At puberty, neurokinin B (NKB) and kisspeptin (Kiss1) may help to amplify GnRH secretion, but their precise roles remain ambiguous. We tested the hypothesis that NKB and Kiss1 are induced as a function of pubertal development, independently of the prevailing sex steroid milieu. We found that levels of Kiss1 mRNA in the arcuate nucleus (ARC) are increased prior to the age of puberty in GnRH/sex steroid-deficient hpg mice, yet levels of Kiss1 mRNA in wild-type mice remained constant, suggesting that sex steroids exert a negative feedback effect on Kiss1 expression early in development and across puberty. In contrast, levels of Tac2 mRNA, encoding NKB, and its receptor (NK3R; encoded by Tacr3) increased as a function of puberty in both wild-type and hpg mice, suggesting that during development Tac2 is less sensitive to sex steroid-dependent negative feedback than Kiss1. To compare the relative responsiveness of Tac2 and Kiss1 to the negative feedback effects of gonadal steroids, we examined the effect of estradiol (E(2)) on Tac2 and Kiss1 mRNA and found that Kiss1 gene expression was more sensitive than Tac2 to E(2)-induced inhibition at both juvenile and adult ages. This differential estrogen sensitivity was tested in vivo by the administration of E(2). Low levels of E(2) significantly suppressed Kiss1 expression in the ARC, whereas Tac2 suppression required higher E(2) levels, supporting differential sensitivity to E(2). Finally, to determine whether inhibition of NKB/NK3R signaling would block the onset of puberty, we administered an NK3R antagonist to prepubertal (before postnatal d 30) females and found no effect on markers of pubertal onset in either WT or hpg mice. These results indicate that the expression of Tac2 and Tacr3 in the ARC are markers of pubertal activation but that increased NKB/NK3R signaling alone is insufficient to trigger the onset of puberty in the mouse.

No abstract

Glioblastoma multiforme (GBM) is a highly aggressive brain tumor associated with poor prognosis and resistance to current therapies. ONCR-1 is a novel oncolytic herpes simplex virus type-1 vector in development for the treatment of GBM. ONCR-1 utilizes a unique conditional-lethal strategy in which miR124 binding sites are inserted into the ICP4 locus to prevent viral replication in neuronal cells while preserving one copy of the γ34.5 gene and enabling potent cytotoxicity in tumor cells. ONCR-1 is armed with a matrix metalloproteinase 9 (MMP9) transgene to facilitate viral spread in the tumor extracellular matrix. Further, ONCR-1 expresses a UL16 binding protein 3 (ULBP3) transgene to activate NK and CD8+ T cells via the NKG2D receptor and promote antitumor immune responses. In human and murine tumor cells in vitro, ONCR-1 evoked potent cytotoxicity. ONCR-1 increased proteolytic MMP in cell supernatants and ULBP3 cell surface expression with no effect on related NKG2D ligands. In mice bearing subcutaneous or orthotopic human U251 GBM tumors, ONCR-1 administered intratumorally (3x105-3x106 PFU QDx1) inhibited tumor growth and prolonged survival. Ex vivo analysis of immune cells in subcutaneous tumors showed significant increases in virally evoked NKG2D+ NK cells with ONCR-1 treatment. In mice bearing dual flank subcutaneous syngeneic A20 tumors, ONCR-1 significantly inhibited tumor growth in injected tumors (1x105-3x105 PFU, Q2Dx3) and evoked potent systemic antitumor immune responses as evidenced by significant inhibition of distal noninjected left flank tumors. IFNγ production from isolated splenocytes cultured in the absence and presence of A20 tumor cells was significantly augmented in ONCR-1-treated animals, providing further evidence of an enhanced antitumor immune response. Moreover, in mice bearing subcutaneous A20 tumors in which ONCR-1 evoked complete tumor regression, complete protection from rechallenge with A20 tumor cells was observed. Depletion of NK cells or CD8+ T cells in mice resulted in inhibition of ONCR-1-mediated antitumor effects in both injected and noninjected murine A20 tumors, supporting an immune mechanism of action. Our preclinical studies demonstrate that ONCR-1 evokes tumor cell killing by direct oncolysis and by enhancing antitumor immune responses. ONCR-1 represents a novel clinical candidate for the treatment of GBM. Citation Format: Alexandra Hicks, Paola Grandi, Michael Paglia, Jingzang Miu, Cecilia Kwong, Jacqueline Gursha, Michael Ball, Weiguo Yao, Daniel Wambua, Terry Farkaly, Kyle Grant, Laura Viggiano Salta, Lorenz Ponce, Joseph Glorioso, Christophe Queva, Mitchell Finer. ONCR-1, a novel herpes simplex virus expressing MMP9 and ULBP3 transgenes, evokes potent oncolysis and development of antitumor immune responses [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 4698.

Oncolytic viruses, through cancer cell-selective lysis and accompanying release of danger signals that promote immune activation, have demonstrated antitumor efficacy in monotherapy of metastatic melanoma and have shown promising activity in combination with checkpoint inhibitors. We present here the activity of ONCR-148, a recombinant oncolytic Herpes Simplex Virus (oHSV) designed to be a safe and efficacious therapy for the treatment of solid tumors. Neuron-specific miR attenuation of ICP4 gene inhibits viral replication in neurons while preserving its potent oncolytic activity in tumor cells. The antitumor potency of the virus is enhanced by incorporating in its genome a transgene expressing fibroblast activation protein (FAP)- and CD3- bispecific antibody that recruits and re-directs the cytolytic activity of T cells toward FAP-expressing stromal cells. FAP is a cell-surface serine protease that is almost exclusively found in fibroblasts within tumor stroma. However, systemic administration of a FAP-CD3 bispecific antibody was not tolerated due to the toxicity toward FAP-expressing bone marrow mesenchymal cells (Tran et al., 2013. J.Exp.Med, 210(6), 1125-1135). Thus, the local intra-tumoral delivery of FAP-CD3 is an attractive approach to promote tumor lysis and T cell activation against cancerous lesions. To test the in vivo activity of ONCR-148, as FAP is poorly expressed in syngeneic mouse tumor models, we developed a bilateral mouse FAP-expressing MC38 syngeneic colon carcinoma model (MC38-FAP), allowing to test local antitumor activity in oHSV-injected tumors and abscopal efficacy mediated by immune cells in the non-injected tumors. Intra-tumoral administration of ONCR-148 in the MC38-FAP model resulted in tumor growth inhibition of 60% (p<0.03) and 46% (p<0.01) on the injected (ipsilateral) and non-injected (contralateral) tumor, respectively. We then investigated additional transgenes that could potentiate the antitumor activity of ONCR-148. We combined in vivo ONCR-148 with ONCR-153, an oHSV armed with the NK and T cell activating cytokine IL-12 and the chemokines CXCL10 and CCL4, allowing for expansion and recruitment of T cells and antigen presenting cells in the tumor milieu. Combined intra-tumoral administration of ONCR-148 and ONCR-153 increased the antitumor response to 90% on the injected tumor (p<0.0001), leading to complete tumor remission in 5 out of 9 treated mice and to 80% on distant tumors (p<0.0001, with no complete responses observed).These data indicate that oHSV expressing bispecific antibodies, while being well tolerated, is efficacious in injected tumors and mediate systemic antitumor immune response. The efficacy can be potentiated by a local co-expression of immunostimulatory cytokines. Such data support the further development of oHSV-1 armed with CD3 bispecific antibodies for the therapy of patients with metastatic cancer. Citation Format: Agnieszka Denslow, Brian B. Haines, Michael S. Ball, Jacqueline Gursha, Daniel Wambua, Cecilia Kwong, Lingxin Kong, Allison Colthart, Prajna Behera, Peter Grzesik, Jennifer Lee, Terry Farkaly, Caitlin Goshert, Edward M. Kennedy, Lorena Lerner, Christophe Quéva. Development of ONCR-148, a miR-attenuated oncolytic HSV-1 designed to potently activate antitumor T cell response [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 1452.

No abstract

We have previously described the generation of autologous T Cell Receptor Fusion Construct (TRuC™) T cells which are engineered to express a fusion protein comprised of an antibody-derived binder tethered to the extracellular domain of the CD3ε signaling subunit. Upon integration of the TRuC into the T cell receptor (TCR), it targets specific tumor surface antigens independent of HLA and uses the complete receptor complex to trigger a comprehensive T cell response. Here, we report about engineering of off-the-shelf TRuC-T cells directed against mesothelin (MSLN). To eliminate the alloreactivity of α/β T cells and reduce the risk of graft-versus-host-disease (GvHD), the TRAC locus is knocked-out. To enable the re-assembly of the TCR, the endogenous TCRα and β subunits are replaced with fusion proteins comprised of antibody-derived binders fused to the TCRγ and δ constant domains. Allogeneic anti-MSLN TRuC-T cells upregulate activation markers, secrete robust cytokines, and lyse tumor cells in an antigen-specific manner without allo-reactivity. In comparison with control autologous TRuC-T cells targeting mesothelin (TC-210), allogeneic TRuC-Ts demonstrate extended tumor clearance in NSG xenograft models due to their enhanced expansion and persistence. To reduce host rejection and further boost the persistence of the allogeneic TRuC-T cells, we eliminated MHC class I surface expression by B2M gene knockout and over-expressed membrane-bound IL15 tethered to its receptor alpha. In summary we have engineered persistence enhanced, allogeneic TRuC-T cells that maintain the signaling properties of the TCR complex with improved efficacy compared to donor-matched autologous TRuC-T cells. Citation Format: Julie Donaghey, Cecilia Kwong, Allison Powell, Troy Patterson, Ella Liberzon, Richard Decker, Julio Gomez Rodriguez, Darby Kreienberg, Jennifer Bian, Urmi Patankar, Holly Horton, Jian Ding, Robert Hofmeister, Dario Gutierrez, Robert Tighe. Engineering off-the-shelf anti mesothelin t-cell receptor fusion construct (TRuC™) t-cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 557.

No abstract

Although oncolytic viruses have shown clinical efficacy for local treatment of cancerous tumors, the ability to induce immune-mediated regression to visceral lesions is not robust. Immune checkpoint modulation has been efficacious in a fraction of cancers associated with an inflamed microenvironment but is associated with toxicity due to nonspecific T-cell activation. Therefore, combining these two strategies has the exciting potential to be an effective cancer therapy. Oncolytic virus can be genetically modified to decrease pathogenicity to normal cells and to increase their lytic potential and ability to stimulate antitumor immunity, thus improving the risk-benefit ratio for cancer patients. Oncorus oHSV (ONCR) are novel oncolytic herpes simplex virus type-1 vectors developed for the treatment of solid tumors. ONCR utilizes a unique conditional-lethal strategy in which tissue-specific miRs binding sites are inserted into early genes essential for viral replication and in the neurovirulence gene ICP34.5. This strategy curtails viral replication in normal cells, including neurons, and limits the expression of g34.5 to tumor cells, thus promoting potent viral replication in presence of type I interferon. Fifteen immunostimulatory transgenes and combination thereof were systematically evaluated in dual flank syngeneic mouse tumor models to test both for oncolysis and abscopal efficacy on the contralateral noninjected tumor. Among the selected transgenes, IL-12 was found to elicit the most potent efficacy on the ipsilateral and contralateral tumors. We demonstrated that the replication of ONCR and the expression of IL-12 was limited to the injected tumors. By contrast, induction of systemic immunity as assessed by IFNg production in contralateral tumors and in plasma was observed. ONCR expressing IL-12 elicited efficacy in multiple subcutaneous and metastatic models. Additional payloads designed to stimulate antigen presentation, recruitment of immune cells and inhibition of tumor immune suppression were identified as further enhancing IL-12 activity. Data supporting the selection of a suite of payloads cloned into a single ONCR vector will be presented. ONCR represents a new class of oncolytic virus that promote antitumor responses through a multiprong mechanism of action dependent on selective tumor cell killing, the induction of systemic antitumor immunity and reversion of immune suppression. Citation Format: Lorena Lerner, Edward M. Kennedy, Terry Farkaly, Allison Colthart, Caity Montagna, Prajna Behera, Judith Jacques, Peter Grzesik, Jennifer Lee, Agnieszka Denslow, Jacqueline Gursha, Brian Haines, Michael Ball, Daniel Wambua, Cecilia Kwong, Lingxin Kong, Michael Paglia, Laura Viggiano Salta, Lorenz Ponce, Caroline Webb, Mitchell Finer, Christophe Quéva. microRNA attenuated oHSV-1 armed with multiple immunomodulatory payloads mediates robust and selective antitumor immune response in preclinical tumor models [abstract]. In: Proceedings of the AACR Special Conference on Tumor Immunology and Immunotherapy; 2018 Nov 27-30; Miami Beach, FL. Philadelphia (PA): AACR; Cancer Immunol Res 2020;8(4 Suppl):Abstract nr B23.