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Originally published In Press as doi:10.1074/jbc.M411020200 on October 26, 2004

J. Biol. Chem., Vol. 280, Issue 1, 54-63, January 7, 2005
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Conformational HER-2/neu B-cell Epitope Peptide Vaccine Designed to Incorporate Two Native Disulfide Bonds Enhances Tumor Cell Binding and Antitumor Activities*

Naveen K. Dakappagari{ddagger}§||||, Kenneth D. Lute§, Sharad Rawale{ddagger}, Joan T. Steele¶, Stephanie D. Allen||, Gary Phillips**, R. Todd Reilly{ddagger}{ddagger}, and Pravin T. P. Kaumaya{ddagger}§¶||§§¶¶¶¶

From the Departments of {ddagger}Obstetrics and Gynecology, §Integrated Biomedical Sciences Graduate Program, Chemistry Biology Interface Program, ||Ohio State Biochemistry Program, Columbus, Ohio 43210, **Center for Biostatistics, {ddagger}{ddagger}Department of Oncology, Sidney Kimmel Comprehensive Cancer Center at Johns Hopkins, Johns Hopkins School of Medicine, Baltimore, Maryland 21231, and the §§Molecular Virology, Immunology and Medical Genetics and ¶¶Arthur G. James Comprehensive Cancer Center, The Ohio State University, Columbus, Ohio 43210

Received for publication, September 24, 2004 , and in revised form, October 22, 2004.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
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.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
HER-2/neu or erbB-2 is a 185-kDa transmembrane protein and member of the human epidermal growth factor receptor (EGFR)1 family. HER-2/neu proto-oncogene is expressed by a variety of tissues and progenitor cells, participating in cellular proliferation and differentiation (1, 2). 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 (3, 4). The demonstration of pre-existent HER-2/neu-specific T-cells as well as HER-2/neu-specific antibodies in breast cancer patients (5–8) 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 (9–12). 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 (13–15).

Passive immunotherapies such as the treatment with the humanized anti-HER-2/neu monoclonal antibody Trastuzumab have produced promising results in a clinical setting (16, 17). However, there remain a number of concerns such as repeated treatments and associated costs, limited duration of therapeutic effectiveness, and possibly undesired immunogenicity. Therefore, a therapeutic approach capable of inducing active specific immunity would prove to be highly advantageous, offering sustained protection at a lower cost, preventive therapy, and long term immunity.

The rationale for active immunization stems from observations made both clinically and in animal models, suggesting that specific immunotherapy may be a more desirable way to target HER-2 positive cancers. One of the earliest observations supporting the possibility of active immunotherapy was reported by Tang et al. (18), whose studies indicated that there was a correlation between HER-2 gene amplification and the presence of dense lymphocytic infiltration in breast tumors (18). This observation re-enforced the idea that there may be an endogenous immune response to tumor despite immunological tolerance.

Vaccine strategies based on peptides have targeted either the cytolytic CD8+ T lymphocyte arm of the immune response or the induction of tumor-specific humoral immune response. Several cytolytic CD8+ T lymphocyte epitopes of HER-2/neu have been defined and tested in clinical trials (19, 20). Our laboratory has focused on elucidating the antigenic potential of the ECD of HER-2/neu by mapping several regions of the human protein and testing their in vitro and in vivo biological activities. We reported the anti-tumor properties of a chimeric B-cell epitope sequence 628–647 that incorporates a promiscuous T-cell epitope (MVF) (12, 21). This peptide construct was highly immunogenic in both outbred rabbits and inbred mice, prevented the development of HER-2/neu-overexpressing mammary tumors in 83% of vaccinated rat neu transgenic mice (12), and was tested in an National Institutes of Health NCI sponsored Phase Ib clinical trial at the Ohio State University James Cancer Hospital. We have extended our studies by defining several other important regions of the extracellular domain of HER-2 as potential targets and a multivalent construct is presently being tested in a Phase I trial (OSU 0105).

It is well established from three-dimensional structures of antigen-antibody complexes that antigenic epitopes are conformational and, as such, vaccine design should attempt to incorporate such parameters so that antibodies of high affinity can be elicited. Our original peptide 628–647 was designed to avoid the complications of disulfide pairings. Now that the S-S pairings in human EGFR has been defined (22), and knowing the homology of EGFR to HER-2, we hypothesize that a new construct (626–649) with the known S-S pairing should elicit higher affinity antibodies than our originally designed construct, as the new construct should mimic the native structure better. Engineering peptides to adopt defined structure offers two important advantages. First, a structurally defined peptide is more desirable because it will present a single, stable determinant, unlike synthetic linear peptides that exist in a multitude of different conformations in solution. As such, a more focused response is elicited giving rise to a restricted, perhaps single population of peptide antibodies. Second, conformational peptide epitopes will give rise to antibodies of high affinity, able to cross-react more effectively with the native receptor, thereby killing the tumor or neutralizing the virus.

The pairing of disulfide bonds is an effective means of conformationally stabilizing peptides. There are 50 cysteine residues in the extracellular domain of HER-2 and EGFR, all of which are conserved between the two members (23, 24). A striking similarity is the presence of two cysteine-rich clusters, in which the spatial distribution of cysteine residues is virtually identical between the two receptors. The sequences of cysteine clusters are rather hydrophilic and were hypothesized to form a specific conformation capable of ligand binding and signal transmission (25). All of the 25 disulfide bond connections in the extracellular domain of EGFR have been experimentally determined (22). Although there are a total of 50 cysteine residues across the HER-2 extracellular domain, only a few cysteines present in the membrane proximal region were found to spontaneously mutate both in rat neu transgenic mice and a small percentage of human tumors (26, 27). These mutations were later shown to cause spontaneous dimerization of the receptor and when the mutant receptors were expressed as transgenes in mice, it lead to accelerated tumor formation and early death (24). These and other studies (28, 29) appear to indicate that the cysteines in the membrane proximal region appear to have special significance in the biology of the receptor. Therefore, we have focused our studies on a peptide sequence (amino acids 626–649) from this membrane proximal region. Therefore, it is possible to conformationally constrain the HER-2 B-cell epitope peptides by introducing the natural disulfide bonds. The introduced disulfide bonds may stabilize the secondary structure in the peptides and enable them to more closely mimic the native receptor. Antibodies generated against such conformationally constrained peptides would most likely have greater affinity for native HER-2.

Based upon the known EGFR disulfide bond structure (22) and its homology to HER-2, we redesigned HER-2 MVF-(628–647) by extending it two amino acids at each terminus. This newly designed sequence, designated HER-2-MVF626–649SS, contains four cysteine residues at 626, 630, 634, and 642 with the disulfide bonds between residues 626 and 634, and residues 630 and 642. To determine whether the cyclized epitope was able to mimic the native HER-2 better than the non-cyclized epitope MVF626–649NC, we studied the immunogenicity of both epitopes in outbred ICR mice. We evaluated the cross-reactivity of the two peptide antibodies by flow cytometry using human HER-2 overexpressing breast cancer cells, BT474. We assessed their direct and indirect antitumor activities by testing their antiproliferative capacity, as well as their ability to mediate ADCC and induce IFN-{gamma} production, in vitro. Last, we determined the immunogenicity and immunoprotective effects of each vaccine in a mouse tumor burden model using an inbred mouse strain (FVB/n).

We report here the successful design, synthesis, and characterization of a highly immunogenic conformationally defined HER-2 B-cell epitope, MVF-(626–649). The doubly disulfidepaired construct elicited high affinity antipeptide antibodies for native HER-2 compared with the corresponding uncyclized linear epitope. Additionally the antibodies elicited by the conformational epitope mediated higher levels of IFN-{gamma} production by human PBMCs and demonstrated enhanced antibody-dependent cell lysis in vitro. Furthermore, a statistically significant reduction in tumor growth was observed in mice vaccinated with the disulfide-paired epitope relative to mice receiving either the linear construct or MVF control peptide (p = 0.001).


   EXPERIMENTAL PROCEDURES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
RESULTS
 DISCUSSION
 REFERENCES
 
Synthesis and Characterization of Conformational and Linear Peptides—HER-2 B-cell epitope 626–649 was synthesized co-linearly with a promiscuous TH epitope derived from the measles virus fusion protein (amino acids 288–302). Peptide synthesis was performed on a Milligen/Biosearch 9600 peptide solid-phase synthesizer (Bedford, MA) using Fmoc/t-But chemistry. Preloaded Fmoc-Ser-CLEAR ACID resin (0.36 mmol/g) (Peptides International, Louisville, KY) was used for the synthesis. The B-cell epitope (HER-2-(626–649)) was assembled by choosing regioselective side chain protection on Cys residues as: Cys(Acm) on 630 and 642, Cys(Trt) on 626 and 634. Also, MVF TH epitope with four reside linker (GPSL) was incorporated for independent folding and was assembled on the B-cell epitope. Both peptides were cleaved from the resin using the cleavage reagent B (trifluoroacetic acid:phenol:water: TIS, 90:4:4:2) and crude peptides were purified as reported earlier (30). Intamolecular disulfide bonds were formed using either iodine oxidation as described by Soll et al. (31) or platinum oxidation (32). Linear peptide was generated by dithiothreitol reduction. Cyclic and linear peptides were further purified by semi-preparative RP-HPLC and characterized by electrospray ionization mass spectroscopy (ESI-MS) (Campus Chemical Instrumentation Center, The Ohio State University, Columbus, OH). Disulfide bridge formation was further confirmed by Maleimide-PEO2-biotin reaction with cyclic and linear peptide and subsequent analysis using ESI-MS.

Circular Dichroism (CD) Measurements—Aqueous solutions for CD were prepared by dissolving the freeze-dried peptide in the appropriate amount of water to give a final concentration of 0.5 mM and used as stock solution for further dilution. CD spectra were recorded on an AVIV model 62A DS CD instrument (Lakewood, NJ) as reported earlier (30). Mean residue ellipticity ([{theta}]M,{lambda}) values were calculated according to the equation, [{theta}]M,{lambda} = ({theta} x 100 x Mr)/(n x c x l). Where {theta} is the recorded ellipticity (deg), Mr the molecular weight of the peptide, n the number of residues in the peptide, c the peptide concentration (mg/ml), and l the path length of the cuvette.

Peptide Immunization and Antibody Purification—All animals were purchased from Harlan (Indianapolis, IN). Female ICR (outbred) and FVB/n (inbred) mice, 6–8 weeks old, were immunized with either disulfide-paired (MVF626–649SS) or unpaired (MVF626–649NC) chimeric peptides dissolved in H2O with 100 µg of a muramyl dipeptide adjuvant, nor-MDP (N-acetylglucosamine-3 yl-acetyl- L-alanyl-D-isoglutamine). Peptides were emulsified (50:50) in Montanide ISA 720 vehicle. The same dose of booster injections was administered twice at 3 and 6 weeks. Sera were collected by retro-orbital bleeding at 1 and 3 weeks after each immunization for determination of antibody titers. High-titered sera were purified on a protein A/G-agarose column (Pierce, Rockford, IL) and eluted antibodies were concentrated and exchanged in phosphate-buffered saline using 100-kDa cut-off centrifuge filter units (Millipore, Bedford, MA). The concentration of antibodies was determined by Coomassie plus protein assay reagent kit (Pierce).

ELISA—Antibody titers were determined as previously described (12). Ab titers were defined as the reciprocal of the highest serum dilution with an absorbance of 0.2 or greater after subtracting the background.

Mouse Isotyping—Mouse sera were isotyped using Mouse Typer Sub-Isotyping Kit (Bio-Rad). The assay was performed according to the manufacturer's instructions, except that a 1/500 dilution of goat anti-rabbit IgG horseradish peroxidase conjugate was used.

Cell Lines and Abs—All cell culture media, fetal calf serum (FCS), and supplements were purchased from Invitrogen. The human breast tumor cell lines, BT-474 (HER-2high; ~2 x 106 molecules/cell) and MDA-468 (HER-2low; 10,000–50,000 molecules/cell) were purchased from American Type Culture Collection (Manassas, VA) and maintained according to the supplier's guidelines. HER-2 mAb Ab-2 (clone 9G6) was purchased from Neomarkers (Fremont, CA). Humanized mouse mAb HerceptinTM was generously provided by Genentech (San Francisco, CA).

Flow Cytometry—5 x 105 BT-474 or MDA-468 cells were titrated with 2, 10, 20, and 50 µg of purified peptide vaccine-induced ICR mouse Abs to establish saturation. Normal mouse Ig (negative control) and HER-2-specific mouse monoclonal antibody Ab-2 (positive control) were used as controls. The cells were incubated in 100 µl of phosphate-buffered saline, 2% FCS for 1 h at 4 °C, washed once in phosphate-buffered saline and then incubated with fluoroscein isothiocyanate-labeled secondary antibody (1:50 dilution) for 30 min at 4 °C in 100 µl of phosphate-buffered saline, 1% FCS. Cells were washed, fixed in 1% formaldehyde, and analyzed by Coulter ELITE flow cytometer (Coulter, Hialeah, FL). A total of 10,000 cells were counted for each sample and final processing was performed. Debris, cell clusters, and dead cells were gated out by light scattered assessment before single parameter histograms were drawn and smoothened.

Anchorage-independent Growth Assay—Into each of four 60-mm culture dishes, 1 x 103 BT-474 cells were suspended in 1 ml of Dulbecco's modified Eagle's/F-12 culture media containing 0.18% agarose. The culture media was supplemented with 10% FCS, 2 mM L-glutamine and contained 1% penicillin-streptomycin. This suspension was overlaid onto a 6-ml feeder layer consisting of 0.24% agarose in conditioned Dulbecco's modified Eagle's/F-12 media (as described above). The cultures received purified peptide vaccine-induced ICR mouse Abs or Herceptin at 10 µg/ml, or were left untreated, and then were incubated at 37 °C. Cultures were fed weekly with 1 ml of conditioned media containing the same dose of antibody. After 21 days the cultures were stained by treating them with 1 ml of Hanks' balanced salt solution containing 1 mg/ml p-iodonitrotetrazolium violet. The next day colonies were counted under low field magnification. The colonies were counted in six random fields for each treatment and averaged to give the final colony count (±S.E.).

IFN-{gamma} Release Assay—96-Well plates were layered with 50,000 BT-474 or MDA-468 human breast cancer cells. After 24 h the tumor cells were coated with normal mouse IgG or purified peptide vaccine-induced ICR mouse Abs at 10 µg/ml and incubated for 2 h. Wells were washed of any unbound antibody and 2 x 105 purified human PBMCs (American Red Cross, Columbus, OH) were added to each well. After 3 days, cell-free supernatants were harvested and IFN-{gamma} levels determined by a sandwich ELISA according to the manufacturer's instructions (BD Biosciences). Results are reported in picograms/ml and represent the mean ± S.E. of duplicate samples.

Antibody-dependent Cell-mediated Cytotoxicity Assay (ADCC)—PBMCs from normal human donors obtained by density gradient centrifugation in Ficoll-Hypaque (Amersham Biosciences) were washed twice in RPMI 1640, 5% FCS and then serially diluted in 96-well plates to give effector to target ratios of 100:1, 20:1, and 4:1. Effector cells were incubated in the presence of interleukin-2 at a concentration of 15 ng/ml. The following day target cells received 2 µg/well of Protein A/G purified peptide vaccine-induced ICR mouse Abs or the humanized mouse monoclonal Herceptin. BT-474 and MDA-468 target cells (HER-2high and HER-2low, respectively) were labeled with 100 µCi/1 x 106 cells of Na51CrO4 (PerkinElmer Life Sciences) and incubated for 1 h at 37 °C. After three washings 5 x 103 target cells were delivered to each well to give a final volume of 0.2 ml/well. The cells were incubated for 4 h at 37 °C, after which time 75 µl of cell-free supernatants were harvested and radioactivity determined using a {gamma}-counter. To assess nonspecific lysis effector, target cells were co-incubated in the presence of normal mouse antibodies. Cytotoxicity was calculated by the formula: (%) lysis = (A-B)/(C-B) x 100, where A represents 51Cr (cpm) from test supernatants, B represents 51Cr (cpm) from target alone in culture (spontaneous release), and C represents maximum 51Cr release from target cells lysed with 5% Triton X-100. Results represent the average (±S.E.) of triplicate samples and have normal mouse Abs subtracted.

Tumor Challenge and Evaluation of Tumor Development—The neu-expressing mouse mammary carcinoma cell line NT2.5, derived from a spontaneous mammary tumor that developed in an FVB/n rat neu transgenic mouse (FVB/n202), was used for tumor challenge. These cells were maintained in Dulbecco's modified Eagle's medium Nutrient Mixture F-12 supplemented with 10% FCS and 0.01 mg/ml insulin. Groups of FVB/n mice (n = 7–10) were challenged with 5 x 106 NT2.5 cells subcutaneously (lower abdomen) 10 days after final immunization. Mice were monitored twice weekly for the presence of palpable tumors. Tumors were measured with calipers in a blinded fashion and tumor volume was calculated by the formula (long measurement x short measurement2)/2.

Statistical Analysis—Tumor growth over time was analyzed using the Stata's® XTGEE (cross-sectional generalized estimating equations) model that fits general linear models that allow you to specify within animal correlation structure. This model is used to account for the correlation in the response variable when you have repeated measures over time on the same subject (mouse). This approach works well when the data are fairly balanced (a relatively small number of missing values) and are measured at a common set of times on many experimental units. The model includes terms for both treatment group and time. Treatment by time interaction is used to calculate the differences in the slopes of each group. This difference is tested to see if it is statistically significant. A test for the difference in the intercepts was not done because biologically these should all be zero at the beginning of the treatment program. The statistical model used in the XTGEE analysis is shown as: Y = {beta}o + {beta}1; treatment + {beta}2; day + {beta}3. (treatment · day) + {epsilon}0, where Y = log transformed tumor volumes, {beta}i values = regression coefficients; {epsilon}0 = error term, which is normally distributed with mean equal to zero and variance {sigma}2. The model assumes that the data are normally distributed and that volume is a continuous linear variable. Log transformation of the volume addresses both of these issues. The slopes by treatment of the log transformed tumor volumes were calculated and compared to determine whether there was a statistically significant difference between treatments. A rough Bonferroni correction is used to keep the overall significance level at 5%. However, Bonferroni's method gives results that are too conservative for this many multiple comparisons and thus an {alpha} = 0.01 was used to determine whether individual differences are statistically significant (i.e. p value ≤ {alpha}) between control groups compared with vaccine-treated groups of animals. The results of the above regression are transformed back into their original units. The exponential slope is used to calculate doubling time along with their 95% confidence intervals. Doubling time is the number of days required for the size of the tumor to double. The general form of the exponential equation used to calculate doubling time is shown as, volume (mm3) = Vo exp(m·days), where Vo = initial volume and m = exponential slope. The model fits a y intercept greater than 0 even though the biological intercept is 0 at the beginning of the study. The initial volumes generated by the model were not used to compare the treatment effect. Also no attempt was made to force the model through the origin.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
DISCUSSION
 REFERENCES
 
Selection, Design, and Characterization of Conformational and Linear Chimeric Peptides—Sequence 628–647, which we have studied extensively (12, 21), has been redesigned based on the disulfide bond pairings identified in Abe et al. (22). With this information, we have extended the sequence (626–649) by two residues at both the N and C terminus, such that disulfide pairings between Cys-626 and Cys-634, and Cys-630 and Cys-642 could be selectively carried out. Selective side chain protection was critical to achieve the correct disulfide pairing. Thus side chain protection for residues 626 and 634 was trityl, which would be conveniently removed upon cleavage of the peptide. The side chain of the cysteine residues at 630 and 642 were protected by ACM, which can be selectively removed and cyclized by oxidation (I2 or Pt) after the first cyclization. Intramolecular disulfide bridges constrain the conformation in peptides and lead to stabilization of the secondary structure. Although many different oxidation methods such as air oxidation, oxidized glutathione (GSSG), dimethyl sulfoxide (Me2SO), iodine, ethoxycarbonylsulfenyl chloride (SceCl), potassium ferricyanide (K3Fe(CN)6), and trans-dichlorotetracynoplatinate(IV) ([Pt-(CN)4Cl2]2) have been used for this purpose, thiol oxidation still remains a significant challenge (31). We explored the use of two different cyclization protocols (I2 or platinum oxidation) and each method was evaluated on the basis of yield, purity, cost, and overall utility. We initially performed the oxidation reaction using [Pt(CN)4Cl2]2-. In the typical oxidation reaction the partially protected peptide and 75 eq [Pt(CN)4Cl2]2- was dissolved in phosphate buffer at pH 5.8 and stirred. The reaction was monitored by reverse-phase HPLC. Even though [Pt(CN)4Cl2]2- reagent was reported as an efficient oxidant for the rapid and quantitative formation of intramolecular peptide disulfide bonds, low yields and side products resulted as indicated by mass spectroscopy analysis. Iodine oxidation in our hands gave a product that was higher in purity and yield, thus the method of choice in these studies. Dithiothreitol reduction was utilized to generate the corresponding linear construct (MVF626–649NC).

Selective disulfide formation was performed at a high dilution. The peptide was cleaved from the resin using reagent B (trifluoroacetic acid:phenol:water:TIS, 90:4:4:2). The S-Acm groups remained bound to the peptide, whereas all other protecting groups were cleaved (Fig. 1). Both peptides were purified by preparative RP-HPLC and characterized by ESI-MS. Iodine oxidation was carried out in acetic acid and the first disulfide bond was formed in 1 h. S-Acm removal was accelerated by addition of 20% water. The mass spectra of crude product showed the complete removal of the ACM groups. The lyophilized peptide was purified by semi-preparative RP-HPLC and fractions were pooled together, lyophilized, and characterized by ESI-MS. The completion of the oxidation reaction was confirmed by maleimide-PEO2-Biotin reaction in which a significant mass shift would be obtained and that could be analyzed by mass spectroscopy. PEO-maleimide reacts with free thiol to form addition product. Fully oxidized peptide or linear peptide MVF HER-2-(626–649) and maleimide-PEO2-Biotin (50 eq) in water was stirred for 12 h at room temperature and analyzed by ESI-MS. The linear peptide MVF HER-2-(626–649) has four thiol groups and shows addition of four PEO groups (4 x 526.62), whereas fully oxidized peptide remains unchanged (Fig. 1).



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  		FIG. 1.
Chimeric MVF-(626–649) epitope, sequence, and strategy for selective oxidation. Regioselective cysteine protection of Acm was utilized to ensure desired intramolecular disulfide bond formation. Mass spectroscopic analysis of the cyclization reaction was utilized to demonstrate complete oxidation. Epitopes were treated with PEO-maleimide. This biotinylation agent (Mr = 525.62) selectively attacks free sulfhydryl groups and can therefore be used to determine the completion of disulfide pairing.

 
Secondary Structural Characteristics of Cyclized and Non-cyclized Peptide Constructs—Circular dichroism (CD) has been used for analysis of secondary structural elements in peptides constrained with disulfide bonds (33–35). Positive and negative features near 200 nm are representative of {beta}-sheet and extended coil conformations, respectively (35). CD spectra of epitopes MVF626–649NC and MVF626–649SS in water show significant differences at 100 µM concentration (Fig. 2). Peptide MVF626–649NC at 100 µM concentration in water shows CD ellipticity minima at 209 nm (molar ellipticity -7117.72), whereas peptide MVF626–649SS at 100 µM concentration in water shows CD ellipticity minima at 202 nm (molar ellipticity -11453.62). Epitope MVF626–649SS is constrained by two intramolecular disulfide bonds (626–634 and 630–642) and this would give rise to different hydrodynamic and spectroscopic properties, whereas linear epitope MVF626–649NC is largely unstructured. Therefore, it is more likely the antibodies generated by the disulfide-paired peptide would cross-react better with the native receptor and lead to more potent antitumor activities.



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  		FIG. 2.
Secondary structural characteristics of chimeric MVF-(626–649) epitopes. CD spectroscopy measurements were performed using 100 µM solution of MVF626–649SS or MVF626–649NC in water. Peptide MVF626–649NC shows CD ellipticity minima at 209 nm, whereas peptide MVF626–649SS shows CD ellipticity minima at 202 nm, demonstrating significant differences in secondary structure.

 
Immunogenicity of Cyclized and Non-cyclized Peptide Constructs in ICR Mice—Groups of ICR mice (n = 5) were immunized with either linear, disulfide-paired or MVF control peptide. As shown in Fig. 3 both the linear and the disulfide-paired peptide elicited high-titered antibodies. Titers >200,000 3 weeks after the third immunization were elicited from three of the cyclized and two of the non-cyclized mice. MVF-specific antibodies were not detectable in MVF-immunized control ICR mice.



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  		FIG. 3.
Antibody responses to peptide constructs in ICR mice. Direct ELISAs were performed to determine the immunogenicity of the cyclized and linear constructs in ICR mice (outbred). Each treatment group consisted of 5 mice and the results of individual mice are shown. Ab titers were defined as the reciprocal of the highest serum dilution with an absorbance of 0.2 or greater after subtracting the background. 1y+3w indicates the titer of blood drawn 3 weeks after the first immunization. Preimmune and Th cell epitope (MVF) sera were used as negative controls (data not shown).

 
Cyclized Construct Enhances Cross-reactivity of Antibodies to Native HER-2—To determine whether antibodies elicited by the linear and disulfide-paired peptides show differences in their ability to recognize the HER-2 receptor, we tested the binding of ICR purified antibodies to HER-2 overexpressing human breast cancer cell line BT474. Fig. 4 shows that both the cyclized and noncyclized peptide antibodies were able to bind the native receptor, however, antibodies from the sera of mice immunized with the conformationally constrained peptide (MVF626–649SS) demonstrated improved binding as compared with the linear peptide (MVF626–649NC). The cyclized antibodies displayed more than 5-fold greater binding when compared with non-cyclized antibodies (Fig. 4, mean log fluorescence 2.29 and 1.56, respectively). The cyclized and the non-cyclized antibodies did not bind HER-2 negative breast cancer cell line MDA-468 (data not shown).



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  		FIG. 4.
Peptide-specific antibodies recognize human breast cancer cells overexpressing HER-2. Flow cytometry was used to assess whether antibodies from ICR mice induced by the linear and disulfide-paired peptides exhibit differences in their ability to recognize native HER-2. BT-474 human breast cancer cells (HER-2high) were treated with 10 µg/ml normal mouse Ig (negative control), mouse monoclonal Ab-2 (positive control), or peptide antibodies raised in ICR mice. The bold line histogram indicates unstained cells; dotted line histogram indicates normal mouse Ig (negative); and shaded histograms indicate MVF626NC (black), MVF626SS (light gray), and Ab-2 (dark gray).

 
Both Cyclized and Non-cyclized Peptide Antibodies Exhibit Antiproliferative Effects in Vitro—A direct mechanism by which anti-HER-2 antibodies can exert their anti-tumor effects is by interfering and/or inhibiting growth. Because we showed that the antibodies elicited to the conformational epitope demonstrated superior tumor cell binding, we were interested in finding whether improved antibody binding would equate into enhanced antiproliferative effects. The anchorage independent growth assay is a more reliable measure of the ability of cells to grow in vivo. In this assay (Fig. 5) the Abs of ICR mice immunized with the linear epitope (MVF626–649NC) showed significantly higher growth inhibition relative to what was observed for animals treated with the conformational epitope (MVF626–649SS) (58 and 31% inhibition, respectively). We also studied anchorage-dependent growth inhibition by the bioreduction of 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide as an indicator of proliferation. The antibodies elicited by both cyclized and non-cyclized peptide constructs showed similar inhibitory profiles, giving around 20% inhibition (data not shown).



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  		FIG. 5.
Peptide antibodies demonstrate antiproliferative effects on HER-2 positive human breast cancer cells in vitro. The anchorage-independent growth assay demonstrates the effects of peptide antibodies from ICR mice on BT-474 (HER-2high) and MDA-468 (HER-2low) cell growth. 1 x 103 cells received 10 µg/ml Herceptin, peptide antibodies, or no treatment for 3 weekly treatments. Cells were stained with p-iodonitrotetrazolium violet and colonies were counted under low field magnification. The colonies were counted in six random fields for each treatment and averaged to give the final colony count (±S.E.).

 
Cyclized Peptide Antibodies Mediate Improved Antitumor Activity—IFN-{gamma} is a potent antitumor cytokine known to significantly retard the growth of tumors (36–38) and is an indirect mechanism by which some antibodies could further exert their influence on tumor growth. We measured the ability of peptide antibodies to activate the release of IFN-{gamma} by coating HER-2 positive and negative tumor cells with peptide antibodies from ICR mice and culturing them in the presence of human PBMCs. The cells were then incubated for 72 h after which time cell-free supernatants were harvested and IFN-{gamma} levels determined by sandwich ELISA. As seen in Fig. 6, both cyclized and non-cyclized peptide antibodies cause HER-2 overexpressing cells to produce at least seven times the amount of IFN-{gamma} compared with cells incubated with preimmune antibodies. The cyclized peptide antibodies (MVF626–649SS) induced ~25% higher levels of IFN-{gamma} compared with linear peptide antibodies (MVF626–649NC). The levels of IFN-{gamma} observed in HER-2 negative cells was similar to that observed for preimmune-treated cells (negative control).



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  		FIG. 6.
Peptide Ab-coated tumor cells stimulate IFN-{gamma} release in human PBMCs. BT-474 (HER-2high) and MDA-468 (HER-2low) were incubated with peptide antibodies from ICR mice or preimmune IgG and cultured with 2 x 105 human PBMCs for 3 days. Cell-free supernatants were harvested and the amount of IFN-{gamma} was determined by ELISA. Results are reported in pg/ml and represent the mean (±S.E.) of duplicate samples.

 
Cyclized Peptide Antibodies Exhibit Enhanced ADCC Specific Lysis—Because antibodies can kill tumor cells by engaging mononuclear cells in the blood, we also measured the ability of antipeptide antibodies to mediate ADCC. We first measured the isotypes of antibodies elicited from the two constructs in mice and found that IgG1 and IgG2 were the major isoforms. Antibodies formed from the MVF626–649SS construct contained 57% IgG1 and 42% IgG2 (both IgG2a and IgG2b), whereas the MVF626–649NC elicited antibodies that had 61% IgG1 and 38% IgG2 (data not shown). These two isotypes are known to mediate ADCC using human PBMCs as effectors (39, 40). As indicated in Fig. 7 cyclized peptide antibodies demonstrated twice the level of specific lysis as the non-cyclized antibodies (11 and 5.6%, respectively) at an effector to target ratio of 100 to 1 with the human breast cancer cell line BT-474. This relationship was observed at every effector to target ratio. Herceptin was used as a positive control.



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  		FIG. 7.
Conformational peptide antibodies demonstrate improved ADCC. Target cell line BT-474 (HER-2high) was coated with peptide antibodies from ICR mice, normal mouse IgG, or Herceptin and cultured with human PBMC effector cells in the presence of interleukin-2 to give an effector:target (E:T) ratio of 100:1, 20:1, and 4:1. The results represent the mean (±S.E.) of triplicate samples after subtracting nonspecific normal mouse IgG lysis (see "Experimental Procedures" for calculation).

 
Cyclized Peptide Antibodies Are Highly Immunogenic, Immunoprotective, and Reduce Tumor Burden in FVB/n Mice—To better understand the potential clinical benefit associated with introducing conformational constraints into B-cell epitope vaccines, we initially studied the immunogenicity of both cyclized and non-cyclized constructs in FVB/n mice that could then be challenged with the cell line NT2.5, derived from a spontaneous mammary tumor isolated from FVB/n202 transgenic mice (originally described by Guy et al. (41)). As a consequence of neu overexpression these mice develop spontaneous mammary adenocarcinomas in a manner similar to that observed in human breast cancer patients (9), and are therefore a suitable model for human breast cancer studies.

Fig. 8A shows four of five FVB/n mice receiving the disulfidepaired construct had titers greater than 100,000, whereas only one mouse that received the non-cyclized construct had a titer greater than 100,000. The inbred mouse strain generated higher titers to the cyclized construct compared with the linear construct. MVF-specific antibodies were not detectable in MVF-immunized control FVB/n mice.



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  		FIG. 8.
Improved immunogenicity and immunoprotective effects of vaccination with a conformational epitope in FVB/n mice. A, direct ELISAs were performed to determine the immunogenicity of the cyclized and linear constructs in FVB/n mice (inbred). Each treatment group consisted of 5 mice and the results of individual mice are shown. Ab titers were defined as the reciprocal of the highest serum dilution with an absorbance of 0.2 or greater after subtracting the background. 1y+3w indicates the titer of blood drawn 3 weeks after the first immunization. Preimmune and Th cell epitope (MVF) sera were used as negative controls (data not shown). B, groups of FVB/n mice were challenged with 5 x 106 NT2.5 cells subcutaneously (lower abdomen) 2 weeks after the final immunization. Tumor volumes were calculated by the formula (long measurement x short measurement2)/2. Tumor measurements were taken twice weekly until day 33. Results are reported as mm3 and represent the mean (±S.E.) of groups (n = 7–10) of mice.

 
To determine whether there was an immunoprotective effect conferred upon animals immunized with the conformationally restrained B-cell epitope, we vaccinated groups of FVB/n mice with each of the following constructs: HER-2 MVF626–649SS (conformationally restrained), HER-2 MVF626–649NC (conformationally unrestricted), or MVF control peptide. The mean tumor volumes over time for each of the three treatment groups are shown in Fig. 8B. There is a statistical difference in mean tumor volume ({alpha} = 0.10) at day 33 comparing MVF626–649SS to MVF immunized mice and MVF626–649SS to MVF626–649NC immunized mice (p = 0.024 and p = 0.033, respectively).

Over time (from day 1 to day 33) the tumor growth shows an exponential trend in the growth rate so a logarithmic transformation was performed on the data and a normal probability plot generated. Two of the three comparisons were statistically significant at {alpha} = 0.01. Mice immunized with the conformationally restrained epitope, MVF626–649SS, demonstrated a statistically significant reduction in growth rate compared with mice receiving MVF626–649NC or MVF control peptide (p = 0.001). The results of the above regression were transformed back into their original units and used to calculate doubling time (Table I). Doubling time is defined as the number of days required for the size of the tumor to double.


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  		TABLE I
Effect of peptide vaccination on the doubling time of tumors Groups of FVB/n mice were immunized with MVF control peptide, MVF626NC, or MVF626SS. The mice were challenged 10 days after final immunization with 5 x 106 NT2.5 cells. Doubling time is defined as the number of days required for the size of the tumor to double. The 95% confidence interval for the estimated doubling time is interpreted as saying that if this experiment were repeated 100 times, it is expected that out of the 100 confidence intervals calculated, 95 would contain the true doubling time. See "Experimental Procedures" for calculations of the doubling time.

 
As seen in Table I, MVF626–649SS vaccinated mice (constrained epitope) had the longest doubling time (6.63 days), thereby demonstrating the greatest ability to delay tumor growth. The rates of growth observed in mice immunized with either MVF626–649NC or MVF control peptide were similar (4.31 and 4.48 days, respectively), and were not found to be statistically different.


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
REFERENCES
 
Although there have been a substantial number of cancer studies describing the use of peptide vaccines, the majority of these strategies focus on the use of putative T-cell epitopes derived from tumor antigens. However, the development of a highly efficacious cancer vaccine will require not only the use of T-cell vaccines, but also highly engineered molecularly defined B-cell epitopes. As such the aim of our studies has centered on the identification and biological evaluation of B-cell epitopes of the HER-2 oncoprotein. In an effort to improve antibody affinity and cross-reactivity to human HER-2/neu, we are exploring the utility of engineering structurally constrained B-cell epitopes. Previously the design of structurally defined epitopes was largely dependent upon information obtained from computer-aided analysis and molecular modeling approaches. However, with the crystal structure of HER-2 having been solved (42, 43), we are now poised to exploit these developments. However, in the present study, we have relied on the knowledge of disulfide pairings from Abe et al., (22), because the published crystal structure has omitted the membrane proximal region (amino acids 620–656).

Various studies have illustrated the benefits associated with introducing conformational constraints. The rational design of peptides that incorporate certain secondary and tertiary structural characteristics has had important implications in vaccine development (30, 44, 45). Recently, Calvo et al. (46), investigated the use of conformational epitopes from the human papillomavirus type 16-E7 oncoprotein as a diagnostic tool for identifying the presence of HPV16 in cervical cancer patients. They synthesized a series of conformational peptides as well as their linear counterparts and demonstrated that sera isolated from patients with invasive cervical carcinoma showed strong reactivity to an {alpha}-helical peptide. Similarly, Cabezas et al. (47), demonstrated that antibodies directed against a cyclized peptide derived from the V5 loop of HIV glycoprotein gp120, but not its linear counterpart, were able to bind recombinant gp120. Moreover only the cyclized peptide was able to elicit virus-neutralizing antibodies in vaccinated rabbits.

More recently, Riemer et al. (50) reported the utility of the cysteine-constrained peptide library for the identification of mimotopes capable of reacting with Herceptin. Although, the mimotopes bear no direct homology to HER-2, they have been shown to mimic the conformation of B-cell epitopes recognized by Herceptin. Immunization of Balb/c mice with one of their selected mimotopes conjugated to tetanus toxoid resulted in antibodies recognizing Her-2/neu as well as causing internalization of the receptor in vitro. However, the antitumor activity and efficacy of these mimotopes has not been evaluated in an active immunization setting. These studies illustrate the potential clinical efficacy of establishing structural compatibility between peptide antibodies and native protein. Therefore, it would be interesting to compare the anti-tumor activities of Herceptin reactive mimotopes with our disulfide bond constrained peptide as both antibodies target the therapeutically relevant membrane proximal region but in a non-overlapping manner. Our 626–649 construct is downstream of the herceptin binding domain and was clipped off in the published three-dimensional structure. Of more relevance to the mimotope publication is our ongoing work based on the published structure of Her-2/neu with Herceptin Fab (42) in designing, testing, and evaluating an engineered, conformationally relevant triple disulfide-bonded epitope of Her-2/neu encompassing sequence 563–616 that mediates the interaction with Herceptin.

The presence of HER-2 in many cancers, its low levels of expression in normal tissues of adults (48), and its surface-exposed extracellular domain makes it an attractive target for immunotherapy, as well as a useful tool for the discovery of novel molecularly defined epitopes. The extracellular domain contains 50 cysteine residues, and two cysteine-rich clusters that have been hypothesized to play a role in both ligand binding and signal transduction (25). Moreover the disulfide bond structure has been determined, and the 25 disulfide pairings mapped (22). This creates the possibility of identifying and synthesizing putative B-cell epitopes for vaccine use. We report here the identification of a highly immunogenic conformationally constrained B-cell epitope, MVF626–649SS.

To systematically assess the biological effects of cyclization we also synthesized its linear counterpart, MVF626–649NC, and evaluated the two epitopes in parallel fashion. An important requirement for vaccines is that they should be capable of demonstrating protection in an outbred population. We therefore evaluated the immunogenicity of the peptides initially in outbred animals, ICR mice, and eventually also in inbred FVB/n mice as a challenge model. Both epitopes proved to be highly immunogenic as demonstrated by antibody titers.

An important factor in developing peptide vaccines is that the antibodies generated from a synthetic peptide vaccine recognize the native protein. Antibodies to the cyclized peptide bound to the native protein better than the linear peptide as established by flow cytometry. Although the cyclized epitope led to an antibody population that showed improved binding to HER-2, this did not translate into a more potent anti-proliferative effect on breast cancer cells overexpressing HER-2. Knowledge gained from the crystal structure of the HER-2 extracellular domain (42) in complex with Herceptin antibody indicates that the conformational state of the membrane proximal region of HER-2 is very critical for receptor dimerization and signaling. Although, the antibodies induced by linear peptide bind the tumor cells less better than the disulfide-linked peptide, it is likely that the linear peptide is producing more of the antibodies, which are capable of binding and interfering better with an antigen conformation required for receptor signaling. Based on our previous findings (12, 20) as well as in the present work, we believe antibodies elicited by the linear peptide are superior at mediating direct growth inhibition, whereas the antibodies produced by the disulfide-linked peptide are better at mediating the indirect anti-tumor effects such as ADCC and IFN-{gamma} release by human PBMC. The potent indirect effects combined with more modest direct effects appear to be very effective in the treatment of established tumors, a situation more relevant to the treatment of human cancer patients with pre-existing tumor burden. Antibodies against HER-2 can have varied effects on tumor growth, some having no effect, others promoting or inhibiting growth (49). The cyclized epitope caused a 25% increase in the release of IFN-{gamma} compared with the linear epitope. Antibodies generated against the cyclized epitope showed an increase in cytotoxicity against HER-2 overexpressing cells compared with the linear construct as measured by ADCC. These findings demonstrate that the constrained epitope is better able to kill tumor cells by mediating ADCC and increasing the production of IFN-{gamma}, an antitumor cytokine that appreciably impedes the growth of tumors.

An in vivo tumor model necessitated the use of FVB/n mice for immunogenicity and tumor challenge studies. The mice generated higher titers to the cyclized construct compared with the linear construct. It is interesting to note that the outbred population of mice generated similar levels of titers for both the cyclized and linear construct, whereas the inbred strain generated higher titers for the cyclized construct. The difference in the antibody response may be because of differences in the B-cell repertoire between the inbred mice and outbred mice. In addition, vaccinated mice were challenged with a tumor cell line derived from a spontaneous mammary tumor that developed in an FVB/n rat neu transgenic mouse (FVB/n202). These tumors from transgenic mice have a conspicuous similarity to tumors seen in patients with breast cancer (9). Mice that were immunized with the cyclized epitope showed a reduction in tumor volume after 33 days compared with mice immunized with the linear or control constructs and demonstrated a statistically significant reduction in growth rate compared with mice receiving MVF626–649NC or MVF control peptide (p = 0.001). The distinction between preventing neu-mediated oncogenic signals in nascent tumors, as we have shown before (12), and the antibody-mediated elimination of a pre-existing tumor burden (our current model) is important. One would expect that the inhibition of neu signaling is key in the spontaneous tumor prevention model, but the established tumors may have evolved to be less dependent on neu signal transduction to maintain the transformed phenotype. In the latter case immune-mediated mechanisms for tumor killing become more important (i.e. ADCC, cytolytic CD8+ T lymphocyte, etc). These data argue that combining our vaccine with a more T-cell-directed vaccine may be more efficacious.

We conclude that constraining the 626–649 epitope via disulfide bonds produced antibodies with better anti-tumor activity than the linear epitope. With the crystal structure of the extracellular domain of HER-2 bound to Herceptin available (42), we are currently investigating the anti-tumor properties of other structurally engineered peptide epitopes spanning residues 563–616 delineated from the structure of the HER-2-Herceptin complex.


   FOOTNOTES
 
* This work was supported by National Institutes of Health NCI Grant CA 84356 (to P. T. P. K.). 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. Back

|||| Present address: Alexion Antibody Technologies, Inc., 3985 Sorrento Valley Blvd., Suite A, San Diego, CA 92121. Back

¶¶ To whom reprint requests should be addressed: The Ohio State University, Suite 316 Medical Research Facility, 420 W. 12th Ave., Columbus, OH 43210. Tel.: 614-292-7028; Fax: 614-292-1135; E-mail: Kaumaya.1{at}osu.edu.

1 The 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. Back


   ACKNOWLEDGMENTS
 
We thank Kary Green-Church for assistance with the mass spectroscopy analysis and the Ohio State University Comprehensive Cancer Center Analytical Cytometry Laboratory for assistance with flow cytometry.



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J. T. Garrett, S. Rawale, S. D. Allen, G. Phillips, G. Forni, J. C. Morris, and P. T. P. Kaumaya
Novel Engineered Trastuzumab Conformational Epitopes Demonstrate In Vitro and In Vivo Antitumor Properties against HER-2/neu
J. Immunol., June 1, 2007; 178(11): 7120 - 7131.
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