well Capacity.

Thin-film photodiodes (TFPD) monolithically integrated on the Si Read-Out Integrated Circuitry (ROIC) are promising imaging platforms when beyond-silicon optoelectronic properties are required. Although TFPD device performance has improved significantly, the pixel development has been limited in terms of noise characteristics compared to the Si-based image sensors. Here, a thin-film-based pinned photodiode (TF-PPD) structure is presented, showing reduced kTC noise and dark current, accompanied with a high conversion gain (CG). Indium-gallium-zinc oxide (IGZO) thin-film transistors and quantum dot photodiodes are integrated sequentially on the Si ROIC in a fully monolithic scheme with the introduction of photogate (PG) to achieve PPD operation. This PG brings not only a low noise performance, but also a high full well capacity (FWC) coming from the large capacitance of its metal-oxide-semiconductor (MOS). Hence, the FWC of the pixel is boosted up to 1.37 Me- with a 5 µm pixel pitch, which is 8.3 times larger than the FWC that the TFPD junction capacitor can store. This large FWC, along with the inherent low noise characteristics of the TF-PPD, leads to the three-digit dynamic range (DR) of 100.2 dB. Unlike a Si-based PG pixel, dark current contribution from the depleted semiconductor interfaces is limited, thanks to the wide energy band gap of the IGZO channel material used in this work. We expect that this novel 4 T pixel architecture can accelerate the deployment of monolithic TFPD imaging technology, as it has worked for CMOS Image sensors (CIS).

Image sensors using thin-film absorbers.

Image sensors are must-have components of most consumer electronics devices. They enable portable camera systems, which find their way into billions of devices annually. Such high volumes are possible thanks to the complementary metal-oxide semiconductor (CMOS) platform, leveraging wafer-scale manufacturing. Silicon photodiodes, at the core of CMOS image sensors, are perfectly suited to replicate human vision. Thin-film absorbers are an alternative family of photoactive materials, distinguished by the layer thickness comparable with or smaller than the wavelength of interest. They allow design of imagers with functionalities beyond Si-based sensors, such as transparency or detectivity at wavelengths above Si cutoff (e.g., short-wave infrared). Thin-film image sensors are an emerging device category. While intensive research is ongoing to achieve sufficient performance of thin-film photodetectors, to our best knowledge, there have been few complete studies on their integration into advanced systems. In this paper, we will describe several types of image sensors being developed at imec, based on organic, quantum dot, and perovskite photodiode and show their figures of merit. We also discuss the methodology for selecting the most appropriate sensor architecture (integration with thin-film transistor or CMOS). Application examples based on imec proof-of-concept sensors are demonstrated to showcase emerging use cases.

Antigen-dependent IL-12 signaling in CAR T cells promotes regional to systemic disease targeting.

Chimeric antigen receptor (CAR) T cell therapeutic responses are hampered by limited T cell trafficking, persistence, and durable anti-tumor activity in solid tumors. However, these challenges can be largely overcome by relatively unconstrained synthetic engineering strategies. Here, we describe CAR T cells targeting tumor-associated glycoprotein-72 (TAG72), utilizing the CD28 transmembrane domain upstream of the 4-1BB co-stimulatory domain as a driver of potent anti-tumor activity and IFNγ secretion. CAR T cell-mediated IFNγ production facilitated by IL-12 signaling is required for tumor cell killing, which is recapitulated by engineering an optimized membrane-bound IL-12 (mbIL12) molecule in CAR T cells. These T cells show improved antigen-dependent T cell proliferation and recursive tumor cell killing in vitro, with robust in vivo efficacy in human ovarian cancer xenograft models. Locoregional administration of mbIL12-engineered CAR T cells promotes durable anti-tumor responses against both regional and systemic disease in mice. Safety and efficacy of mbIL12-engineered CAR T cells is demonstrated using an immunocompetent mouse model, with beneficial effects on the immunosuppressive tumor microenvironment. Collectively, our study features a clinically-applicable strategy to improve the efficacy of locoregionally-delivered CAR T cells engineered with antigen-dependent immune-modulating cytokines in targeting regional and systemic disease.

A reservoir of stem-like CD8+ T cells in the tumor-draining lymph node preserves the ongoing antitumor immune response.

"Stem-like" TCF1+ CD8+ T (TSL) cells are necessary for long-term maintenance of T cell responses and the efficacy of immunotherapy, but, as tumors contain signals that should drive T cell terminal differentiation, how these cells are maintained in tumors remains unclear. In this study, we found that a small number of TCF1+ tumor-specific CD8+ T cells were present in lung tumors throughout their development. Yet, most intratumoral T cells differentiated as tumors progressed, corresponding with an immunologic shift in the tumor microenvironment (TME) from "hot" (T cell inflamed) to "cold" (non­T cell inflamed). By contrast, most tumor-specific CD8+ T cells in tumor-draining lymph nodes (dLNs) had functions and gene expression signatures similar to TSL from chronic lymphocytic choriomeningitis virus infection, and this population was stable over time despite the changes in the TME. dLN T cells were the developmental precursors of, and were clonally related to, their more differentiated intratumoral counterparts. Our data support the hypothesis that dLN T cells are the developmental precursors of the TCF1+ T cells in tumors that are maintained by continuous migration. Last, CD8+ T cells similar to TSL were also present in LNs from patients with lung adenocarcinoma, suggesting that a similar model may be relevant in human disease. Thus, we propose that the dLN TSL reservoir has a critical function in sustaining antitumor T cells during tumor development and in protecting them from the terminal differentiation that occurs in the TME.

Inducible de novo expression of neoantigens in tumor cells and mice.

Inducible expression of neoantigens in mice would enable the study of endogenous antigen-specific naïve T cell responses in disease and infection, but has been difficult to generate because leaky antigen expression in the thymus results in central T cell tolerance. Here we develop inversion-induced joined neoantigen (NINJA), using RNA splicing, DNA recombination and three levels of regulation to prevent leakiness and allow tight control over neoantigen expression. We apply NINJA to create tumor cell lines with inducible neoantigen expression, which could be used to study antitumor immunity. We also show that the genetic regulation in NINJA mice bypasses central and peripheral tolerance mechanisms and allows for robust endogenous CD8 and CD4 T cell responses on neoantigen induction in peripheral tissues. NINJA will enable studies of how T cells respond to defined neoantigens in the context of peripheral tolerance, transplantation, autoimmune diseases and cancer.

Method for Malaria Diagnosis Based on Extractions of Samples Using Non-Invasive Techniques: An Opportunity for the Nursing Clinical Practice.

Malaria has been for millennia one of the best known and most destructive diseases affecting humans. Its high impact has aroused great interest for the development of new effective and reliable diagnostic techniques. Recently it has been recently published that hairs from mammal hosts are able to capture, hold and finally remove foreign DNA sequences of Leishmania parasites. The aim of this study was to check if Plasmodium falciparum (P. falciparum) DNA remains stable in blood samples deposited in Whatman paper after suffering different transport and storage conditions, and to compare the sensitivity of these results with those offered by thick a smear and Rapid Diagnostic Test, and besides to examine whether P. falciparum DNA would be detected and quantified by Real time quantitative PCR (qPCR) from hairs of people with different types of malaria. P. falciparum Histidine Repeat Protein II (pHRP-II) antigen detection and P. falciparum DNA were detected in 18 of 19 dry blood samples adhered to Whatman paper (94.74%), besides, Plasmodium DNA was also detected in seven out of 19 hair samples analyzed (36.84%), remaining stable until analysis for several months under the exposure to different environmental conditions. Although the sensitivity of PCR for the diagnosis of malaria in hair samples is not as high as blood analysis, the study of Plasmodium DNA presence in blood and hair could constitute a complementary tool with numerous advantages in sample collection, transport and storage. We suggest that the method could be also applied to medical, forensic and paleo-parasitological diagnosis, not only for malaria but also for searching many other pathogens in hair samples.

Application of qPCR method to hair and cerumen samples for the diagnosis of canine leishmaniosis in Araçatuba, Brazil.

Visceral leishmaniosis (VL) remains a serious public health problem in Brazil. Dogs are the main hosts of the parasite, developing canine leishmaniosis (CanL), hence the importance of an accurate diagnosis of the animals. Recently, the application of qPCR method to non-invasive samples obtained from dogs with CanL has shown high sensitivity. Thus, we analyzed by qPCR blood, hair (from healthy zones and cutaneous lesions) and cerumen of 16 dogs with confirmed leishmaniosis from Araçatuba, a Brazilian endemic area. Cerumen-qPCR showed the highest sensitivity (87.5%), followed by hair (lesions: 78.57%, healthy skin: 62.5%), and blood (68.75%). We also analyzed blood, hair and cerumen of 5 healthy dogs from a non-endemic area, obtaining 100% of specificity in all samples. The use of cerumen and hair for qPCR analysis provides high reliability, taking into account the sensitivity and total specificity of the method. The non-invasive sampling procedure without the need of specific conditions of storage and transport support the usefulness of hair and cerumen for the diagnosis of CanL.

A large-scale field randomized trial demonstrates safety and efficacy of the vaccine LetiFend® against canine leishmaniosis.

Canine leishmaniosis is a zoonotic disease caused by Leishmania infantum. Extensive research is currently ongoing to develop safe and effective vaccines to protect from disease development. The European Commission has granted a marketing authorization for LetiFend®, a new vaccine containing recombinant Protein Q. The efficacy of LetiFend® vaccination in a large-scale dog population of both sexes, different breeds and ages in endemic areas is reported in this multicenter, randomized, double-blind, placebo-controlled field trial. Dogs (n = 549) living in France and Spain were randomly selected to receive a single subcutaneous dose of LetiFend® or placebo per year, and were naturally exposed to two L. infantum transmission seasons. Clinical examinations, blood and lymphoid organ sampling to evaluate serological, parasitological and disease status of the dogs were performed at different time points during the study. LetiFend® was very well tolerated and clearly reduced the incidence of clinical signs related to leishmaniosis. The number of confirmed cases of leishmaniosis was statistically significantly lower in the vaccine group. The number of dogs with parasites was close to be significantly reduced in the vaccine group (p = 0.0564). Re-vaccination of seropositive dogs demonstrated to be safe and not to worsen the course of the disease. The likelihood that a dog vaccinated with LetiFend® develops a confirmed case or clinical signs of leishmaniosis in areas with high pressure is, respectively, 5 and 9.8 time less than that for an unvaccinated dog. Thus, the overall efficacy of the LetiFend® vaccine in the prevention of confirmed cases of leishmaniosis in endemic areas with high disease pressure was shown to be 72%. In conclusion, this field trial demonstrates that LetiFend® is a novel, safe and effective vaccine for the active immunization of non-infected dogs from 6 months of age in reducing the risk of developing clinical leishmaniosis after natural infection with Leishmania infantum.

First detection of Leishmania kDNA in canine cerumen samples by qPCR.

Nowadays, searching for alternative non-invasive methods for molecular diagnosis of canine visceral leishmaniosis is getting increasingly important. We previously described the presence of Leishmania kinetoplast DNA (kDNA) in canine hair; in this case we hypothesized whether foreign DNA might be present in cerumen of dogs with leishmaniosis, and be detected by Real time quantitative PCR (qPCR). A population of 38 dogs that lived in Leishmania endemic areas was divided in two groups: A (33 dogs with confirmed leishmaniosis by serological techniques) and B (5 healthy dogs). Blood, lymph node, bone marrow and cerumen samples from all animals were tested for the presence of parasite kDNA. Our method was 100% specific, and in dogs from group A, Leishmania infantum kDNA was detected and quantified in the 100% of lymph node samples, in 90.9% of cerumen samples, in 88.5% of the bone marrow samples and in 57.6% of the blood samples. The qPCR-cerumen is a new non-invasive method that shows a high potential for the diagnosis of zoonotic visceral leishmaniosis.

First detection of Leishmania infantum kinetoplast DNA in hair of wild mammals: application of qPCR method to determine potential parasite reservoirs.

The data presented in this paper describe the application of a method for a reliable and non-invasive diagnosis of leishmaniosis in wild reservoirs, based on the detection of Leishmania infantum kinetoplast DNA (kDNA) in hair samples by Real Time PCR (qPCR). The study has been performed on 68 ear/leg hair samples from 5 different wild species (Vulpes vulpes, Canis lupus, Martes foina, Rattus norvegicus and Erinaceus europaeus) from several geographic areas of West and North Spain. The presence of Leishmania kDNA was detected in 14 of the 68 analyzed samples, being the highest quantity of DNA observed in foxes. This is the first report of the presence of Leishmania in a hedgehog. The kDNA remained stable under the exposure of hair to different environmental conditions (freezing or high temperature, ultraviolet rays or treatment with tanning salts). This detection method could constitute a suitable alternative for the search of the parasite in wild hosts, due to the numerous advantages that hair samples present for collection, transport and storage processes.

Detection and chronology of parasitic kinetoplast DNA presence in hair of experimental Leishmania major infected BALB/c mice by Real Time PCR.

Hair can accumulate foreign chemical or biological substances. Recently, it has been reported that parasite DNA can also be detected in the hair of Leishmania infantum infected dogs. The aim of this work has been to find out whether parasite DNA incorporates in the hair of Leishmania major experimentally infected animals. For this purpose, a group of 4 BALB/c mice, intradermally inoculated in both ears with 1000 L. major V1 strain promastigote forms, was monitored for parameters associated to the infection during 35 days. Weekly, ear swelling was measured, and hair samples from ears and leg were collected. Blood samples were obtained before challenge and at day 35 post infection, when parasite load was measured in ear, lymph node and spleen by limit dilution. Ear swelling and other parameters observed in the infected mice were consistent with those described for this model. The presence of parasite kinetoplast DNA (kDNA) was detected by Real Time PCR in all ear and leg hair samples at the final timepoint. These data suggests that hair is a specialized tissue in the sequestration and removal of foreign DNA. Detection of DNA in hair could be, therefore, a useful tool to chronologically record the infection process during experimental mice assays.

Arginase I induction during Leishmania major infection mediates the development of disease.

In a previous work, we demonstrated that the induction of arginase I favored the replication of Leishmania inside macrophages. Now we have analyzed the differential expression of this enzyme in the mouse model of L. major infection. Ours results show that arginase I is induced in both susceptible and resistant mice during the development of the disease. However, in BALB/c-infected tissues, the induction of this protein parallels the time of infection, while in C57BL/6 mice, the enzyme is upregulated only during footpad swelling. The induction of the host arginase in both strains is mediated by the balance between interleukin-4 (IL-4) and IL-12 and opposite to nitric oxide synthase II expression. Moreover, inhibition of arginase reduces the number of parasites and delays disease outcome in BALB/c mice, while treatment with l-ornithine increases the susceptibility of C57BL/6 mice. Therefore, arginase I induction could be considered a marker of disease in leishmaniasis.