MAbs against viral antigens
Given the potential antiviral effect of the antibodies, viruses have evolved multiple mechanisms to protect themselves from antibody binding. One of these, the viral receptor glycosylation, is widely shared among different viruses. Carbohydrates are poorly immunogenic and, therefore, do not stimulate the response of type B lymphocytes and simultaneously hide the underlying protein structures. HCV E2 protein contains up to 11 potential N-linked glycosylation sites. Specific glycans mask the CD81-binding site and, therefore, nAb epitopes [
48]. Lipid shielding may represent an additional strategy used by HCV to evade the antibody response. Current data suggest that key neutralizing epitopes are less accessible on LVPs. More recently, HCV has been found capable of direct cell-to-cell transmission, which is largely resistant to antibody neutralization [
49,
50]. HIV envelope protein is also glycosilated and changes occur in the frequency and position of glycans HIV gp120; these 'evolving glycan shields' have been shown to decrease sensitivity to antibody neutralization [
51]. Other factors of antibody escape for HIV are: trimerization of the gp 120 and gp 41 that can shield vulnerable epitopes better exposed on the individual monomeric subunits; kinetic and spatial constraints that impede antibodies from accessing potentially vulnerable sites during receptor binding and membrane fusion process; the variable loops of gp120 that are a prime target for nAbs, which usually have a very narrow breadth of reactivity [
52]. Finally, the high mutation rate of many viruses, including HIV and HCV, which undergo rapid antigenic variation, allows them to escape neutralization, constituting a significant hurdle for nAbs development.
All these problems may be counter-balanced by selecting nAbs which target conserved and more accessible areas of viral particles, and/or by using mixtures of nAbs which target various key epitopes. In fact, it has been demonstrated that combination therapy with mAb cocktails prevents escape variants for many viruses, including influenza [
53], coronavirus [
54] and LCMV [
55], and that broad neutralization in the sera of most of some individual HIV infected donors can be associated with single or four to five principal specificities [
56].
Recent studies have indicated that nAbs play a critical role in HCV disease outcome. Viral clearance was associated with a rapid induction of neutralizing antibodies in the early phase of infection with some evidence that these antibodies are broadly reactive [
57,
58]. In contrast, chronic HCV infection was characterized by absent or low-titer neutralizing antibodies in the early phase of infection and the persistence of infection, despite the induction of cross-neutralizing antibodies in the later phase of infection. Current understanding of the nAb response raised against HCV suggests that E2 is the major target, and that multiple epitopes within E2 may be targeted by both linear-and conformation-dependent antibodies. Predominantly, these neutralization epitopes overlap with CD81-binding sites and clearly demonstrate a role in inhibition of entry. Currently, one of these mAbs, MBL-HCV1, is being investigated in clinical trials in the prevention of liver re-infection after transplantation, for which novel antiviral preventive and therapeutic strategies are urgently needed. In fact, re-infection of the graft is universal, being characterized by accelerated progression of liver disease; IFN-based therapies exhibit enhanced adverse effects and limited efficacy in these patients [
59,
60]. MBL-HCV1 is a fully human monoclonal antibody isolated from transgenic mice and directed to a highly conserved linear epitope of HCV E2 glycoprotein. It is able to neutralize pseudoviruses from multiple HCV genotypes and has demonstrated efficacy in preventing HCV genotype-1 infection in HCV naïve chimpanzees. A phase I open-labeled, dose escalation study was performed in healthy adult volunteers starting with 1 mg/kg and escalating to 3, 10, 30 and 50 mg/kg after a 10-day post-infusion safety review. MBL-HCV1 was well-tolerated without any seriously adverse effect event. Based on the favorable safety, tolerability and pharmacokinetics data, a phase II study of MBL-HCV1 in chronically infected HCV patients undergoing liver transplantation has been planned [
61].
In the context of HIV disease, despite intensive study over two decades, only a small number of broadly neutralizing mAbs have been identified from infected patients and little is known about their activity
in vivo. These antibodies are able to inhibit viral entry of most primary HIV isolates
in vitro [
17,
62‐
64] and the exceptionally high level of mutation found in their genes may reflect chronic immune responses to HIV and persistent hypermutation and selection [
65]. A number of trials evaluating different formulations of anti-HIV monoclonal antibodies are now in progress.
The first trial assessed a chimeric monoclonal antibody CGP 47,439 to the V3 loop of the HIV-1 envelope gp120 over 21 weeks [
66,
67]. Subsequent studies evaluated the kinetics of monoclonal antibody F105 directed to the CD4-binding site of gp120 [
68,
69], a humanized antibody binding to the V3 epitope GPGRAF [
70]. Finally, a humanized mAb, KD-247 is under evaluation in clinical trials. Its epitope was mapped to 6 aa, IGPGRA, at the tip of the V3 loop of Envelope protein and demonstrates cross-neutralizing activity against HIV-1 isolates in clade B [
71].
A drug based on the mAb cocktail mode is also currently in clinical development. In this regard it has already been observed that in HIV neutralization assays the effectiveness of a mix of broadly neutralizing antibodies increased synergistically compared to the effect of the individual antibody. The synergy effect was relatively weak, with a maximum of two- to four-fold enhancement, between antibody pairs, thereby increasing neutralization titers about 10-fold in triple and quadruple antibody combinations [
72]. However, the use of antibodies in the cocktail mode, as an approach to improve their effectiveness, is already recognized for other pathogens or toxins. In the case of tetanus toxin, it has been reported that combining the action of three out of four antibodies increased the neutralizing activity up to 200 times [
73]. In the case of botulinum toxin, neutralizing activity has been reported up to 20,000 times higher when using a mixture of three monoclonal antibodies [
74]. Instead, other studies have demonstrated that the combination of two potent neutralizing mAbs against HIV, VRC01 and PG9, although not synergistic, can mediate additive neutralization viral activity and provides an improved neutralization coverage of 90% to 97% of viral strains by combining independent epitope targeting [
75].
In a proof-of-concept passive immunization trial with humans, it has been demonstrated that a cocktail of the three broadly neutralizing mAbs - 2G12, 4E10 and 2F5 - was able to delay viral rebound in patients whose infections were fully suppressed by antiretroviral treatment before administration of the antibodies [
76]. Interestingly, the main antiviral effect observed was primarily attributable to the 2G12 antibody, a mAb that binds to a non-continuous epitope composed of glycosylation residues distributed over the envelope protein gp120 [
64], whereas the other two mAbs, 4E10 [
77] and 2F5 [
78], recognize two adjacent highly conserved epitopes on the membrane-proximal ectodomain of the HIV-1 envelope protein gp41. In earlier phase I clinical trials, safety and tolerability were demonstrated [
79,
80]. During a long-term multiple dose phase II clinical trial, high doses of the three neutralizing antibodies were given in combination to 14 HIV-1-infected individuals at weekly intervals over three months. Pharmacokinetic analysis revealed that repeated infusions at high dose levels were well tolerated by the patients and did not elicit an endogenous immune response against the monoclonal antibodies. The antibodies showed distribution and elimination kinetics similar to those seen for other human-like antibodies, though monoclonal antibody 2G12 had a significantly longer elimination half-life (21.8 +/- 7.2 days) than monoclonal antibodies 4E10 (5.5 +/- 2.2 days) and 2F5 (4.3 +/- 1.1 days) [
81]. Furthermore, analyses of the emergence of mutations conferring resistance to these three mAbs were performed. Sequence analysis of the 2G12 epitope relevant N-glycosylation sites of viruses derived from 13 patients demonstrated that mutations in these sites are associated with resistance.
In vitro selection experiments with isolates of four of these individuals corroborated the
in vivo finding that virus strains rapidly escape 2G12 pressure. Importantly,
in vitro selection with 2F5 and 4E10 demonstrated that resistance to these nAbs can be difficult to achieve and can lead to selection of variants with impaired infectivity [
82]. Moreover, generation of viruses resistant to the triple-combination was a slower process characterized by recurrent loss of virus replication; some generated triple-resistant viruses seemed to be impaired in their replicative fitness, and none of the patients developed detectable viruses that escaped neutralization by all three mAbs within the 77-day observation period [
83].
As is true with all mAbs designed for infectious disease, the development of a successful vaccine would reduce the need for them. However, given the scarcity of drugs in the field of virology and given the slow progress on the HIV vaccine front, the development and use of microbicides, compounds that could be applied topically to prevent HIV transmission, is one of the possible strategies to counter the spread of HIV. In this regard, mAbs could be proposed as suitable components of microbicides to fight HIV entry at mucosal surface. A safety study of P2G12 mAb administered vaginally in healthy women has been completed. P2G12 is the broadly neutralizing 2G12 mAb manufactured from tobacco plants [
84]. Most mAbs in clinical trials have been produced using a system called Chinese Hamster Ovary cell (CHO-Cell) fermentation [
85], including 2G12 used along with 2F5 and 4E10 antibodies as a cocktail. The CHO-Cell fermentation production method is very expensive and cannot produce enough mAbs on a scale required for the global market; therefore, plant manufacture of such mAbs may hopefully offer some solutions to lower production costs and improve output. The process yields five grams of purified antibody from 250 kg of tobacco and production costs could be 10 to 100 times lower than when using conventional bioreactors. This study has been designed to confirm the safety of a vaginally delivered mAb P2G12 derived from plants and manufactured to Good Manufacturing Practice (a quality standard used for the manufacture of medicinal products). The medicine is the first plant-produced antibody to be greenlit for clinical testing by Britain's Medicines and Healthcare products Agency (MHRA). It took about a year to get that agency's stamp of approval because it required assurances that the drugs did not contain allergenic plant sugars or pesticides. No matter how it is produced, P2G12 antibody has not been shown to actually prevent HIV-1 infection in clinical trials; thus a version made from tobacco plants would not see approval any time soon. P2G12 would also likely be just one ingredient in a cocktail of plant-produced antibodies [
84].
MAbs against host receptors
To eliminate or reduce the development of escape variants it has been proposed that targeting the conserved cellular receptors of the virus may open new avenues for a viable antibody therapy for HIV infection. HIV entry into CD4+ T cells requires the presence of a co-receptor, either CCR5 or CXCR4, on the target cell. Thus, based on this hypothesis, mAbs directed against CD4 and against the co-receptor CCR5, have been developed and are being analyzed in clinical trials.
CD4 functions as a co-receptor, physically associating with the TCR during Ag recognition by binding to a non-polymorphic component of the major histocompatibility complex (MHC) class II molecules on the surface of the antigen-presenting cell. Ibalizumab, a humanized mAb, binds CD4 on T cell surface away from the binding site for MHC class II molecules. It does not inhibit gp120 binding to CD4 but appears to exert its antiviral property by post-binding conformational effects that prevent CD4-bound gp120 from interacting with CCR5 or CXCR4 [
19,
86]. By contrast, other monoclonal antibodies, that competitively inhibit gp120 binding, interfere with MHC class II immune function [
87,
88]. The reported human experience with Ibalizumab consists of three clinical trials. During phase I study, it was observed that peak mean reductions in viral load occurred later in the higher dose cohorts, whereas the extent and duration of viral suppression correlated with the degree of CD4+ cell coating by ibalizumab, which was maintained longer in the higher dose cohorts, with a duration of 15 to 34 days. Peak increases in CD4 counts at one day after infusion, well before the peak declines in viral load; this suggests that the increase may have been due to redistribution of CD4+ cells from lymphoid tissue rather than regeneration of CD4+ cells in the setting of viral suppression. A multidose study demonstrated continued safety over an extended treatment period and provided data on the development of ibalizumab resistance. Resistance testing showed reduced susceptibility relative to baseline. Resistant isolates remained dependent on CD4 for viral entry, suggesting that resistance did not develop through the use of alternative receptors. Genotypic analysis was unable to identify mutations, diagnostic of ibalizumab resistance. Consistent with the allosteric mechanism of ibalizumab's anti-HIV-1 effect, the development of resistance is associated with a reduction in the maximum percentage inhibition rather than the shift in the IC50 characteristic of competitive inhibitors [
89,
90]. The half-life of IgGs under normal physiological circumstances is two to three weeks [
91]. In contrast, the average half-life of ibalizumab is 3 to 3.5 days [
89]. This is consistent with observations of other anti-CD4 antibodies, in which internalization or shedding of the receptor results in more rapid antibody degradation. A randomized, double-blind, placebo controlled, phase IIa study has evaluated the ibalizumab efficacy, the results showing a considerable viral load reduction with respect to the placebo arm [
92].
CCR5 is a chemokine receptor that mediates activation and migration of T cells and other leukocytes. CCR5-using (R5) viruses typically mediate transmission and then predominate through the progression to symptomatic disease. Viruses can use an alternative chemokine receptor, CXCR4, either exclusively or in addition to CCR5. The CXCR4-using virus can be present initially, but tends to result in an increasing proportion of subjects in the later stages of the disease [
93]. CCR5 co-receptor antagonists represent an emerging antiretroviral treatment class and the first to target a host molecule.
Currently, two anti-CCR5 mAbs are being investigated. One of these is CCR5mAb004, a fully human IgG4 monoclonal antibody with robust activity against a diverse panel of HIV-1 isolates; it synergizes
in vitro with other ARV classes and appears safe and effective in reducing HIV viral load. High levels of receptor occupancy were observed for 14 to 28 days with the highest dose cohorts, suggesting the potential for weekly, fortnightly or even monthly dosing [
94].
The other anti-CCR5 mAb is PRO 140, a humanized mAb that also synergizes with small-molecule CCR5 antagonists in laboratory studies [
95]. PRO140 is being investigated in two modes of administration: the classical intravenous (IV) form, and subcutaneous (SC) form. The trial involving SC administration is the first to bear the proof of concept for a mAb administered subcutaneously in HIV-1 infected subjects as a potent and long-acting antiretroviral agent.
An IV form of PRO 140 tested as monotherapy in HIV-1 subjects with only R5 virus detectable [
96] demonstrated potent and prolonged antiviral activity, with a 1.83 log10 mean reduction in HIV-1 RNA and safety relative to placebo. The successive randomized, double-blind, placebo-controlled IIa trial examined the antiviral activity, tolerability and pharmacokinetics of single intravenous infusions of up to 10-mg/kg of mAbs. All PRO 140-treated subjects treated with 10 mg/kg experienced a 1-log10-unit reduction in HIV-1 RNA level, there being just one exception; a post-study analysis using the enhanced-sensitivity Trofile assay determined that this subject had dual/mixed virus at screening. There was no change in co-receptor tropism or emergence of PRO 140-resistant virus during the course of this study, supporting the view that PRO 140 broadly inhibits R5 HIV-1 with a high barrier to resistance. The maximum tolerated dose of IV PRO 140 has not been determined, suggesting a sizeable margin of safety for PRO 140 SC administration study [
97].
The study involving PRO 140 SC administration showed virologic suppression between successive doses and no changes in R5 viral susceptibility to PRO 140 following three weeks of monotherapy, indicating no adaptation of virus to use CCR5 in the presence of drug. Pharmacokinetic data suggest the possibility of a drug regimen administered fortnightly for HIV infected individuals. Proteins and other macromolecules drain from SC sites into both blood capillaries and the lymphatic system. In animals, proteins with molecular weights of greater than 16,000 daltons have been observed to drain primarily into the lymphatic system following SC administration [
98]. Such proteins transit through lymph fluid and typically are not absorbed significantly into the blood until they reach the thoracic duct. Since the molecular weight of PRO 140 is approximately 150,000 daltons, a substantial amount of SC PRO 140 can be expected to drain into the lymphatic system and potentially encounter CCR5+ cells in lymphoid tissues prior to reaching the bloodstream. For these reasons, serum concentrations may not provide a full picture of the overall exposure following SC dosing of PRO 140. SC infusion is currently used by individuals with primary immunodeficiency to self-administer at home significantly larger amounts (approximately 11 grams) and volumes (approximately 70 mL total, up to 15 mL/site) of the weekly SC-administered immunoglobulin [
99]. Self-administration of 324 mg SC PRO 140 would be much simpler in comparison. Therefore, SC PRO 140 offers the potential for significant dose-dependent HIV-1 RNA suppression and may offer greater convenience for many patients in terms of patient self-administration [
100].
The SC injection mode was chosen in order to evaluate PRO140 safety and efficacy as an adjunct to an oral antiretroviral regimen in HIV-infected injection drug users with viral rebound and documented poor adherence to the previous antiretroviral regimen. Therefore, a phase IIb, national, multicenter, randomized, double-blind, placebo-controlled study was initiated and is currently recruiting participants. Given the complications that arise from the occurrence of drug resistances, the use of antibodies together with combined therapy increases the drug number and, therefore, the therapeutic opportunities. In particular, in the case of CCR5 inhibitors, one report has demonstrated that resistance to CCR5 inhibitors may increase the sensitivity of the resistant virus to certain neutralizing antibodies [
101].
Compared to CCR5, CXCR4-based blocking agents as therapy against HIV are less attractive due to the crucial role of CXCR4 in many biological processes, and the absence to date of known naturally occurring mutations leading to the inactivation of CXCR4 gene in humans. Moreover, one major problem is linked to the fact that, whereas R5 viruses are found on their own in 50% or more of patients, viruses that using CXCR4 co-receptor (X4) usually are present mixed together with R5 viruses; therefore, the use of CXCR4 specific mAbs could result in only little or transient effect on the overall viremia, also complicating the evaluation of pharmacological activity. However, antibodies against CXCR4 might still provide some benefits for some HIV positive patients when co-administrated with CCR5 antagonists, if the safety of such combinations is established [
93].
There is a pressing need for antiviral agents that are effective against multiple classes of viruses. Broad specificity might be achieved by targeting phospholipids that are widely expressed on infected host cells or on viral envelopes. Phosphatidylserine (PS), the most abundant anionic phospholipid of the plasma membrane, is segregated at the inner leaflet of the plasma membrane of resting mammalian cells. Loss of PS asymmetry occurs during apoptosis, cell injury, cell activation and malignant transformation, and results from inhibition of the translocases or activation of PS exporters, or lipid scrambling enzymes, such as scramblases. After enveloped viruses replicate within the host cell, they create their 'envelope' by carrying along part of the host cell's membrane upon exiting. As a result, the target phospholipid becomes exposed on the surface of the virus as well as on the infected host cell [
102].
Bavituximab is the first in a new class of patented antibody therapeutics that target and, preferentially bind, to these exposed phospholipids. It has demonstrated broad therapeutic potential across multiple oncology indications and represents a new approach to treating viral disease, too. Bavituximab is currently being evaluated in randomized phase II clinical trials for non-small cell lung cancer and pancreatic cancer, for therapy of chronic HCV infection and for HIV/HCV co-infection. The therapeutic effect of bavituximab appears to be due to ADCC of tumor and virus-infected cells. Since PS exposure is an early event during virus infection, ADCC may limit virus spread. Furthermore, in the infectious disease setting, bavituximab causes opsonization and clearance of infectious virus from the bloodstream, leaving less virus to infect other tissues. Three completed phase I HCV clinical trials have shown that bavituximab is generally safe and well-tolerated. Reductions in serum HCV RNA levels were also observed. A randomized phase II clinical trial with previously untreated HCV genotype-1 infected patients was designed to determine the early virologic response (EVR) rate after 12 weeks of therapy with bavituximab in combination with the antiviral drug ribavirin and safety profile versus pegylated IFN-α-2a and ribavirin. The results show that the combination of bavituximab with ribavirin has a better safety profile than an IFN-containing regimen. However, the EVR development in the bavituximab-containing arm was later than the IFN-containing group; therefore, a longer-term evaluation is needed to adequately compare their effectiveness. In addition, the lower dose level appears to be more active in HCV patients than the high dose does. Such results suggest that future studies evaluating longer bavituximab treatment durations at or around the lower dose level in combination with ribavirin and potentially direct acting antivirals in certain patient populations may hold promise as IFN-free HCV therapeutic regimens [
103].
Targeting PS on cells infected with multiple different viruses and on virions themselves is a promising antiviral strategy. Although resistance has developed in monotherapy trials with ibalizumab (an anti-CD4 antibody), host-derived antigen, such as anionic phospholipids, on virus-infected cells are independent of the viral genome and as a consequence the acquisition of drug resistance should be theoretically less problematic than with agents that target virus-encoded components.
Immunomodulatory mAbs
Since the discovery of PD-1 as an inhibitory receptor associated with T-cell dysfunction, the roles of various inhibitory receptors on virus-specific CD8+ T cells have been extensively studied in human chronic viral infections, such as HCV, HBV and HIV infections. As blocking the inhibitory receptors
in vitro restores the functions of virus-specific T cells, novel HIV and HCV treatments based on blockade of several immune checkpoint molecules are being investigated. In particular, mAbs interfering with two major inhibitory networks of the B7:CD28 family, namely the PD-1 and CTLA-4 pathways [
104], are currently being studied in clinical trials, to evaluate their safety and efficacy. These mAbs recognize the PD-1 or CTLA-4 receptor and neutralize the binding with their respective ligands.
The PD-1:PD-L1 pathway delivers inhibitory signals which regulate T cell activation. As a result it performs a key role in various processes, namely in multiple tolerance checkpoints that prevent autoimmunity, in the suppressive tumor microenvironment, in the immune-mediated tissue damage, in host defenses aimed at eradicating microbial pathogens and tumors and finally, in T cell exhaustion that contributes to both lack of viral control during chronic infections and to T cell unresponsiveness [
105]. In cancers, a strong correlation between increased PD-L1 expression on tumors and a negative survival prognosis in patients has already been observed. Various studies indicate that mAbs targeting the PD-1 signaling pathway reinvigorate antigen-specific T-cell responses and promote an immune response to fight tumors [
106]. In HCV infection the relationship between the PD-1 expression and the outcome of the acute HCV infection was questioned; subsequently, recent studies have shown that the progression of acute HCV infection to the chronic stage is associated with a high level of PD-1 on HCV-specific CD8+ T cells, whereas the clearance of HCV infection is associated with lower levels of PD-1 expression [
36].
Given these premises, MDX-1106, a fully human antibody also known as ONO-4538, and CT-011, a humanized antibody, both interacting with PD-1 receptor, are being developed as a treatment for cancer disease and for therapy of chronic HCV infection [
107]. To date, most clinical experience with PD-1 blockade has been gained with MDX-1106 in the tumor setting. Drug-related grade 3 or 4 toxic effects occurred in 14% of patients, in whom there were drug-related adverse events of special interest, those with potential immune-related causes; they included pneumonitis, vitiligo, colitis, hepatitis, hypophysitis and thyroiditis. Pneumonitis (3%) ranged from isolated radiographic abnormalities to progressive, diffuse infiltrates associated with clinical symptoms in a small number of patients. Although three deaths occurred, mild-to-moderate pneumonitis was managed successfully with either observation or glucocorticoids. However, objective responses were observed in approximately one in four to one in five patients with non-small-cell lung cancer, melanoma, or renal-cell cancer; overall, an adverse-event profile does not appear to preclude its use [
108]. Besides these studies, an ongoing phase I safety trial with active hepatitis C genotype 1 infected patients has been designed to assess the safety and tolerability profile of MDX-1106 [
109]. Clinical studies to evaluate the use of CT-011 in HCV disease have also been initiated [
110].
CTLA-4 is up-regulated on activated T cells and inhibits T cell activation by reducing the production of IL-2 and arresting cell cycle progression. CTLA-4 has also been shown to have an impact on T cell responses in animal tumor models and humans [
111,
112]. Human trials that used a blocking anti-CTLA-4 mAb demonstrated a reduction in tumor mass and clinical benefit in a substantial minority of treated subjects. Studies of the role for CTLA-4 in chronic infections have produced mixed results. In chronic HIV infection, many studies indicate that impaired CD4+ T cell function is associated with viral persistence [
113], although the function of CTLA-4 in causing HIV persistence by suppressing T cell function remains unclear [
114]. On the other hand, CTLA-4's role in chronic HCV infection seems to be more defined. The HCV-specific CD8+ T cells found in the livers of chronic HCV patients overexpressed not only PD-1, but also CTLA-4. Co-expression of PD-1 and CTLA-4 was observed in liver-infiltrating lymphocytes, but not in peripheral blood lymphocytes [
36], suggesting the phenotypic differences of virus-specific CD8+ T cells in different
in vivo compartments. PD-1 and CTLA-4 expressing HCV-specific T cells were profoundly dysfunctional [
115].
Tremelimumab is a fully human IgG2 mAb directed against CTLA-4. While a phase II study for HIV disease with this drug has been withdrawn prior to enrollment, clinical trials for HCV disease are still underway. Tremelimumab binds to activated T lymphocytes and results in inhibition of B7-CTLA-4-mediated down-regulation of T-cell activation. It also acts as an IL-2 stimulant. It was generated, using XenoMouse technology (Figure
2), as an anticancer agent and is currently in worldwide phase III development for malignant melanoma, phase II development for colorectal cancer, gastrointestinal cancer, gynecological cancer and non-small cell lung cancer in the US and other countries. It is also being investigated for prostate, breast and pancreatic cancer in various countries. As for anti-PD-1 antibodies, immune-related adverse effects of tremelimumab are of special interest because of its presumed mechanism of action. Most of the experience in identifying and managing CTLA-4 treatment-related side effects has derived from studies in cancer, particularly in melanoma. These effects mainly include colitis/diarrhea, dermatitis, hepatitis and endocrinopathies; uveitis, nephritis and inflammatory myopathy also have been occasionally reported. These unique side effects are likely a direct result of breaking immune tolerance upon CTLA-4 blockade; they are generally mild, reversible and manageable, following specific treatment guidelines that include symptomatic therapies or systemic corticosteroids [
116]. In December 2008, Pfizer initiated a phase II trial in patients with late-stage unresectable liver cancer who also have hepatitis C infections. The primary endpoint of this single-armed study is the ability of tremelimumab to produce tumor responses among HCV-infected patients with hepatocellular carcinoma and to produce changes in hepatitis C viral load. The first results indicate that tremelimumab demonstrated an excellent safety profile, with a promising antitumor efficacy against HCC in 17 patients, as well as an intense antiviral activity. In fact, a significant and progressive decline in serum HCV viral load was observed, this being associated with an increase in anti-HCV immune response in 76% of patients [
117].
Since there are multiple levels of immunoregulation, a synergistic use of antibodies against different checkpoint molecules might represent the next stage in immunotherapy for chronic infectious diseases, as evidenced from
ex vivo studies about the combined PD-1/CTLA-4 blockade in HCV disease [
36]. Furthermore, because the host mechanisms that inhibit T cell activity are common and conserved aside from specific virus-encoded immune evasion strategies, the antibodies targeting inhibitory receptors may prove extremely versatile drugs potentially effective against multiple classes of viruses.