Electrical Stimulation for Immune Modulation in Cancer Treatments

Immunotherapy is becoming a very common treatment for cancer, using approaches like checkpoint inhibition, T cell transfer therapy, monoclonal antibodies and cancer vaccination. However, these approaches involve high doses of immune therapeutics with problematic side effects. A promising approach to reducing the dose of immunotherapeutic agents given to a cancer patient is to combine it with electrical stimulation, which can act in two ways; it can either modulate the immune system to produce the immune cytokines and agents in the patient’s body or it can increase the cellular uptake of these immune agents via electroporation. Electrical stimulation in form of direct current has been shown to reduce tumor sizes in immune-competent mice while having no effect on tumor sizes in immune-deficient mice. Several studies have used nano-pulsed electrical stimulations to activate the immune system and drive it against tumor cells. This approach has been utilized for different types of cancers, like fibrosarcoma, hepatocellular carcinoma, human papillomavirus etc. Another common approach is to combine electrochemotherapy with immune modulation, either by inducing immunogenic cell death or injecting immunostimulants that increase the effectiveness of the treatments. Several therapies utilize electroporation to deliver immunostimulants (like genes encoded with cytokine producing sequences, cancer specific antigens or fragments of anti-tumor toxins) more effectively. Lastly, electrical stimulation of the vagus nerve can trigger production and activation of anti-tumor immune cells and immune reactions. Hence, the use of electrical stimulation to modulate the immune system in different ways can be a promising approach to treat cancer.

Cell membrane-anchored and tumor-targeted IL-12 (attIL12)-T cell therapy for eliminating large and heterogeneous solid tumors

BackgroundAdoptive T-cell transfer has become an attractive therapeutic approach for hematological malignancies but shows poor activity against large and heterogeneous solid tumors. Interleukin-12 (IL-12) exhibits potent antitumor efficacy against solid tumors, but its clinical application has been stalled because of toxicity. Here, we aimed to develop a safe approach to IL-12 T-cell therapy for eliminating large solid tumors.MethodsWe generated a cell membrane-anchored IL-12 (aIL12), a tumor-targeted IL-12 (ttIL12), and a cell membrane-anchored and ttIL-12 (attIL12) and a cell membrane-anchored and tumor-targeted ttIL-12 (attIL12) armed T cells, chimeric antigen receptor-T cells, and T cell receptor-T (TCR-T) cells with each. We compared the safety and efficacy of these armed T cells in treating osteosarcoma patient-derived xenograft tumors and mouse melanoma tumors after intravenous infusions of the armed T cells.ResultsattIL12-T cell infusion showed remarkable antitumor efficacy in human and mouse large solid tumor models. Mechanistically, attIL12-T cells targeted tumor cells expressing cell-surface vimentin, enriching effector T cell and interferon γ production in tumors, which in turn stimulates dendritic cell maturation for activating secondary T-cell responses and tumor antigen spreading. Both attIL12- and aIL12-T-cell transfer eliminated peripheral cytokine release and the associated toxic effects.ConclusionsThis novel approach sheds light on the safe application of IL-12-based T-cell therapy for large and heterogeneous solid tumors.

Ubiquitin Activating Enzyme UBA6 Regulates Th1 and Tc1 Cell Differentiation

Ubiquitination is a crucial mechanism in regulating the immune response, setting the balance between immunity and tolerance. Here, we investigated the function of a poorly understood alternative branch of the ubiquitin-activating E1 enzyme UBA6 in activating immune cells. UBA6 expression levels were elevated in T cells by toll-like receptor agonists and anti-CD3/28 antibody stimulation, but not in dendritic cells, macrophages, B cells, and natural killer cells. Additionally, we generated T cell-specific UBA6-deficient mice and found that UBA6-deficient CD4 and CD8 T cells elevated the production of interferon-gamma (IFN-γ). Moreover, the transfer of UBA6-deficient CD4 and CD8 T cells in RAG1-knockout mice exacerbated the development of multi-organ inflammation compared with control CD4 and CD8 T cell transfer. In human peripheral blood CD4 and CD8 T cells, basal levels of UBA6 in lupus patients presented much lower than those in healthy controls. Moreover, the IFN-γ production efficiency of CD4 and CD8 T cells was negatively correlated to UBA6 levels in patients with lupus. Finally, we found that the function of UBA6 was mediated by destabilization of IκBα degradation, thereby increasing NF-κB p65 activation in the T cells. Our study identifies UBA6 as a critical regulator of IFN-γ production in T cells by modulating the NF-κB p65 activation pathway.

Pro-Resolving Factors Released by Macrophages After Efferocytosis Promote Mucosal Wound Healing in Inflammatory Bowel Disease

Nonresolving inflammation is a critical driver of several chronic inflammatory diseases, including inflammatory bowel diseases (IBD). This unresolved inflammation may result from the persistence of an initiating stimulus or from the alteration of the resolution phase of inflammation. Elimination of apoptotic cells by macrophages (a process called efferocytosis) is a critical step in the resolution phase of inflammation. Efferocytosis participates in macrophage reprogramming and favors the release of numerous pro-resolving factors. These pro-resolving factors exert therapeutic effects in experimental autoimmune arthritis. Here, we propose to evaluate the efficacy of pro-resolving factors produced by macrophages after efferocytosis, a secretome called SuperMApo, in two IBD models, namely dextran sodium sulfate (DSS)-induced and T cell transfer-induced colitis. Reintroducing these pro-resolving factors was sufficient to decrease clinical, endoscopic and histological colitis scores in ongoing naive T cell-transfer-induced colitis and in DSS-induced colitis. Mouse primary fibroblasts isolated from the colon demonstrated enhanced healing properties in the presence of SuperMApo, as attested by their increased migratory, proliferative and contractive properties. This was confirmed by the use of human fibroblasts isolated from patients with IBD. Exposure of an intestinal epithelial cell (IEC) line to these pro-resolving factors increased their proliferative properties and IEC acquired the capacity to capture apoptotic cells. The improvement of wound healing properties induced by SuperMApo was confirmed in vivo in a biopsy forceps-wound colonic mucosa model. Further in vivo analysis in naive T cell transfer-induced colitis model demonstrated an improvement of intestinal barrier permeability after administration of SuperMApo, an intestinal cell proliferation and an increase of α-SMA expression by fibroblasts, as well as a reduction of the transcript coding for fibronectin (Fn1). Finally, we identified TGF-β, IGF-I and VEGF among SuperMApo as necessary to favor mucosal healing and confirmed their role both in vitro (using neutralizing antibodies) and in vivo by depleting these factors from efferocytic macrophage secretome using antibody-coated microbeads. These growth factors only explained some of the beneficial effects induced by factors released by efferocytic macrophages. Overall, the administration of pro-resolving factors released by efferocytic macrophages limits intestinal inflammation and enhance tissue repair, which represents an innovative treatment of IBD.

Carboxylesterase-1 assisted targeting of HDAC inhibitors to mononuclear myeloid cells in Inflammatory Bowel Disease

Background and Aims Histone deacetylase inhibitors (HDACi) exert potent anti-inflammatory effects. Because of the ubiquitous expression of HDACs, clinical utility of HDACi is limited by off-target effects. Esterase-sensitive motif (ESM) technology aims to deliver ESM-conjugated compounds to human mononuclear myeloid cells, based on their expression of carboxylesterase 1 (CES1). This study aims to investigate utility of an ESM-tagged HDACi in inflammatory bowel disease (IBD). Methods CES1 expression was assessed in human blood, in vitro differentiated macrophage and dendritic cells and Crohn's disease (CD) colon mucosa by mass cytometry, quantitative PCR and immunofluorescence staining respectively. ESM-HDAC528 intracellular retention was evaluated by mass spectrometry. Clinical efficacy of ESM-HDAC528 was tested in DSS-induced colitis and T cell transfer colitis models using transgenic mice expressing human CES1 under the CD68 promotor. Results CES1 mRNA was highly expressed in human blood CD14 + monocytes, in vitro differentiated and LPS stimulated macrophages and dendritic cells. Specific hydrolysis and intracellular retention of ESM-HDAC528 in CES1 + cells was demonstrated. ESM-HDAC528 inhibited LPS-stimulated IL-6 and TNF-α production 1000 times more potently than its control, HDAC800, in CES1 high monocytes. In healthy donors peripheral blood, CES1 expression was significantly higher in CD14 ++CD16 - monocytes compared to CD14 +CD16 ++ monocytes. In CD inflamed colon, a higher number of mucosal CD68 + macrophages expressed CES1 compared to non-inflamed mucosa. In vivo, ESM-HDAC528 reduced monocyte differentiation in the colon and significantly improved colitis in a T cell transfer model, whilst having limited potential in ameliorating DSS-induced colitis. Conclusions We demonstrate that monocytes and inflammatory macrophages specifically express CES1, and can be preferentially targeted by ESM-HDAC528 to achieve therapeutic benefit in IBD.

Intracolonic Neuropeptide Y Y1 Receptor Inhibition Attenuates Intestinal Inflammation in Murine Colitis and Cytokine Release in IBD Biopsies

We have demonstrated that neuropeptide Y (NPY) can regulate pro-inflammatory signaling in the gut via cross-talk with the pro-inflammatory cytokine tumor necrosis factor (TNF). Here, we investigated if selective blocking of NPY receptors, NPY1R or NPY2R, using small molecule non-peptide antagonists (BIBP-3222 for NPY1R and BIIE-0246 for NPY2R) in the colon could attenuate intestinal inflammation by lowering TNF levels (BIBP - N-[(1R)]-4-[(Aminoiminomethyl)amino-1-[[[(4-hydroxyphenyl)methyl]amino]carbonyl]butyl-α-phenylbenzeneacetamide; BIIE - N-[(1S)-4-[(Aminoiminomethyl)amino]-1-[[[2-(3,5-dioxo-1,2-diphenyl-1,2,4-triazolidin-4-yl)ethyl]amino]carbonyl]butyl]-1-[2-[4-(6,11-dihydro-6-oxo-5H-dibenz[b,e]azepin-11-yl)-1-piperazinyl]-2-oxoethyl]-cyclopentaneacetamide). Colitis was induced using dextran sodium sulfate in drinking water for 7 days, or by adoptive T-cell transfer in RAG-/- mice. Colonic biopsies from healthy subjects (n = 10) and IBD patients (n = 34, UC = 20, CD = 14) were cultured ex vivo in presence or absence of NPY antagonists (100 µM, 20 h), and cytokine release into culture supernatants was measured by ELISA. Intracolonic administration of BIBP (but not BIIE) significantly reduced clinical, endoscopic, and histological scores, and serum TNF, interleukin (IL)-6, and IL-12p70 in DSS colitis; it also significantly attenuated histological damage and serum IL-6 in T-cell colitis (P < .05). Intracolonic administration of BIBP significantly reduced TNF and interferon (IFN)-γ release from UC biopsies, whereas BIIE downregulated only IFN-γ (P < .05). BIBP significantly reduced TNF and interferon (IFN)-γ release from UC biopsies, whereas BIIE downregulated only IFN-γ (P < .05). Our data suggest a promising therapeutic value for NPY1R inhibition in alleviating intestinal inflammation in UC, possibly as enemas to IBD patients.

The Microenvironment of Small Intestinal Neuroendocrine Tumours Contains Lymphocytes Capable of Recognition and Activation after Expansion

Traditionally, immune evasion and immunotherapy have been studied in cancers with a high mutational load such as melanoma or lung cancer. In contrast, small intestinal neuroendocrine tumours (SINETs) present a low frequency of somatic mutations and are described as genetically stable tumours, rendering immunotherapies largely unchartered waters for SINET patients. SINETs frequently metastasise to the regional lymph nodes and liver at the time of diagnosis, and no curative treatments are currently available for patients with disseminated disease. Here, we characterised the immune landscape of SINET and demonstrated that tumour-infiltrating lymphocytes (TILs) can be expanded and activated during autologous tumour challenge. The composition of lymphocyte subsets was determined by immunophenotyping of the SINET microenvironment in one hepatic and six lymph node metastases. TILs from these metastases were successfully grown out, enabling immunophenotyping and assessment of PD-1 expression. Expansion of the TILs and exposure to autologous tumour cells in vitro resulted in increased T lymphocyte degranulation. This study provides insights into the largely unknown SINET immune landscape and reveals the anti-tumour reactivity of TILs, which might merit adoptive T cell transfer as a feasible treatment option for patients with SINET.

Identification and Targeting of Mutant Peptide Neoantigens in Cancer Immunotherapy

In recent decades, adoptive cell transfer and checkpoint blockade therapies have revolutionized immunotherapeutic approaches to cancer treatment. Advances in whole exome/genome sequencing and bioinformatic detection of tumour-specific genetic variations and the amino acid sequence alterations they induce have revealed that T cell mediated anti-tumour immunity is substantially directed at mutated peptide sequences, and the identification and therapeutic targeting of patient-specific mutated peptide antigens now represents an exciting and rapidly progressing frontier of personalized medicine in the treatment of cancer. This review outlines the historical identification and validation of mutated peptide neoantigens as a target of the immune system, and the technical development of bioinformatic and experimental strategies for detecting, confirming and prioritizing both patient-specific or “private” and frequently occurring, shared “public” neoantigenic targets. Further, we examine the range of therapeutic modalities that have demonstrated preclinical and clinical anti-tumour efficacy through specifically targeting neoantigens, including adoptive T cell transfer, checkpoint blockade and neoantigen vaccination.