Molecular chaperones are essential for maintaining cellular functions by assisting protein folding and translocation or by preventing protein misfolding and aggregation Citation[1]. Studies over the last two decades support the concept of intracellular chaperone molecules as carriers of the antigenic repertoire of cancer cells, which is attributed to their intrinsic property to interact with polypeptide chains Citation[2]. Autologous tumor-derived, chaperone–peptide complex preparations therefore provide an innovative immunotherapeutic approach to treatment of cancers. However, only limited improvement in clinical outcome has been achieved using this approach Citation[3]. Recombinant chaperone complex vaccines, which are built on the superior client or antigen-holding capability of large stress proteins (Hsp110 and Grp170), present an alternative vaccine strategy that effectively targets defined tumor antigens. Engineering a modified immunochaperone that is able to concurrently promote antigen cross-presentation and innate immune responses may lead to the development of a new generation of chaperone-based vaccine therapy with improved clinical feasibility and vaccine efficacy.
Mechanisms underlying chaperone-based vaccine therapies
During the past decade, much has been learned regarding the molecular basis for chaperone-mediated immune regulation that permits their use as vaccine adjuvants. A key feature is their ability to efficiently direct associated polypeptides into the endogenous antigen presentation pathway for cross-presentation by professional APCs such as dendritic cells (DCs). It has been well documented that chaperone interaction with various receptors on APCs is critical for enhanced internalization and cross-presentation of antigens carried by chaperone molecules. Several chaperone-binding structures on APCs, such as CD91 and scavenger receptors, including SRA, LOX-1 and SREC, have been described Citation[4]. Unexpectedly, our studies using SRA knockout mice revealed that SRA acts as an immunosuppressor capable of attenuating antitumor immune responses generated by chaperone-based vaccines Citation[5,6]. It is likely that the different receptors may result in distinct immune outcomes upon binding to their ligands (e.g., chaperones). To selectively target chaperone-associated antigen to the receptors truly responsible for chaperone-facilitated T-cell cross-priming, the exact activity of individual receptors in the context of chaperone-based vaccination needs to be determined using both in vitro and in vivo systems.
Independently from their antigen shuttling and presentation functions, chaperone molecules have been shown to promote the phenotypic and functional maturation of APCs (e.g., DCs and macrophages) by upregulating costimulatory molecules and inducing secretion of cytokines or chemokines. This highlights the potential of extracellular chaperones in bridging innate and adaptive immunity. While there is convincing evidence supporting the intrinsic immunostimulatory properties of chaperones Citation[7], it should be noted that these molecules do not stimulate an innate immune response as strongly as pathogen-associated molecular patterns (PAMPs), such as lipopolysaccharides.
Autologous tumor-derived, chaperone–peptide complex vaccines
Due to their inherent chaperoning functions, certain chaperone molecules extracted from cancer cells can carry a tumor-specific, immunogenic peptide fingerprint Citation[8]. This offers personalized cancer vaccine therapy and bypasses the need for the identification of tumor-associated antigens. Autologous tumor-derived, chaperone–peptide complexes have been extensively studied over the last two decades and have been proven to be effective in treating a number of malignant diseases in preclinical cancer models. Although the results from multiple Phase I/II clinical trials of Gp96/Grp94-peptide complex (also known as vitespen, formerly HSPPC-96) were promising, the Phase III trial failed to demonstrate overall survival benefits Citation[3]. Introspective analysis revealed that vitespen treatment has benefited only subsets of early-stage melanoma and kidney cancer patients, which also appeared to depend on the doses of vaccine delivered to these patients. Clinical trials with the Gp96/Grp94-based vaccine vitespen in glioblastoma patients are ongoing (ClinicalTrials.gov identifiers: NCT00293423, NCT00905060 Citation[101]). Another related approach to generating chaperone-based autologous vaccines utilizes chaperone-rich lysates Citation[9], which are also being clinically tested as part of an immunotherapy regimen. In addition, Hsp70-peptide complex as an autologous vaccine can also be prepared from tumor–DC fusion cells that allow optimization of antigen processing and peptide loading Citation[10].
Recombinant large chaperone–protein antigen complex vaccines
The clinical applications of autologous chaperone–peptide vaccines were hampered by the limited access to human tumor specimens and a complicated procedure for vaccine preparation. As a result, many patients were excluded from clinical trials due to insufficient quantity of vaccines Citation[11]. Taking advantage of the exceptional client- or antigen-holding capability of the large chaperones Hsp110 and Grp170, we have developed a recombinant chaperone–protein complex vaccine that is generated by complexing defined tumor protein antigen to these molecules under heat shock conditions in vitro Citation[12]. These large chaperones are highly efficient in promoting cross-presentation of associated protein antigen and eliciting a robust cytotoxic T-lymphocyte response to tumors Citation[12–15]. The ancient chaperoning function has been shown to be a major factor that determines the effective T-cell cross-priming activity of these large chaperones Citation[15]. Although the reconstituted chaperone–protein complex vaccine may lack the personalized nature of autologous chaperone–peptide vaccines, the protein antigen that contains a large reservoir of potential peptides can potentially stimulate polyepitope-directed T and B cells. Importantly, this synthetic platform can be used to develop a multivalent vaccine targeting many different antigen targets Citation[16]. As an ‘off-the-shelf’ product that can be manufactured in large quantities, this vaccine may be used as adjuvant therapy for patients with completely resected disease or those at high risk for recurrence of cancer. A Phase I clinical trial of recombinant Hsp110–gp100 chaperone vaccine for the treatment of human melanoma is underway at the Roswell Park Cancer Institute (NY, USA) in collaboration with the US National Cancer Institute.
Incorporating a ‘danger’ signal into chaperone-based vaccine regimens
Upregulation of costimulatory molecules and production of inflammatory cytokines during the activation of DCs are crucial for the effective priming of T cells reactive with tumor antigens. It is well established that engaging the pathogen-sensing signaling pathways in APCs with PAMPs can potentiate antigen presentation and adaptive immunity. Emerging evidence shows that chaperones’ interactions with PAMPs synergistically stimulate an innate immune response Citation[17,18]. Our recent study demonstrates that Grp170 binds highly efficiently to CpG oligodeoxynucleotides, an agonist for Toll-like receptor 9, and amplifies innate immunity that confers protection from Listeria monocytogenes Citation[18]. Although earlier studies support the notion that extracellular chaperones possess direct immunostimulatory activity, the innate immune response generated by these molecules of mammalian origin may not be sufficient to functionally activate APCs that have captured antigens. It is conceivable that incorporation of a pathogen-associated ‘danger’ signal into the recombinant chaperone vaccine regimen would lead to improved antitumor efficacy.
We recently constructed a chimeric chaperone by strategically fusing the flagellin-derived, NF-κB-stimulating sequence to Grp170 Citation[19]. This multifunctional molecule, termed Flagrp170, has two distinct features that are essential for chaperone-based vaccine therapy: promoting cross-presentation of associated tumor antigens and concurrently augmenting functional activation of DCs via engaging the NF-κB signaling pathway Citation[20]. Introducing this chimeric chaperone into DCs strongly activates them, as indicated by substantial elevation of CD40 and CD86, as well as production of IL-12. The study of this chimeric immunochaperone was carried out in the setting of in situ vaccination by delivering a Flagrp170-encoding adenovirus Citation[19]. Intratumoral administration of Flagrp170 preferentially induces the Th1 polarization of the tumor microenvironment (TME) with characteristics of high levels of cytokine IL-12 and IFN-γ, increased recruitment of CD8+ and NK cells to the tumor sites. The systemic antitumor immunity leads to profound inhibition of both treated tumors and distant metastases in several preclinical cancer models. Although flagellin and Flagrp170 induce similar levels of IL-12 in the TME, the Flagrp170 is much more efficient than flagellin in provoking an effective cytotoxic T-lymphocyte response to tumors, reinforcing a pivotal role of chaperoning by Grp170 in the stimulation of the adaptive arm of the immune system. Our results are consistent with the view that coupling tumor antigens and an immunostimulating ‘danger’ signal into the same vaccine delivery cargo is crucial for optimal antigen cross-presentation by DCs. This modified large chaperone represents a novel immune modulator that can be exploited to overcome the immunosuppressive mechanism in the TME for improved treatment outcomes. A recombinant large chaperone–protein complex vaccine based on this chimeric chaperone is currently under investigation.
Future opportunities for chaperone-based vaccines
Extensive studies over the last two decades have demonstrated that molecular chaperoning represents an innovative platform for the development of safe and effective vaccine therapies to promote immune control of cancers. A better understanding of the mechanism of chaperone action in immune modulation is expected to provide new opportunities for improving and optimizing chaperone-based cancer immunotherapy. Given their highly efficient capability in promoting antigen cross-presentation, integrating a strong ‘danger’ signal or PAMP that further stimulates the innate arm of an immune response into autologous or recombinant chaperone vaccines should lead to enhanced therapeutic potency. With their proven safety profile, it is feasible to test chaperone-based vaccines either in adjuvant settings or in conjunction with conventional treatment modalities (e.g., radiotherapy or chemotherapy) in clinical trials. To overcome the immunoregulatory processes involved in maintenance of host immune homeostasis and/or tumor-induced immunosuppressive mechanisms that dampen the effectiveness of vaccines or immunotherapy, other approaches targeting immune checkpoint molecules or immunosuppressors (e.g., blockade of CTLA-4 or Treg), should also be a focus of future research on chaperone-based vaccines.
Financial & competing interests disclosure
The work was supported in part by NIH research grants (CA129111, CA154708 and CA099326), Department of Defense grant W81XWH-10-PCRP-SIDA and Harrison Scholarship. The authors have no other relevant affiliations or financial involvement with any organization or entity with a financial interest in or financial conflict with the subject matter or materials discussed in the manuscript apart from those disclosed.
No writing assistance was utilized in the production of this manuscript.
References
- Lindquist S, Craig EA. The heat-shock proteins. Annu. Rev. Genet. 22, 631–677 (1988).
- Srivastava P. Interaction of heat shock proteins with peptides and antigen presenting cells: chaperoning of the innate and adaptive immune responses. Annu. Rev. Immunol. 20, 395–425 (2002).
- Srivastava PK, Callahan MK, Mauri MM. Treating human cancers with heat shock protein–peptide complexes: the road ahead. Expert Opin. Biol. Ther. 9(2), 179–186 (2009).
- Calderwood SK, Theriault J, Gray PJ, Gong J. Cell surface receptors for molecular chaperones. Methods 43(3), 199–206 (2007).
- Wang XY, Facciponte J, Chen X, Subjeck JR, Repasky EA. Scavenger receptor-A negatively regulates antitumor immunity. Cancer Res. 67(10), 4996–5002 (2007).
- Qian J, Yi H, Guo C et al. CD204 suppresses large heat shock protein-facilitated priming of tumor antigen gp100-specific T cells and chaperone vaccine activity against mouse melanoma. J. Immunol. 187(6), 2905–2914 (2011).
- Henderson B, Calderwood SK, Coates AR et al. Caught with their PAMPs down? The extracellular signalling actions of molecular chaperones are not due to microbial contaminants. Cell Stress Chaperones 15(2), 123–141 (2010).
- Murshid A, Gong J, Calderwood SK. Heat-shock proteins in cancer vaccines: agents of antigen cross-presentation. Expert Rev. Vaccines 7(7), 1019–1030 (2008).
- Graner M, Raymond A, Akporiaye E, Katsanis E. Tumor-derived multiple chaperone enrichment by free-solution isoelectric focusing yields potent antitumor vaccines. Cancer Immunol. Immunother. 49(9), 476–484 (2000).
- Enomoto Y, Bharti A, Khaleque AA et al. Enhanced immunogenicity of heat shock protein 70 peptide complexes from dendritic cell–tumor fusion cells. J. Immunol. 177(9), 5946–5955 (2006).
- Oki Y, Younes A. Heat shock protein-based cancer vaccines. Expert Rev. Vaccines 3(4), 403–411 (2004).
- Wang XY, Chen X, Manjili MH, Repasky E, Henderson R, Subjeck JR. Targeted immunotherapy using reconstituted chaperone complexes of heat shock protein 110 and melanoma-associated antigen gp100. Cancer Res. 63(10), 2553–2560 (2003).
- Manjili MH, Henderson R, Wang XY et al. Development of a recombinant HSP110-HER-2/neu vaccine using the chaperoning properties of HSP110. Cancer Res. 62(6), 1737–1742 (2002).
- Manjili MH, Wang XY, Chen X et al. HSP110-HER2/neu chaperone complex vaccine induces protective immunity against spontaneous mammary tumors in HER-2/neu transgenic mice. J. Immunol. 171(8), 4054–4061 (2003).
- Park JE, Facciponte J, Chen X et al. Chaperoning function of stress protein grp170, a member of the hsp70 superfamily, is responsible for its immunoadjuvant activity. Cancer Res. 66(2), 1161–1168 (2006).
- Wang XY, Sun X, Chen X et al. Superior antitumor response induced by large stress protein chaperoned protein antigen compared with peptide antigen. J. Immunol. 184(11), 6309–6319 (2010).
- Warger T, Hilf N, Rechtsteiner G et al. Interaction of TLR2 and TLR4 ligands with the N-terminal domain of Gp96 amplifies innate and adaptive immune responses. J. Biol. Chem. 281(32), 22545–22553 (2006).
- Zuo D, Yu X, Guo C et al. Molecular chaperoning by glucose-regulated protein 170 in the extracellular milieu promotes macrophage-mediated pathogen sensing and innate immunity. FASEB J. 26(4), 1493–1505 (2012).
- Yu X, Guo C, Yi H et al. A multifunctional chimeric chaperone serves as a novel immune modulator inducing therapeutic antitumor immunity. Cancer Res. 73(7), 2093–2103 (2013).
- Andreakos E, Williams RO, Wales J, Foxwell BM, Feldmann M. Activation of NF-κB by the intracellular expression of NF-κB-inducing kinase acts as a powerful vaccine adjuvant. Proc. Natl Acad. Sci. USA 103(39), 14459–14464 (2006).
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- ClinicalTrials.gov. www.clinicaltrials.gov