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MTL-CEBPA Combined with Immunotherapy Or RFA Enhances Immunological Anti-Tumor Response in Preclinical Models

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MTL-CEBPA Combined with Immunotherapy Or RFA Enhances Immunological Anti-Tumor Response in Preclinical Models International Journal of Molecular Sciences Article MTL-CEBPA Combined with Immunotherapy or RFA Enhances Immunological Anti-Tumor Response in Preclinical Models Kai-Wen Huang 1,2,* , Choon Ping Tan 3, Vikash Reebye 3,4, Cheng Ean Chee 5, Dimitris Zacharoulis 6, Robert Habib 3, David C. Blakey 3, John J. Rossi 7, Nagy Habib 3,4,* and Mikael H. Sodergren 4 1 Hepatitis Research Center, Department of Surgery, National Taiwan University Hospital, College of Medicine, Taipei 100, Taiwan 2 Centre of Mini-Invasive Interventional Oncology, National Taiwan University Hospital, Taipei 100, Taiwan 3 MiNA Therapeutics Ltd., London W12 0BZ, UK; [email protected] (C.P.T.); [email protected] (V.R.); [email protected] (R.H.); [email protected] (D.C.B.) 4 Department of Surgery and Cancer, Imperial College London, London W12 0NN, UK; [email protected] 5 Department of Haemato-Oncology, National University Cancer Institute, Singapore 119077, Singapore; [email protected] 6 Department of General Surgery, University Hospital of Larissa, 41110 Larissa, Greece; [email protected] 7 Department of Molecular and Cellular Biology, Beckman Research Institute of City of Hope, Duarte, CA 91010, USA; [email protected] * Correspondence: [email protected] (K.-W.H.); [email protected] (N.H.); Tel.: +44-(0)20-3313-8574 (N.H.); Fax: +44-(0)20-3313-3212 (N.H.) Abstract: The transcription factor CEBPA is a master regulator of liver homeostasis, myeloid cell Citation: Huang, K.-W.; Tan, C.P.; differentiation and is downregulated in several oncogenic diseases. MTL-CEBPA is a small activating Reebye, V.; Chee, C.E.; Zacharoulis, RNA drug which upregulates gene expression of CEBPA for treatment of hepatocellular carcinoma D.; Habib, R.; Blakey, D.C.; Rossi, J.J.; (HCC). We investigate whether MTL-CEBPA has immune modulatory effects by combining MTL- Habib, N.; Sodergren, M.H. CEBPA with an anti-PD-1 checkpoint inhibitor (CPI) and/or radiofrequency ablation (RFA) in two MTL-CEBPA Combined with preclinical models. First, mice with two flanks of HCC tumors (BNL) were treated with combinations Immunotherapy or RFA Enhances of RFA (right flank), anti-PD-1 or MTL-CEBPA. The reduction of the left flank tumors was most Immunological Anti-Tumor Response in Preclinical Models. Int. J. Mol. Sci. pronounced in the group treated with RFA+anti-PD1+MTL-CEBPA and 7/8 animals responded. 2021, 22, 9168. https://doi.org/ This was the only group with a significant increase in CD8+ and CD49b+/CD45+ tumor infiltrating 10.3390/ijms22179168 lymphocytes (TIL). Second, a combination of anti-PD-1+MTL-CEBPA was tested in a CT26 colon cancer model and this treatment significantly reduced tumor size, modulated the tumor immune Academic Editor: Marieke F. Fransen microenvironment and increased TILs. These data suggest a clinical role for combination treatment with CPIs, RFA and MTL-CEBPA through synergistic priming of the immune tumor response, Received: 25 July 2021 enabling RFA and CPIs to have a pronounced anti-tumor effect including activity in non-treated Accepted: 23 August 2021 tumors in the case of RFA. Published: 25 August 2021 Keywords: CEBPA; abscopal; immunotherapy; PD-1 Publisher’s Note: MDPI stays neutral with regard to jurisdictional claims in published maps and institutional affil- iations. 1. Introduction The transcription factor CEBPA (CCAAT/enhancer-binding protein alpha) is a leucine zipper protein which acts as a master regulator of liver homeostasis, including albumin production, and multiple oncogenic processes including cell cycle control, proliferation Copyright: © 2021 by the authors. and angiogenesis. It is also a master regulator of the hematopoietic myeloid cell lineage, Licensee MDPI, Basel, Switzerland. in which it primes and activates the myeloid gene expression program by binding to This article is an open access article promoters or enhancers of myeloid-related genes [1,2]. Myeloid lineage specific genetic distributed under the terms and conditions of the Creative Commons ablation of CEBPA has been shown to promote tumor growth by increasing the number Attribution (CC BY) license (https:// and tumor infiltration of myeloid-derived suppressor cells (MDSC) leading to a pro- creativecommons.org/licenses/by/ angiogenic, immune suppressive and pro-tumorigenic environment [3]. Furthermore, 4.0/). Int. J. Mol. Sci. 2021, 22, 9168. https://doi.org/10.3390/ijms22179168 https://www.mdpi.com/journal/ijms Int. J. Mol. Sci. 2021, 22, 9168 2 of 15 CEBPA protein is found to be downregulated in the peritoneal macrophages of tumor- bearing mice [4]. MTL-CEBPA comprises a double stranded RNA payload formulated inside a SMARTICLES®liposomal nanoparticle to specifically target and upregulate the CEBPA gene [5]. In a hepatocellular carcinoma (HCC) cell line transfected with MTL- CEBPA, increased levels of both CEBPA and albumin mRNA in addition to a 3-fold increase in albumin secretion and 50% decrease in cell proliferation was observed [6,7]. In a cirrhotic rat model with multifocal HCC, the i.v. injection of a formulated small activating RNA (saRNA) to CEBPA decreased the tumor burden by 80%, as well as increasing circulating albumin up to 30% [8–10]. MTL-CEBPA is the first saRNA and the first drug targeting CEBPA to enter phase 1 clinical trials, where it has been shown to be associated with limited toxicity and has shown promising initial clinical response in patients with advanced HCC [11,12]. The impact on circulating white blood cell populations suggest that the mechanism may be through a significant immuno-modulatory effect on the tumor immune microenvironment (TIME) through an impact on myeloid cells. The PD-1 inhibitor Nivolumab, which causes the activation of T-cells and cell-mediated immune responses against tumor cells, has shown clinical activity in a range of tumors. It gained accelerated FDA approval for second-line treatment of HCC based on a subgroup of the CHECKMATE-040 trial [13]. Patients treated with Nivolumab showed an overall re- sponse rate of 14.3% (95% CI: 9.2, 20.8), with 3 complete responses and 19 partial responses. Response duration ranged from 3.2 to 38.2+ months; 91% of responders had responses lasting 6 months or longer and 55% had responses lasting 12 months or longer. Radiofrequency ablation (RFA) is the process by which a tumor is destroyed using heat generated by a high frequency alternating current and applied through an electrode tip. RFA is one of the standard treatment options for HCC in clinical practice and is associated with a significant survival benefit [14]. Following RFA, the localized coagulation necrosis of the tumor remains in the body and provides proinflammatory signals to induce the release of large amounts of cellular debris, a source of tumor antigens, which can trigger a host- adaptive immune response against the tumor [15]. Evidence suggests that tumor thermal ablation induces modulation of both innate and adaptive immune systems, inducing anti- tumor immune responses through efficient loading of dendritic cells, enhanced antigen presentation and an amplified tumor-specific T-cell response [16–20]. We hypothesize that the oncological efficacy of MTL-CEBPA may be enhanced by combination treatment with either PD-1 inhibition, RFA therapy or a combination of RFA and PD-1 through synergism of immuno-modulatory response. The aim of this study was to evaluate the therapeutic response of combination therapy of MTL-CEBPA and anti-PD-1 therapy with or without RFA in two distinct mouse syngeneic cancer models and to characterize changes in the immune infiltrate following treatment. 2. Materials and Methods 2.1. Mice For the BNL model, BALB/c mice were purchased from BioLasco Co. (Taipei, Taiwan) and animal studies were performed in compliance with approval from the Institutional Animal Care and Use Committee of College of Medicine, National Taiwan University. For the CT26 anti-tumor study carried out by Alderley Park Ltd, Alderley Park, Macclesfield, Cheshire, UK under UK Home Office legislation. Female Balb/c mice (6 weeks old) were purchased from Envigo UK. 2.2. Tumor Cell Lines BALB/c-derived murine hepatocellular carcinoma cell line BNL 1ME A.7R.1 (BNL; ATCC, Manassas, VA, USA) was cultured in DMEM supplemented with 10% fetal bovine serum (FBS) and antibiotics (penicillin 100 units/mL, streptomycin 100 µg/mL and ampho- tericin 25 µg/mL) (Gibco BRL, Gaithersburg, MD, USA). The murine colon tumor cell line CT26.WT (CRL-2638) was grown in RPMI-1640 media with 10% FCS and 2 mM glutamine ◦ at 37 C and 5% CO2. Int. J. Mol. Sci. 2021, 22, 9168 3 of 15 2.3. BNL Model Experimental Groups and Treatment Schedule 64 male BABL/c mice were implanted in bilateral flanks by subcutaneous (s.c.) in- jection of 50 µL of BNL cell suspension containing 5 × 105 cells. Mice were randomly allocated to one of the following 8 experimental groups (8 animals/group): 1. Control 2. RFA 3. Anti-PD1 4. MTL-CEBPA 5. Anti-PD1+ MTL-CEBPA 6. RFA + anti-PD1 7. RFA+ MTL-CEBPA 8. RFA+ Anti-PD1+ MTL-CEBPA Four weeks after cancer-cell injection, when the tumor reached the diameter of ~1.5 × 1.5 cm, one of the bilateral tumors was treated by RFA at day 0. MTL-CEBPA (3 mg/kg) was given by i.v. injection on days 0, 2 and 5 post RFA treatment. Anti-PD-1 (RMP1-14, BioXCell, West Lebanon, NH, USA), at 200 µg/mouse/dose, was given intraperitoneally (i.p.) on days 0, 2 and 5 post RFA treatment. The tumor sizes were assessed using microcalipers, and the tumor volumes were calculated using the following equation: volume = length × (width)2 × 0.5. Mice were sacrificed on day 7. 2.4. Radiofrequency Ablation (RFA) Treatment Animals were anaesthetized with (i.p.) injection of Ketamine/Xylazine solution and positioned prone.
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