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-γ 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. 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-γ 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. HER-2/neu or erbB-2 is a 185-kDa transmembrane protein and member of the human epidermal growth factor receptor (EGFR) 1The abbreviations used are: EGFR, epidermal growth factor receptor; HER-2, human epidermal growth factor receptor; MVF, measles virus fusion protein (amino acids 288–302); ADCC, antibody dependent cell mediated cytotoxicity; MVF626–649SS, cyclized chimeric HER-2 B-cell epitope 626–649; MVF626–649NC, linear chimeric HER-2 B-cell epitope 626–649; PBMC, peripheral blood mononuclear cell; Ab, antibody; Fmoc, N-(9-fluorenyl)methoxycarbonyl; RP-HPLC, reverse phase-high performance liquid chromatography; ELISA, enzyme-linked immunosorbent assay; FCS, fetal calf serum; ESI-MS, electrospray ionization mass spectroscopy. family. HER-2/neu proto-oncogene is expressed by a variety of tissues and progenitor cells, participating in cellular proliferation and differentiation (1Kim J.Y. Sun Q. Oglesbee M. Yoon S.O. J. Neurosci. 2003; 23: 5561-5571Crossref PubMed Google Scholar, 2Vinter-Jensen L. APMIS Suppl. 1999; 93: 1-42PubMed Google Scholar). HER-2/neu overexpression is associated with markedly aggressive forms of cancer with a worse prognosis of several malignancies, including breast, ovary, pancreas, lung, and prostate (3Hynes N.E. Stern D.F. Biochim. Biophys. Acta. 1994; 1198: 165-184Crossref PubMed Scopus (1001) Google Scholar, 4Scholl S. Beuzeboc P. Pouillart P. Ann. Oncol. 2001; 12: S81-S87Abstract Full Text PDF PubMed Scopus (143) Google Scholar). The demonstration of pre-existent HER-2/neu-specific T-cells as well as HER-2/neu-specific antibodies in breast cancer patients (5Disis M.L. Smith J.W. Murphy A.E. Chen W. Cheever M.A. J. Cancer Res. 1994; 54: 1071-1076Google Scholar, 6Disis M.L. Bernhard H. Gralow J.R. Hand S.L. Emery S.R. Calenoff E. Cheever M.A. Ciba Found. Symp. 1994; 187 (discussion 207-111): 198-207PubMed Google Scholar, 7Fisk B. Blevins T.L. Wharton J.T. Ioannides C.G. J. Exp. Med. 1995; 181: 2109-2117Crossref PubMed Scopus (509) Google Scholar, 8Disis M.L. Cheever M.A. Adv. Cancer Res. 1997; 71: 343-371Crossref PubMed Google Scholar) has energized the development of an immunotherapeutic approach. The fact that patients are capable of mounting even weak immune responses is extremely promising for it suggests that it may be possible to overcome tolerance through vaccination. Substantial evidence exists that immunizing with peptides derived from self-antigens may be an effective means of circumventing tolerance (9Reilly R.T. Gottlieb M.B. Ercolini A.M. Machiels J.P. Kane C.E. Okoye F.I. Muller W.J. Dixon K.H. Jaffee E.M. Cancer Res. 2000; 60: 3569-3576PubMed Google Scholar, 10Ercolini A.M. Machiels J.P. Chen Y.C. Slansky J.E. Giedlen M. Reilly R.T. Jaffee E.M. J. Immunol. 2003; 170: 4273-4280Crossref PubMed Scopus (85) Google Scholar, 11Disis M.L. Grabstein K.H. Sleath P.R. Cheever M.A. Clin. Cancer Res. 1999; 5: 1289-1297PubMed Google Scholar, 12Dakappagari N.K. Douglas D.B. Triozzi P.L. Stevens V.C. Kaumaya P.T. Cancer Res. 2000; 60: 3782-3789PubMed Google Scholar). This finding has important implications in HER-2 cancer therapy; it suggests that vaccination may be an effective means of either initiating or boosting antitumor immunity. Equally promising, the autoimmunity provoked by vaccination seems to be tumor-restricted as signs of autoimmune disease are not detected in other tissues expressing physiological levels of HER-2 (13Disis M.L. Gralow J.R. Bernhard H. Hand S.L. Rubin W.D. Cheever M.A. J. Immunol. 1996; 156: 3151-3158PubMed Google Scholar, 14Jasinska J. Wagner S. Radauer C. Sedivy R. Brodowicz T. Wiltschke C. Breiteneder H. Pehamberger H. Scheiner O. Wiedermann U. Zielinski C.C. Int. J. Cancer. 2003; 107: 976-983Crossref PubMed Scopus (46) Google Scholar, 15Disis M.L. Gooley T.A. Rinn K. Davis D. Piepkorn M. Cheever M.A. Knutson K.L. Schiffman K. J. Clin. Oncol. 2002; 20: 2624-2632Crossref PubMed Scopus (381) Google Scholar). Passive immunotherapies such as the treatment with the humanized anti-HER-2/neu monoclonal antibody Trastuzumab have produced promising results in a clinical setting (16Slamon D.J. Leyland-Jones B. Shak S. Fuchs H. Paton V. Bajamonde A. Fleming T. Eiermann W. Wolter J. Pegram M. Baselga J. Norton L. N. Engl. J. Med. 2001; 344: 783-792Crossref PubMed Scopus (9401) Google Scholar, 17Vogel C.L. Cobleigh M.A. Tripathy D. Gutheil J.C. Harris L.N. Fehrenbacher L. Slamon D.J. Murphy M. Novotny W.F. Burchmore M. Shak S. Stewart S.J. Press M. J. Clin. Oncol. 2002; 20: 719-726Crossref PubMed Scopus Google Scholar). a of such as and associated of and a capable of specific to be a and immunity. The for from both and in that specific may be a to target HER-2 of the the of by B. P. P. A. V. Pouillart P. S. J. PubMed Scopus Google that a HER-2 and the of in breast tumors B. P. P. A. V. Pouillart P. S. J. PubMed Scopus Google Scholar). This the that may be an immune response to tumor on peptides have either the of the immune response or the induction of immune epitopes of HER-2/neu have and in clinical P. S. L. W. 2000; PubMed Google Scholar, K.L. Schiffman K. M.L. J. Clin. 2001; 107: PubMed Scopus Google Scholar). has on the antigenic of the of HER-2/neu by several of the human protein and in vitro and in vivo We the anti-tumor of a chimeric B-cell epitope that a promiscuous T-cell epitope (MVF) N.K. Douglas D.B. Triozzi P.L. Stevens V.C. Kaumaya P.T. Cancer Res. 2000; 60: 3782-3789PubMed Google Scholar, N.K. J. R. Kaumaya P.T. J. Immunol. 2003; 170: PubMed Scopus Google Scholar). This peptide in both outbred and inbred the development of tumors in of vaccinated mice N.K. Douglas D.B. Triozzi P.L. Stevens V.C. Kaumaya P.T. Cancer Res. 2000; 60: 3782-3789PubMed Google and in an of clinical the Cancer We have by several other important of the extracellular domain of HER-2 as and a is in a is well from three-dimensional of that antigenic epitopes are conformational as design to such that antibodies of high be peptide designed to the of disulfide that the pairings in human has M. J. D. J. Full Text Full Text PDF PubMed Scopus Google and the of to HER-2, we that a with the elicit antibodies designed as the mimic the native structure peptides to structure two important a peptide is it a linear peptides that in a of in a response is to a of peptide antibodies. conformational peptide epitopes to antibodies of high to with the native thereby the tumor or the The of disulfide bonds is an effective means of are in the extracellular domain of HER-2 and EGFR, of are the two L. T.L. Y.C. Chen E. A. J. T.A. J. U. A. A. PubMed Scopus Google Scholar, Muller W.J. U. S. A. 1996; 93: PubMed Scopus Google Scholar). is the of two in the of is the two The of are and to a specific capable of and T. S. T. K. N. N. T. K. PubMed Scopus Google Scholar). of the disulfide in the extracellular domain of have determined M. J. D. J. Full Text Full Text PDF PubMed Scopus Google Scholar). are a of the HER-2 extracellular a in the region to both in mice and a of human tumors Muller W.J. J. 1999; PubMed Scopus Google Scholar, Muller W.J. 1994; PubMed Scopus Google Scholar). to of the receptor and the expressed as in it to tumor and Muller W.J. U. S. A. 1996; 93: PubMed Scopus Google Scholar). and other T. Davis T. U. S. A. 2001; PubMed Scopus Google Scholar, A. Chen J. Q. R. J. 2002; Full Text Full Text PDF PubMed Scopus Google Scholar) to that the in the region to have in the of the we have on a peptide (amino acids from this it is possible to the HER-2 B-cell epitope peptides by the natural disulfide The introduced disulfide bonds may the structure in the peptides and to closely mimic the native such peptides have for native the disulfide structure M. J. D. J. Full Text Full Text PDF PubMed Scopus Google Scholar) and to HER-2, we HER-2 by it two acids This designed sequence, and with the disulfide bonds and and and To the cyclized epitope to mimic the native HER-2 the epitope MVF626–649NC, we the immunogenicity of both epitopes in outbred We the cross-reactivity of the two peptide antibodies by flow human HER-2 overexpressing breast cancer cells, We and antitumor activities by as well as to ADCC and in we determined the immunogenicity and of in a tumor an inbred We the and of a HER-2 B-cell The doubly disulfidepaired high antibodies for native HER-2 compared with the corresponding uncyclized linear epitope. the antibodies by the conformational epitope mediated levels of by human and enhanced cell in a statistically significant reduction in tumor growth observed in mice vaccinated with the disulfide-paired epitope to mice receiving either the linear or control peptide (p = 0.001). and of and B-cell epitope with a promiscuous epitope derived from the measles virus fusion protein (amino acids on a peptide used for the The B-cell epitope by on on and on and epitope with for and on the B-cell epitope. peptides from the the and peptides as R. Sun M. Kaumaya P.T. J. Full Text Full Text PDF PubMed Scopus Google Scholar). disulfide bonds either as by R. J. 2000; PubMed Scopus Google Scholar) or T. J. 1999; PubMed Scopus Google Scholar). peptide by and linear peptides by and by electrospray ionization mass The by with and linear peptide and for by the peptide in the of to a of and used as for on an as R. Sun M. Kaumaya P.T. J. Full Text Full Text PDF PubMed Scopus Google Scholar). to the = is the the of the the of in the the peptide and the of the and from and with either disulfide-paired or chimeric peptides in with of a in The of administered and by and for of antibody on a protein and antibodies and in The of antibodies determined by protein determined as N.K. Douglas D.B. Triozzi P.L. Stevens V.C. Kaumaya P.T. Cancer Res. 2000; 60: 3782-3789PubMed Google Scholar). as the of the with an of or the The to the that a of and cell fetal calf and from The human breast tumor cell BT-474 and from and to the HER-2 from by BT-474 or cells with and of peptide to and monoclonal antibody used as The cells in of for in and with antibody for in of in and by flow of cells for and cell and cells by and of BT-474 cells in of The with FCS, and This a of in The peptide or or and with of the of the by with of The The in for treatment and to the with BT-474 or human breast cancer the tumor cells with or peptide and for of antibody and human to and levels determined by a to the are in and the of from human by in in and in to to target of and cells in the of a of The target cells of peptide or the humanized monoclonal BT-474 and target cells and with cells of and for target cells to well to a of The cells for of and determined a To target cells in the of antibodies. by the = represents from represents from target in and represents from target cells with the of and have and of cell line derived from a tumor that in an used for tumor cells in with and of mice = with cells for the of with in a and tumor by the growth the that linear that to This is used to for the in the response have on the This well the are of and are a of on The for both treatment and by is used to the in the of This is to it is statistically for the in the not biologically be the of the treatment The used in the is = treatment = tumor = = is with to and The that the are and that is a linear of the both of The by treatment of the tumor and compared to a statistically significant is used to the results that are for this and an = used to are statistically significant control compared with of The results of the are into The is used to with is the of for the of the tumor to The of the used to is = = and = The a even the is the of the The by the not used to the treatment to the through the and of and we have N.K. Douglas D.B. Triozzi P.L. Stevens V.C. Kaumaya P.T. Cancer Res. 2000; 60: 3782-3789PubMed Google Scholar, N.K. J. R. Kaumaya P.T. J. Immunol. 2003; 170: PubMed Scopus Google has on the disulfide pairings in M. J. D. J. Full Text Full Text PDF PubMed Scopus Google Scholar). this we have the by two both the and such that disulfide pairings and and and be to the disulfide for and be of the The of the and by be and cyclized by or the disulfide the in peptides and to of the such as and have used for this a significant R. J. 2000; PubMed Scopus Google Scholar). We the of two or and on the of and We the the the peptide and in and The by as an for the and of peptide disulfide and as by mass in a that in and the of in reduction to the corresponding linear disulfide a high The peptide from the The to the other peptides by and by in and the disulfide in by of The mass of showed the of the The peptide by and and by The of the by in a significant mass be and that be by mass spectroscopy. with free to peptide or linear peptide and in for and by The linear peptide has and of peptide of and has used for of in peptides with disulfide bonds J.R. K.H. J. 2003; PubMed Scopus Google Scholar, C. J. Res. 2001; PubMed Scopus Google Scholar, J. 2001; PubMed Scopus Google Scholar). and are of and J. 2001; PubMed Scopus Google Scholar). of epitopes and in significant in peptide in is by two disulfide bonds and and this to and linear epitope is it is the antibodies by the disulfide-paired peptide with the native receptor and to antitumor of and in of mice = with either disulfide-paired or control in both the linear and the disulfide-paired peptide antibodies. the from of the cyclized and two of the antibodies not in control of to antibodies by the linear and disulfide-paired peptides in to the HER-2 we the of antibodies to HER-2 overexpressing human breast cancer cell line that both the cyclized and peptide antibodies to the native antibodies from the of mice with the peptide as compared with the linear peptide The cyclized antibodies compared with antibodies and The cyclized and the antibodies did not HER-2 breast cancer cell line not and in by antibodies anti-tumor is by we showed that the antibodies to the conformational epitope superior tumor cell we in finding antibody into enhanced The growth is a of the of cells to in this the of mice with the linear epitope showed growth inhibition to observed for with the conformational epitope and We growth inhibition by the of as an of The antibodies by both cyclized and peptide constructs showed inhibition not is a antitumor to the growth of tumors C. W. D. A. Int. J. Cancer. PubMed Scopus Google Scholar, A. R. Res. 1996; Google Scholar, K. B. Norton J. Res. 1996; Full Text PDF PubMed Scopus Google Scholar) and is an by antibodies on tumor We the of peptide antibodies to the of by HER-2 and tumor cells with peptide antibodies from mice and in the of human The cells for and levels determined by in both cyclized and peptide antibodies HER-2 overexpressing cells to the of compared with cells with antibodies. The cyclized peptide antibodies levels of compared with linear peptide antibodies The levels of observed in HER-2 cells to that observed for cells ADCC antibodies tumor cells by mononuclear cells in the we the of antibodies to We the of antibodies from the two constructs in mice and that and the from the and and the antibodies that and not two are to ADCC human as E. O. A. M. U. S. A. PubMed Scopus Google Scholar, B. T. R. J.R. M. Cancer Res. 1997; Google Scholar). in cyclized peptide antibodies the of specific as the antibodies and an to target of to with the human breast cancer cell line This observed to target used as a and in the clinical associated with conformational into B-cell epitope we the immunogenicity of both cyclized and constructs in mice that be with the cell line derived from a tumor from mice by M.A. M. Muller W.J. U. S. A. PubMed Scopus Google a of overexpression mice in a to that observed in human breast cancer patients (9Reilly R.T. Gottlieb M.B. Ercolini A.M. Machiels J.P. Kane C.E. Okoye F.I. Muller W.J. Dixon K.H. Jaffee E.M. Cancer Res. 2000; 60: 3569-3576PubMed Google and are a for human breast cancer of mice receiving the disulfidepaired that the a The inbred to the cyclized compared with the linear antibodies not in control To an with the B-cell we vaccinated of mice with of the HER-2 HER-2 or control The tumor for of the treatment are in is a in tumor = to mice and to mice (p = and = to the tumor growth an in the growth a on the and a of the statistically significant = with the MVF626–649SS, a statistically significant reduction in growth compared with mice receiving or control peptide (p = 0.001). The results of the into and used to is as the of for the of the tumor to of peptide vaccination on the of tumors of mice with control or The mice with is as the of for the of the tumor to The for the is as that this it is that of the the for of the in a in vaccinated mice the thereby the to tumor The of growth observed in mice with either or control peptide and and not to be statistically have a of cancer the of peptide the of on the of T-cell epitopes derived from tumor the development of a cancer not the of T-cell but B-cell such the of has on the and of B-cell epitopes of the HER-2 an to improve antibody and cross-reactivity to human we are the of B-cell the design of epitopes dependent from and with the structure of HER-2 K. A.M. D.J. 2003; PubMed Scopus Google Scholar, M. M. 2003; Full Text Full Text PDF PubMed Scopus Google we are to in the we have on the of disulfide pairings from M. J. D. J. Full Text Full Text PDF PubMed Scopus Google the structure has the region (amino acids have the associated with conformational The design of peptides that and tertiary has important implications in development R. Sun M. Kaumaya P.T. J. Full Text Full Text PDF PubMed Scopus Google Scholar, P.T. D.B. J. E. PubMed Scopus Google Scholar, P.T. A.M. E. J. Full Text PDF PubMed Google Scholar). J.C. D. O. J. Med. 2003; PubMed Scopus Google the of conformational epitopes from the human as a for the of in cancer a of conformational peptides as well as linear and that from patients with showed to an E. M. 2000; PubMed Scopus (46) Google that antibodies a cyclized peptide derived from the of but not linear to the cyclized peptide to elicit antibodies in vaccinated M. Wagner S. A. L. Pehamberger H. Scheiner O. Zielinski C.C. E. J. Immunol. PubMed Scopus Google Scholar) the of the peptide for the of capable of with the to HER-2, have to mimic the of B-cell epitopes by of mice with of to in antibodies as well as of the receptor in the antitumor and of has not in an the clinical of peptide antibodies and native protein. it be to the anti-tumor activities of with disulfide peptide as both antibodies target the relevant region but in a is of the domain and in the three-dimensional to the is on the structure of with K. A.M. D.J. 2003; PubMed Scopus Google Scholar) in and an relevant epitope of that the with The of HER-2 in levels of in tissues of C. Slamon D.J. 5: Google and extracellular domain it an target for as well as a for the of The extracellular domain and two that have to a in both and T. S. T. K. N. N. T. K. PubMed Scopus Google Scholar). the disulfide structure has and the disulfide pairings M. J. D. J. Full Text Full Text PDF PubMed Scopus Google Scholar). This the of and B-cell epitopes for We the of a B-cell To the of we linear MVF626–649NC, and the two epitopes in important for vaccines is that be capable of in an outbred We the immunogenicity of the peptides in outbred and in inbred mice as a epitopes to be as by antibody important factor in peptide vaccines is that the antibodies from a peptide the native protein. to the cyclized peptide to the native protein the linear peptide as by flow cytometry. the cyclized epitope to an antibody that showed to HER-2, this did not into a on breast cancer cells overexpressing from the structure of the HER-2 extracellular domain K. A.M. D.J. 2003; PubMed Scopus Google Scholar) in complex with antibody that the conformational of the region of HER-2 is for receptor and the antibodies by linear peptide the tumor cells the disulfide-linked it is that the linear peptide is of the are capable of and with an antigen for receptor on N.K. Douglas D.B. Triozzi P.L. Stevens V.C. Kaumaya P.T. Cancer Res. 2000; 60: 3782-3789PubMed Google Scholar, K.L. Schiffman K. M.L. J. Clin. 2001; 107: PubMed Scopus Google Scholar) as well as in the we antibodies by the linear peptide are superior growth the antibodies produced by the disulfide-linked peptide are the anti-tumor such as ADCC and by human The with to be effective in the treatment of a relevant to the treatment of human cancer patients with tumor HER-2 have on tumor or growth Cancer Immunol. 2002; PubMed Scopus Google Scholar). The cyclized epitope a in the of compared with the linear epitope. the cyclized epitope showed an in HER-2 overexpressing cells compared with the linear as by that the epitope is to tumor cells by ADCC and the of an antitumor that the growth of in vivo tumor the of mice for immunogenicity and tumor The mice to the cyclized compared with the linear is to that the outbred of mice levels of for both the cyclized and linear the inbred for the cyclized The in the antibody response may be of in the B-cell the inbred mice and outbred vaccinated mice with a tumor cell line derived from a tumor that in an tumors from mice have a to tumors in patients with breast cancer (9Reilly R.T. Gottlieb M.B. Ercolini A.M. Machiels J.P. Kane C.E. Okoye F.I. Muller W.J. Dixon K.H. Jaffee E.M. Cancer Res. 2000; 60: 3569-3576PubMed Google Scholar). that with the cyclized epitope showed a reduction in tumor compared with mice with the linear or control constructs and a statistically significant reduction in growth compared with mice receiving or control peptide (p = 0.001). The in as we have N.K. Douglas D.B. Triozzi P.L. Stevens V.C. Kaumaya P.T. Cancer Res. 2000; 60: 3782-3789PubMed Google and the of a tumor is that the inhibition of is in the tumor but the tumors may have to be dependent on to the the for tumor important ADCC, that with a may be We that the epitope disulfide bonds produced antibodies with anti-tumor the linear epitope. the structure of the extracellular domain of HER-2 to K. A.M. D.J. 2003; PubMed Scopus Google we are the anti-tumor of other peptide epitopes from the structure of the We for with the mass and the Cancer for with flow cytometry.
No takes yet. Share an insight, caveat, or question.
Dakappagari et al. (2004) studied this question.
Synapse has enriched 5 closely related papers on similar clinical questions. Consider them for comparative context: