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Hepatoma Res 2021;7:3.10.20517/2394-5079.2020.107© The Author(s) 2021.
Open AccessReview

The immune microenvironment and progression of immunotherapy and combination therapeutic strategies for hepatocellular carcinoma

1Department of Pathology, School of Basic Medical Sciences & State Key Laboratory of Reproductive Medicine & Key Laboratory of Antibody Technique of National Health Commission, Nanjing Medical University, Nanjing 211166, China.

2Interdisciplinary Innovation Institute for Medicine and Engineering, Southeast University-Nanjing Medical University, Nanjing 211189, China.

3Sir Run Run Hospital, Nanjing Medical University, Nanjing 21116, China.

4Department of Medical Oncology, The Affiliated Cancer Hospital of Zhengzhou University, Zhengzhou 450008, China.

#Contribute equally to this work.

Correspondence Address: Dr. Hong-Ping Xia, Department of Pathology, School of Basic Medical Sciences & State Key Laboratory of Reproductive Medicine & Key Laboratory of Antibody Technique of National Health Commission, Nanjing Medical University, Nanjing 211166, China. E-mail: xiahongping@njmu.edu.cn ; Dr. Xiao-Bing Chen, Department of Medical Oncology, The Affiliated Cancer Hospital of Zhengzhou University, Zhengzhou 450008, China. E-mail: xiaobingchen01@163.com ; Dr. Fu-Bing Wu, Sir Run Run Hospital, Nanjing Medical University, Nanjing 21116, China. E-mail: wfbwkx@163.com

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    © The Author(s) 2021. Open Access This article is licensed under a Creative Commons Attribution 4.0 International License (https://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, sharing, adaptation, distribution and reproduction in any medium or format, for any purpose, even commercially, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made.

    Abstract

    Hepatocellular carcinoma (HCC) accounts for 75%-85% of all primary liver cancers and is the leading cause of cancer-related deaths. China accounts for almost half of the global incidence and deaths of HCC. The poor response of chemotherapeutics and targeted drugs may be due to the drug resistance, heterogeneity of HCC, severe chronic liver damage and cirrhosis. Restoration of the liver microenvironment changes caused by chronic injury is crucial. Immunotherapy recently seems to show promise for the treatment of HCC induced by inflammatory injury. However, the unique liver immune system and resident immune tolerance state also pose a challenge for HCC immunotherapy. Different combinations of strategies have been developed for enhancement of HCC treatment. Here, we will discuss the immune microenvironment and progression of immunotherapy and combination therapeutic strategies for HCC.

    Introduction

    Hepatocellular carcinoma (HCC) accounts for 75%-85% of all primary liver cancers. Due to the rapid progression of HCC, the lack of effective treatment programs, and poor prognosis makes it the fourth leading cause of cancer-related deaths[1]. Due to regional differences in medical diagnosis and treatment, more than half of the new cases and deaths of HCC each year occur in the Asia-pacific region. Patients with early HCC in Europe and United States can be diagnosed and effectively treated in time[2]. More than 70% of HCC patients do not benefit from medical therapy. The vast majority of HCC patients present with an advanced stage at diagnosis, and the most effective surgical programs are often challenging to implement. In the past ten years, dozens of promising chemotherapeutics have failed the phase III trial, with only sorafenib demonstrating a low objective response rate and a slight increase in survival[3]. Research on targeted drugs for cell proliferation, metastasis and angiogenesis are encouraging, such as regorafenib and lenvatinib, although the overall survival rate remains dissatisfactory[4]. The ineffectiveness of chemotherapeutics and targeted drugs may be due to drug resistance and heterogeneity of HCC. HCC is usually accompanied by severe chronic liver damage and cirrhosis. Hence, anti-HCC drugs require a good balance of therapeutic response and drug toxicity and this often limits the application of highly active compounds with high toxicity[5,6]. Therefore, restoration of the liver microenvironment caused by chronic injury should be incorporated in the holistic management of HCC. In recent years, immunotherapy has been used in the treatment of various solid tumors. This was observed through the checkpoint inhibition of programmed cell death 1/programmed cell death ligand 1 (PD-1/PD-L1) and cell toxic T lymphocyte-associated protein 4 (CTLA-4) while improving the tumor immune microenvironment which seems to be particularly relevant for the treatment of HCC. However, the unique liver immune system and resident immune tolerance state make it different from other organs. Besides, continuous matrix remodeling of the malignant hepatocyte transformation caused by chronic inflammation and scars has created an immunosuppressive microenvironment that promotes the development of HCC, posing a challenge for HCC immunotherapy[7].

    The immune microenvironment of the liver and HCC

    The liver has a unique immune regulation and balance mechanism. On one hand, the portal vein system is directly exposed to gastrointestinal pathogens and requires an effective immune response. On the other hand, it needs to deal with a large number of harmless blood antigens and maintain the immune tolerance of the liver[8]. In most cases, the liver is in a physiological immune tolerance state[9]. Most non-parenchymal cells, such as live sinusoidal endothelial cells (LSEC), Kupffer cells (KCs) and hepatic dendritic cells (HDC), gather in the liver sinusoids. It constitutes the physiological basis of the liver’s immunosuppressive microenvironment[10,11]. LSEC has the dual functions of immune surveillance and immune tolerance. It acts as an antigen-presenting cell (APC) to present pathogens or tumor antigens[12]. At the same time, it inhibits the excessive responses of DC and T lymphocytes to bacterial antigens from the portal system[13-15]. KCs maintain immune tolerance by engulfing pathogenic microorganisms derived from the intestine, secreting inhibitory factors (such as IL-10 and prostaglandins) and activating the proliferation of regulatory T cells (Tregs)[16-19]. Besides, HDC is also a component of the liver immune tolerance by reducing the expression of MHC II and co-stimulatory molecules[20]. In summary, this immune-tolerant physiological environment creates a huge obstacle to the host’s anti-tumor immunity.

    The pathogenesis of HCC is characterized by destruction of the sinusoidal structure by a viral infection and inflammatory injury, impairment of immune surveillance and immune tolerance functions leading to liver cirrhosis and liver cancer[6,10]. The high-risk factors of HCC (hepatitis virus, alcohol, aflatoxin, etc.) drive hepatocyte DNA damage, endoplasmic reticulum stress and necrosis, which in turn leads to the formation of regenerative nodules, proliferative nodules and ultimately HCC[21]. HCC has abundant immune cell infiltration, which is the immune response of the host trying to clear the tumor. Unfortunately, this immune response is often dysregulated[22]. Tumor-infiltrating lymphocytes (TILs) account for a high proportion of HCC[23,24], but these ineffective TILs often prove to be insufficient to control tumor growth[25]. The increased FoxP3+ Treg may impair the effector function of CD8+ T cells, which exacerbates the immunosuppressive microenvironment in HCC and is associated with a poor prognosis[26]. In addition, adaptive immune cells (such as CD8 + T cells, Th17 cells and B cells) can also stimulate the development of HCC[27,28]. There are a large number of bone marrow-derived suppressor cells (MDSCs) and Tregs in the microenvironment of HCC, which evade immune surveillance through a variety of mechanisms, such as the expressing high-levels of SOCS3 and IL-10 to limit immune cell activation[29] and secretion fibrosis factor TGF-β. This is used to build an environment of immunosuppression and drug resistance[30], and directly down-regulates the expression of T cells or NK cell activation ligands (MHC class I and NKG2D, etc.)[31,32]. Therefore, the immunosuppressive environment of HCC is an arduous challenge to the host’s immune system, which makes immunotherapy a promising approach for HCC treatment in the future.

    The strategies of HCC immunotherapy

    According to the immunological basis of HCC, we divide current HCC immunotherapy into four categories, including immune checkpoint inhibitors, oncolytic virus therapy, HCC vaccine and chimeric antigen receptor T (CAR-T) cell therapy [Figure 1]. Due to the destruction of the HCC sinusoidal structure, it is difficult for LSEC and HDC to complete the antigen presentation process. Therefore, the specific DC vaccine is obtained by impinging tumor-associated antigen (TAA) or tumor lysate into DC in vitro. It activates cytotoxic T lymphocytes (CTLs) through major histocompatibility complex (MHC) class II-TCR antigen presentation and CD40/CD80/CD86-CD28 interaction. CTLs recognize and destroy tumor cells containing HCC-related antigens on MHC class I molecules. In addition to blocking the antigen presentation process, cancer cells will evade CTLs by upregulating immune checkpoint ligands, such as PD-L1 binding to the PD-1 receptor on the surface of CTLs to exhaust it, and CTLA-4 blocking the interaction between CD40/CD80/CD86 and CD28. Therefore, antibodies against PD-L1/PD-1 and CTLA-4 are used for immune checkpoint inhibitor therapy of HCC. The other alternative is more direct, by cloning in vitro chimeric antigen receptor T cells that can target specific antigen genes [such as Glypican 3 (GPC3) or alpha-fetoprotein (AFP)] related antigens to directly kill tumor cells. Finally, genetically engineered oncolytic virus therapy can also selectively replicated in tumors, killing cancer cells while stimulating antigen presentation and adaptive anti-tumor immune responses.

    Figure 1. The immune microenvironment and immunotherapy for HCC. We divide current HCC immunotherapy into four categories, namely immune checkpoint inhibitors, oncolytic virus therapy, HCC vaccine and chimeric antigen receptor T (CAR-T) cell therapy. HCC: hepatocellular carcinoma

    Immune checkpoint inhibitors

    Tumor cells express a variety of immunosuppressive ligands on their surface, which bind to the indicated inhibitory receptors of activated T cells involved in the anti-tumor response. This process in turn reduces the intensity of the anti-tumor immune response, thereby evading immune surveillance[33]. Drugs that block these immunosuppressive targets to eliminate tumor immune escape are called immune checkpoint inhibitors (ICI). PD-1 is a member of the CD28 superfamily and is expressed on the surface of T cells and B cells. Its activation will lead to the phosphorylation of ITSM (Immunoreceptor tyrosine-based switch motif) in the cytoplasm of the cell, inhibiting energy metabolism in T cells, thereby hindering cell cloning proliferation and secretion of cytokines. In order to avoid the killing of T cells, tumor cells highly express PD-L1 and release the PD-L1 into the peripheral blood, which causes the exhaustion of T cells and the loss of tumor antigen presentation ability of myeloid immune cells[34]. Therefore, targeted inhibition of the interaction of PD-1 and PD-L1 is of great significance for the treatment of HCC.

    Nivolumab, as the first PD-1 targeted drug to be used in clinical practice, was initially used in the treatment of melanoma, and its objective response rate and one-year survival rate were 40.0% and 72.9%, respectively[35]. Subsequently, Nivolumab was tried to treat advanced HCC. Among 144 HCC patients, 20% showed a good response to nivolumab, and 3 of them achieved complete remission (CR), highlighting the potential of Nivolumab to treat advanced HCC[36]. Another anti-PD-1 targeted drug Pembrolizumab has also shown effectiveness in the treatment of advanced HCC, with an objective response rate and a one-year survival rate of 17% and 54%[37]. In fact, a single ICI is not satisfactory for the treatment of advanced HCC. The current ICI therapy is mostly performed in a variety of combinations (for example, anti-PD-L1 antibody plus anti-CTLA-4 antibody), which is more effective than a single agent. In the absence of targetable lymphocytes in the tumor microenvironment, inhibition of PD-1/PD-L1 cannot stimulate cancer immunity, and inhibition of the CTLA-4 can cause CD8 + T cells to proliferate in the lymph nodes and infiltrate the tumor tissue, thereby enhancing the efficacy of anti-tumor. In fact, combination therapy of molecularly targeted drugs and immune checkpoint inhibitors has received considerable attention. For example, immunosuppressive cytokines that cause the immunosuppressive liver environment of patients with liver cancer, such as interleukin (IL)-10, transforming growth factor (TGF)-β and vascular endothelial growth factor (VEGF) molecular targeted drugs[38,39]. Table 1 shows the ongoing use of ICI in combination with various interventions (such as kinase inhibitors, cytokine or receptor inhibitors, and embolotherapy).

    Table 1

    Clinical trials of immune checkpoint inhibitors for HCC

    Clinical trials identifierTargetStatusActive treatmentNPrimary endpoints or outcomesRef.
    Single immune checkpoint inhibitors
       NCT03630640PD-1Recruiting Phase 2Nivolumab50OS, 2 years
       NCT03383458PD-1Recruiting, Phase 3Nivolumab530Recurrence-free Surviva, 49 months; OS, 7 years;
    Time to recurrence, 49 months
       NCT04161911PD-1Completed Phase 3Nivolumab1,426OS, 7.75 years
    Combination of immune checkpoint Inhibitors
       NCT03222076CTLA-4
    PD-1
    Recruiting Phase 2Ipilimumab
    Nivolumab
    45AEs, 5 years
       NCT03682276CTLA-4
    PD-1
    Recruiting, Phase I/IIIpilimumab
    Nivolumab
    32AEs, 127 Days;
    Delay to surgery, 89 Days
       NCT03510871PD-1
    CTLA-4
    Not yet recruiting, Phase IINivolumab
    Ipilimumab
    40The percentage of subjects with tumor shrinkage, 4 years
    Combination of Immune Checkpoint Inhibitors with Tyrosine kinase inhibitor
       NCT04310709Multikinase
    PD-1
    Recruiting, Phase IIRegorafenib
    Nivolumab
    42ORR, 6 months[40-42]
       NCT04170556Multikinase
    PD-1
    Recruiting, Phase IIRegorafenib
    Nivolumab
    60AEs, 24 months
       NCT03299946Multikinase
    PD-1
    Active, not recruiting, Phase ICabozantinib
    Nivolumab
    15AEs, 4 years
       NCT03841201Multikinase
    PD-1
    Recruiting, Phase IILenvatinib
    Nivolumab
    50ORR, 6 months
       NCT03418922Multikinase
    PD-1
    Active, not recruiting Phase 1Lenvatinib
    Nivolumab
    30DLTs, 28 days
       NCT03006926Multikinase
    PD-1
    Phase 1; Active, not recruitingLenvatinib
    Pembrolizumab
    104AEs, 3 years;
    DLT, 21 days;
    ORR, 3 years
       NCT02856425Multikinase PD-1Phase 1; RecruitingNintedanib
    Pembrolizumab
    18MTD, 24 months
       NCT02572687PD-L1
    VEGF
    Phase 1; Active, not recruitingRamucirumab
    MEDI4736
    114DLTs, 28 days
       NCT02576509Raf-1
    PD-1
    Active, not recruiting, Phase IIISorafenib
    Nivolumab
    743OS, 41 months
       NCT02988440Raf1
    PD-1
    Phase 1; CompletedPDR001
    Sorafenib
    20AEs, 30 days;
    DLT, 8 weeks;
       NCT03893695ALK-1
    PD-1
    Recruiting Phase 1 Phase 2GT90001 Nivolumab20DLTs, 28 days
       NCT03059147PI3k
    PD-1
    Active, not recruiting, Phase IISF1126
    Nivolumab
    14DLTs, 56days
       NCT03655613C-Met
    PD-1
    Recruiting Phase 1 Phase 2APL-101
    Nivolumab
    119DLTs, 35 days
       NCT02795429PD-1+cMetPhase 1/2; Active, not recruitingPDR001
    INC280
    90DLT, 42 days;
    ORR, 3 years
    Combination of immune checkpoint inhibitors with Cytokine/receptor inhibitor
       NCT02423343TGFβR1
    PD-1
    Active, not recruiting Phase 1 Phase 2Galunisertib
    Nivolumab
    75MTD, 6 months
       NCT04123379PD-1
    CCR2/CCR5
    Recruiting Phase 2Nivolumab BMS-813160
    BMS-986253
    50Primary pathologic response: 2 years; Significant tumor necrosis: 2 years
    Combination of immune checkpoint inhibitors with embolotherapy
       NCT03033446Embolotherapy PD-1Recruiting, Phase IIRadioembolization
    Nivolumab
    40ORR, 8 weeks
       NCT03380130PD-1
    Embolotherapy
    Active, not recruiting Phase 2Nivolumab SIR-Spheres40AEs, 2 years[43,44]
       NCT03572582PD-1
    Embolotherapy
    Active, not recruiting Phase 2Nivolumab
    TACE
    49ORR, 42 months
       NCT04268888PD-1
    Embolotherapy
    Recruiting Phase 2 Phase 3Nivolumab and TACE/TAE522OS: 2 years;
    TTTP
    Multiple combination therapy
       NCT01658878PD-1
    Raf-1
    CTLA-4
    multikinase
    Active, not recruiting, Phase I/IINivolumab
    Sorafenib
    Ipilimumab
    Cabozantinib
    1,097AEs, 100 days;
    ORR, 6 months
    [45,46]
       NCT04039607PD-1
    CTLA-4
    Raf-1
    VEGFR/FGFR
    Recruiting, Phase IIINivolumab
    Ipilimumab
    Sorafenib
    lenvatinib
    1,084OS, 4 years
       NCT04472767PD-1
    CTLA-4
    Multikinase
    Embolotherapy
    Not yet recruiting, Phase IINivolumab
    Ipilimumab
    Cabozantinib
    Transarterial Chemoembolization
    35Percentage of Progression-free Survival, 6 Months;
    Complete Response Rate, 1 year
       NCT04050462PD-1
    Multikinase
    IL-8
    Not yet recruiting, Phase IINivolumab
    Cabiralizumab
    BMS-986253
    74ORR, 6 years
       NCT03071094Oncolytic therapy
    PD-1
    Active, not recruiting, Phase I/IIPexastimogene Devacirepvec;
    Nivolumab
    30DLTs, 4Weeks;
    ORR, 6 months
       NCT03897543PD-1
    INK T cells Agonist
    Recruiting Phase 1 Phase 2Nivolumab
    ABX196
    48AEs, 1 year

    Oncolytic virus therapy

    The oncolytic virus can specifically host in cancer cells, replicate and destroy the cell structure and hence was not initially classified as immunotherapy. Subsequent studies confirmed that oncolytic viruses could induce anti-cancer immune responses and immunogenic cancer cell death, making them a form of immunotherapy[47]. Compared with traditional therapies, oncolytic virus therapy is safer, has the selective specificity of host cancer cells, and continuously self-replicates to lyse cancer cells[48]. In the tumor microenvironment, pathogen-associated molecular patterns (PAMP) of oncolytic viruses can be recognized by pattern recognition receptors (PRR) of immune cells, such as through TLR or MDA5 activation of macrophages or dendritic cells[49,50]. As a secondary effect, oncolytic viruses enhance the recognition and presentation of tumor antigens, and activate the infiltration of cytotoxic T cells into tumors[51]. Therefore, oncolytic virus therapy is a very interesting method to overcome HCC immunosuppression. Currently, oncolytic virus therapies used for HCC include dsDNA or ssRNA viruses, such as measles vaccine virus (MeV), herpes simplex virus (HSV), adenovirus (Adenovirus) and vaccinia virus (VV), etc., which are used to engineer infection vectors[52]. For example, inserting the overexpression sequence of granulocyte-macrophage colony-stimulating factor (GM-CSF) into the oncolytic virus sequence, GM-CSF recruits myeloid cells in the periphery to enhance the immune response in the tumor microenvironment[53]. So far, preclinical studies for HCC oncolytic virus therapy have been very encouraging. We have compiled preclinical studies on HCC oncolytic therapy for the past ten years, as shown in Table 2.

    Table 2

    Representative Oncolytic therapy used in preclinical studies

    Virus strainModificationTherapeutic geneHCC cell linesAnimal modelDoseRef.
    Recombinant VSV-NDV, L289AReplaced of hemagglutinin-neuraminidase (HN)NoneHepG2 Huh7NOD.CB17-prkdcscid/NCrCrl (NOD-SCID).107 TCID50,IV[54]
    Getah-like alphavirus, M1Insertion of valosin-containing protein (VCP) inhibitorsXBP1Hep3BHep3B xenografts,
    Nonhuman primate Macaca fascicularis.
    5 × 105 PFUs, IV
    1 × 109 PFUs, IV
    [55]
    HSV, d0-GFPMutated in glycoprotein K and glycoprotein BNoneHuh7, SMMC7721, QGY7703, L-02, BEL7404, GSG7701, HCCLM3, MHHC97H, H22Huh7 and Hep3B xenografts BALB/c.1 × 107 PFU, IV
    Ad5Insertion of Golgi protein 73 (GP73) promoter and sphingosine kinase 1 (SphK1)-short hairpin RNA (shRNA)SphK1Huh7, HL-7702Huh7 xenografts BALB/c nude mice.6 × 108 PFU, IT
    Recombinant influenza viral, PR8deletion in NS and insertion of h GM-CSFhGM-CSFMDCK, A549, SMCC7721,HepG2HepG2 xenografts BALB/c nude mice.2 × 109 PFU, IT[56]
    MeV, MV-EdmNoneNoneCC-LM3, MHCC-97HLM3 xenografts BALB/c nude mice.5 × 106 PFU, IT[57]
    Ad, Ad-spInsertion of Vestigial-Like Family Member 4 (VGLL4)VGLL4Hep3B, Huh-7Huh-7 xenografts BALB/c nude mice.5 × 108 PFU, IT[58]
    HSV, HSV T-01α47 and γ34.5 loci are deleted and the LacZ gene replaces the ICP6 geneNoneHuH-7, Li-7 JHH-1, JHH2, JHH5, JHH6, JHH7, HLE, HLF, PLC/PRF/5, huH-1Hepa1-6 xenografts BALB/c nude mice.2 × 106 PFU, IT[59]
    Ad, Ad-ΔBInsertion of ING4 and TRAILING4 and TRAILHep3BHep3B xenografts BALB/c nude mice.1 × 1010 PFU, IV[60]
    Ad, Ad-wnt-E1A(Δ24bp)-TSLC1Insertion of TSLC1Wnt and Rb pathwayMHCC-97H, PLC/PRF/5PLC/PRF/5 xenografts BALB/c nude mice.6 × 108 PFU, IT[61]
    Ad, OAV SG655-mGMPInsertion of 11R-P53 and GM-CSF11R-P53 and GM-CSFHep3B-C, ECCG5ECCG5 xenografts BALB/c miceUnknow[62]
    Ad, Ad-ΔB/TRAIL and Ad-ΔB/IL-12Mutated in E1A and deleted in E1B regions. Insertion of hTRAIL or hIL-12hTRAIL or hIL-12Hep3B and HuH7Athymic nude mice, orthotopic model2 × 108 PFU, IV 1 × 1010 PFU, IV[63]
    MeV, (Res + MeV)Encoding of GFP as a marker gene and SCD as suicide geneNoneHepG2 and Hep3BNo animal model usedVarious MOIs[64]
    VV, GLV-2b-372Deletion of TK and insertion of TurboFP635 geneNoneHuh-7, Hep G2, SNU-449, and SNU-739Athymic nude mice Huh-7 xenograft1 × 105 PFU, IT[65]
    VV, GLV-1 h68Deletion of TK and insertion of Renilla luciferasegreenNoneHuh-7, Hep 3B, SNU-449 and SNU-739No animal model usedVarious MOIs[66]
    Ad, TelomelysinhTERT inserted upstream of the E1 genehTERTHuman: Huh-7, Hep3B, PLC5, HA22T, HCC36, and HepG2 Mouse: Hepa-1c1c7 and Hepa 1-6HBx transgenic mice, orthotopic model1.25 × 108 PFU, IT
    6.25 × 108 PFU, IT
    3.0 × 108 PFU, IT
    [67]
    HSV, G47ΔICP47 and γ34.5-deletionNoneHepG2, HepB, SMMC-7721, BEL-7404, and BEL-7405Balb/c nude mice SMMC-7721, BEL-7404 xenograft2 × 107 PFU, IT[68]
    HSV, LCSOVViral glycoprotein H gene linked with liver-specific apolipoprotein E (apoE)-AAT promoter. miR-122a complimentary sequence to the 3ʹ untranslated region (3ʹUTR). miR-124a and let-7 also inserted at 3ʹ UTRmiR122, miR-124a and let-7HuH-7, HepG2, and Hep3BHsd: athymic (nu/nu) mice, Hep3B xenograft5 × 106 PFU, IT[69]
    VV, GLV-1 h68Deletion of TK and insertion of Renilla luciferasegreen fluorescent protein (Ruc-GFP), β-galactosidase, β-glucuronidaseNoneHuH7 and PLC/PRF/5Athymic Nude-Foxn1nu HuH7 and PLC xenografts5 × 106 PFU, IV[70]
    Ad, SG7011let7TInsertion of eight copies of let-7 target sites (let7T) into the 39 untranslated region of E1AmiRNA, let-7HepG2, Hep3B, PLC/PRF/5, and Huh7Hep3B and SMMC-7721 xenografts BALB/c nude mice.5 × 108 PFU, IT[71]
    VV, JX-963Deletion of TK and VGF, insertion of h GM-CSFhGM-CSFNoneImmunocompetent, orthotopic, NZW rabbits VX2 tumor modelVarious PFU, IV[72]

    Although many preclinical research attempts have been made in oncolytic therapy in recent years, there are still very few programs that have entered the clinical stage. At present, the only HCC oncolytic virus entering clinical research is JX-549, with VV as an engineered vector. VV has the stability and efficiency of intravenous administration, is widely used in the safety of live vaccines, has the advantages of immune-inducing activity and better editability, and has become a carrier of various engineered tumor-melting viruses[73-75]. The thymidine kinase gene (TK) gene of JX-594 (also known as PexaVec; Jennerex Inc.) was deleted to make it more specific for cancer cell infection. In addition, hGM-CSF and β-galactosidase were inserted to enhance its immunostimulatory activity and replication capabilities[73,76,77]. JX-594 showed complete tumor response and systemic efficacy in a phase I clinical study[78]. In the phase II trial, low-dose JX-594 has significant anti-cancer effect and immune activation ability[79], but this requires earlier interventional therapy[80]. Currently, a large-scale 600-person multicenter Phase 3 trial is still in progress (NCT02562755). More clinical studies of HCC oncovirus are shown in Table 3.

    Table 3

    Clinical trials of oncolytic viral therapy for HCC

    Clinical trials identifierStatusActive treatmentnPrimary end points or outcomesRef.
    NCT03071094Active, not recruiting.
    Phase 1 and 2 trials
         JX-594;
         Nivolumab
    30DLTs, 4 weeks;
    ORR, 6 months
    NCT02562755Active, not recruiting.
    Phase 3 trials
         JX-594;
         Sorafenib
    600OS, 53 months
    NCT00554372Completed. Phase 2 trials     JX-59430mRECIST v1.0 criterion;
    Choi criterion. 4 weeks
    [81]
    NCT01387555Completed. Phase 2b trials     JX-594;129OS, 21 months[82]
    NCT00629759Completed.
    Phase 1 trials
         JX-59414MTD, Safety evaluation throughout study participation[83]

    HCC vaccine

    Tumor vaccine is a treatment program to increase the specificity of tumor antigens, mainly antigen peptide vaccines and DCs vaccines, which are used to stimulate specific immune responses. The clinical trials of therapeutic vaccines for HCC are summarized in Table 4. At present, there are relatively few registered clinical trials for DCs vaccines in HCC, partly because of the unsatisfactory results of previous clinical trials of such vaccines[86]. On the other hand, the tumor heterogeneity of HCC also limits the development of a single antigen peptide or DCs vaccine. With the development of large-scale DNA sequencing technology, patient-specific multi-target peptide or DCs vaccine is still a promising strategy for the treatment of HCC. DC, as professional antigen-presenting cells (APC), recognize, process and present TAA. Allogeneic DC vaccines can provide T cells with antigens and co-stimulatory molecules needed for immune response. In short, DCs are mobilized from peripheral blood and their expansion is stimulated with GM-CSF to produce DCs for reinfusion. Prior to this, DC needs to be exposed to TAA to trigger the specificity of the vaccine[87]. DCs can be transduced with DNA or RNA encoding known TAA, or directly co-cultured with patient tumor lysate[88]. Phase I clinical studies have shown that the allogeneic DCs vaccine can produce a specific immune response in 73% of HCC patients[89].

    Table 4

    Clinical trials of therapeutic vaccines for HCC

    Clinical trials identifierStatusActive treatmentnPrimary endpoints or outcomesRef.
    NCT04248569Recruiting, Phase IDNAJB1-PRKACA peptide vaccine, Nivolumab, Ipilimumab.12DLTs, 4 weeks; Fold change in interferon-producing DNAJB1-PRKACA-specific CD8+ and CD4+ T cells, 12 weeks;
    NCT03674073Recruiting, Phase INeoantigen Vaccines; Microwave Ablation24CTCAE v4.0, 1 year
    NCT02409524Completed, Phase IIIndividualized anti-cancer vaccine (CRCL-AlloVax)15OS, 12 weeks
    NCT01974661Completed, Phase ICOMBIG-DC vaccine (ilixadencel).18Registration of adverse events. 0.5 years[84]
    NCT03203005Completed, Phase I/IIIMA970A vaccine; CV8102 adjuvant; Cyclophosphamide.22Registration of adverse events, 2 years; Immunogenicity, 2 years
    NCT00005629Completed, Phase I/IIAlpha-fetoprotein peptide-pulsed autologous dendritic cell vaccine6Safety, 1 month
    NCT00022334Completed, Phase I/IIAlpha-fetoprotein peptide-pulsed autologous dendritic cell vaccine33DLT and MTD, 1 year
    NCT04147078Recruiting, Phase INeoantigen-primed dendritic cell (DC) cell vaccine80DFS, 5 years
    NCT04251117Recruiting, Phase, I/IIaPersonalized neoantigen DNA vaccine (GNOS-PV02) and plasmid-encoded IL-12 (INO-9012) in combination with pembrolizumab (MK-3475)12CTCAE v5.0, 2 years
    Immunogenicity, 2 years
    NCT02089919Completed, Phase I/IICancer stem cell vaccine40Adverse events. 3 months[85]
    NCT00028496Completed, Phase IRecombinant fowlpox-CEA(6D)/TRICOM vaccine48DLT and MTD, 56 days.
    NCT03942328Recruiting, Phase IAutologous dendritic cells and Prevnar vaccine26Adverse events. 1 year
    NCT02232490Recruiting, Phase IIIHepcortespenlisimut-L (V5) therapeutic vaccine120Changes in plasma AFP, 3 months

    Chimeric antigen receptor T cell therapy

    In addition to immune checkpoint inhibitors, oncolytic viruses and vaccines, adoptive therapy using genetically modified T cells have also become one of the potential immunotherapy options for HCC. T cells can be engineered to express a chimeric antigen receptor (CAR), which is composed of a T cell receptor CD3ζ chain and co-stimulatory receptors (e.g., CD28 and TNFRSF9) to form an antigen recognition domain[90]. The antigen recognition domain endows CAR-T cells with specificity for tumor-associated antigens, which shows promise in the treatment of HCC. Besides, CAR-T cells have a strong adaptive immunity and can recognize antigens that are not present in MHC molecules. CAR-T cell therapy has been used in the preclinical treatment of a variety of solid tumors, but there are few clinical studies on HCC, and more are still in the preclinical research stage. Like the HCC vaccine, the technical difficulty lies in the choice of tumor-specific antigens[91]. CD133 is expressed by cancer stem cells derived from various epithelial cells and is an attractive cancer treatment target. CAR-T cells targeting CD133 have shown the feasibility of treating advanced HCC, with controllable toxicity and effective activity[92]. Glypican-3 (GPC3) is a member of the heparan sulfate glycoprotein family and belongs to a transmembrane glycoprotein. It plays an important role in cell proliferation, differentiation and metastasis. CAR-T cells targeting glypican-3 can inhibit the growth of HCC[93,94]. Besides, there are HCC recognition antigens such as NKG2D[95] and CD147[96] for CAR-T cell transformation. In addition, the CAR of CAR-T cells can be inserted into the expression of a variety of cytokine genes to overcome the immunosuppressive effects of the HCC microenvironment[97,98]. The clinical trials of CAR-T cell therapy for liver cancer are summarized in Table 5.

    Table 5

    Clinical trials of Chimeric antigen receptor T cell therapy for liver cancer

    No.TitleStatusConditionsInterventionsURL
    1Study evaluating the efficacy and safety With CAR-T for liver cancerUnknown statusLiver neoplasmsBiological: EPCAM-targeted CAR-T cellshttps://ClinicalTrials.gov/show/NCT02729493
    2Clinical study of ET1402L1-CAR T cells in AFP expressing hepatocellular carcinomaTerminatedHepatocellular carcinoma|liver cancerBiological: autologous ET1402L1-CART cellshttps://ClinicalTrials.gov/show/NCT03349255
    3T cells co- expressing a second generation glypican 3-specific chimeric antigen receptor with cytokines interleukin-21 and 15 as immunotherapy for patients with liver cancer (TEGAR)WithdrawnHepatocellular carcinoma|hepatoblastomaGenetic: TEGAR T cells|drug: cytoxan|drug: fludarabinehttps://ClinicalTrials.gov/show/NCT04093648
    4Glypican 3-specific chimeric antigen receptor expressed in t cells for patients with pediatric solid tumors (GAP)RecruitingLiver CancerGenetic: GAP T cells| drug: cytoxan|drug: fludarahttps://ClinicalTrials.gov/show/NCT02932956
    5Safety and Efficacy of CEA-targeted CAR-T therapy for relapsed/refractory CEA+ cancerRecruitingSolid Tumor|Lung CancerBiological: CEA CAR-T cellshttps://ClinicalTrials.gov/show/NCT04348643
    6Autologous CAR-T/TCR-T cell immunotherapy for solid malignanciesRecruitingEsophagus cancer|hepatoma|glioma|gastric cancerBiological: CAR-T/TCR-T cells immunotherapyhttps://ClinicalTrials.gov/show/NCT03941626
    7A Study of MG7 redirected autologous T cells for advanced MG7 positive liver metastases (MG7-CART)Unknown statusLiver MetastasesBiological: MG7-CARThttps://ClinicalTrials.gov/show/NCT02862704
    8A Study of CD147-targeted CAR-T by hepatic artery infusions for very advanced hepatocellular carcinomaRecruitingAdvanced hepatocellular carcinomaBiological: CD147-CARThttps://ClinicalTrials.gov/show/NCT03993743
    9CAR-T hepatic artery infusions and Sir-Spheres for liver metastasesCompletedLiver MetastasesBiological: anti-CEA CAR-T cells|Device: Sir-Sphereshttps://ClinicalTrials.gov/show/NCT02416466
    10CAR-T hepatic artery infusions or pancreatic venous infusions for CEA-expressing liver metastases or pancreas cancerActive, not recruitingLiver MetastasesBiological: anti-CEA CAR-T cellshttps://ClinicalTrials.gov/show/NCT02850536
    11Hepatic transarterial administrations of NKR-2 in patients with unresectable liver metastases from colorectal cancerActive, not recruitingColon Cancer Liver MetastasisBiological: NKR-2 cellshttps://ClinicalTrials.gov/show/NCT03370198
    12Dose escalation and dose expansion phase I study to assess the safety and clinical activity of multiple doses of NKR-2 administered concurrently with FOLFOX in colorectal cancer with potentially resectable liver metastasesActive, not recruitingColon Cancer Liver MetastasisBiological: NKR-2 cellshttps://ClinicalTrials.gov/show/NCT03310008
    13Interleukin-15 armored Glypican 3-specific chimeric antigen receptor expressed in T cells for pediatric solid tumorsNot yet recruitingLiver Cancer|Rhabdomyosarcoma, et al.Genetic: AGAR T cells|drug: cytoxan|drug: fludarahttps://ClinicalTrials.gov/show/NCT04377932
    14Treatment of relapsed and/or chemotherapy refractory advanced malignancies by CART133CompletedLiver Cancer|Pancreatic Cancer, et al.Biological: anti-CD133-CAR vector-transduced T cellshttps://ClinicalTrials.gov/show/NCT02541370
    15Autologous CAR-T/TCR-T cell immunotherapy for malignanciesRecruitingSolid tumorsBiological: CAR-T cell immunotherapyhttps://ClinicalTrials.gov/show/NCT03638206
    16A study of chimeric antigen receptor T cells combined with interventional therapy in advanced liver malignancyUnknown statusCarcinoma, Hepatocellular|Pancreatic Cancer, et al.Drug: CAR-T cellhttps://ClinicalTrials.gov/show/NCT02959151
    17A clinical research of CAR T cells targeting EpCAM positive cancerRecruitingHepatic Carcinoma, et al.Biological: CAR-T cell immunotherapyhttps://ClinicalTrials.gov/show/NCT03013712
    18NKG2D-based CAR T-cells immunotherapy for patient with r/r NKG2DL+ solid tumorsNot yet recruitingHepatocellular Carcinoma|Glioblastoma, et al.Biological: NKG2D-based CAR T-cellshttps://ClinicalTrials.gov/show/NCT04270461
    19GPC3-T2-CAR-T cells for immunotherapy of cancer with GPC3 expressionRecruitingHepatocellular Carcinoma, et al.Biological: GPC3 and/or TGF-beta targeting CAR-T cellshttps://ClinicalTrials.gov/show/NCT03198546
    20NKG2D CAR-T(KD-025) in the treatment of relapsed or refractory NKG2DL+ tumorsNot yet recruitingSolid Tumor|Hepatocellular Carcinoma, et al.Drug: KD-025 CAR-T cellshttps://ClinicalTrials.gov/show/NCT04550663
    21GPC3-CAR-T Cells for the hepatocellular carcinomaNot yet recruitingHepatocellular CarcinomaBiological: GPC3-CAR-T cellshttps://ClinicalTrials.gov/show/NCT04506983
    22CAR-T cell immunotherapy for HCC targeting GPC3WithdrawnGPC3 Positive Hepatocellular CarcinomaBiological: CAR-T cell immunotherapyhttps://ClinicalTrials.gov/show/NCT02723942
    23Clinical Study on the efficacy and safety of c-Met/PD-L1 CAR-T cell injection in the treatment of HCCUnknown statusPrimary Hepatocellular CarcinomaBiological: c-Met/PD-L1 CAR-T cell injectionhttps://ClinicalTrials.gov/show/NCT03672305
    24A study of GPC3 redirected autologous T cells for advanced HCCUnknown statusCarcinoma, HepatocellularDrug: TAI-GPC3-CART cellshttps://ClinicalTrials.gov/show/NCT02715362
    25GPC3-targeted CAR-T cell for treating GPC3 positive advanced HCCRecruitingHepatocellular CarcinomaBiological: CAR-T cell immunotherapyhttps://ClinicalTrials.gov/show/NCT04121273
    26A Study of GPC3-targeted T cells by intratumor injection for advanced HCC (GPC3-CART)Unknown statusCarcinoma, HepatocellularDrug: GPC3-CART cellshttps://ClinicalTrials.gov/show/NCT03130712
    27Phase I/II study of anti-Mucin1 (MUC1) CAR T cells for patients with MUC1+ advanced refractory solid tumorUnknown statusHepatocellular Carcinoma, et al.Biological: anti-MUC1 CAR T cellshttps://ClinicalTrials.gov/show/NCT02587689
    28Anti-GPC3 CAR T for treating patients with advanced HCCCompletedHepatocellular CarcinomaBiological: anti-GPC3 CAR Thttps://ClinicalTrials.gov/show/NCT02395250
    29Anti-GPC3 CAR-T for treating GPC3-positive advanced hepatocellular carcinoma (HCC)Unknown statusHepatocellular CarcinomaBiological: retroviral vector-transduced autologous T cells to express anti-GPC3 CARs|drug: fludarabine|drug: cyclophosphamidehttps://ClinicalTrials.gov/show/NCT03084380
    30Clinical study of redirected autologous T cells with a chimeric antigen receptor in patients with malignant tumorsActive, not recruitingHepatocellular Carcinoma, et al.Genetic: CAR-CD19 T cell|genetic: CAR-BCMA T cell|genetic: CAR-GPC3 T cell|genetic: CAR-CLD18 T cell|drug: fludarabine|drug: cyclophosphamidehttps://ClinicalTrials.gov/show/NCT03302403
    31A clinical research of CAR T cells targeting CEA positive colorectal cancer (CRC)Not yet recruitingStage III Colorectal Cancer|Colorectal Cancer Liver MetastasisBiological: Anti-CEA-CAR Thttps://ClinicalTrials.gov/show/NCT04513431
    32Study of anti-CEA CAR-T + chemotherapy vs. chemotherapy alone in patients with CEA+ pancreatic cancer & liver metastasesNot yet recruitingMalignant tumor of pancreas metastatic to liverBiological: anti-CEA CAR-T cells|drug: gemcitabine/nab paclitaxel|drug: NLIR+FU/FA|drug: capecitabinehttps://ClinicalTrials.gov/show/NCT04037241
    33Glypican 3-specific chimeric antigen receptor expressing T cells for hepatocellular carcinoma (GLYCAR)RecruitingHepatocellular CarcinomaGenetic: GLYCAR T cells|drug: cytoxan|drug: fludarabinehttps://ClinicalTrials.gov/show/NCT02905188
    344th generation chimeric antigen receptor T cells targeting glypican-3RecruitingAdvanced Hepatocellular CarcinomaDrug: CAR-GPC3 T cellshttps://ClinicalTrials.gov/show/NCT03980288
    35PD-1 antibody expressing CAR-T cells for EGFR family member positive advanced solid tumor (lung, liver and stomach)Unknown statusPD-1 Antibody|CAR-T cells|advanced solid tumorBiological: HerinCAR-PD1 cellshttps://ClinicalTrials.gov/show/NCT02862028
    36Chimeric antigen receptor T cells targeting glypican-3RecruitingHepatocellular carcinomaBiological: CAR-GPC3 T cellshttps://ClinicalTrials.gov/show/NCT03884751
    37A clinical study in patients with high-risk recurrent primary hepatocellular carcinoma using autologous TILsActive, not recruitingHepatic CarcinomaDrug: tumor infiltrating lymphocytehttps://ClinicalTrials.gov/show/NCT04538313
    38CAR-GPC3 T cells in patients with refractory hepatocellular carcinomaCompletedHepatocellular CarcinomaGenetic: CAR-GPC3 T cellshttps://ClinicalTrials.gov/show/NCT03146234

    The current combination of therapeutic strategies for HCC

    Currently, there are many immunotherapy and other target therapy drugs approved by the Food and Drug Administration (FDA) of The United States of America (USA) for liver cancer treatment, including Atezolizumab, Avastin (Bevacizumab), Bevacizumab, Cabometyx (Cabozantinib-S-Malate), Cyramza (Ramucirumab), Keytruda (Pembrolizumab), Lenvatinib Mesylate, Lenvima (Lenvatinib Mesylate), Nexavar (Sorafenib Tosylate), Nivolumab, Opdivo (Nivolumab), Pemazyre (Pemigatinib), Pembrolizumab, Pemigatinib, Ramucirumab, Regorafenib, Sorafenib Tosylate, Stivarga (Regorafenib), Tecentriq (Atezolizumab). Single agent therapy has historically shown poor results in HCC, leading to trials of combination therapy for a more efficacious outcome. For example, the FDA has approved Opdivo (nivolumab) + Yervoy (ipilimumab) based on the CheckMate 040 trial, atezolizumab + bevacizumab for patients with advanced HCC based on the IMbrave150 (NCT03434379) study. The CheckMate 040 is a multicentered, open-labelled, multicohort, phase 1/2 study. The result showed that nivolumab + ipilimumab had manageable safety, promising objective response rate, and durable responses. The arm A regimen (4 doses nivolumab 1 mg/kg + ipilimumab 3 mg/kg every 3 weeks then nivolumab 240 mg every 2 weeks) received accelerated approval in the US based on this study[99]. The IMbrave150a study is a global, open-labelled, phase 3 trial for patients with unresectable HCC who had not previously received systemic treatment. The study included 336 patients in the atezolizumab + bevacizumab group and 165 patients in the sorafenib group. The result showed that atezolizumab + bevacizumab resulted in better overall (overall survival at 12 months was 67.2% vs. 54.6%) and progression-free survival (6.8 months vs. 4.3 months) outcomes than sorafenib[100]. There are many different combinations of immune checkpoint inhibitors with other different therapeutic strategies under investigation. Some of the combination clinical trials are concluded in the Table 1.

    Conclusion and prospect

    Immunotherapy is a revolution in HCC treatment. Significant responses have been observed in various tumor types with immunotherapy, especially immune checkpoint inhibitors and CAR-T cells. However, it is clear that not all HCC patients are sensitive to current immunotherapy, and even in those who do respond, the effect is difficult to last. Lots of data indicate that most HCCs are immunosuppressive tumors. Therefore, ongoing research using a multifaceted approach to enhance the activity of the immune environment remain underway to enhance current immunotherapy strategies.

    Declarations

    Authors’ contributions

    Drafted the outline of this review: Feng ZY, Xia HP

    Drafted the manuscript: Feng ZY, Xu FG, Liu Y, Xu HJ, Wu FB, Chen XB, Xia HP

    Finalized the manuscript: Chen XB, Xia HP

    Availability of data and materials

    Not applicable.

    Financial support and sponsorship

    This work was supported by grants from the National Natural Science Foundation of China (82072739), The Recruitment Program of Overseas High-Level Young Talents, “Innovative and Entrepreneurial Team” [No.(2018) 2015], Science and Technology Development Fund of Nanjing Medical University and Chinese Foundation for Hepatitis Prevention and Control-TianQing Liver Disease Research Fund (TQGB20190164, TQGB20200139).

    Conflicts of interest

    All authors declared that there are no conflicts of interest.

    Ethical approval and consent to participate

    Not applicable.

    Consent for publication

    Not applicable.

    Copyright

    © The Author(s) 2021.

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    Cite This Article

    Feng ZY, Xu FG, Liu Y, Xu HJ, Wu FB, Chen XB, Xia HP. The immune microenvironment and progression of immunotherapy and combination therapeutic strategies for hepatocellular carcinoma. Hepatoma Res 2021;7:3. http://dx.doi.org/10.20517/2394-5079.2020.107

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    • Divya Prasanna Kumar   
      This review sheds light on the use of immunotherapy as a therapeutic approach for the treatment of HCC. In addition, the need for combination therapy has been explained in brief by providing details of the ongoing clinical trials. 

      25 Jan 2021 09:29

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