magnetic-fluorescent-bead-based-dual-reporter-flow-analysis-pdl1-vaxx

Magnetic Fluorescent Bead-Based Dual-Reporter Flow Analysis of PDL1-Vaxx Peptide Vaccine-Induced Antibody Blockade of the PD-1/PD-L1 Interaction

The inhibition of checkpoint receptors (PD-1, PD-L1, and CTLA-4) with monoclonal antibodies has shown great benefit in clinical trials for treating cancer ...

The Ohio State University Wexner Medical Center

One of the major risk factors for asthma development is exposure to environmental allergens. House dust mites (HDM) can induce DNA damage, resulting in asthma. Resveratrol (RES) produced by several plants, has anti‑apoptotic properties and may affect a variety of biological processes. The aim of the present study was to investigate the protective role of RES against apoptosis in bronchial epithelial cells. C57BL/6J mice treated with HDM exhibited high levels of cell apoptosis, while RES significantly reversed this process. Induced DNA damage was more severe in the HDM group vs. the HDM combined with RES group. This result was confirmed by immunostaining and western blot analysis of the protein expression of the DNA damage‑related gene γH2AX, which was highly induced by HDM. In addition, treatment with RES protected bronchial epithelial cells exposed to HDM from DNA damage. RES decreases reactive oxygen species levels to inhibit oxidative DNA damage in bronchial epithelial cells. Furthermore, compared with the HDM group, induced cell apoptosis could be attenuated by RES in the group of combined treatment with RES and HDM. A DNA repair inhibitor augmented DNA damage and apoptosis in bronchial epithelial cells, whereas RES significantly attenuated cell apoptosis through inhibiting DNA damage.

Resveratrol decreases cell apoptosis through inhibiting DNA damage in bronchial epithelial cells.

Immunotherapy with monoclonal antibodies to checkpoint inhibitors against the PD-1/PD-L1 signaling pathway is a landmark achievement in cancer therapy. Some anti-PD-1 inhibitors such as nivolumab and pembrolizumab have shown clinical success, in a percentage of patients with prolonged survival rates. However, adverse effects accompany these benefits. In this case, strategies with lower toxicity and increased specificity are urgently required. Cancer vaccines have the ability to stimulate the native immune system and in particular, an engineered B-cell epitope can elicit high-affinity polyclonal antibodies with similar efficacy to PD-1 monoclonal antibodies in murine animal models. We have previously designed and synthesized a unique B-cell vaccine, PD1-Vaxx [MVF-PD-1(92-110)], and we have tested the immunogenicity and antitumor properties in CT26 colon cancer BALB/c syngeneic mice model. This manuscript provides results from comprehensive preclinical pharmacology studies encompassing primary and secondary pharmacodynamics, biodistribution, and safety studies. The results from these preclinical studies support the use of PD1-Vaxx in a first-in-human clinical trial in patients with non-small cell lung cancer (NSCLC). A phase I trial in patients with NSCLC has commenced.

Preclinical Studies of a Novel Human PD-1 B-Cell Peptide Cancer Vaccine PD1-Vaxx From BALB/c Mice to Beagle Dogs and to Non-Human Primates (Cynomolgus Monkeys).

Blockade of checkpoint receptors with monoclonal antibodies against CTLA-4, PD-1 and PD-L1 has shown great clinical success in several cancer subtypes, yielding unprecedented responses albeit a significant number of patients develop resistance and remain refractory. Both PD-1/PD-L1 and HER-2 signaling pathway inhibitors have limited efficacy and exhibits significant toxicities that limit their use. Ongoing clinical studies support the need for rationale combination of immuno-oncology agents to make a significant impact in the lives of cancer patients. We introduce the development of a novel chimeric PD-L1 B-cell peptide epitope vaccine (amino acid 130-147) linked to a "promiscuous" T cell measles virus fusion (MVF) peptide (MVF-PD-L1(130); PDL1-Vaxx) or linked to tetanus toxoid (TT3) TT3-PD-L1 (130) via a linker (GPSL). These vaccine constructs are highly immunogenic and antigenic in several syngeneic animal models. The PD-L1 vaccines elicited high titers of polyclonal antibodies that inhibit tumor growth in multiple syngeneic cancer models, eliciting antibodies of different subtypes IgG1, IgG2a, IgG2b and IgG3, induced PD-1/PD-L1 blockade, decreased proliferation, induced apoptosis and caused ADCC of tumor cells. The PDL1-Vaxx induces similar inhibition of tumor growth versus the standard anti-mouse PD-L1 antibody in both syngeneic BALB/c and C57BL/6J mouse models. The combination of PDL1-Vaxx with HER-2 vaccine B-Vaxx demonstrated synergistic tumor inhibition in D2F2/E2 carcinoma cell line. The anti-PDL1-Vaxx block PD-1/PD-L1 interaction and significantly prolonged anti-tumor responses in multiple syngeneic tumor models. The combination of HER-2 vaccine (B-Vaxx) with either PDL1-Vaxx or PD1-Vaxx demonstrated synergistic tumor inhibition. PDL1-Vaxx is a promising novel safe checkpoint inhibitor vaccine.

A newly discovered PD-L1 B-cell epitope peptide vaccine (PDL1-Vaxx) exhibits potent immune responses and effective anti-tumor immunity in multiple syngeneic mice models and (synergizes) in combination with a dual HER-2 B-cell vaccine (B-Vaxx).

The multifunctional protein, voltage-dependent anion channel 1 (VDAC1), is located on the mitochondrial outer membrane. It is a pivotal protein that maintains mitochondrial function to power cellular bioactivities via energy generation. VDAC1 is involved in regulating energy production, mitochondrial oxidase stress, Ca transportation, substance metabolism, apoptosis, mitochondrial autophagy (mitophagy), and many other functions. VDAC1 malfunction is associated with mitochondrial disorders that affect inflammatory responses, resulting in an up-regulation of the body's defensive response to stress stimulation. Overresponses to inflammation may cause chronic diseases. Mitochondrial DNA (mtDNA) acts as a danger signal that can further trigger native immune system activities after its secretion. VDAC1 mediates the release of mtDNA into the cytoplasm to enhance cytokine levels by activating immune responses. VDAC1 regulates mitochondrial Ca transportation, lipid metabolism and mitophagy, which are involved in inflammation-related disease pathogenesis. Many scientists have suggested approaches to deal with inflammation overresponse issues via specific targeting therapies. Due to the broad functionality of VDAC1, it may become a useful target for therapy in inflammation-related diseases. The mechanisms of VDAC1 and its role in inflammation require further exploration. We comprehensively and systematically summarized the role of VDAC1 in the inflammatory response, and hope that our research will lead to novel therapeutic strategies that target VDAC1 in order to treat inflammation-related disorders.

Mitochondrial VDAC1: A Potential Therapeutic Target of Inflammation-Related Diseases and Clinical Opportunities.

 Endoplasmic reticulum (ER) stress regulates mucus hypersecretion, and may activate downstream factors via TBK1 signaling to induce gene expression. However, it remains unclear whether ER stress promotes airway mucus secretion through the TBK1 pathway. We aimed to investigate the role of the TBK1 pathway in the regulation of MUC5AC expression in a mouse model of house dust mite (HDM)-induced allergic asthma.  Mice with HDM-induced asthma and human bronchial epithelial BEAS-2B cells were treated with amlexanox, an anti-allergy drug (25 μM), or 4-PBA (10 mM). Tissue and cell samples were collected. Tissue samples were stained with hematoxylin and eosin (H&E) or periodic acid Schiff (PAS) to evaluate pathology. Protein expression was analyzed by western blotting and immunofluorescence.  Mice exposed to HDM presented ER stress and hypersecretion of mucus Muc5ac from airway epithelial cells ( < 0.001). Similar results were observed in BEAS-2B cells following exposure to HDM. Both  and  studies revealed that HDM-induced ER stress induced MUC5AC overexpression via TBK1 signaling. Amlexanox and 4-PBA markedly reduced mucus production and weakened the TBK1 signal, which mediates MUC5AC hypersecretion.  TBK1 plays a pivotal role in HDM-induced ER stress, leading to overproduction of MUC5AC in the asthmatic airway epithelium. The overproduction of MUC5AC can be significantly decreased by inhibiting TBK1 or ER stress using 4-PBA. These findings highlight potential target-specific therapies for patients with chronic allergic asthma.

Linlin Guo

Department of Obstetrics & Gynecology

The Ohio State University Wexner Medical Center

Magnetic Fluorescent Bead-Based Dual-Reporter Flow Analysis of PDL1-Vaxx Peptide Vaccine-Induced Antibody Blockade of the PD-1/PD-L1 Interaction