Targeted adenovirus-mediated transduction of human T cells in vitro and in vivo

Clinical success in T cell therapy has stimulated widespread efforts to increase safety and potency and to extend this technology to solid tumors. Yet progress in cell therapy remains restricted by the limited payload capacity, specificity of target cell transduction, and transgenic gene expression efficiency of applied viral vectors. This renders complex reprogramming or direct in vivo applications difficult. Here, we developed a synergistic combination of trimeric adapter constructs enabling T cell-directed transduction by the human adenoviral vector serotype C5 in vitro and in vivo. Rationally chosen binding partners showed receptor-specific transduction of otherwise non-susceptible human T cells by exploiting activation stimuli. This platform remains compatible with high-capacity vectors for up to 37 kb DNA delivery, increasing payload capacity and safety because of the removal of all viral genes. Together, these findings provide a tool for targeted delivery of large payloads in T cells as a potential avenue to overcome current limitations of T cell therapy

NK cells with tissue-resident traits shape response to immunotherapy by inducing adaptive antitumor immunity

T cell-directed cancer immunotherapy often fails to generate lasting tumor control. Harnessing additional effectors of the immune response against tumors may strengthen the clinical benefit of immunotherapies. Here, we demonstrate that therapeutic targeting of the interferon-γ (IFN-γ)-interleukin-12 (IL-12) pathway relies on the ability of a population of natural killer (NK) cells with tissue-resident traits to orchestrate an antitumor microenvironment. In particular, we used an engineered adenoviral platform as a tool for intratumoral IL-12 immunotherapy (AdV5-IL-12) to generate adaptive antitumor immunity. Mechanistically, we demonstrate that AdV5-IL-12 is capable of inducing the expression of CC-chemokine ligand 5 (CCL5) in CD49a+ NK cells both in tumor mouse models and tumor specimens from patients with cancer. AdV5-IL-12 imposed CCL5-induced type I conventional dendritic cell (cDC1) infiltration and thus increased DC-CD8 T cell interactions. A similar observation was made for other IFN-γ-inducing therapies such as Programmed cell death 1 (PD-1) blockade. Conversely, failure to respond to IL-12 and PD-1 blockade in tumor models with low CD49a+ CXCR6+ NK cell infiltration could be overcome by intratumoral delivery of CCL5. Thus, therapeutic efficacy depends on the abundance of NK cells with tissue-resident traits and, specifically, their capacity to produce the DC chemoattractant CCL5. Our findings reveal a barrier for T cell-focused therapies and offer mechanistic insights into how T cell-NK cell-DC cross-talk can be enhanced to promote antitumor immunity and overcome resistance

NK cells with tissue-resident traits shape response to immunotherapy by inducing adaptive antitumor immunity

T cell-directed cancer immunotherapy often fails to generate lasting tumor control. Harnessing additional effectors of the immune response against tumors may strengthen the clinical benefit of immunotherapies. Here, we demonstrate that therapeutic targeting of the interferon-γ (IFN-γ)-interleukin-12 (IL-12) pathway relies on the ability of a population of natural killer (NK) cells with tissue-resident traits to orchestrate an antitumor microenvironment. In particular, we used an engineered adenoviral platform as a tool for intratumoral IL-12 immunotherapy (AdV5-IL-12) to generate adaptive antitumor immunity. Mechanistically, we demonstrate that AdV5-IL-12 is capable of inducing the expression of CC-chemokine ligand 5 (CCL5) in CD49a; +; NK cells both in tumor mouse models and tumor specimens from patients with cancer. AdV5-IL-12 imposed CCL5-induced type I conventional dendritic cell (cDC1) infiltration and thus increased DC-CD8 T cell interactions. A similar observation was made for other IFN-γ-inducing therapies such as Programmed cell death 1 (PD-1) blockade. Conversely, failure to respond to IL-12 and PD-1 blockade in tumor models with low CD49a; +; CXCR6; +; NK cell infiltration could be overcome by intratumoral delivery of CCL5. Thus, therapeutic efficacy depends on the abundance of NK cells with tissue-resident traits and, specifically, their capacity to produce the DC chemoattractant CCL5. Our findings reveal a barrier for T cell-focused therapies and offer mechanistic insights into how T cell-NK cell-DC cross-talk can be enhanced to promote antitumor immunity and overcome resistance

Malignant tissues produce divergent antibody glycosylation of relevance for cancer gene therapy effectiveness

Gene therapy approaches now allow for the production of therapeutic antibodies by healthy or cancerous human tissues directly in vivo, and, with an increasing number of gene delivery methods available, the cell type for expression can be chosen. Yet, little is known about the biophysical changes introduced by expressing antibodies from producer cells or tissues targeted by gene therapy approaches, nor about the consequences for the type of glycosylation. The effects of different glycosylation on therapeutic antibodies have been well studied by controlling their glycan compositions in non-human mammalian production cells, i.e., Chinese hamster ovary cells. Therefore, we investigated the glycosylation state of clinically approved antibodies secreted from cancer tissues frequently targeted by in vivo gene therapy, using native mass spectrometry and glycoproteomics. We found that antibody sialylation and fucosylation depended on the producer tissue and the antibody isotype, allowing us to identify optimal producer cell types according to the desired mode of action of the antibody. Furthermore, we discovered that high amounts (>20%) of non-glycosylated antibodies were produced in cells sensitive to the action of the produced antibodies. Different glycosylation in different producer cells can translate into an altered potency of in-vivo produced antibodies, depending on the desired mode of action, and can affect their serum half-lives. These results increase our knowledge about antibodies produced from cells targeted by gene therapy, enabling development of improved cancer gene therapy vectors that can include in vivo glycoengineering of expressed antibodies to optimize their efficacies, depending on the desired mode of action

Altered microvascular density in patients with systemic lupus erythematosus treated with hydroxychloroquine—an optical coherence tomography angiography study

Purpose!#!To evaluate the retinal microvascular density using optical coherence tomography angiography (OCTA) in patients with systemic lupus erythematosus (SLE) treated with hydroxychloroquine (HCQ).!##!Methods!#!Nineteen eyes of 19 patients with SLE (study group) without HCQ retinopathy and 19 eyes of 19 healthy subjects (control group) were included in this study. The study group was divided into patients using HCQ for > 5 years (high-risk group) and < 5 years (low-risk group). The VD data of the 3 × 3 mm OCT angiogram of the superficial capillary plexus (SCP) and the choriocapillaris (VD-CC), the foveal avascular zone (FAZ) area and the central retinal thickness (CRT) were extracted and analyzed.!##!Results!#!VD in the en face SCP was significantly reduced in the high-risk group and the low-risk group compared with that in the control group (p < 0.001, p = 0.001) and in the high-risk group compared with the low-risk group (p = 0.007). Correlation analysis between the cumulative dose of HCQ and the VD of the study group revealed a negative correlation, but no statistical significance (p = 0.074). However, a significant positive correlation was observed for the low-risk group (p = 0.035). In patients with SLE, VD-CC was lower (p = 0.042) and the FAZ area larger (p = 0.019). CRT showed no difference between the groups (p = 0.183).!##!Conclusion!#!In this study, SLE patients showed a reduced VD in both groups. In patients treated with HCQ < 5 years, HCQ might have a protective effect on retinal microvasculature. Analysis of retinal microvascular density using OCTA could be useful in the diagnosis and monitoring of vascular alteration in patients with SLE

Modular Adapters Utilizing Binders of Different Molecular Types Expand Cell-Targeting Options for Adenovirus Gene Delivery

Efficient and cell-specific delivery of DNA is essential for the effective and safe use of gene delivery technologies. Consequently, a large variety of technologies have been developed and applied in a wide range of $ex vivo$ and $in vivo$ applications, including multiple approaches based on viral vectors. However, widespread success of a technology is largely determined by the versatility of the method and the ease of use. The rationally designed adapter technology previously developed redirects widely used human adenovirus serotype 5 (HAdV-C5) to a defined cell population, by binding and blocking the adenoviral knob tropism while simultaneously allowing fusions of an N-terminal retargeting module. Here we expand modularity, and thus applicability of this adapter technology, by extending the nature of the cell-binding portion. We report successful receptor-specific transduction mediated by a retargeting module consisting of either a DARPin, a single-chain variable fragment (scFv) of an antibody, a peptide, or a small molecule ligand. Furthermore, we show that an adapter can be engineered to carry more than one specificity, allowing dual targeting. Specific HAdV-C5 retargeting was thus demonstrated to human epidermal growth factor receptor 2 (HER2), human folate receptor α, and neurotensin receptor 1, effective at vector concentrations as low as a multiplicity of infection of 2.5. Therefore, we report a modular design which allows plug-and-play combinations of different binding modules, leading to efficient and specific mono- or dual-targeting while circumventing tedious optimization procedures. This extends the technology to combinational applications of cell-specific binding, supporting research in gene therapy, synthetic biology, and biotechnology

iMATCH: an integrated modular assembly system for therapeutic combination high-capacity adenovirus gene therapy

Adenovirus-mediated combination gene therapies have shown promising results in vaccination or treating malignant and genetic diseases. Nevertheless, an efficient system for the rapid assembly and incorporation of therapeutic genes into high-capacity adenoviral vectors (HCAdVs) is still missing. In this study, we developed the iMATCH (integrated modular assembly for therapeutic combination HCAdVs) platform, which enables the generation and production of HCAdVs encoding therapeutic combinations in high quantity and purity within 3 weeks. Our modular cloning system facilitates the efficient combination of up to four expression cassettes and the rapid integration into HCAdV genomes with defined sizes. Helper viruses (HVs) and purification protocols were optimized to produce HCAdVs with distinct capsid modifications and unprecedented purity (0.1 ppm HVs). The constitution of HCAdVs, with adapters for targeting and a shield of trimerized single-chain variable fragment (scFv) for reduced liver clearance, mediated cell- and organ-specific targeting of HCAdVs. As proof of concept, we show that a single HCAdV encoding an anti PD-1 antibody, interleukin (IL)-12, and IL-2 produced all proteins, and it led to tumor regression and prolonged survival in tumor models, comparable to a mixture of single payload HCAdVs in vitro and in vivo. Therefore, the iMATCH system provides a versatile platform for the generation of high-capacity gene therapy vectors with a high potential for clinical development

The SHREAD gene therapy platform for paracrine delivery improves tumor localization and intratumoral effects of a clinical antibody

The goal of cancer-drug delivery is to achieve high levels of therapeutics within tumors with minimal systemic exposure that could cause toxicity. Producing biologics directly in situ where they diffuse and act locally is an attractive alternative to direct administration of recombinant therapeutics, as secretion by the tumor itself provides high local concentrations that act in a paracrine fashion continuously over an extended duration (paracrine delivery). We have engineered a SHielded, REtargeted ADenovirus (SHREAD) gene therapy platform that targets specific cells based on chosen surface markers and converts them into biofactories secreting therapeutics. In a proof of concept, a clinically approved antibody is delivered to orthotopic tumors in a model system in which precise biodistribution can be determined using tissue clearing with passive CLARITY technique (PACT) with high-resolution three-dimensional imaging and feature quantification within the tumors made transparent. We demonstrate high levels of tumor cell-specific transduction and significant and durable antibody production. PACT gives a localized quantification of the secreted therapeutic and allows us to directly observe enhanced pore formation in the tumor and destruction of the intact vasculature. In situ production of the antibody led to an 1,800-fold enhanced tumor-to-serum antibody concentration ratio compared to direct administration. Our detailed biochemical and microscopic analyses thus show that paracrine delivery with SHREAD could enable the use of highly potent therapeutic combinations, including those with systemic toxicity, to reach adequate therapeutic windows