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Epitope-Specific Antibody Response to HT-1080 Fibrosarcoma Cells by Mimotope Immunization1

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Epitope-Specific Antibody Response to HT-1080 Fibrosarcoma Cells by Mimotope Immunization1 Vol. 6, 3629–3635, September 2000 Clinical Cancer Research 3629 Epitope-specific Antibody Response to HT-1080 Fibrosarcoma Cells by Mimotope Immunization1 Mikhail Popkov, Salwa Sidrac-Ghali, genic in humans severely limits their clinical applications, Valery Alakhov, and Rosemonde Mandeville2 especially when repeated administrations are required. It was therefore suggested that passive immunization using human- Institut National Recherche Scientifique, Institute Armand-Frappier, University of Quebec, Laval, Quebec, H7V 1B7 Canada [M. P., ized or reshaped mouse mAbs could have clinical applica- S. S-G., V. A.]; Supratek Pharma Inc., Laval, Quebec, H7V 1B7 tion, and several of these mAbs are now in clinical trials (2). Canada [V. A.]; Department of Biological Sciences, University of Ideally, antibody humanization should not diminish the Quebec in Montreal, Montreal, Quebec, H3C 3P8 Canada [R. M.]; specificity and affinity of the antibody toward the antigen, and Biophage Inc., 6100 Royalmount Avenue, Montreal, Quebec, H4P 2R2 Canada [R. M.] whereas immunogenicity must be completely eliminated. However, it has become apparent that the accomplishment of both aims is usually time-consuming and costly. Another ABSTRACT approach is the use of synthetic peptides (mimotopes) corre- Mouse monoclonal antibody (mAb) BCD-F9, which sponding to the sequence of the native antigen that can induce recognizes an unknown antigen found on the surface of antibodies in vivo (3–5). Furthermore, protective immune many tumor cells, was used to screen a phage display library reactions were elicited by mimotope immunization for sev- expressing random peptide decamers. The phage that was eral infectious agents (6–8). Therefore, the use of peptide selected encoded the unique sequence GRRPGGWWMR, mimotopes might be a valuable approach in developing vac- representing the peptide capable of binding to the BCD-F9 cines for the induction of a defined antibody response to mAb. The peptide was synthesized and found to specifically cancer antigens. inhibit the binding of mAb to HT-1080 fibrosarcoma cells. Phage display technology is a powerful tool for identi- Alanine mutagenesis of the sequence encoding this peptide fying peptide structures that mimic natural epitopes, includ- indicated that three residues, PXXWW, were critical for its ing both linear and conformational epitopes expressed on a binding to the BCD-F9 mAb. Polyclonal antibodies gener- variety of cell types (9–13). Phage peptide libraries consist of ated by immunization of rabbits with the synthetic peptide filamentous phages displaying random peptides of defined GRRPGGWWMR (anti-mimotope antiserum or AM-F9) length on their surface. The peptides are usually fused to the bound specifically to HT-1080 cells and inhibited the bind- phage minor coat protein pIII (14–15), which is expressed at ing of the BCD-F9 mAb to these cells. Using an experimental low density (3–5 molecules/phage particle), or to the major animal model in which CD-1 nude mice are inoculated i.v. coat protein pVIII (16), which is represented at a higher copy with HT-1080 cells, develop lung metastasis, and die within number. Such libraries have been screened successfully with 30 days, we have shown that AM-F9 could significantly a variety of mAbs, and the peptides selected have been shown prolong the life span of these animals. Our results suggest to mimic linear, assembled, and nonpeptidic epitopes (12, 13, that a peptide mimotope can potentially be used as a novel 17). In all of these methodologies, the selection of mimotopes immunotherapy to induce a beneficial antitumor response. (molecules of the repertoire able to bind to the ligate) does not necessarily require that the original ligand be known. The INTRODUCTION mimotopes have been shown to effectively induce a specific immune response directed against the epitope recognized by Since the development of the hybridoma approach (1), a 3 the mAb used for the affinity selection of phage clones (4, 7), large number of rodent mAbs with specificity for antigens of which has suggested a new way to induce epitope-specific therapeutic interest have been generated and characterized. antibody responses against unknown epitopes (5, 18, 19). However, the fact that rodent antibodies are highly immuno- However, there are limited data on whether the antibodies generated by mimotopes can recognize native antigens on tumor cells, and much less is known about the antitumor Received 4/4/00; revised 6/21/00; accepted 6/21/00. activity of anti-mimotope antibodies. The costs of publication of this article were defrayed in part by the Recent findings in a nude xenograft mouse model have payment of page charges. This article must therefore be hereby marked shown that mAb BCD-F9 (20) administrated i.v. is able to advertisement in accordance with 18 U.S.C. Section 1734 solely to reduce the growth and metastasis of human HT-1080 tumor indicate this fact. cells.4 Because of the ability of BCD-F9 to recognize a wide 1 M. P. was supported by scholarships from National Sciences and Engineering Research Council of Canada and Fonds par la Formation de variety of neoplastic cell lines as opposed to normal tissues (21), Chercheurs et l’aide a`la recherche. 2 To whom requests for reprints should be addressed, at Biophage Inc., 6100 Royalmount Avenue, Montreal, Quebec, Canada H4P 2R2. Phone: (514) 496-1488; Fax: (514) 496-1521; E-mail: Rosemonde.Mandeville@ nrc.ca. 4 M. Popkov, S. Sidrac-Ghali, Y. Lusignan, S. Lemieux, and R. 3 The abbreviations used are: mAb, monoclonal antibody; KLH, keyhole Mandeville. Inhibition of tumor growth and metastasis of human fibro- limpet hemocyanin. sarcoma HT-1080 cells by mAb BCD-F9, submitted for publication. Downloaded from clincancerres.aacrjournals.org on September 23, 2021. © 2000 American Association for Cancer Research. 3630 Peptide Inducing Anticancer Immune Response this mAb could potentially be used for antitumor immunother- To test the ability of each phage to be recognized by the apy. In this study, we used phage-displayed peptide libraries to BCD-F9 mAb, approximately 108 virions of a given mutant identify ligands mimicking an epitope for BCD-F9 presented on were incubated for1hatroom temperature with the biotinylated the cancer cells, and we analyzed the anti-cancer activity of antibody previously immobilized on microtiter plates as de- antiserum generated against the discovered ligand. scribed above. After 10 washes, phages were eluted with 0.1 M glycine/HCl (pH 2.2), neutralized, and titered on strain K91 (15). The percentage of attachment was calculated as the num- MATERIALS AND METHODS ber of eluted phages divided by the input phages ϫ 100. Cell Line and mAb. The human fibrosarcoma cell line Synthetic Peptides. The mimotope sequence GRRPGG- HT-1080 was purchased from the American Type Culture Col- WWMR (designated M-F9) was synthesized as linear free pep- lection (Manassas, VA) and maintained in DMEM supple- tides by standard solid-phase method 9-fluorenylmethhoxycar- mented with 10% fetal bovine serum. The cells were cultured at bonyl chemistry and TFA (24). The purity of the peptides was 37°C in 5% CO2 under a humidified atmosphere. assessed by reverse phase high-pressure liquid chromatography The BCD-F9 mAb was obtained from the fusion of NS-1 and mass spectrometry. The complete peptide GRRPGGWW- myeloma cells with spleen cells from BALB/c mice hyperimmu- MRAASYC contains five additional residues at the COOH nized with the human breast carcinoma cell line BT-20 (20). terminus. Three residues (AAS) represent the linker fusing the BALB/c mice were inoculated i.p. with hybridoma, and the peptide and gIII protein as expressed on the phage, and a Tyr BCD-F9 mAb was purified from ascetic fluid using a protein residue was added for possible radiolabeling, and a Cys residue G-Sepharose column (Pharmacia, Baie-Durfe, Quebec, Canada). was added for coupling to KLH. P-␥2 peptide was synthesized Phage Libraries and Biopanning. BCD-F9 ligands as a NVSKEPGGWWKGDYC sequence corresponding to the were selected from random phage libraries expressing linear native PLC-␥2 sequence, except that the COOH-terminal Cys (pIII-10aa) or circular (pIII-10aa.Cys) decapeptides fused to pIII residue was added for coupling. For immunization, the peptides filamentous bacteriophage fd (22). were conjugated to KLH via the COOH terminus as described The BCD-F9 mAb was biotinylated by incubation of 100 previously (3). ␮g of the antibody with 5 ␮g of NHS-LC-biotin (Pierce, Rock- Rabbit Immunization. New Zealand White female rab- ␮ ␮ ford, IL) in 50 lof0.1M NaHCO3 for2hatroom temperature, bits were given a primary i.m. immunization with 100 gofthe followed by dialysis against PBS (23). For panning, Nunc peptide-KLH solution emulsified 1:1 in Freund’s complete ad- Maxisorb microtiter plates were coated with streptavidin at 20 juvant and subsequently boosted with 150 ␮g of antigen emul- ␮ g/ml in 0.1 M NaHCO3 overnight at 4°C and then blocked with sified 1:1 in Freund’s incomplete adjuvant at biweekly intervals. 350 ␮l of blocking solution (1% powdered milk in PBS) for 1 h The rabbits were boosted three times and bled 5 days after the at room temperature. The biotinylated BCD-F9 mAb was di- last boost at week 7. The serum samples from rabbits were luted to 10 ␮g/ml in blocking solution, and 25 ␮l were added to tested by ELISA as described previously (3). The synthetic each well. The mAb was bound to the plate for2hatroom peptides M-F9 and P-␥2 were used as capture antigens (1 ␮g/ temperature, and the wells were washed six times with PBS. well). All ELISA experiments were performed at least twice in Then, 1010 phages were added in 50 ␮l of 0.1% milk/PBS and triplicates. bound to the mAb for1hatroom temperature. The plates were Cell Binding and Inhibition Assays.
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