Peptide Vaccines and Cancer Immunotherapy
The main efforts of our laboratory in the past two decades have been the development of unique peptide vaccines and peptidomimetic therapeutic approaches (Figure 2) for targeting viral, bacterial and cancer antigens. The novelty of our approach resides in a hypothesis-driven basic research in vaccinology with an incremental approach involving the elucidation of several basic immunological and structural concepts that could eventually be translated to the clinic for the benefit of cancer patients. These innovations include:
â–ª The ability to predict biologically active immune epitopes and confirm their properties in vitro and in vivo;
â–ª The engineering of unique B-cell epitope peptides that can recapitulate the exquisite native structure of the tumor antigen;
▪ The design of uniquely inhibitory peptide mimics that can block receptor–ligand interactions;
â–ª The idea of combining the B-cell epitopes into chimeric constructs that incorporate a 'promiscuous' T-cell activating species. Vaccination with these novel HER-2 chimeric immunogens results in the production of highly efficacious native-like antipeptide antibodies that can delay, prevent and/or eradicate tumor growth and metastasis with increased efficacy and little or no toxicity;
▪ In order to complement and enhance our vaccine approach, we have been developing 'nonimmunogenic' peptide therapeutics that target both HER-2 and VEGF pathways. This strategy involves the design and synthesis of conformational VEGF peptide mimics that are aimed at disrupting the receptor–ligand interactions with enhanced stability and efficacy;
â–ª The concept of 'tritherapy', which is the combination of immunotherapy, angiotherapy and metronomic chemotherapeutic drugs.
(Enlarge Image)
Figure 2.
Combination of HER-2 vaccines and VEGF peptide mimics.
HER-2 vaccines and VEGF peptide inhibitors developed in our laboratories to inhibit signaling pathways. Antibodies elicited by immunization with MVF-HER-2 (266–296) vaccine bind to domain II of HER-2 and, similarly, anti-MVF-HER-2 (597–626) binds domain IV of HER-2, providing dual inhibition of homo/heterodimerization, and consequently downstream signaling, shutting down the PI3K and MAPK pathways, thereby preventing cancer growth and metastasis. On the other hand, VEGF peptide mimics P3 and P4, which are designed to directly block VEGF binding to VEGFR-2, inhibit intracellular phosphorylation of the tyrosine kinase domain, which reduces angiogenesis.
EGFR: EGF receptor; P: Phosphate; VEGFR: VEGF receptor.
There are several strategies to design immunogenic B-cell epitopes of unknown proteins (reviewed by Kaumaya et al.). First, we have pioneered a unique approach of identifying antigenic epitopes on the surface of proteins whose 3D structure and whose antigen–antibody complex is unknown. It relies on predicting epitopes using correlates of antigenicity such as hydrophilicity, flexibility, hydrophobic index, amphiphilicity and secondary structural propensities. Computer algorithms are available to predict potential B-cell epitopes and we have successfully applied this technology to several targets including HTLV-1, HER-2, VEGF and others. For a large protein, several antigenic epitopes can be prioritized and these peptides are then synthesized by solid-phase peptide chemistry as chimeric immunogens with an appropriate T-cell epitope.
Second, if the 3D structure of the antigen is unknown, prediction of the secondary structures by several criteria, such as α-helices, β-turn and sheet, and loop regions, can be made to aid the design. In that respect, knowledge of protein folding, structure and dynamics can be used to design the B-cell epitopes to fold into stable supersecondary structures (αα, αβ, βαβ and β-turns and loops). When the crystallographic structures of the antigen–antibody complex are known, the engineering of a selective B-cell epitope to mimic the structure of the immunogenic binding site on the tumor antigen is greatly facilitated.
The third aspect is to construct a chimeric peptide containing a promiscuous T-cell epitope that could be used as an immunogen to elicit high-titered and high-affinity antibodies that are able to recognize the protein. We have pioneered a strategy that is accomplished by selecting an appropriate T-cell epitope. Several of these promiscuous T-cell epitopes are known, and in our previous in-depth studies we have found the measles virus fusion (MVF) protein and the TT epitopes to be the most efficacious ones. The most important consideration in the successful design of a peptide vaccine is that the B-cell epitope must be covalently linked to the 'helper' T-cell epitope at either the N- or C-terminus (Figure 3). In our vaccine approach, we preferentially used the T-cell epitope at the N-terminus.
(Enlarge Image)
Figure 3.
Chimeric B- and T-cell epitopes.
B- and T-cell epitopes are colinearly synthesized with a GPSL flexible linker. The linker is flexible, allowing the two epitopes to fold or adopt different conformations independent of each other.
The size of the B-cell epitope should minimally be approximately 18 amino acid residues (˜900 Å), the size of an antigen–antibody binding region, to ensure the peptide propensity to adopt a defined structure. The helper T-cell should ideally be a promiscuous epitope (˜18 residues), the linker should be approximately four residues and the final construct should consist of between 40 and 60 amino acid residues. In some cases, the B-cell epitope can be greater than 18 residues to accommodate a larger binding site, as demonstrated by the HER-2–trastzumab or the HER-2–pertuzumab interface. The chimeric B- and T-cell epitope strategy can fully stimulate the immune system to produce high-affinity antibodies that can specifically target cancer cells and, most importantly, establish an immunological memory in order to prevent cancer relapse.
Traditionally, peptide sequences are coupled to larger carrier proteins, such as bovine serum albumin, TT or keyhole limpet hemocyanin, in order to induce immunogenicity. This approach is fraught with difficulties as the resulting conjugate is subject to processing by the immune cells and the resulting B-cell epitope may not represent the exact epitope identity or configuration, resulting in low-affinity antibodies. Additionally, the coupling procedures are not an exact science in the sense that they cannot be duplicated from batch to batch, resulting in an unpredictable immune response. Other problems are epitope suppression by the carrier protein and poorly characterized constructs due to ambiguity during the chemical coupling reaction. A strategy that involves B- and helper T-cell epitopes is MHC-restricted by the fact that T-helper epitopes are recognized by only a few MHC class II alleles at most. We overcame those problems by proposing the use of 'universal' or promiscuous T-helper epitopes that bind to several MHC haplotypes.
The chimeric peptide is presented intact to the immune cells without processing, yielding high-titered and high-affinity protective polyclonal antibodies. This strategy avoids the proteolysis by APC, normally associated with peptides coupled to large carrier proteins in which the authenticity of the epitopes is destroyed resulting in a poorly immunogenic vaccine. Unlike antigens presented to MHC class I, which are restricted to peptides 8–10-mers, the ends of the groove of MHC class II are open, allowing larger peptides to bind. This is the major advantage over other strategies that focus on generating antibodies in vaccine development. Other active immunization regimens, such as activation of CTLs for many different types of cancer, have been exclusively and extensively used without much success so far, but there is hope that newer methods of delivery and adjuvants may lead to improved vaccines activating the cellular arm of the immune system.
An ideal peptide vaccine should consist of an appropriate tumor antigen B-cell epitope or CTL epitope covalently linked to a universally immunogenic T-helper epitope. In addition, the peptide vaccine must be formulated with an adjuvant or cytokines, which are essential in the development of a good immune response. Adjuvants are usually defined as compounds that increase and/or moderate the intrinsic immunogenicity of an antigen. Generally speaking, adjuvants function in three basic ways: they cause the slow release of an antigen; they modulate the immune response; and they increase the presentation of the antigen to immune cells such as antigen-presenting cells. Poorly soluble aluminum salts (aluminum phosphate, aluminum hydroxide and alum [KAl(SO4)2·12H2O]), as well as calcium phosphate and AS04 (FENDrix®, GlaxoSmithKline, Brentford, UK), are the only vaccine adjuvants currently licensed in the USA. While generally considered quite safe, there are several areas in which these adjuvants are inadequate. Despite the wide use and acceptance of alum, there are a series of problems. Adjuvants that can stimulate Th1 cytokine-dependent IgG2a and IgG2b antibodies have been developed. Oil emulsions, such as MF59, which is composed of squalene, polyoxyethylene and muramyl tripeptide, have been shown to dominantly induce IgG2a production via Th1 immune response. QS-21, which is a purified saponin, also induces IgG2a responses via cell-mediated immunity. Another group of adjuvants that are microbial-derived has also been shown to activate Th1 responses and cause IgG2 production. A TLR-2 ligand known as macrophage-activating protein-2, which is a purified derivative from mycoplasma, causes IgG2a production, while virosomes, which are stabilized lipid complexes with viral proteins, can also activate IgG2a and IgA responses. A bacterial DNA CpG, which is also a TLR-9 ligand, can also cause Ig2a production when delivered with TT. Some cytokines that are used as adjuvants include IL-2, IL-6 and IL-12, which are all known to stimulate T-cell production and cause the production of IgG2a antibodies.
In most of our work, dating back to 2000, we have adopted the use of muramyl dipeptide (N-acetyl-glucosamine-3-yl-acetyl L-alanyl-D-isoglutamine [nor-MDP]) as an adjuvant as it was used in a WHO vaccine. Nor-MDP, a ubiquitous constituent of bacterial cell walls, is recognized by APCs and activates many different cell types, including macrophages, leukocytes, mastocytes, endothelial cells and fibroblasts, inducing the secretion of cytokines such as IL-1, B-cell growth factor and fibroblast-activating factor. The muramyl dipeptide, when emulsified in a squalene-in-water emulsion ISA 720 (SEPPIC, Paris, France), preferentially activates the humoral arm of the immune system.
The final puzzle is solved by vaccination with the chimeric immunogen that will elicit endogenous production of high-titer anti-HER-2 polyclonal antibodies. Such polyclonal antibodies may effectively inhibit function of the target molecule without the risk of tumor escape, as may occur with a mAb directed at a single epitope. The goal of the strategy is active specific immunotherapy providing prolonged therapeutic benefit by eliciting antibodies that could delay, prevent and/or eradicate tumor growth and metastasis with potentially increased efficacy and the generation of immunologic memory. Additional advantages include the avoidance of the need for multiple and expensive infusions that usually lead to severe toxicities.
The advantage of active immunotherapy over passive immunotherapy with mAbs, such as trastuzumab, pertuzumab and bevacizumab, is a fundamental aspect of our work. The half-life of IgG, administered intravenously, can range from 5 to 21 days. Thus, repeated treatments with trastuzumab are necessary – patients typically receive the mAb every 3 weeks. The repeated treatment with trastuzumab raises the cost of passive immunotherapy with this mAb to approximately US$140,000 per year.
An Innovative Peptide Approach to Cancer Treatment
The main efforts of our laboratory in the past two decades have been the development of unique peptide vaccines and peptidomimetic therapeutic approaches (Figure 2) for targeting viral, bacterial and cancer antigens. The novelty of our approach resides in a hypothesis-driven basic research in vaccinology with an incremental approach involving the elucidation of several basic immunological and structural concepts that could eventually be translated to the clinic for the benefit of cancer patients. These innovations include:
â–ª The ability to predict biologically active immune epitopes and confirm their properties in vitro and in vivo;
â–ª The engineering of unique B-cell epitope peptides that can recapitulate the exquisite native structure of the tumor antigen;
▪ The design of uniquely inhibitory peptide mimics that can block receptor–ligand interactions;
â–ª The idea of combining the B-cell epitopes into chimeric constructs that incorporate a 'promiscuous' T-cell activating species. Vaccination with these novel HER-2 chimeric immunogens results in the production of highly efficacious native-like antipeptide antibodies that can delay, prevent and/or eradicate tumor growth and metastasis with increased efficacy and little or no toxicity;
▪ In order to complement and enhance our vaccine approach, we have been developing 'nonimmunogenic' peptide therapeutics that target both HER-2 and VEGF pathways. This strategy involves the design and synthesis of conformational VEGF peptide mimics that are aimed at disrupting the receptor–ligand interactions with enhanced stability and efficacy;
â–ª The concept of 'tritherapy', which is the combination of immunotherapy, angiotherapy and metronomic chemotherapeutic drugs.
(Enlarge Image)
Figure 2.
Combination of HER-2 vaccines and VEGF peptide mimics.
HER-2 vaccines and VEGF peptide inhibitors developed in our laboratories to inhibit signaling pathways. Antibodies elicited by immunization with MVF-HER-2 (266–296) vaccine bind to domain II of HER-2 and, similarly, anti-MVF-HER-2 (597–626) binds domain IV of HER-2, providing dual inhibition of homo/heterodimerization, and consequently downstream signaling, shutting down the PI3K and MAPK pathways, thereby preventing cancer growth and metastasis. On the other hand, VEGF peptide mimics P3 and P4, which are designed to directly block VEGF binding to VEGFR-2, inhibit intracellular phosphorylation of the tyrosine kinase domain, which reduces angiogenesis.
EGFR: EGF receptor; P: Phosphate; VEGFR: VEGF receptor.
HER-2 Peptide Design Strategy
There are several strategies to design immunogenic B-cell epitopes of unknown proteins (reviewed by Kaumaya et al.). First, we have pioneered a unique approach of identifying antigenic epitopes on the surface of proteins whose 3D structure and whose antigen–antibody complex is unknown. It relies on predicting epitopes using correlates of antigenicity such as hydrophilicity, flexibility, hydrophobic index, amphiphilicity and secondary structural propensities. Computer algorithms are available to predict potential B-cell epitopes and we have successfully applied this technology to several targets including HTLV-1, HER-2, VEGF and others. For a large protein, several antigenic epitopes can be prioritized and these peptides are then synthesized by solid-phase peptide chemistry as chimeric immunogens with an appropriate T-cell epitope.
Design of Conformation-dependent Antigenic Determinants
Second, if the 3D structure of the antigen is unknown, prediction of the secondary structures by several criteria, such as α-helices, β-turn and sheet, and loop regions, can be made to aid the design. In that respect, knowledge of protein folding, structure and dynamics can be used to design the B-cell epitopes to fold into stable supersecondary structures (αα, αβ, βαβ and β-turns and loops). When the crystallographic structures of the antigen–antibody complex are known, the engineering of a selective B-cell epitope to mimic the structure of the immunogenic binding site on the tumor antigen is greatly facilitated.
Active Immunotherapy With Chimeric B-cell Peptide & Promiscuous T-cell Epitopes
The third aspect is to construct a chimeric peptide containing a promiscuous T-cell epitope that could be used as an immunogen to elicit high-titered and high-affinity antibodies that are able to recognize the protein. We have pioneered a strategy that is accomplished by selecting an appropriate T-cell epitope. Several of these promiscuous T-cell epitopes are known, and in our previous in-depth studies we have found the measles virus fusion (MVF) protein and the TT epitopes to be the most efficacious ones. The most important consideration in the successful design of a peptide vaccine is that the B-cell epitope must be covalently linked to the 'helper' T-cell epitope at either the N- or C-terminus (Figure 3). In our vaccine approach, we preferentially used the T-cell epitope at the N-terminus.
(Enlarge Image)
Figure 3.
Chimeric B- and T-cell epitopes.
B- and T-cell epitopes are colinearly synthesized with a GPSL flexible linker. The linker is flexible, allowing the two epitopes to fold or adopt different conformations independent of each other.
The size of the B-cell epitope should minimally be approximately 18 amino acid residues (˜900 Å), the size of an antigen–antibody binding region, to ensure the peptide propensity to adopt a defined structure. The helper T-cell should ideally be a promiscuous epitope (˜18 residues), the linker should be approximately four residues and the final construct should consist of between 40 and 60 amino acid residues. In some cases, the B-cell epitope can be greater than 18 residues to accommodate a larger binding site, as demonstrated by the HER-2–trastzumab or the HER-2–pertuzumab interface. The chimeric B- and T-cell epitope strategy can fully stimulate the immune system to produce high-affinity antibodies that can specifically target cancer cells and, most importantly, establish an immunological memory in order to prevent cancer relapse.
Advantages of Chimeric Peptides as Vaccines
Traditionally, peptide sequences are coupled to larger carrier proteins, such as bovine serum albumin, TT or keyhole limpet hemocyanin, in order to induce immunogenicity. This approach is fraught with difficulties as the resulting conjugate is subject to processing by the immune cells and the resulting B-cell epitope may not represent the exact epitope identity or configuration, resulting in low-affinity antibodies. Additionally, the coupling procedures are not an exact science in the sense that they cannot be duplicated from batch to batch, resulting in an unpredictable immune response. Other problems are epitope suppression by the carrier protein and poorly characterized constructs due to ambiguity during the chemical coupling reaction. A strategy that involves B- and helper T-cell epitopes is MHC-restricted by the fact that T-helper epitopes are recognized by only a few MHC class II alleles at most. We overcame those problems by proposing the use of 'universal' or promiscuous T-helper epitopes that bind to several MHC haplotypes.
The chimeric peptide is presented intact to the immune cells without processing, yielding high-titered and high-affinity protective polyclonal antibodies. This strategy avoids the proteolysis by APC, normally associated with peptides coupled to large carrier proteins in which the authenticity of the epitopes is destroyed resulting in a poorly immunogenic vaccine. Unlike antigens presented to MHC class I, which are restricted to peptides 8–10-mers, the ends of the groove of MHC class II are open, allowing larger peptides to bind. This is the major advantage over other strategies that focus on generating antibodies in vaccine development. Other active immunization regimens, such as activation of CTLs for many different types of cancer, have been exclusively and extensively used without much success so far, but there is hope that newer methods of delivery and adjuvants may lead to improved vaccines activating the cellular arm of the immune system.
Enhancing Immunogenicity of Peptide Vaccines
An ideal peptide vaccine should consist of an appropriate tumor antigen B-cell epitope or CTL epitope covalently linked to a universally immunogenic T-helper epitope. In addition, the peptide vaccine must be formulated with an adjuvant or cytokines, which are essential in the development of a good immune response. Adjuvants are usually defined as compounds that increase and/or moderate the intrinsic immunogenicity of an antigen. Generally speaking, adjuvants function in three basic ways: they cause the slow release of an antigen; they modulate the immune response; and they increase the presentation of the antigen to immune cells such as antigen-presenting cells. Poorly soluble aluminum salts (aluminum phosphate, aluminum hydroxide and alum [KAl(SO4)2·12H2O]), as well as calcium phosphate and AS04 (FENDrix®, GlaxoSmithKline, Brentford, UK), are the only vaccine adjuvants currently licensed in the USA. While generally considered quite safe, there are several areas in which these adjuvants are inadequate. Despite the wide use and acceptance of alum, there are a series of problems. Adjuvants that can stimulate Th1 cytokine-dependent IgG2a and IgG2b antibodies have been developed. Oil emulsions, such as MF59, which is composed of squalene, polyoxyethylene and muramyl tripeptide, have been shown to dominantly induce IgG2a production via Th1 immune response. QS-21, which is a purified saponin, also induces IgG2a responses via cell-mediated immunity. Another group of adjuvants that are microbial-derived has also been shown to activate Th1 responses and cause IgG2 production. A TLR-2 ligand known as macrophage-activating protein-2, which is a purified derivative from mycoplasma, causes IgG2a production, while virosomes, which are stabilized lipid complexes with viral proteins, can also activate IgG2a and IgA responses. A bacterial DNA CpG, which is also a TLR-9 ligand, can also cause Ig2a production when delivered with TT. Some cytokines that are used as adjuvants include IL-2, IL-6 and IL-12, which are all known to stimulate T-cell production and cause the production of IgG2a antibodies.
In most of our work, dating back to 2000, we have adopted the use of muramyl dipeptide (N-acetyl-glucosamine-3-yl-acetyl L-alanyl-D-isoglutamine [nor-MDP]) as an adjuvant as it was used in a WHO vaccine. Nor-MDP, a ubiquitous constituent of bacterial cell walls, is recognized by APCs and activates many different cell types, including macrophages, leukocytes, mastocytes, endothelial cells and fibroblasts, inducing the secretion of cytokines such as IL-1, B-cell growth factor and fibroblast-activating factor. The muramyl dipeptide, when emulsified in a squalene-in-water emulsion ISA 720 (SEPPIC, Paris, France), preferentially activates the humoral arm of the immune system.
Advantages of Active Immunotherapy
The final puzzle is solved by vaccination with the chimeric immunogen that will elicit endogenous production of high-titer anti-HER-2 polyclonal antibodies. Such polyclonal antibodies may effectively inhibit function of the target molecule without the risk of tumor escape, as may occur with a mAb directed at a single epitope. The goal of the strategy is active specific immunotherapy providing prolonged therapeutic benefit by eliciting antibodies that could delay, prevent and/or eradicate tumor growth and metastasis with potentially increased efficacy and the generation of immunologic memory. Additional advantages include the avoidance of the need for multiple and expensive infusions that usually lead to severe toxicities.
The advantage of active immunotherapy over passive immunotherapy with mAbs, such as trastuzumab, pertuzumab and bevacizumab, is a fundamental aspect of our work. The half-life of IgG, administered intravenously, can range from 5 to 21 days. Thus, repeated treatments with trastuzumab are necessary – patients typically receive the mAb every 3 weeks. The repeated treatment with trastuzumab raises the cost of passive immunotherapy with this mAb to approximately US$140,000 per year.