Epidermal growth factor receptor peptide vaccination induces cross-reactive immunity to human EGFR, HER2, and HER3.

Current treatments for tumors expressing epidermal growth factor receptor (EGFR) include anti-EGFR monoclonal antibodies, often used in conjunction with the standard chemotherapy, radiation therapy, or other EGFR inhibitors. While monoclonal antibody treatment is efficacious in many patients, drawbacks include its high cost of treatment and side effects associated with multiple drug infusions. As an alternative to monoclonal antibody treatments, we have focused on peptide-based vaccination to trigger natural anti-tumor antibodies. Here, we demonstrate that peptides based on a region of the EGFR extracellular domain IV break immune tolerance to EGFR and elicit anti-tumor immunity. Mice immunized with isoforms of EGFR peptide p580-598 generated anti-EGFR antibody and T-cell responses. Iso-aspartyl (iso-Asp)-modified EGFR p580 immune sera inhibit in vitro growth of EGFR overexpressing human A431 tumor cells, as well as promote antibody-dependent cell-mediated cytotoxicity (ADCC). Antibodies induced by Asp and iso-Asp p580 bound homologous regions of the EGFR family members HER2 and HER3. EGFR p580 immune sera also inhibited the growth of the human tumor cell line MDA-MB-453 that expresses HER2 but not EGFR. Asp and iso-Asp EGFR p580 induced antibodies were also able to inhibit the in vivo growth of EGFR-expressing tumors. These data demonstrate that EGFR peptides from a region of the EGFR extracellular domain IV promote anti-tumor immunity, tumor cell killing, and antibodies that are cross reactive with ErbB family members.

Long-term seizure suppression and optogenetic analyses of synaptic connectivity in epileptic mice with hippocampal grafts of GABAergic interneurons.

Studies in rodent epilepsy models suggest that GABAergic interneuron progenitor grafts can reduce hyperexcitability and seizures in temporal lobe epilepsy (TLE). Although integration of the transplanted cells has been proposed as the underlying mechanism for these disease-modifying effects, prior studies have not explicitly examined cell types and synaptic mechanisms for long-term seizure suppression. To address this gap, we transplanted medial ganglionic eminence (MGE) cells from embryonic day 13.5 VGAT-Venus or VGAT-ChR2-EYFP transgenic embryos into the dentate gyrus (DG) of adult mice 2 weeks after induction of TLE with pilocarpine. Beginning 3-4 weeks after status epilepticus, we conducted continuous video-electroencephalographic recording until 90-100 d. TLE mice with bilateral MGE cell grafts in the DG had significantly fewer and milder electrographic seizures, compared with TLE controls. Immunohistochemical studies showed that the transplants contained multiple neuropeptide or calcium-binding protein-expressing interneuron types and these cells established dense terminal arborizations onto the somas, apical dendrites, and axon initial segments of dentate granule cells (GCs). A majority of the synaptic terminals formed by the transplanted cells were apposed to large postsynaptic clusters of gephyrin, indicative of mature inhibitory synaptic complexes. Functionality of these new inhibitory synapses was demonstrated by optogenetically activating VGAT-ChR2-EYFP-expressing transplanted neurons, which generated robust hyperpolarizations in GCs. These findings suggest that fetal GABAergic interneuron grafts may suppress pharmacoresistant seizures by enhancing synaptic inhibition in DG neural circuits.

Differentiation and functional incorporation of embryonic stem cell-derived GABAergic interneurons in the dentate gyrus of mice with temporal lobe epilepsy.

Cell therapies for neurological disorders require an extensive knowledge of disease-associated neuropathology and procedures for generating neurons for transplantation. In many patients with severe acquired temporal lobe epilepsy (TLE), the dentate gyrus exhibits sclerosis and GABAergic interneuron degeneration. Mounting evidence suggests that therapeutic benefits can be obtained by transplanting fetal GABAergic progenitors into the dentate gyrus in rodents with TLE, but the scarcity of human fetal cells limits applicability in patient populations. In contrast, virtually limitless quantities of neural progenitors can be obtained from embryonic stem (ES) cells. ES cell-based therapies for neurological repair in TLE require evidence that the transplanted neurons integrate functionally and replace cell types that degenerate. To address these issues, we transplanted mouse ES cell-derived neural progenitors (ESNPs) with ventral forebrain identities into the hilus of the dentate gyrus of mice with TLE and evaluated graft differentiation, mossy fiber sprouting, cellular morphology, and electrophysiological properties of the transplanted neurons. In addition, we compared electrophysiological properties of the transplanted neurons with endogenous hilar interneurons in mice without TLE. The majority of transplanted ESNPs differentiated into GABAergic interneuron subtypes expressing calcium-binding proteins parvalbumin, calbindin, or calretinin. Global suppression of mossy fiber sprouting was not observed; however, ESNP-derived neurons formed dense axonal arborizations in the inner molecular layer and throughout the hilus. Whole-cell hippocampal slice electrophysiological recordings and morphological analyses of the transplanted neurons identified five basic types; most with strong after-hyperpolarizations and smooth or sparsely spiny dendritic morphologies resembling endogenous hippocampal interneurons. Moreover, intracellular recordings of spontaneous EPSCs indicated that the new cells functionally integrate into epileptic hippocampal circuitry.

Cell-Selective Adeno-Associated Virus-Mediated SCN1A Gene Regulation Therapy Rescues Mortality and Seizure Phenotypes in a Dravet Syndrome Mouse Model and Is Well Tolerated in Nonhuman Primates.

Dravet syndrome (DS) is a developmental and epileptic encephalopathy caused by monoallelic loss-of-function variants in the SCN1A gene. SCN1A encodes for the alpha subunit of the voltage-gated type I sodium channel (NaV1.1), the primary voltage-gated sodium channel responsible for generation of action potentials in GABAergic inhibitory interneurons. In these studies, we tested the efficacy of an adeno-associated virus serotype 9 (AAV9) SCN1A gene regulation therapy, AAV9-REGABA-eTFSCN1A, designed to target transgene expression to GABAergic inhibitory neurons and reduce off-target expression within excitatory cells, in the Scn1a+/- mouse model of DS. Biodistribution and preliminary safety were evaluated in nonhuman primates (NHPs). AAV9-REGABA-eTFSCN1A was engineered to upregulate SCN1A expression levels within GABAergic inhibitory interneurons to correct the underlying haploinsufficiency and circuit dysfunction. A single bilateral intracerebroventricular (ICV) injection of AAV9-REGABA-eTFSCN1A in Scn1a+/- postnatal day 1 mice led to increased SCN1A mRNA transcripts, specifically within GABAergic inhibitory interneurons, and NaV1.1 protein levels in the brain. This was associated with a significant decrease in the occurrence of spontaneous and hyperthermia-induced seizures, and prolonged survival for over a year. In NHPs, delivery of AAV9-REGABA-eTFSCN1A by unilateral ICV injection led to widespread vector biodistribution and transgene expression throughout the brain, including key structures involved in epilepsy and cognitive behaviors, such as hippocampus and cortex. AAV9-REGABA-eTFSCN1A was well tolerated, with no adverse events during administration, no detectable changes in clinical observations, no adverse findings in histopathology, and no dorsal root ganglion-related toxicity. Our results support the clinical development of AAV9-REGABA-eTFSCN1A (ETX101) as an effective and targeted disease-modifying approach to SCN1A+ DS.