HER-2 is a member of the ErbB family of receptor tyrosine kinases (RTKs) associated with aggressive forms of several human cancers and a well-established target for both passive and active immunotherapy. VEGF is overexpressed in many different types of cancer and hence both VEGF and its receptors (VEGFR1 and VEGFR2) are prime targets for tumor-directed anti-angiogenic intervention. Agents targeting RTKs for cancer therapy include antibodies that block RTK ligands or the receptors themselves, as well as small-molecule inhibitors that inhibit the intracellular catalytic domain of RTKs. Many FDA-approved therapies targeting both HER-2 (Trastuzumab, Herceptin, Pertuzumab, Omnitarg) and VEGF (Bevacizumab, Avastin) have significant toxicities and are associated with the development of resistance. Clinical applications of monoclonal antibody-based therapy in general is limited by a number of concerns such as the frequency of treatments, associated costs, limited duration of action, undesired immunogenicity, and significant risks of carditoxicity. Similarly small-molecule RTK inhibitors such as sunitinib, which have entered clinical trials alone or in combination with radiotherapy or chemotherapy, show problems of efficacy, development of resistance and unacceptable safety profiles which altogether hamper their clinical progress.
Immunization or treatment with peptides offers the opportunity of stimulating the body’s immune response leading to immunological memory. Peptides are relatively safe, non toxic, cheaper than antibodies and highly specific. The only drawback associated with peptides is their relatively limited stability (owing to degradation by proteases). This can however be overcome by using D-amino acids, which cannot be recognized by proteases. Thus, peptides can be synthesized with a reversal of the peptide chirality and using D-amino acids, resulting to a topographical equivalent of the parent peptide.
The overexpression of HER-2 is associated with increased expression of VEGF at both the RNA and protein levels in human breast cancer cells, and exposure of HER-2 positive cells to trastuzumab significantly decreases VEGF.25
SHC, a downstream adaptor protein of the HER-2 signaling pathway, has been identified as a critical angiogenic switch for VEGF production.26
This suggests that the effects of HER-2 on tumor cell behavior may be mediated in part through the stimulation of angiogenesis. A two-pronged approach to target cancer cells by co-immunizing with defined tumor-associated antigens and angiogenesis-associated antigens has been shown to exert synergistic anti-tumor effects.27-29
Altogether, these observations indicate that combination therapy targeting both HER-2 and VEGF constitutes a superior strategy as compared with either monotherapy, since antiangiogenic therapy alone tends to only delay tumor growth15
while targeting HER-2 and VEGF will abrogate two distinct tumorigenic pathways.
We evaluated the antiproliferative effects of the antibodies raised by HER-2 and VEGF peptides, alone or combined, on different cell lines. Trastuzumab has been shown to act only on HER-2 positive cells and we made similar observations (), whereby little inhibition was observed with the (HER-2 low) MDA-468 cell line as compared with the (HER-2 high) BT-474 cell line. The anti-peptide antibodies were effective in inhibiting both cancer cell lines. The HER-2–266–296 peptide antibody showed some inhibitory effects on the HER-2-low cell line (MDA-468) () and this is probably due to the fact that the peptide was synthesized using the pertuzumab epitope. Indeed, antibodies raised against this peptide should be able to function like pertuzumab, hence having inhibitory effects in cells independent of HER-2. We also evaluated the in vitro effects of the combination treatment with both HER-2 and VEGF anti-peptide antibodies on cell proliferation and viability, finding that the combination regimen produces greater anti-tumor effects than either treatment alone ().
HER-2 is known to dimerize with its partner HER-1 and HER-3 leading to receptor phosphorylation and intracellular signaling. Pertuzumab mainly functions by sterically blocking HER-2 from binding to its partners and is therefore classified as a dimerization inhibitor.30,31
We therefore investigated the effects of antibodies raised by HER-2 and VEGF peptides on HER-2 phosphorylation, finding an amelioration in phosphorylation inhibition from less than 35% in the case of single treatments to about 75% in the case of the combination regimen (). One of the main mode of action of antibodies is to cause ADCC, so we also evaluated the ability of our anti-peptide antibodies to cause ADCC against BT-474 cells. We observed that the anti-peptide antibodies were able to cause ADCC and their effects were comparable to that of the positive control trastuzumab (). Also in the case of anti-HER-2 and anti-VEGF peptide antibodies in combination, there was an increase in ADCC as compared with single treatments as well as to trastuzumab. We have previously shown the anti-angiogenic effects of our VEGF peptide mimics in different angiogenesis assays32
and here we illustrated further their ability to inhibit microvascular outgrowth in the mouse aortic ring assay (). We also showed that the VEGF peptide mimics are able to prevent the VEGF-mediated induction of VE-cadherin in matrigel plugs that were implanted in mice ().
In order to evaluate the effects of peptide administration in vivo, we used a transplantable tumor mouse model. BALB/c mice were immunized with the HER-2 peptide before being challenged with TUBO cells and treated with VEGF peptides. We observed significant differences between treated and control groups a delay in tumor growth and development, and a decrease in tumor weight. Vaccination with MVF-HER-2 266–296 followed by the administration of VEGF-P4 produced the best results, and 40% of the mice in this group remained tumor-free at the end of the experiment (). The VEGF peptide treatment also appeared to cause a decrease in blood flow to the tumors, contributing to its anti-tumor effects (). Tumor sections stained for actively dividing cells and blood vessels showed a marked reduction in positively stained cells following the vaccination, which was exacerbated with VEGF peptides were administered post-treatment (Figs. Seven and 8). These results strongly suggest that tumor growth and development can be strongly inhibited by simultaneously targeting the tumors and their blood supply. This is also because while tumor cells are genetically unstable (thus constantly changing and hence baing prone to developing resistance), the tumor vasculature is genetically stable.33
Thus, active immunization with HER-2 peptide epitopes and treatment with VEGF peptide mimics is a better strategy than immunization alone. Also, the D-amino acid-based peptide produced greater inhibitory effects, probably due to its longer half-life in vivo.
In conclusion, our results show the potential synergy between immunotherapy with HER-2 peptide vaccines and antiangiogenic therapy with VEGF inhibitors that are able to prevent or delay tumor growth. This combination strategy targeting different aspects of the tumor microenvironment show enhanced efficacy as compared with indivual treatments. The simultaneous and sequential strategy to inhibit different signaling pathways shows great promise for the design and development of effective anti-tumor and anti-angiogenic therapies against cancer. Foremost in those strategies lies the intuitive and rational design of effective peptide molecules that mimic the corresponding native structure for high efficacy inhibition of both antigen:antibody and receptor ligand interactions. Such molecules are safe and non-toxic and might offer great advantages in the treatment and management of cancers.