T cell receptor (TCR) T cell therapy is a promising cancer treatment modality. However, its successful development for epithelial cancers may depend on the identification of high-avidity TCRs directed against tumor-restricted target antigens. The human papillomavirus (HPV) E7 antigen is an attractive therapeutic target that is constitutively expressed by HPV+ cancers but not by healthy tissues. It is unknown if genetically engineered TCR T cells that target E7 can mediate regression of HPV+ cancers. We identified an HPV-16 E7-specific, HLA-A*02:01-restricted TCR from a uterine cervix biopsy from a woman with cervical intraepithelial neoplasia. This TCR demonstrated high functional avidity, with CD8 coreceptor-independent tumor targeting. Human T cells transduced to express the TCR specifically recognized and killed HPV-16+ cervical and oropharyngeal cancer cell lines and mediated regression of established HPV-16+ human cervical cancer tumors in a mouse model. These findings support the therapeutic potential of this approach and established the basis for an E7 TCR gene therapy clinical trial in patients with metastatic HPV+ cancers (NCT02858310).

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Impairment of endothelial barrier function is implicated in many vascular and inflammatory disorders. One prevalent mechanism of endothelial dysfunction is an increase in reactive oxygen species under oxidative stress. Previous reports have demonstrated that hydrogen peroxide (H 2 O 2 ), a highly stable reactive oxygen species that modulates physiological signaling pathways, also enhances endothelial permeability, but the mechanism of this effect is unknown. Here, we identify the actin-binding protein myristoylated alanine-rich C-kinase substrate (MARCKS) as a key mediator of the H 2 O 2 -induced permeability change in bovine aortic endothelial cells. MARCKS knockdown and H 2 O 2 treatment alter the architecture of the actin cytoskeleton in endothelial cells, and H 2 O 2 induces the phosphorylation and translocation of MARCKS from the cell membrane to the cytosol. Using pharmacological inhibitors and small interference RNA constructs directed against specific proteins, we uncover a signaling cascade from Rac1 to Abl1, phospholipase Cγ1, and PKCδ that is triggered by H 2 O 2 and leads to MARCKS phosphorylation. Our findings establish a distinct role for MARCKS in the regulation of H 2 O 2 -induced permeability change in endothelial cells, and suggest potential new therapeutic targets for the treatment of disorders involving oxidative stress and altered endothelial permeability.

Sartoretto JL, Jin BY, Bauer M, Gertler FB, Liao R, Michel T. Regulation of VASP phosphorylation in cardiac myocytes: differential regulation by cyclic nucleotides and modulation of protein expression in diabetic and hypertrophic heart. Am J Physiol Heart Circ Physiol 297: H1697-H1710, 2009. First published September 4, 2009 doi:10.1152/ajpheart.00595.2009.-Vasodilator-stimulated phosphoprotein (VASP) is a major substrate for cyclic nucleotide-dependent kinases that has been implicated in cardiac pathology, yet many aspects of VASP's molecular regulation in cardiomyocytes are incompletely understood. In these studies, we explored the role of VASP, both in signaling pathways in isolated murine myocytes, as well as in a model of cardiac hypertrophy in VASP null mice. We found that the ␤-adrenergic agonist isoproterenol promotes the rapid and reversible phosphorylation of VASP at Ser157 and Ser239. Forskolin and the cAMP analog 8-(4-chlorophenylthio)-cAMP promote a similar pattern of VASP phosphorylation at both sites. The effects of isoproterenol are blocked by atenolol and by compound H-89, an inhibitor of the cAMP-dependent protein kinase. By contrast, phosphorylation of VASP only at Ser239 is seen following activation of particulate guanylate cyclase by atrial natriuretic peptide, or following activation of soluble guanylate cyclase by sodium nitroprusside, or following treatment of myocytes with cGMP analog. We found that basal and isoproterenol-induced VASP phosphorylation is entirely unchanged in cardiomyocytes isolated from either endothelial or neuronal nitric oxide synthase knockout mice. In cardiomyocytes isolated from diabetic mice, only basal VASP phosphorylation is increased, whereas, in cells isolated from mice subjected to ascending aortic constriction (AAC), we found a significant increase in basal VASP expression, along with an increase in VASP phosphorylation, compared with cardiac myocytes isolated from sham-operated mice. Moreover, there is further increase in VASP phosphorylation in cells isolated from hypertrophic hearts following isoproterenol treatment. Finally, we found that VASP null mice subjected to transverse aortic constriction develop cardiac hypertrophy with a pattern similar to VASP ϩ/ϩ mice. Our findings establish differential receptor-modulated regulation of VASP phosphorylation in cardiomyocytes by cyclic nucleotides. Furthermore, these studies demonstrate for the first time that VASP expression is upregulated in hypertrophied heart. signal transduction; endothelial nitric oxide synthase; neuronal nitric oxide synthase; phosphoprotein VASODILATOR-STIMULATED PHOSPHOPROTEIN (VASP) is the archetypal member of a family of actin-binding proteins termed the ENA/VASP protein family (34). This protein family includes the Drosophila protein Enabled (ENA), its mammalian homolog MENA, the ENA/VASP-like protein Evl, and VASP (13). MENA/VASP proteins are implicated in actin polymerization, and these proteins appear to modulate diverse cellular responses, including cell migration (26). VASP ...

mice; pooled data are also shown. * , P Ͻ 0.05; ** , P Ͻ 0.01; *** , P Ͻ 0.001.

T cell receptor (TCR) gene-engineered T cells have shown promise in the treatment of melanoma and synovial cell sarcoma, but their application to epithelial cancers has been limited. The identification of novel therapeutic TCRs for the targeting of these tumors is important for the development of new treatments. Here, we describe the preclinical characterization of a TCR directed against Kita-Kyushu Lung Cancer Antigen-1 (KK-LC-1, encoded by CT83 ), a cancer germline antigen with frequent expression in human epithelial malignancies including gastric cancer, breast cancer, and lung cancer. Gene-engineered T cells expressing the KK-LC-1 TCR (KK-LC-1 TCR-Ts) demonstrated recognition of CT83 + tumor lines in vitro and mediated regression of established CT83+ xenograft tumors in immunodeficient mouse models. Cross-reactivity studies based on experimental determination of the recognition motifs for the target epitope did not demonstrate cross-reactivity against other human proteins. CT83 gene expression studies in 51 non-neural tissues and 24 neural tissues showed expression restricted exclusively to germ cells. CT83 was however expressed by a range of epithelial cancers, with the highest expression noted in gastric cancer. Collectively, these findings support the further investigation and clinical testing of KK-LC-1 TCR-Ts for gastric cancer and possibly other malignancies. Electronic supplementary material The online version of this article (10.1186/s40425-019-0678-x) contains supplementary material, which is available to authorized users.

T cell receptor (TCR) gene therapy can mediate tumor regression in patients; however, successful treatment of epithelial cancers has been limited. We studied the targeting of Kita-Kyushu lung cancer antigen 1 (KK-LC-1), a cancer germline antigen expressed in multiple epithelial cancers, with TCR gene-engineered T cells. We thoroughly vetted KK-LC-1 expression in both cancerous and healthy tissue by pulling data from BioGPS and cBioPortal and analyzing a broad range of patient samples, primary cancer lines, and healthy tissue by both qRT-PCR and fluorescence in situ hybridization. Our findings show that KK-LC-1 is expressed in numerous epithelial cancers including breast, lung, cervical, ovarian, melanoma, and prostate whereas expression in healthy tissue is limited to immune-privileged sites including the epididymis and testis. We identified an HLA-A*01:01-restricted KK-LC-152-60-specific TCR isolated from the tumor infiltrating lymphocytes of a patient with metastatic cervical adenocarcinoma. T cells from two healthy donors were transduced with the KK-LC-1 TCR and tested for in vitro targeting of tumor cell lines. The KK-LC-1 TCR recognized the HLA-A*01:01+, KK-LC-1+ cell lines 4156, EKVX, and PC-3, as determined by IFN-γ release. The HLA-A*01:01- cell lines, HeLA, DU-145, and MDA-MD-468, were initially not recognized by the KK-LC-1 TCR; however, following stable transduction with HLA-A*01:01, these cell lines were targeted. The HLA-A*01:01+, KK-LC-1- cell lines, PC-3 and 3748, were not recognized by the KK-LC-1 TCR. To assess the TCR’s potential for off-target activity, we used amino acid scanning to determine the recognition motif, allowing for subsequent targeted in silico searches to identify peptides containing the same recognition motif. Using NCBI Blast and the ScanProsite database, 10 peptides with homologous essential but different non-essential residues were identified. A retroviral vector encoding the KK-LC-152-60 TCR was used to genetically engineer peripheral blood T cells. No peptide recognition by the TCR was observed when dendritic cells were pulsed with the individual peptides and co-cultured with KK-LC-1 TCR-transduced T cells. Finally, we assessed if human genetically engineered T cells expressing the KK-LC-1 TCR could mediate in vivo tumor regression in an NSG mouse model. Genetically engineered T cells expressing the KK-LC-1 TCR induced tumor regression in both a melanoma and cervical cancer line in a dose-dependent manner. Collectively, these findings indicate that KK-LC-1 is a suitable target for TCR gene therapy and they provide preclinical support for testing of the KK-LC-1 TCR in a phase I clinical trial. Citation Format: Bridget Marcinkowski, Carylinda Serna, Benjamin Jin, Scott Norberg, Christian Hinrichs. Preclinical characterization of a KK-LC-1-specific T cell receptor for the treatment of epithelial cancers [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 1429.