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Clinical Cancer Research Vol. 6, 3629-3635, September 2000
© 2000 American Association for Cancer Research

Experimental Therapeutics, Preclinical Pharmacology

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

Mikhail Popkov, Salwa Sidrac-Ghali, Valery Alakhov and Rosemonde Mandeville2

Institut National Recherche Scientifique, Institute Armand-Frappier, University of Quebec, Laval, Quebec, H7V 1B7 Canada [M. P., S. S-G., V. A.]; Supratek Pharma Inc., Laval, Quebec, H7V 1B7 Canada [V. A.]; Department of Biological Sciences, University of Quebec in Montreal, Montreal, Quebec, H3C 3P8 Canada [R. M.]; and Biophage Inc., 6100 Royalmount Avenue, Montreal, Quebec, H4P 2R2 Canada [R. M.]


   ABSTRACT
Top
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Mouse monoclonal antibody (mAb) BCD-F9, which recognizes an unknown antigen found on the surface of many tumor cells, was used to screen a phage display library expressing random peptide decamers. The phage that was selected encoded the unique sequence GRRPGGWWMR, representing the peptide capable of binding to the BCD-F9 mAb. The peptide was synthesized and found to specifically inhibit the binding of mAb to HT-1080 fibrosarcoma cells. Alanine mutagenesis of the sequence encoding this peptide indicated that three residues, PXXWW, were critical for its binding to the BCD-F9 mAb. Polyclonal antibodies generated by immunization of rabbits with the synthetic peptide GRRPGGWWMR (anti-mimotope antiserum or AM-F9) bound specifically to HT-1080 cells and inhibited the binding of the BCD-F9 mAb to these cells. Using an experimental animal model in which CD-1 nude mice are inoculated i.v. with HT-1080 cells, develop lung metastasis, and die within 30 days, we have shown that AM-F9 could significantly prolong the life span of these animals. Our results suggest that a peptide mimotope can potentially be used as a novel immunotherapy to induce a beneficial antitumor response.


   INTRODUCTION
Top
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Since the development of the hybridoma approach (1) , a large number of rodent mAbs3 with specificity for antigens of therapeutic interest have been generated and characterized. However, the fact that rodent antibodies are highly immunogenic in humans severely limits their clinical applications, especially when repeated administrations are required. It was therefore suggested that passive immunization using humanized or reshaped mouse mAbs could have clinical application, and several of these mAbs are now in clinical trials (2) . Ideally, antibody humanization should not diminish the specificity and affinity of the antibody toward the antigen, whereas immunogenicity must be completely eliminated. However, it has become apparent that the accomplishment of both aims is usually time-consuming and costly. Another approach is the use of synthetic peptides (mimotopes) corresponding to the sequence of the native antigen that can induce antibodies in vivo (3, 4, 5) . Furthermore, protective immune reactions were elicited by mimotope immunization for several infectious agents (6, 7, 8) . Therefore, the use of peptide mimotopes might be a valuable approach in developing vaccines for the induction of a defined antibody response to cancer antigens.

Phage display technology is a powerful tool for identifying peptide structures that mimic natural epitopes, including both linear and conformational epitopes expressed on a variety of cell types (9, 10, 11, 12, 13) . Phage peptide libraries consist of filamentous phages displaying random peptides of defined length on their surface. The peptides are usually fused to the phage minor coat protein pIII (14, 15) , which is expressed at low density (3–5 molecules/phage particle), or to the major coat protein pVIII (16) , which is represented at a higher copy number. Such libraries have been screened successfully with a variety of mAbs, and the peptides selected have been shown to mimic linear, assembled, and nonpeptidic epitopes (12 , 13 , 17) . In all of these methodologies, the selection of mimotopes (molecules of the repertoire able to bind to the ligate) does not necessarily require that the original ligand be known. The mimotopes have been shown to effectively induce a specific immune response directed against the epitope recognized by the mAb used for the affinity selection of phage clones (4 , 7) , which has suggested a new way to induce epitope-specific antibody responses against unknown epitopes (5 , 18 , 19) . 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 activity of anti-mimotope antibodies.

Recent findings in a nude xenograft mouse model have shown that mAb BCD-F9 (20) administrated i.v. is able to reduce the growth and metastasis of human HT-1080 tumor cells.4 Because of the ability of BCD-F9 to recognize a wide variety of neoplastic cell lines as opposed to normal tissues (21) , this mAb could potentially be used for antitumor immunotherapy. In this study, we used phage-displayed peptide libraries to identify ligands mimicking an epitope for BCD-F9 presented on the cancer cells, and we analyzed the anti-cancer activity of antiserum generated against the discovered ligand.


   MATERIALS AND METHODS
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Cell Line and mAb.
The human fibrosarcoma cell line HT-1080 was purchased from the American Type Culture Collection (Manassas, VA) and maintained in DMEM supplemented with 10% fetal bovine serum. The cells were cultured at 37°C in 5% CO2 under a humidified atmosphere.

The BCD-F9 mAb was obtained from the fusion of NS-1 myeloma cells with spleen cells from BALB/c mice hyperimmunized with the human breast carcinoma cell line BT-20 (20) . BALB/c mice were inoculated i.p. with hybridoma, and the BCD-F9 mAb was purified from ascetic fluid using a protein G-Sepharose column (Pharmacia, Baie-Durfe, Quebec, Canada).

Phage Libraries and Biopanning.
BCD-F9 ligands were selected from random phage libraries expressing linear (pIII-10aa) or circular (pIII-10aa.Cys) decapeptides fused to pIII filamentous bacteriophage fd (22) .

The BCD-F9 mAb was biotinylated by incubation of 100 µg of the antibody with 5 µg of NHS-LC-biotin (Pierce, Rockford, IL) in 50 µl of 0.1 M NaHCO3 for 2 h at room temperature, followed by dialysis against PBS (23) . For panning, Nunc Maxisorb microtiter plates were coated with streptavidin at 20 µg/ml in 0.1 M NaHCO3 overnight at 4°C and then blocked with 350 µl of blocking solution (1% powdered milk in PBS) for 1 h at room temperature. The biotinylated BCD-F9 mAb was diluted to 10 µg/ml in blocking solution, and 25 µl were added to each well. The mAb was bound to the plate for 2 h at room temperature, and the wells were washed six times with PBS. Then, 1010 phages were added in 50 µl of 0.1% milk/PBS and bound to the mAb for 1 h at room temperature. The plates were washed 12 times with PBS to remove nonspecific phages, whereas bound phages were eluted by treatment with 50 µl of 0.1 M glycine/HCl buffer (pH 2.2) containing 1 mg/ml BSA. Neutralization of the eluate, titration, and amplification on agar medium were carried out essentially as described previously (15) . The binding and elution steps were repeated four times. Viral DNA was sequenced with fUSE 32P primer 5'-TGAATTTTCTGTATGAGG-3' (kindly provided by Dr. George Smith, University of Missouri, Columbia, MO) by using the Sequenase T7 kit (Pharmacia) as recommended by the supplier.

Alanine Substitutions and Phage Attachment Assay.
Mutants of the phage selected for its binding to the BCD-F9 mAb were produced as described previously (18) . Briefly, a series of complementary oligonucleotides in which a given non-Ala residue of DNA encoding the peptide was changed to encode Ala were used in the construction of mutant phages. Complementary oligonucleotides {gamma}2for (5'-TGGCTTCTAAAGAGCCGGGGGGGTGGTGGAAGGGGGCGGCCTCTG-3') and {gamma}2rev (5'-AGGCCGCCCCCTTCCACCACCCCCCCGGCTCTTTAGAAGCCACGT-3') were used in the construction of phage {gamma}2. All mutants were purified and verified by DNA sequencing as described above.

To test the ability of each phage to be recognized by the BCD-F9 mAb, approximately 108 virions of a given mutant were incubated for 1 h at room temperature with the biotinylated antibody previously immobilized on microtiter plates as described 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 number of eluted phages divided by the input phages x 100.

Synthetic Peptides.
The mimotope sequence GRRPGGWWMR (designated M-F9) was synthesized as linear free peptides by standard solid-phase method 9-fluorenylmethhoxycarbonyl chemistry and TFA (24) . The purity of the peptides was assessed by reverse phase high-pressure liquid chromatography and mass spectrometry. The complete peptide GRRPGGWWMRAASYC contains five additional residues at the COOH terminus. Three residues (AAS) represent the linker fusing the peptide and gIII protein as expressed on the phage, and a Tyr residue was added for possible radiolabeling, and a Cys residue was added for coupling to KLH. P-{gamma}2 peptide was synthesized as a NVSKEPGGWWKGDYC sequence corresponding to the native PLC-{gamma}2 sequence, except that the COOH-terminal Cys residue was added for coupling. For immunization, the peptides were conjugated to KLH via the COOH terminus as described previously (3) .

Rabbit Immunization.
New Zealand White female rabbits were given a primary i.m. immunization with 100 µg of the peptide-KLH solution emulsified 1:1 in Freund’s complete adjuvant and subsequently boosted with 150 µg of antigen emulsified 1:1 in Freund’s incomplete adjuvant at biweekly intervals. The rabbits were boosted three times and bled 5 days after the last boost at week 7. The serum samples from rabbits were tested by ELISA as described previously (3) . The synthetic peptides M-F9 and P-{gamma}2 were used as capture antigens (1 µg/well). All ELISA experiments were performed at least twice in triplicates.

Cell Binding and Inhibition Assays.
For the preparation of cells, 10 ml of PBS were added to HT-1080 cell culture, and the cells were washed, harvested by incubation with PBS containing 0.5 mM EDTA, and transferred to 15-ml centrifuge tubes. Viable cells were then counted using the trypan blue dye exclusion technique. The concentration of cells was adjusted to 107 cells/ml, and 100 µl were used for each sample. The BCD-F9 mAb (1 µg) was then added to the sample tubes and incubated on ice for 45 min, washed twice with 2 ml of BSA/PBS, and centrifuged for 5 min at 1500 rpm. A total of 10 µl of antibody [goat antimouse IgG FITC-labeled antibody (Roche Diagnostics, Laval, Quebec, Canada) diluted 1:25 with PBS] was added to the sample, incubated on ice for 45 min, and washed twice with BSA/PBS. The cells were fixed using 0.5 ml of 1% paraformaldehyde in PBS, and the percentage of cells binding the antibody was analyzed on a Epics XL-MCL flow cytometer (Coulter, Hialeah, FL) equipped with a 488 nm argon laser.

For phage inhibition assay, 0.5 µg of the BCD-F9 mAb was preincubated for 2 h at room temperature with 100 µl of BSA/PBS containing various concentrations of phage particles F9, {gamma}2, and W7 expressing the VCDWWGWGIC peptide. Aliquots were then added to HT-1080 cells (106 cells/sample), and the cells were treated as described above.

For peptide inhibition assay, 0.5 µg of the BCD-F9 mAb was preincubated for 2 h at room temperature with 100 µl of BSA/PBS containing various concentrations of the synthetic peptides M-F9, P-{gamma}2, and VCDWWGWGIC. Aliquots were then added to HT-1080 cells (106 cells/sample), and the cells were treated as described above.

For antisera inhibition assay, HT-1080 cells (106 cells/sample) were preincubated for 45 min on ice with different dilutions of antisera: (a) AM-F9; (b) AP-{gamma}2; or (c) preimmune rabbit serum. One µg of the BCD-F9 mAb was added to the sample tubes, and treatment proceeded as described above.

Experimental Metastasis.
Confluent monolayers of HT-1080 cells were harvested by incubation with PBS containing 0.5 mM EDTA. Viable cells were counted by the trypan blue exclusion dye method. Aliquots containing >95% viable cells were used in this experiment. The cells (2 x 107cells/ml) were suspended in PBS, and 0.1 ml of the suspension was injected i.v. into the tail vein of CD-1 nude mice (day 0). Rabbit preimmune serum (100 µl) or polyclonal antisera AM-F9 (100 µl) was administered i.v. in 0.2 ml of PBS on days 1–2, 5–9, 12–16, and 19–21, and the lungs were recovered from recently deceased animals. The lungs were fixed in 10% buffered formalin, embedded in paraffin, sectioned, and stained with H&E for routine histological examination by light microscopy.

Statistical Analysis.
The Cox-Mantel log-rank test was performed using INSTAT software. P < 0.05 was considered significant.


   RESULTS
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Identification of BCD-F9-specific Mimotopes.
To search for a peptide that is able to bind with the BCD-F9 mAb, we screened two phage peptide libraries. One of these libraries displays linear decapeptides that are fused to the minor coat protein pIII, whereas the second has additional two cysteines flanking the random decapeptide sequence, leading to formation of circular peptides through the creation of a disulfide bridge. After four rounds of biopanning using the BCD-F9 mAb as a target, we observed an approximately 33,000-fold increase in the number of eluted phages in the case of the linear library (Fig. 1)Citation . The circular library showed a marginal enrichment of about 100-fold after the third round of selection. Therefore, no further manipulations were continued with this library.



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  		Fig. 1. Selection of phages that bind to the BCD-F9 mAb. Phages from linear (•) or circular ({circ}) decapeptide libraries were bound to mAb-coated microtiter wells as described in "Materials and Methods" and eluted with glycine/HCl buffer (pH 2.2). Enrichment (ni/n1) was calculated as the total number of phages recovered after elution (ni, number of transducing units recovered after i round of selection) divided by the number of transducing units recovered after the first round of selection (n1). Data represent mean values from plating in triplicate.

 
After the fourth round of selection with the linear library, 16 independent phage isolates were amplified and sequenced. All isolates yielded the identical DNA sequence encoding the amino acid sequence GRRPGGWWMR. Eight additional independent isolates were chosen from the second round and sequenced. All of the phages from the second round encoded the same sequence obtained from the fourth-round isolates. The comparison of the identified peptide sequence with the protein sequences available in the GenBank showed the homology of five amino acid residues (PGGWW) to six SH3 domaincontaining proteins (National Center for Biotechnology Information accession numbers AAD29953, AAC78610, Q15811, AAD29952, NP_003015, and NP_002652). These proteins are all intracellular signal transducers; therefore, it is unlikely that they contain the BCD-F9-reactive epitope.

Determination of Critical Amino Acids Necessary for Binding to the BCD-F9 mAb.
To determine which residues were critical for binding, a series of mutant phages were constructed in which residues inside the PGGWW block were individually changed to Ala. Phage {gamma}2 expressing the sequence corresponding to that of PLC-{gamma}2, one of the six proteins identified in the GenBank (National Center for Biotechnology Information accession number NP_002652), was also constructed to determine the effect of the remaining residues NH2- or COOH-terminal to this block. Each of the resulting phages was then tested for its ability to bind to the BCD-F9 mAb. Results shown in Fig. 2Citation indicate that mutations of three residues, Pro (fourth position), Trp (seventh position), and Trp (eighth position), led to a significant reduction in binding of the peptide to the BCD-F9 mAb. The remaining residues had no effect on the peptide binding when changed all at once in phage {gamma}2. We did not change two Gly residues inside the PGGWW block mainly because it has been shown previously that Gly residues do not usually contribute directly to the binding but rather serve as structural linkers to position the critical residues (18) .



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  		Fig. 2. Binding of Ala phage mutants and phage {gamma}2 to the BCD-F9 mAb. The effect of Ala substitutions of three residues in the peptide GRRPGGWWMR (Pro in the fourth position from the NH2 terminus, Trp in the seventh position, and Trp in the eighth position) is shown. F9 phage served as a positive control for binding to the BCD-F9 mAb. The percentage of attachment is the number of eluted phages divided by the input phage x 100. Data represent mean values from plating in triplicate.

 
Binding of Phage F9 and Mimotope (M-F9) to the BCD-F9 mAb.
To confirm the specificity of the peptide represented on phage F9, we analyzed its ability to inhibit binding of the BCD-F9 mAb to HT-1080 cells i.e., cells that express a high level of the BCD-F9-specific antigen. Phages {gamma}2 and W7 were used as controls mainly because they show the highest and the lowest binding to the BCD-F9 mAb, respectively (Fig. 3)Citation . BCD-F9 was incubated with the serial dilutions of phages and then assayed for its ability to bind to HT-1080 cells. The results shown in Fig. 3ACitation demonstrate that phage F9 induced a 47% inhibition at a concentration of 1013 virions/ml, whereas phage {gamma}2 was less potent, inducing a 38% inhibition at the same virion concentration. The phage W7 did not show any specific binding to the BCD-F9 mAb.



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  		Fig. 3. Inhibition of BCD-F9 binding to HT-1080 cells by phages and by synthetic peptides. Binding of BCD-F9 to HT-1080 cells was studied in the presence of the competing phages (A) F9 (•), {gamma}2 ({blacksquare}), and W7 ({circ}) or in the presence of the competing peptides (B) M-F9 (•), P-{gamma}2 ({blacksquare}), and VCDWWGWGIC irrelevant peptide ({circ}) as described in "Materials and Methods." The Y axis shows inhibition (percentage) of binding of BCD-F9 to HT-1080 cells as a function of the phage (A) or of the free peptide (B) concentration (X axis). The data represent the means ± SD from three independent experiments.

 
A peptide corresponding to the sequence presented on phage F9 (M-F9 peptide) was then synthesized and tested for its ability to bind to the BCD-F9 mAb. We also synthesized a peptide corresponding to phage {gamma}2 (P-{gamma}2 peptide) and an unrelated (VCDWWGWGIC) peptide. These three peptides were tested for their ability to bind to the BCD-F9 mAb. Briefly, BCD-F9 was incubated with the peptides at various concentrations of the synthetic peptides and then assayed for its ability to bind to HT-1080 cells. Fig. 3BCitation shows that M-F9 bound specifically to the antibody and blocked its subsequent binding to HT-1080 cells. The highest potency of the M-F9 peptide was IC50 = 2 x 10-6 M, as compared with the P-{gamma}2 peptide that showed an inhibitory effect only at concentrations higher than 100 µM. The peptide VCDWWGWGIC had no inhibitory effect (Fig. 3B)Citation .

Induction of an Epitope-specific Immune Response.
To investigate whether the mimotope represents an immunogenic structure corresponding to the natural epitope expressed on cancer cells, rabbits were immunized with either the M-F9 peptide or the P-{gamma}2 peptide conjugated with KLH. After a primary immunization and three boosts, the sera from individual rabbits were collected and tested for anti-peptide antibodies using an ELISA technique. All of the sera collected showed an ability to bind to peptides at 1:2000 dilution, whereas the preimmune serum showed no anti-peptide reactivity. We then investigated the specificity of the rabbit anti-mimotope antisera by inhibiting the binding of the BCD-F9 mAb to HT-1080 cells. As shown in Fig. 4Citation , serum from the rabbit immunized with M-F9 peptide (AM-F9) inhibited the binding of BCD-F9 to HT-1080 cells by 75%, suggesting that the rabbit anti-mimotope antiserum was directed against the epitope of these tumor cells. In contrast, the serum from the rabbit immunized with the P-{gamma}2 peptide, as well as rabbit pre-immune serum, did not inhibit the binding of the BCD-F9 mAb to HT-1080 cells.



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  		Fig. 4. Inhibition of binding of the BCD-F9 mAb to HT-1080 cells by polyclonal antisera. The binding of BCD-F9 to HT-1080 cells was studied in the presence of the competing polyclonal antisera AM-F9 ({blacksquare}), AP-{gamma}2 (), and pre-immune serum ({square}) as described in "Materials and Methods." The Y axis shows inhibition (percentage) of binding of BCD-F9 to HT-1080 cells as a function of the rabbit polyclonal antiserum dilution (X axis). The data represent the means ± SD from three independent experiments.

 
Effect of AM-F9 on Experimental Lung Metastasis.
Finally, we examined the antitumor activity of AM-F9 in an experimental metastasis model. In this model, HT-1080 cells injected i.v. into CD-1 nude mice induce the formation of metastatic foci in the lung, and 100% of the animals die within 30 days after inoculation. After 50 days of observation, the mean survival time values were calculated for groups of mice injected with AM-F9 and mice treated with pre-immune serum. These values were 47 ± 2 and 25 ± 1, respectively (Fig. 5)Citation . The animals treated with AM-F9 had significantly prolonged life span (T/C = 188%), suggesting that the antitumor activity of AM-F9 was not dependent on the nonspecific action of the rabbit serum. Histological examination of the lungs showed diffuse metastasis in the case of control animals and more localized metastatic foci in the case of animals treated with anti-mimotope antisera. In-life observations showed that mice treated with AM-F9 appeared as healthy as the control mice during the duration of the experiment, suggesting that no severe side effects were caused by AM-F9.



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  		Fig. 5. Survival analysis of HT-1080 tumor-bearing nude mice. HT-1080 cells were inoculated i.v. into CD-1 nude mice on day 0. Rabbit preimmune serum ({circ}) or AM-F9 (•) was administered i.v. on days 1–2, 5–9, 12–16, and 19–21. Each group comprised five mice. Statistical significance (P < 0.05 versus pre-immune serum) was calculated using the Cox-Mantel log-rank test.

 

   DISCUSSION
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
Several authors have demonstrated previously that peptide mimotopes can be obtained from phage epitope libraries (9, 10, 11) or by chemical synthesis (25, 26, 27) and that these sequences bind antibodies raised against native structures. In fact, several studies have shown that peptide mimotopes can be found for most, if not all, anti-carbohydrate mAbs (28, 29, 30) . Peptide epitopes have also been shown to mimic glycosphingolipids and oligonucleotide structures (30, 31, 32) . In the case of mimotope immunization, several investigators showed that synthetic peptides could induce an epitope-specific immune response in vivo (3, 4, 5) . Furthermore, protective immune reactions by mimotope immunizations have been shown for several infectious agents (6, 7, 8) , viral antigens (33) , and pollen allergens (34) . The larger issue of whether such mimotopes can be exploited to elicit functional antibody response against tumor cells has yet to be fully resolved.

In this study, we chose a well-characterized functional mAb, BCD-F9, and used it to identify peptides that mimic the epitope recognized by this antibody. The BCD-F9 mAb is a murine mAb (IgG2a) that recognizes an antigen present on the surface of many tumor cell lines. Because of its high selectivity toward tumor cells as opposed to normal tissues, this mAb could potentially be used for killing tumor cells (35) . Whereas the tumor antigen that is recognized by BCD-F9 has not been isolated, our unpublished data demonstrate that this mAb recognizes a conformational epitope on Mr 57,000 glycoprotein.4

Although we expected to identify several mimotopes, only one sequence was selected from a linear random decapeptide library, whereas a circular library did not generate any specific phages. Despite the great number of independent clones in these libraries (>109), they cover only a small percentage of all theoretically possible decapeptides. Therefore, it is possible that the circular library does not contain peptides mimicking the most optimal structure of the native epitope. It is also possible that there is a bias against particular sequences because of the need to maintain phage infectivity, resulting in the absence of those sequences from the library.

Mutational analysis indicated that specificity is likely to reside in the PGGWW block of the amino acids (Fig. 2)Citation . This block is common to several SH3 domain-containing regulatory proteins involved in signal transduction (36) . The intracellular localization of these signaling proteins suggests that the F9 peptide mimics the native tumor-specific epitope despite the fact that they have different amino acid sequences. Binding studies and immunization experiments confirmed the specificity of the mimotope for the BCD-F9 mAb. In addition, the mimotope was specifically recognized by BCD-F9 without the phage carrier, indicating that the mimotope alone is responsible for the interaction with BCD-F9, without the involvement of structure entities from the phage particle. Another indication for the correct mimicking of the epitope by F9 peptide was obtained by the immunization experiments. We demonstrated that rabbits immunized with peptide-KLH could elicit an IgG response to the natural antigen on tumor cells.

On the other hand, results obtained with the peptide {gamma}2 were surprising, and we cannot reasonably explain their poor binding. However, we cannot exclude the possibility that the amino acid residues flanking the PGGWW block interfere with binding when the peptide is isolated from the phage carrier. A similar situation has been reported previously in which the peptides were able to mimic the natural epitope only when presented on the phage surface (19 , 34) .

In summary, this report describes the isolation of a mimotope corresponding to the epitope of the BCD-F9 mAb. Inhibition studies with either mimotope-displaying phage particles or synthetic peptides proved the specificity of the isolated mimotope. Immunization of rabbits with the mimotope induced polyclonal antibodies capable of blocking the binding of the BCD-F9 mAb to HT-1080 tumor cells. Finally, we assessed the effect of AM-F9 serum on tumor metastasis in an experimental animal model and showed that i.v. treatment with anti-mimotope antisera (15 injections of 100 µl/mouse) significantly prolonged the life span of nude mice (Fig. 5)Citation . However, the beneficial effects of this passive immunotherapy are only achieved if relatively large amounts of antibody are applied, making this treatment very expensive. Thus, it would be an advantage to replace or combine the passive treatment with an active immunization using a mimotope. The success of such an approach has already been demonstrated with the use of mimotopes as experimental oral anti-IgE vaccines (37) .

The properties of AM-F9 were further investigated in a separate study.5 It was found that (a) treatment with AM-F9 significantly inhibited the growth of HT-1080 tumor cells injected s.c. into CD-1 nude mice, (b) AM-F9 elicited antibody-dependent cellular cytotoxicity by splenic natural killer cells and by peritoneal cells, and (c) AM-F9 mediated complementdependent cytotoxicity.

The results of this study suggest that phage epitope libraries may have broad application for the design of anti-cancer vaccines. We believe that this strategy may allow the design of immunogenic peptides without prior knowledge of the target antigen. A new generation of anti-cancer vaccines could consist of a mixture of mimotopes, including peptides that mimic carbohydrates and complex epitopes, selected using tumor-specific mAbs.


   ACKNOWLEDGMENTS
 
We thank Dr. G. P. Smith for providing the fUSE 32P primer, Dr. F. Shareck for the oligonucleotide synthesis, Dr. J. Hu for peptide synthesis, and L. Labrie and L. Forget for DNA sequence analysis.


   FOOTNOTES
 
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

1 M. P. was supported by scholarships from National Sciences and Engineering Research Council of Canada and Fonds par la Formation de Chercheurs et l’aide à la recherche. Back

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{at}nrc.ca Back

3 The abbreviations used are: mAb, monoclonal antibody; KLH, keyhole limpet hemocyanin. Back

4 M. Popkov, S. Sidrac-Ghali, Y. Lusignan, S. Lemieux, and R. Mandeville. Inhibition of tumor growth and metastasis of human fibrosarcoma HT-1080 cells by mAb BCD-F9, submitted for publication. Back

5 M. Popkov, Y. Lusignan, S. Lemieux, and R. Mandeville. Immunotherapy with antimimotope polyclonal antibodies in a nude mouse xenograft model: mechanisms of action, submitted for publication. Back

Received 4/ 4/00; revised 6/21/00; accepted 6/21/00.


   REFERENCES
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

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