Bacterial Products or Bacteria-derived Adjuvants
Given their potent immunostimulatory capacity, bacteria-derived substances constitute a major potential source of adjuvants. The adjuvant activity is mediated through activation of TLRs that mediate the danger signals activating the host immune defense system.[15] TLRs are part of the innate immune system, and they belong to the pattern-recognition receptors family. The pattern-recognition receptor family is designed to recognize and bind conserved pathogen-associated molecular patterns, which are molecular structures common to a large group of microorganisms. They provide essential requirements for initiating T-cell immunity: antigen uptake, processing and presentation by dendritic cells (DCs) and other antigen-presenting cells (APCs), DC maturation and T-cell activation. However, only a few of these, including TLR-3, -4, -7/8 and -9 agonists, represent promising cancer immunotherapeutics and have been included in the ranked National Cancer Institute's list of immunotherapeutic agents with the highest potential to treat cancer.[1] Most TLRs are expressed on the cell surface, except TLR-3, -7, -8 and -9, which are found within endosomes, and their activation results in transcription of type I interferon (IFN) genes and proinflammatory cytokine genes such as TNF-α, IL-1 and IL-6.[16] TLR activation results in increased numbers of MHC and costimulatory molecules on DCs, essential for efficient antigen presentation and priming of naive T and B cells, therefore contributing to vaccine responses.[17,18] Although the engagement of TLR signaling pathways is a promising mechanism for boosting vaccine responses, some issues related to efficacy and potential vaccine-associated adverse effects may arise from repeated CpG administration and need to be resolved in human trials. Most of the TLR ligands may show serious undesired side effects when administered at a high dose.[19] Moreover, triggering of TLR-9 with CpG oligodeoxynucleotides activates plasmacytoid cells to upregulate the expression of the enzyme indoleamine 2,3-dioxygenase (IDO), a key molecule in tumor-mediated immune tolerance.[20] The increased IDO levels in such APCs might antagonize the antitumor immune response of the host.
TLR-2/4 Ligands
Bacillus Calmette–Guérin Some microbes, such as the strain of bacillus Calmette–Guérin (BCG) originally used as a vaccine against TB, can serve as adjuvants for cancer vaccines.[21] BCG interacts with multiple immunoreceptors, including TLR-2 and -4, as well as the nucleotide binding oligomerization domain-like receptor 2.[22] BCG adjuvant has been extensively evaluated with cellular vaccines in colorectal cancer. Autologous tumor cells mixed with BCG were of significant clinical benefit for patients with stage II colon cancer, as reviewed in.[23] In this section, we discuss three successful or failed vaccine products that have been extensively studied in clinical trials with a BCG adjuvant.
Canvaxin™ Canvaxin™ (CancerVax Corp., CA, USA), a polyvalent whole-cell vaccine admixed with BCG as adjuvant, comprises three melanoma cell lines that express more than 20 characterized antigens.[24] They include MAGE, tyrosinase TA-90, Lewis-X and MART-1. Canvaxin has been tested extensively in Phase II adjuvant trials. There were positive results from Phase II trials in patients with stage III melanoma, in which median overall survival (OS) and 5-year rate of OS were significantly higher in patients who received postoperative adjuvant therapy with Canvaxin vaccine than in patients who did not receive the vaccine.[25] In a matched-pair analysis of patients with stage IV melanoma that compared vaccine and nonvaccine therapies after complete resection of melanoma metastatic to distant sites, the 5-year rate of OS and the median OS were 39% and 38 months, respectively, in vaccine patients, compared with 20% and 19 months, respectively, in nonvaccine patients (p = 0.0009). The median disease-free survival (DFS) was also prolonged in vaccine-treated patients (9.1 vs 4.2 months, p = 0.005). By multivariate analysis, vaccine therapy was the most significant prognostic factor for both OS (p = 0.003) and DFS (p = 0.0007).[26] Responding patients demonstrated a cellular delay-type hypersensitivity response to some of the antigens, suggesting a correlation between immune response and clinical outcome.[27]
Despite promising Phase II data, there was no difference in overall survival between the patients who received Canvaxin and the patients who received placebo in the randomized Phase III trial of BCG administered with Canvaxin in patients with resected stage III and IV melanoma. Better outcomes were instead reported for the BCG-alone arm. Another Phase III study with Canvaxin in melanoma patients (stage IV; n = 496) was also stopped prematurely in 2005. Patients in the vaccine arm of the trial were shown to have worse outcomes than those in the control arm.[28]
Oncovax® Oncovax® (Vaccinogen, Emmen, The Netherlands) represents an autologous whole-cell irradiated live tumor cell vaccine admixed with BCG. The first results after a median follow-up of 6.5 years of adjuvant active specific immunotherapy (ASI) for human colorectal cancer revealed an OS of 65.9% in the ASI group and 51.3% in a control group that received no vaccine.[29] However, no significant benefit was observed in a second larger study in colorectal cancer (stages II and III; n = 205 [ASI] vs n = 207 [control]).[30] This was subsequently attributed to deficiency in the quality control of the multicenter study. A further randomized study performed in The Netherlands and published in 1999 in The Lancet saw no significant benefit of Oncovax immunization in operated stage III colorectal cancer but differences for stage II patients,[31] leading to regulatory approval in The Netherlands. After 6.5 years, 82.5% had survived in the ASI group versus 72.7% in the control group that received no vaccine.[32,33]
GM2 Ganglioside Vaccine The third example is the GM2 ganglioside vaccine. The ganglioside GM2 is a serologically well-defined melanoma antigen and the most immunogenic ganglioside expressed on melanoma cells. Since 1996, the regimen of high-dose IFN-α2b therapy (HDI) has been established as a postsurgical adjuvant therapy in stage IIB and III high-risk melanoma. Owing to the toxicity associated with HDI, vaccine alternatives are of interest. Several studies conducted at the Memorial Sloan–Kettering Cancer Center (NY, USA) have demonstrated that administration of GM2 in combination with BCG induced IgM anti-GM2 antibodies in the majority of patients and that these antibody responses were correlated with improved recurrence-free survival and OS in stage III melanoma patients. The initial randomized Phase III trial conducted showed a trend in the vaccine arm toward improved OS and progression-free survival within GM2 antibody producers.[34] A variety of GM2 vaccine formulations were studied and a commercial vaccine preparation was selected consisting of GM2 coupled to KLH and combined with the QS-21 adjuvant (GMK vaccine, Progenic Pharmaceuticals [NY, USA]). Immunization of melanoma patients with the GMK vaccine has been shown to induce high titers of IgM antibodies in >80% of patients as well as IgG antibodies that had not been previously observed with GM2 plus BCG.[34–37] A larger randomized Phase III trial of an Intergroup mechanism (Intergroup trial E1694) evaluated the efficacy of the GMK vaccine versus HDI in 880 patients with stage IIB/III melanoma (EORTC 18961; n = 880).[38] The trial was closed after interim analysis indicated inferiority of GMK compared with HDI in terms of relapse-free survival and OS. A second Phase III trial was initiated in May 2001, with 1300 patients at immediate risk of recurrence of the disease randomized after surgery to receive either GMK vaccine or the current standard of care. The preliminary data were presented at the 2008 ASCO Annual Meeting.[39] Criteria needed for ending the trial were met after an interim analysis revealed GMK vaccine failed to show effectiveness of relapse-free survival. The GMK vaccine's impact on survival remains unknown in both of these large randomized trials.
Lipopolysaccharide & Derivatives Another important group of compounds derived from the cell wall of Gram-negative bacteria are the lipopolysaccharides or endotoxins. They are potent B-cell mitogens, but also activate T cells to produce IFN-γ and tumor necrosis factor and thereby enhance cellular immune responses. Lipopolysaccharide functions as a vaccine adjuvant but is considered too toxic for clinical use. The major structural element responsible for its toxicity and adjuvant effect is lipid A. In weakly acid conditions, lipid A can be hydrolyzed to obtain monophosphoryl lipid A (MPL® [GSK and other]), a compound which retains the adjuvant activity of lipid A with reduced toxicity.[40]
Monophosphoryl Lipid A Lipid A molecules that target the TLR-4 complex are among the most commonly used vaccine adjuvants. One promising candidate is MPL, a derivative of lipid A from Salmonella minnesota. MPL adjuvant has shown an excellent safety and efficacy profile in several thousands of patients and received regulatory approval in Europe as adjuvant for a hepatitis B vaccine (Fendrix™ [GSK Biologicals, Belgium]). MPL is a component of Melacine (Corixa Corp., Seattle, WA, USA; discussed later) and Cervarix (GSK Biologicals), the preventative GlaxoSmithKline (GSK) HPV vaccine.[41] GSK is currently developing MPL-based vaccines and has developed three different adjuvant systems based on MPL:
AS01: MPL + liposomes + QS-21;
AS02: MPL + oil-in-water (O/W) emulsion + QS-21;
AS04: MPL + aluminum hydroxide/aluminum phosphate.
These formulations and combinations are under evaluation for a wide range of vaccine candidates in clinical trials. Here we describe cancer vaccine candidates using MPL adjuvant in the most advanced stage of development.
MAGE-A3 Vaccine MPL is used as part of a proprietary immunological adjuvant system (AS15, AS02b) in GSK MAGE-A3 vaccine. AS02b consists of MPL and QS-21 in an O/W emulsion. AS15 is a liposomal formulation of CpG, MPL and QS-21. GSK's vaccine bolsters the T-cell immunogenicity of its recombinant MAGE-A3 protein by the addition of AS15, which enhances delivery to APCs and optimizes potent T-cell response to MAGE-A3. Interestingly, while the results from a placebo-controlled, randomized Phase II trial of MAGE-A3/AS02b confirmed an excellent safety profile and suggested clinical benefit with prolonged DFS for patients with resected stage I and II non-small-cell lung cancer (NSCLC), the CpG adjuvant was not added to the vaccine formulation until the initiation of a global Phase III trial of MAGE-A3/AS15.[42] The Phase II trial in patients with metastatic melanoma confirmed a safe vaccination approach and showed superiority of adjuvant AS15 over AS02b suggesting that the addition of TLR-9 agonist CpG to MPL increased vaccine efficacy.[43] A follow-up Phase III study of MAGE-A3/AS15 in NSCLC patients is ongoing.
Stimuvax® MPL adjuvant is also a component of the liposomal Stimuvax® (Merck, Biomira) vaccine targeting MUC-1 in advanced NSCLC. BLP25, BLP25, also called Stimuvax, a lyophilized, liposomal formulation consisting of BLP25 (a 25-amino acid sequence [STAPPAHGVTSAPDTRPAPGSTAPP] that provides MUC1 specificity), MPL and three lipids is a vaccine that was originally developed by scientists at Cancer Research (UK).[44] It was further developed by Biomira and has been tested in small trials. Clinical studies have confirmed that Stimuvax is well tolerated and elicits a cellular immune response in patients with lung cancer.[45] A Phase IIb trial showed that the L-BLP25 vaccine increases survival rates for NSCLC stage IIIb patients with smaller, nonmetastatic NSCLC tumors in the absence of a malignant pleural effusion.[44] Stimuvax is now being tested in a worldwide Phase III clinical trial called Stimulated Targeted Antigenic Responses to NCSLC (START) that will recruit approximately 1300 people with stage III NSCLC from various countries around the world.
Synthetic TLR-4 Agonists Based on these successes, a new generation of synthetic TLR-4 agonists are being developed with similar potential as MPL. A synthetic TLR-4 agonist known as glucopyranosyl lipid A has been evaluated clinically as an adjuvant for a seasonal influenza vaccine[46] and is also being developed as a cancer vaccine adjuvant. Another synthetic TLR-4 agonist being developed is RC-529 (DynaVax, GSK), which is licensed for a HBV vaccine in Europe and ongoing for patients with resected, high-risk melanoma.
Detox B Substances produced by bacteria, such as detox B, are also frequently used as adjuvants. DETOX adjuvant, a Salmonella-derived MPL-A which contains cell wall skeletons from Mycobacterium phlei, has been evaluated in Phase I/II vaccines studies in several cancers. In this section, we discuss some successful or failed cancer trials with the Detox B adjuvant.
Melacine® Melacine® (Corixa, WA, USA and GSK) is amongst the most studied allogeneic vaccines, developed by Malcolm Mitchell. Melacine is registered as a combination of a mechanical tumor lysate comprising two melanoma cell lines that has defined antigens, HER-2/neu and L523S, and an immunological adjuvant, DETOX. The peptidoglycan cell wall of BCG containing the NOD-2 agonist, muramyl dipeptide (the active component of Freund's adjuvant), is also a component of Melacine. The original clinical testing of Melacine began in 1985. In 1988, a Phase II trial in patients with metastatic melanoma showed a 6% response rate with four patients in complete remission for 7–10 years.[47] On the basis of this trial, the vaccine was approved by the Canadian FDA. In melanoma patients, cellular immune responses to vaccination with Melacine or irradiated autologous tumor cells with DETOX correlated with clinical responses.[48]
A Phase II trial in conjunction with IFN-α2b (Intron) has demonstrated promising results that led to the initiation of a second Phase III trial in conjunction with Intron A.[49,50] Meanwhile, none of the Phase III studies with Melacine in the adjuvant setting of melanoma (stages III and IV) have resulted in a significant benefit. The Southwest Oncology Group conducted a Phase III trial to evaluate the efficacy of Melacine as adjuvant therapy in 689 node-negative patients with intermediate thickness melanoma (stage II) and found no benefit from the vaccine therapy.[49–52] However, the data from this trial revealed that a subgroup of vaccine-treated patients with expression of two or more HLA class I antigens (HLA-A2, -A28, -B44, -B45, -C3) had significantly improved 5-year relapse-free survival compared with patients with the same HLA expression but not treated with the vaccine (83 vs 59%; p = 0.0005).[53] These data were not accepted by the US FDA because the comparison of outcome by HLA type was retrospective and not prospective. Thus, a new trial randomizing patients with the HLA-A2 and/or HLA-C3 phenotype will be required before approval in the USA. Most recently, data from a multicenter Phase III trial in which the researchers compared Melacine and low-dose IFN-α with standard HDI adjuvant therapy in 600 stage III melanoma patients were reported. The data showed Melacine plus low-dose IFN-α had an effect comparable to standard HDI therapy, with no significant differences in relapse free survival or OS.[54] The advantage of Melacine over chemotherapy was that it was nontoxic and therefore allowed a better quality of life compared with chemotherapy. For that reason, Melacine is now available on prescription to patients in Canada and is awaiting approval in the USA.
Theratope™ Vaccine (Biomira, Now Oncothyreon) Sialyl-Tn (STn) is a carbohydrate associated with the mucin, MUC1, on a number of human cancer cells and is associated with more aggressive disease. Consequently, STn is an ideal candidate for ASI therapy. Synthetic STn haptens conjugated to KLH (STn–KLH [Theratope]), together with DETOX adjuvant, have been used in pilot vaccination studies for MUC1-expressing epithelial tumors, including metastatic breast cancers. Theratope STn–KLH cancer vaccines have shown safety in breast and ovarian cancer patients and trends towards a decreased risk for relapse and death.[55] Overall, Theratope vaccine has been well tolerated with minimal toxicity. Vaccination after high-dose chemotherapy and autologous stem cell rescue resulted in humoral and cellular immune responses; however, no clinical benefit was demonstrated with this approach.[56,57] The definitive Phase III trial comparing the outcome of patients with metastatic breast cancer receiving vaccinations with Theratope vaccine versus vaccination with the nonspecific immune stimulants KLH and Detox-B stable emulsion (Detox-B, now called Enhanzyn® Immunostimulant [Corixa Corp.]) ended enrolment on 30 March 2001. Over 1000 women with distant metastatic breast cancer were enrolled.[58] Overall, this vaccine showed no improvement in either time to progression or OS.
TLR-3 Agonists
Polyriboinosinic-polyribocytidylic acid (Poly I:C), a double-stranded polyinosinic:polycytidylic acid, is a synthetic analog of viral dsRNA that stimulates endosomal (TLR-3) and/or cytosolic MDA5,[59] leading to increased production of type I IFNs.[59,60] When stabilized with poly-L-lysine and carboxymethylcellulose, it is known as poly ICLC, and is more stable and, in that regard has greater activity. Poly I:C and poly ICLC are potent TLR-3 agonists with vaccine adjuvant potential.[61–70] Poly I:C and derivatives are being studied as vaccine adjuvants in cancer trials as preclinical studies have shown promising results.[71] Several trials with Poly ICLC adjuvants are ongoing for the treatment of patients with various advanced malignancies such as prostate cancer (A Pilot Immunotherapy Study of Combination PSMA and TARP Peptide With Poly ICLC Adjuvant in HLA-A2 (+) Patients With Elevated PSA After Initial Definitive Treatment[201]), resected stage III or stage IV Melanoma (A Phase I Study of Poly ICLC and NY-ESO-1/gp100/MART-1 Peptides Emulsified With Montanide ISA 51 With Escalating Doses of CP 870,893[202]), patients with high-risk melanoma in complete clinical remission (A Phase I/Phase II Open Label Study of the TLR-3 Agonist Poly-ICLC as an Adjuvant for NY-ESO-1 Protein Vaccination With or Without Montanide ISA-51 VG[203]) and triple-negative breast cancer patients (A Pilot Study of a MUCI Peptide and Poly-ICLC Vaccine[204]). These studies are detailed at.[205]
TLR-7/8 Ligands
A new group of immune response modifiers are the synthetic low-molecular weight imidazoquinolines – imiquimod and resiquimod[72] – and other compounds, such as certain guanine nucleoside analogs.[73] These molecules exert their stimulatory effects via TLR-7, inducing cytokine production and upregulating costimulatory molecules and MHC I/II in DCs.[73] To date, several ligands have been characterized as TLR-7 and/or TLR-8 ligands, classified in both synthetic compounds and natural nucleoside structures. Resiquimod is also recognized by TLR-8 and has been shown to be a potent topically active immune response modifier that significantly enhances the cutaneous immune response.[74] Imiquimod, a TLR-7 agonist, is currently FDA approved as a topical medication for genital warts and basal cell skin cancer. Imiquimod's adjuvant activity was evaluated in a clinical trial in combination with FMS-like tyrosine kinase (Flt)-3 ligand, a hematopoietic growth factor that induces expansion and differentiation of all DC progenitors, especially IFN-producing killer and plasmacytoid DC,[75–81] with or without peptides in melanoma patients. Topical application of imiquimod resulted in more cutaneous reactions to peptides and more circulating peptide-specific T cells.[82] Imiquimod's adjuvant activity was also evaluated in transdermal vaccination of a peptide-based vaccine in patients with resected stage II, III or IV melanoma. The vaccine combination showed an excellent safety profile, elicited humoral and cellular NY-ESO-1-specific immune responses in a significant fraction of patients and was easy to administer.[83] A Phase I randomized, double-blind, placebo-controlled study of topical resiquimod as an adjuvant for NY-ESO-1 protein plus Montanide vaccination in patients with tumors that often express NY-ESO-1 is ongoing.
Building on the promising data with imiquimod, synthetic TLR-7/8 agonists with alterated structure have been developed in order to refine properties related to toxicity and specificity. These agonists (like stabilized ssRNA, R-848 and 852A) have now also been tested as immune response modifiers (IRMs) in preclinical models[84] and in clinical trials.[85,86]
TLR-9 Ligands & CpG Oligodeoxynucleotides
Bacterial DNA has unmethylated CpG motifs (uncommon in mammalian DNA), which are recognized by TLR-9 with the concomitant activation of innate immunity and induction of cytokines that promote Th1 immunity.[87] DNA containing CpG motifs is one of the most potent cellular adjuvants.[88] CpG oligodeoxynucleotides (ODN) have shown great promise as adjuvants for cancer vaccines (reviewed in [89]). The activity of CpG as a vaccine adjuvant is under development for vaccines against cancer, as addressed in these recent trials.
In melanoma patients, CpG oligodeoxynucleotide, PF-3512676, mixed with a Melan-A tumor peptide antigen in Montanide (a water-in-oil [W/O] emulsion) stimulated strong and rapid CD8+ T-cell responses reaching a mean of >1% of Melan-A-specific CD8+ T cells.[90] The addition of PF-3512676 to the Melan-A peptide vaccine not only induced a high frequency of CD8+ T cells, but also promoted effector cell differentiation to a level comparable to that seen in Epstein–Barr virus- and cytomegalovirus-specific T cells.[91]
Vaccination of cancer patients with the recombinant tumor antigen, NY-ESO-1, in Montanide without a TLR-9 agonist had been relatively poorly immunogenic, but this approach induced specific CD4+ and cross-primed CD8+ T-cell responses with the use of PF-3512676 in many patients.[92] While studies lacked a concurrent non-CpG control arm, comparisons with vaccines containing antigen/Montanide ISA 51 were suggestive of substantially increased immunogenicity of the CpG-containing formulation.[90] However, CpG did not induce antigen-specific immunity in patients with advanced cancers when administered sequentially after granulocyte–macrophage colony-stimulating factor (GM-CSF) before antigen injection,[93] suggesting that emulsification in Montanide ISA 51 may be necessary for CpG's adjuvant effect.
CpG is incorporated into a MAGE-A3 melanoma vaccine (AS15) that has progressed into Phase III clinical trials for the treatment of patients with early stage completely resected NSCLC whose tumors express the antigen (GSK, discussed above).
Expert Rev Vaccines. 2011;10(8):1207-1220. © 2011 Expert Reviews Ltd.
Cite this: Outlining Novel Cellular Adjuvant Products for Therapeutic Vaccines Against Cancer - Medscape - Aug 01, 2011.
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