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A practical approach to immunotherapy of hepatocellular carcinoma using T cells redirected against hepatitis B virus.

ABSTRACT: Hepatocellular carcinoma (HCC) cells often have hepatitis B virus (HBV)-DNA integration and can be targeted by HBV-specific T cells. The use of viral vectors to introduce exogenous HBV-specific T-cell receptors (TCR) on T cells to redirect their specificity is complex and expensive to implement in clinical trials. Moreover, it raises safety concerns related to insertional mutagenesis and potential toxicity of long-lived HBV-specific T cells in patients with persistent infection. To develop a more practical and safer approach to cell therapy of HCC, we used electroporation of mRNA encoding anti-HBV TCR. Approximately 80% of CD8(+) T cells expressed functional HBV TCR 24 hours postelectroporation, an expression efficiency much higher than that obtained by retroviral transduction (~18%). Antigen-specific cytokine production of electroporated T cells was efficient within 72-hour period, after which the redirected T cells lost their HBV-specific function. Despite this transient functionality, the TCR-electroporated T cells efficiently prevented tumor seeding and suppressed the growth of established tumors in a xenograft model of HCC. Finally, we established a method for large-scale TCR mRNA electroporation that yielded large numbers of highly functional clinical-grade anti-HBV T cells. This method represents a practical approach to cell therapy of HCC and its inherently self-limiting toxicity suggests potential for application in other HBV-related pathologies.Molecular Therapy-Nucleic Acids (2013) 2, e114; doi:10.1038/mtna.2013.43; published online 13 August 2013.

SUBMITTER: Koh S

PROVIDER: S-EPMC3759740 | biostudies-literature | 2013 Aug

REPOSITORIES: biostudies-literature

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A practical approach to immunotherapy of hepatocellular carcinoma using T cells redirected against hepatitis B virus.

Koh Sarene S Shimasaki Noriko N Suwanarusk Rossarin R Ho Zi Zong ZZ Chia Adeline A Banu Nasirah N Howland Shanshan Wu SW Ong Alice Soh Meoy AS Gehring Adam J AJ Stauss Hans H Renia Laurent L Sällberg Matti M Campana Dario D Bertoletti Antonio A

Molecular therapy. Nucleic acids 20130813

Hepatocellular carcinoma (HCC) cells often have hepatitis B virus (HBV)-DNA integration and can be targeted by HBV-specific T cells. The use of viral vectors to introduce exogenous HBV-specific T-cell receptors (TCR) on T cells to redirect their specificity is complex and expensive to implement in clinical trials. Moreover, it raises safety concerns related to insertional mutagenesis and potential toxicity of long-lived HBV-specific T cells in patients with persistent infection. To develop a mor ...[more]

PMID: 23941866

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Project description:BackgroundIn the past decade, the field of tumour immunotherapy has made a great progress. However, the efficacy of immune checkpoint blocking (ICB) in the treatment of hepatocellular carcinoma (HCC) remains limited. Cytotoxic lymphocyte trafficking into tumours is critical for the success of ICB. Therefore, additional strategies that increase cytotoxic lymphocyte trafficking into tumours are urgently needed to improve patient immune responses.MethodsPaired adjacent tissue and cancerous lesions with HBV-associated HCC were subjected to RNA-seq analysis. Bone morphogenetic protein (BMP9), which reflects vessel normalisation, was identified through Cytoscape software, clinical specimens and Gene Expression Omnibus (GEO) datasets for HCC. The functional effects and mechanism of BMP9 on the tumour vasculature were evaluated in cells and animals. An ultrasound-targeted microbubble destruction (UTMD)-mediated BMP9 delivery strategy was used to normalise the vasculature and evaluate therapeutic efficacy mediated by cytotoxic lymphocytes (NK cells) in combination with a PD-L1 antibody in human cancer xenografts of immune-deficient mice.ResultsWe discovered that hepatitis B virus (HBV) infection-induced downregulation of BMP9 expression correlated with a poor prognosis and pathological vascular abnormalities in patients with HCC. BMP9 overexpression in HBV-infected HCC cells promoted intra-tumoural cytotoxic lymphocyte infiltration via vascular normalisation by inhibiting the Rho-ROCK-myosin light chain (MLC) signalling cascade, resulting in enhanced efficacy of immunotherapy. Furthermore, UTMD-mediated BMP9 delivery restored the anti-tumour function of cytotoxic lymphocytes (NK cells) and showed therapeutic efficacy in combination with a PD-L1 antibody in human cancer xenografts of immune-deficient mice.ConclusionsHBV-induced BMP9 downregulation causes vascular abnormalities that inhibit intra-tumoural cytotoxic lymphocyte infiltration, providing a rationale for developing and combining immunotherapy with BMP9-based therapy to treat HBV-associated HCC.

Project description:AimTo develop a novel non-sequencing method for the detection of hepatitis B virus (HBV) pre-S deletion mutants in HBV carriers.MethodsThe entire region of HBV pre-S1 and pre-S2 was amplified by polymerase chain reaction (PCR). The size of PCR products was subsequently determined by capillary gel electrophoresis (CGE). CGE were carried out in a PACE-MDQ instrument equipped with a UV detector set at 254 nm. The samples were separated in 50 μm ID eCAP Neutral Coated Capillaries using a voltage of 6 kV for 30 min. Data acquisition and analysis were performed using the 32 Karat Software. A total of 114 DNA clones containing different sizes of the HBV pre-S gene were used to determine the accuracy of the CGE method. One hundred and fifty seven hepatocellular carcinoma (HCC) and 160 non-HCC patients were recruited into the study to assess the association between HBV pre-S deletion and HCC by using the newly-established CGE method. Nine HCC cases with HBV pre-S deletion at the diagnosis year were selected to conduct a longitudinal observation using serial serum samples collected 2-9 years prior to HCC diagnosis.ResultsCGE allowed the separation of PCR products differing in size > 3 bp and was able to identify 10% of the deleted DNA in a background of wild-type DNA. The accuracy rate of CGE-based analysis was 99.1% compared with the clone sequencing results. Using this assay, pre-S deletion was more frequently found in HCC patients than in non-HCC controls (47.1% vs 28.1%, P < 0.001). Interestingly, the increased risk of HCC was mainly contributed by the short deletion of pre-S. While the deletion ≤ 99 bp was associated with a 2.971-fold increased risk of HCC (95%CI: 1.723-5.122, P < 0.001), large deletion (> 99 bp) did not show any association with HCC (P = 0.918, OR = 0.966, 95%CI: 0.501-1.863). Of the 9 patients who carried pre-S deletions at the stage of HCC, 88.9% (8/9) had deletions 2-5 years prior to HCC, while only 44.4%4 (4/9) contained such deletions 6-9 years prior to HCC.ConclusionCGE is a sensitive approach for HBV pre-S deletion analysis. Pre-S deletion, especially for short DNA fragment deletion, is a useful predictive marker for HCC.

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A practical approach to immunotherapy of hepatocellular carcinoma using T cells redirected against hepatitis B virus.

Publications

A practical approach to immunotherapy of hepatocellular carcinoma using T cells redirected against hepatitis B virus.

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