Clinically relevant immunosuppressants influence UVB-induced tumor size through effects on inflammation and angiogenesis.

Immunosuppressive therapies allow long-term patient and transplant survival, but are associated with increased development of UV-induced skin cancers, particularly squamous cell carcinomas. The mechanisms by which CsA, MMF, tacrolimus (TAC) or sirolimus (SRL), alone or in dual combinations, influence tumor development and progression are not completely understood. In the current study, chronically UV-exposed mice treated with SRL alone or in combination with CsA or TAC developed more tumors than mice treated with vehicle or other immunosuppressants, but the tumors were significantly smaller and less advanced. Mice treated with CsA or TAC developed significantly larger tumors than vehicle-treated mice, and a larger percentage in the CsA group were malignant. The addition of MMF to CsA, but not to TAC, significantly reduced tumor size. Immunosuppressant effects on UVB-induced inflammation and tumor angiogenesis may explain these findings. CsA enhanced both UVB-induced inflammation and tumor blood vessel density, while MMF reduced inflammation. Addition of MMF to CsA reduced tumor size and vascularity. SRL did not affect inflammation, but significantly reduced tumor vascularity. Thus the choice of immunosuppressants has important implications for tumor number, size and progression, likely due to the influence of immunosuppressants on UVB-induced inflammation and angiogenesis.

Peptide vaccines of the HER-2/neu dimerization loop are effective in inhibiting mammary tumor growth in vivo.

Human epidermal growth factor receptor-2 (HER-2)/neu (ErbB2), a member of the epidermal growth factor family of receptors, is overexpressed in 20-30% of breast cancers. It is an attractive target for receptor-directed antitumor therapy using mAbs. Unlike other epidermal growth factor receptor family members, HER-2/neu does not bind a high-affinity ligand, but rather functions as the preferred dimerization partner. Pertuzumab (Omnitarg) is a humanized mAb directed against the HER-2/neu dimerization domain that inhibits receptor signaling. The recent definition of the crystal structure of the HER-2/neu-pertuzumab complex demonstrated that the receptor dimerization region encompassed residues 266-333. Based on the three-dimensional structure of the complex, we have designed three conformational peptide constructs (sequences 266-296, 298-333, and 315-333) to mimic regions of the dimerization loop of the receptor and to characterize their in vitro and in vivo antitumor efficacy. All the constructs elicited high-affinity peptide Abs that inhibited multiple signaling pathways including HER-2/neu-specific inhibition of cellular proliferation and cytoplasmic receptor domain phosphorylation. All the peptide Abs showed Ab-dependent cellular cytotoxicity to varying degrees with the 266-296 constructs being equally effective as compared with Herceptin. The 266-296 peptide vaccine had statistically reduced tumor onset in both transplantable tumor models (FVB/n and BALB/c) and significant reduction in tumor development in two transgenic mouse tumor models (BALB-neuT and VEGF(+/-)Neu2-5(+/-)). The 266-296 construct represents the most promising candidate for antitumor vaccination and could also be used to treat a variety of cancers with either normal or elevated expression of HER-2 including breast, lung, ovarian, and prostate.

Novel engineered trastuzumab conformational epitopes demonstrate in vitro and in vivo antitumor properties against HER-2/neu.

Trastuzumab is a growth-inhibitory humanized Ab targeting the oncogenic protein HER-2/neu. Although trastuzumab is approved for treatment of advanced breast cancer, a number of concerns exist with passive immunotherapy. Treatment is expensive and has a limited duration of action, necessitating repeated administrations of the mAb. Active immunotherapy with conformational B cell epitopes affords the possibility of generating an enduring immune response, eliciting protein-reactive high-affinity anti-peptide Abs. The three-dimensional structure of human HER-2 in complex with trastuzumab reveals that the Ag-binding region of HER-2 spans residues 563-626 that comprises an extensive disulfide-bonding pattern. To delineate the binding region of HER-2, we have designed four synthetic peptides with different levels of conformational flexibility. Chimeric peptides incorporating the measles virus fusion "promiscuous" T cell epitope via a four-residue linker sequence were synthesized, purified, and characterized. All conformational peptides were recognized by trastuzumab and prevented the function of trastuzumab inhibiting tumor cell proliferation, with 563-598 and 597-626 showing greater reactivity. All epitopes were immunogenic in FVB/N mice with Abs against 597-626 and 613-626 recognizing HER-2. The 597-626 epitope was immunogenic in outbred rabbits eliciting Abs which recognized HER-2, competed with trastuzumab for the same epitope, inhibited proliferation of HER-2-expressing breast cancer cells in vitro and caused their Ab-dependent cell-mediated cytotoxicity. Moreover, immunization with the 597-626 epitope significantly reduced tumor burden in transgenic BALB-neuT mice. These results suggest the peptide B cell immunogen is appropriate as a vaccine for HER-2-overexpressing cancers because the resulting Abs show analogous biological properties to trastuzumab.

Conformational HER-2/neu B-cell epitope peptide vaccine designed to incorporate two native disulfide bonds enhances tumor cell binding and antitumor activities.

Cancer vaccines designed to elicit an antibody response that target antigenic sites on a tumor antigen must closely mimic the three-dimensional structure of the corresponding region on the antigen. We have designed a complex immunogen derived from the extracellular domain of human HER-2/neu-(626-649) that represents a three-dimensional epitope. We have successfully introduced two disulfide bonds into this sequence, thereby recapitulating the natural disulfide pairings observed in the native protein. To evaluate the immunogenicity of the doubly cyclized disulfide-linked peptide versus the free uncyclized peptide we examined the induction of antibody responses in both inbred and outbred mice strains, with both constructs eliciting high titered antibodies. The disulfide-paired specific antibodies exhibited enhanced cross-reactivity to HER-2/neu expressed on BT-474 cell line as determined by flow cytometry. The antitumor activities of the disulfidepaired specific antibodies did not improve the in vitro growth inhibition of human breast cancer cells overexpressing HER-2, but showed superior antitumor responses in the context of ADCC and interferon-gamma induction. Inbred mice (FVB/n) vaccinated with the disulfide-paired epitope exhibited a statistically significant reduction in the development of exogenously administered tumors in vivo compared with mice receiving either the free uncyclized or the promiscuous T-cell epitope (MVF) control peptide (p = 0.001). This study demonstrates the feasibility and importance of designing conformational epitopes that mimic the tertiary structure of the native protein for eliciting biologically relevant anti-tumor antibodies. Such approaches are a prerequisite to the design of effective peptide vaccines.