Intratumoral delivery of immune therapy and gene therapy: the next era for cancer therapy.

Cancer immunotherapy is a field that garners significant interest, fueled by the clinical success of immune checkpoint inhibitors. In contrast to conventional cancer therapies, immunotherapies leverage the host's immune system by enhancing innate and adaptive immunity to control cancer progression. Despite these exciting advances, only a subset of patients respond to these drugs, and immunotherapies frequently result in immune-related toxicity.

Amplification of the CXCR3/CXCL9 axis via intratumoral electroporation of plasmid CXCL9 synergizes with plasmid IL-12 therapy to elicit robust anti-tumor immunity.

Clinical studies have demonstrated that local expression of the cytokine IL-12 drives interferon-gamma expression and recruits T cells to the tumor microenvironment, ultimately yielding durable systemic T cell responses. Interrogation of longitudinal biomarker data from our late-stage melanoma trials identified a significant on-treatment increase of intratumoral transcripts that was restricted to responding patients, underscoring the clinical relevance of tumor-infiltrating CXCR3 immune cells. In this study, we sought to understand if the addition of DNA-encodable CXCL9 could augment the anti-tumor immune responses driven by intratumoral IL-12.

Intratumoral Electroporation of Plasmid Encoded IL12 and Membrane-Anchored Anti-CD3 Increases Systemic Tumor Immunity.

Unlabelled: Intratumoral delivery of plasmid IL12 via electroporation (IT-tavo-EP) induces localized expression of IL12 leading to regression of treated and distant tumors with durable responses and minimal toxicity. A key driver in amplifying this local therapy into a systemic response is the magnitude and composition of immune infiltrate in the treated tumor. While intratumoral IL12 typically increases the density of CD3+ tumor-infiltrating lymphocytes (TIL), this infiltrate is composed of a broad range of T-cell subsets, including activated tumor-specific T cells, less functional bystander T cells, as well as suppressive T regulatory cells.

Intratumoral Plasmid IL12 Expands CD8 T Cells and Induces a CXCR3 Gene Signature in Triple-negative Breast Tumors that Sensitizes Patients to Anti-PD-1 Therapy.

Purpose: Triple-negative breast cancer (TNBC) is an aggressive disease with limited therapeutic options. Antibodies targeting programmed cell death protein 1 (PD-1)/PD-1 ligand 1 (PD-L1) have entered the therapeutic landscape in TNBC, but only a minority of patients benefit. A way to reliably enhance immunogenicity, T-cell infiltration, and predict responsiveness is critically needed.

Phase II Trial of IL-12 Plasmid Transfection and PD-1 Blockade in Immunologically Quiescent Melanoma.

Purpose: Tumors with low frequencies of checkpoint positive tumor-infiltrating lymphocytes (cpTIL) have a low likelihood of response to PD-1 blockade. We conducted a prospective multicenter phase II trial of intratumoral plasmid IL-12 (tavokinogene telseplasmid; "tavo") electroporation combined with pembrolizumab in patients with advanced melanoma with low frequencies of checkpoint positive cytotoxic lymphocytes (cpCTL).

Patients And Methods: Tavo was administered intratumorally days 1, 5, and 8 every 6 weeks while pembrolizumab (200 mg, i.

Characterization of abscopal effects of intratumoral electroporation-mediated IL-12 gene therapy.

Intratumoral electroporation-mediated IL-12 gene therapy (IT-pIL12/EP) has been shown to be safe and effective in clinical trials, demonstrating systemic antitumor effects with local delivery of this potent cytokine. We recently optimized our IL-12 gene delivery platform to increase transgene expression and efficacy in preclinical models. Here we analyze the immunological changes induced with the new IT-pIL12/EP platform in both electroporated and distant, non-electroporated lesions.

Development of an adaptive electroporation system for intratumoral plasmid DNA delivery.

Intratumoral electroporation of plasmid DNA encoding the proinflammatory cytokine interleukin 12 promotes innate and adaptive immune responses correlating with anti-tumor effects. Clinical electroporation conditions are fixed parameters optimized in preclinical tumors, which consist of cells implanted into skin. These conditions have little translatability to clinically relevant tumors, as implanted models cannot capture the heterogeneity encountered in genetically engineered mouse models or clinical tumors.

Melanoma treatment with intratumoral electroporation of tavokinogene telseplasmid (pIL-12, tavokinogene telseplasmid).

Tumors evade detection and/or clearance by the immune system via multiple mechanisms. IL-12 is a potent immunomodulatory cytokine that plays a central role in immune priming. However, systemic delivery of IL-12 can result in life-threatening toxicity and therefore has shown limited efficacy at doses that can be safely administered.

ST-11: A New Brain-Penetrant Microtubule-Destabilizing Agent with Therapeutic Potential for Glioblastoma Multiforme.

Glioblastoma multiforme is a devastating and intractable type of cancer. Current antineoplastic drugs do not improve the median survival of patients diagnosed with glioblastoma multiforme beyond 14 to 15 months, in part because the blood-brain barrier is generally impermeable to many therapeutic agents. Drugs that target microtubules (MT) have shown remarkable efficacy in a variety of cancers, yet their use as glioblastoma multiforme treatments has also been hindered by the scarcity of brain-penetrant MT-targeting compounds.

A mitotic kinase scaffold depleted in testicular seminomas impacts spindle orientation in germ line stem cells.

Correct orientation of the mitotic spindle in stem cells underlies organogenesis. Spindle abnormalities correlate with cancer progression in germ line-derived tumors. We discover a macromolecular complex between the scaffolding protein Gravin/AKAP12 and the mitotic kinases, Aurora A and Plk1, that is down regulated in human seminoma.

Gravin orchestrates protein kinase A and β2-adrenergic receptor signaling critical for synaptic plasticity and memory.

A kinase-anchoring proteins (AKAPs) organize compartmentalized pools of protein kinase A (PKA) to enable localized signaling events within neurons. However, it is unclear which of the many expressed AKAPs in neurons target PKA to signaling complexes important for long-lasting forms of synaptic plasticity and memory storage. In the forebrain, the anchoring protein gravin recruits a signaling complex containing PKA, PKC, calmodulin, and PDE4D (phosphodiesterase 4D) to the β2-adrenergic receptor.

Gravin is a transitory effector of polo-like kinase 1 during cell division.

The mitogenic and second-messenger signals that promote cell proliferation often proceed through multienzyme complexes. The kinase-anchoring protein Gravin integrates cAMP and calcium/phospholipid signals at the plasma membrane by sequestering protein kinases A and C with G protein-coupled receptors. In this report we define a role for Gravin as a temporal organizer of phosphorylation-dependent protein-protein interactions during mitosis.

Chk-ing in and Chk-ing out: kinase compartmentalization comes to checkpoint control.

Checkpoints are the sentinels of cell-cycle progression. In this issue of Molecular Cell, Yaffe and colleagues (Reinhardt et al., 2010) show that spatial and temporal resolution of Chk1 and MK2, checkpoint kinases with identical substrate specificity, are necessary to signal different aspects of DNA damage signaling.

Spatial distribution of protein kinase A activity during cell migration is mediated by A-kinase anchoring protein AKAP Lbc.

Protein kinase A (PKA) has been suggested to be spatially regulated in migrating cells due to its ability to control signaling events that are critical for polarized actin cytoskeletal dynamics. Here, using the fluorescence resonance energy transfer-based A-kinase activity reporter (AKAR1), we find that PKA activity gradients form with the strongest activity at the leading edge and are restricted to the basal surface in migrating cells. The existence of these gradients was confirmed using immunocytochemistry using phospho-PKA substrate antibodies.

The role of CKIP-1 in cell morphology depends on its interaction with actin-capping protein.

CKIP-1 is a pleckstrin homology domain-containing protein that induces alterations of the actin cytoskeleton and cell morphology when expressed in human osteosarcoma cells. CKIP-1 interacts with the heterodimeric actin-capping protein in cells, so we postulated that this interaction was responsible for the observed cytoskeletal and morphological effects of CKIP-1. To test this postulate, we used peptide "walking arrays" and alignments of CKIP-1 with CARMIL, another CP-binding protein, to identify Arg-155 and Arg-157 of CKIP-1 as residues potentially required for its interactions with CP.

The shape of things to come: an emerging role for protein kinase CK2 in the regulation of cell morphology and the cytoskeleton.

Protein kinase CK2 is a highly conserved, pleiotropic, protein serine/threonine kinase that is essential for life in eukaryotes. CK2 has been implicated in diverse cellular processes such as cell cycle regulation, circadian rhythms, apoptosis, transformation and tumorigenesis. In addition, there is increasing evidence that CK2 is involved in the maintenance of cell morphology and cell polarity, and in the regulation of the actin and tubulin cytoskeletons.

The pleckstrin homology domain-containing protein CKIP-1 is involved in regulation of cell morphology and the actin cytoskeleton and interaction with actin capping protein.

CKIP-1 is a pleckstrin homology domain-containing protein that interacts with protein kinase CK2. To elucidate the functions of CKIP-1, we generated human osteosarcoma cell lines with tetracycline-regulated expression of Flag-CKIP-1. Flag-CKIP-1 expression resulted in distinct changes in cellular morphology.

The Pleckstrin homology domain of CK2 interacting protein-1 is required for interactions and recruitment of protein kinase CK2 to the plasma membrane.

CKIP-1 is a recently identified interaction partner of protein kinase CK2 with a number of protein-protein interaction motifs, including an N-terminal pleckstrin homology domain. To test the hypothesis that CKIP-1 has a role in targeting CK2 to specific locations, we examined the effects of CKIP-1 on the localization of CK2. These studies demonstrated that CKIP-1 can recruit CK2 to the plasma membrane.

Phosphorylation regulates the stability of the regulatory CK2beta subunit.

Protein kinase CK2 is a protein serine/threonine kinase that exhibits elevated expression in a number of cancers and displays oncogenic activity in mice. The regulatory CK2beta subunit has a central role in assembly of functional tetrameric CK2 complexes where it participates in modulation of catalytic activity and substrate specificity. Since overexpression of CK2beta results in elevated levels of CK2 activity, we investigated the molecular mechanisms that control its degradation since perturbations in these pathways could contribute to elevated CK2 in cancer.