Several immunotherapeutic approaches targeting HER2 have been reported in the literature, including both passive and active immunization [23
]. The major problem in the use of HER2 as a target for active immunotherapy is the presence of immune tolerance to the self-antigen. The present study was conducted to assess the efficacy of two previously selected anti-Id (Ab2) human scFv fragments [21
] to generate an active anti-HER2 immune response. We show that vaccination with anti-Id scFv40 and scFv69 might represent a potential new therapeutic approach for the treatment of patients with HER2-positive tumors. We also demonstrate that the in vivo
anti-tumor effects of the anti-Id scFv69 vaccine are associated with a robust anti-HER2 humoral response through a Th2-dependent mechanism.
First, we demonstrate that sera from scFv40- or scFv69-immunized BALB/c mice contain Ab1' antibodies that strongly inhibit growth of SK-OV-3 cells, suggesting a "trastuzumab-like" biological effect as reported with the in vitro
use of the humanized anti-HER2 trastuzumab (Herceptin®
, ROCHE, Basel, Switzerland) [24
]. When adoptively transferred in nude mice bearing SK-OV-3 Luc tumor cells, these sera efficiently inhibited tumor growth. These results provide proof of the therapeutic potential of Ab1' antibodies. They also demonstrate the implication of the humoral response in the biological effects.
Second, we show that prophylactic vaccination of both virgin or primiparous MMTV.f.huHER2(Fo5) females with anti-Id scFv69 induces anti-HER2 Ab1' immune response followed by inhibition of spontaneous development of palpable tumors. The study by Finkle and colleagues indicated that early treatment with mu4D5 (the murine version of trastuzumab) was of benefit in MMTV.f.huHER2(Fo5)-transgenic females at high risk of developing huHER2-positive breast tumors. Similarly, we show that mice vaccinated with scFv69 (end of treatment at six months of age) were still free of tumors at the age of 14 months and that only one out of seven mice immunized with anti-Id scFv69 developed a tumor. Whereas in the work by Finkle et al. an early and prolonged treatment with mu4D5 was necessary to significantly alter mammary tumor incidence and progression, in our study, four injections of scFv69 were sufficient to elicit almost 100% protection against mammary tumors. Thus, different from the passive approach (mu4D5), active immunization with a therapeutic cancer vaccine may result in a gradual and lasting response, which could abolish tumor development and induce a long-term memory response leading to protection from disease recurrence.
Third, the analysis of the humoral response induced in immunized MMTV.f.huHER2(Fo5) mice shows that both a robust anti-scFv69 (Ab3) Th1 and an anti-HER2 (Ab1') Th2 response were associated with the delay of mammary tumor onset observed in animals treated with anti-Id scFv69. Based on these results, we think that anti-Id scFv69 could efficiently be used for the long term prevention of HER2-positive tumors via its specific anti-HER2 Th2-dependent immune mechanism.
ScFv40 and scFv69 were isolated by phage display, which is a relatively new technique to identify peptides or antibodies that mimic natural epitopes, including conformational B cell epitopes [25
], as it is the case for the epitope recognized by trastuzumab [26
]. The sequence analysis of scFv40 and scFv69 revealed no strong homology between their CDR1, 2 and 3 regions and the regions forming the epitope recognized by trastuzumab. This finding was expected as the epitope targeted by trastuzumab is described to be discontinuous. However, regarding the antigen mimicry capacity of the scFv69 fragment, its surface characteristics should be equivalent to those of the epitope of the selecting Ab1, although their amino acid sequence may differ [27
]. Similarly, the epitope mimics generated by Riemer et al
] by phage display using trastuzumab bear no sequence homology to HER2, but they are effective in mimicking the HER2 antigen. Their sequence was subsequently matched to the third loop of HER2 at the HER2/trastuzumab interface using computational methods [29
The low sequence homology between the CDR regions of scFv40 or scFv69 and the regions forming the HER2-epitope recognized by trastuzumab [26
] could explain the lack of anti-HER2 Th1 response following immunization with scFv69. Indeed, anti-Id scFv are supposed to mimic the three-dimensional rather than the primary sequence of HER2 which is in accordance with their anti-idiotypic nature. Furthermore, the lack of anti-HER2 Th1 response may be beneficial since it might balance the immunosuppressive effect of tumor-specific regulatory T cells (Treg). Depletion or blockade of Treg cells can lead to immune protection from tumor-associated antigens that are expressed as self-antigens [30
]. In our study, we demonstrate that scFv69-immunization leads to a HER2-specific Th2 immune response, suggesting that this type of vaccination could be effective in cancer patients even in the presence of immunosuppressive Treg cells.
Finally, cardiomyopathy has been shown to be a side effect of trastuzumab treatment in 7% of women following treatment with first-line anthracycline therapy and in up to 28% of women when trastuzumab is used concurrently with anthracycline [31
]. In the study by Finkle et al
., human HER2 was reported to be expressed in heart, but no histological or clinical evidence of cardiac or any other organ toxicity were observed following treatment with mu4D5. However, these mice were not treated with other therapeutic agents, such as anthracyclines, before or during the study. In this context, it will be useful to study the effect of immunization with scFv69 on heart function, morphology and histology after treatment with anthracycline and trastuzumab as we believe that the most promising clinical application for anti-Id scFv69-based-vaccines should be as adjuvant therapy after treatment with chemotherapy and trastuzumab. Indeed, integrating a targeted vaccine therapy after induction of a major response with a monoclonal antibody (for example, trastuzumab) could be considered as another therapeutic approach [33