| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
|
|
|
|||||||
|
|||||||||
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
* BioLife Science, and Departments of
Pathophysiology,
Surgery, and
Dermatology, and
¶ Clinical Division of Oncology, Department of Medicine I, Medical University of Vienna, Vienna, Austria; and
|| Department of Pathology, University of Bern, Bern, Switzerland
 |
Abstract |
|---|
 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
|
Introduction |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
30% of invasive breast cancers and 70% of ductal carcinomata in situ (4, 5), in a small percentage of melanomas (6), and in other malignancies originating from various organs, including ovary (4), kidney, colon (7), and bladder (8). Overexpression of Her-2/neu leads to increased responsiveness to stromal growth factors of the EGF family, resulting in potentiated signaling, which ultimately results in malignant proliferation (9). As Her-2/neu contains a large extracellular domain of 110 kDa, it has attracted interest as a target for immunological attack. This view was further strengthened by the discovery of humoral (10) and cellular immune responses against Her-2/neu in some patients with Her-2/neu overexpressing tumors (11). Moreover, Her-2/neu peptide-specific T cells could be isolated from peripheral blood of cancer patients as well as of healthy individuals (12), indicating that tolerance to this self-Ag is not absolute. As a series of mAbs directed against the Her-2/neu receptor became available and their interaction with Her-2/neu overexpressing cells was characterized, differences between their intrinsic actions were demonstrated: depending on epitope specificity, the interaction of Her-2/neu and anti-Her-2/neu Abs was shown to result in either inhibition or enhancement of tumor proliferation (13, 14, 15, 16). This emphasizes that an inappropriately induced immune response could result in detrimental effects. In contrast, a selective immune response toward "inhibitory" epitopes may lead to cancer cell death (17).
In this setting, the phage display technique is an attractive tool to generate mimics of an epitope recognized by a given tumor growth-inhibiting Ab (18). For this purpose, we chose trastuzumab (herceptin), a growth-inhibitory humanized monoclonal anti-Her-2/neu Ab, approved by the U.S. Food and Drug Administration for passive immunotherapy in the treatment of patients with advanced Her-2/neu overexpressing breast cancer. The Ab binds to the extracellular domain of Her-2/neu and inhibits the downstream signaling cascade, resulting in growth inhibition of Her-2/neu overexpressing tumor cells (17). This inhibitory capacity was found to be associated with internalization of the receptor-Ab complex and movement into endocytic vesicles (3, 15, 19). Trastuzumab proved to be successful as monotherapy of pretreated patients (20, 21), monotherapy as first-line treatment of metastatic disease (22), and in combination with various cytotoxic agents including paclitaxel, docetaxel, gemcitabine, vinorelbine, platinum compounds and other drugs (23, 24, 25, 26, 27). Building upon these described impressive clinical effects of passive trastuzumab application, we aimed to generate vaccine constructs for active immunization, with the potential to elicit in vivo production of Abs with immunological and biological properties equivalent to trastuzumab.
|
Materials and Methods |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
The Her-2/neu positive human mammary carcinoma cell line SK-BR-3 (HTB-30; American Type Culture Collection (ATCC), Manassas, VA) was grown in McCoy’s medium (Life Technologies, Inchinnan, U.K.) supplemented with 10% FCS, 1% glutamine, 1% penicillin/streptomycin, and 50 µg/ml gentamicin sulfate. The human mammary cell line MDA-MB-468 (HTB-132; ATCC), which is Her-2/neu-negative, was grown in Leibovitz-15 medium (Life Technologies) and supplemented as previously described.
Membrane fraction cell extracts were prepared as previously described (28). Total cell lysates were made (outlined in Ref. 29) using a modified lysis buffer, containing 20 mM Tris, 150 mM NaCl, 1 mM EDTA, 1 mM EGTA, 1% Triton X-100, and a protease inhibitor mixture (Complete; Roche, Basel, Switzerland), at pH 7.5. The protein concentration was determined photometrically, using bicinchoninic acid (BCA Protein Assay kit; Pierce, Rockford, IL). Extracts were aliquoted and stored at –80°C.
Monoclonal Abs
Trastuzumab (herceptin), a humanized IgG1 mAb, was purchased from Roche (Hertfordshire, U.K.). Rituximab (rituxan), also a humanized IgG1 mAb and directed against CD20, was purchased from Genentech (IDEC Pharmaceuticals, San Diego, CA) and used as an isotype control.
Phage library and biopanning
Three successive rounds of panning with trastuzumab were performed with a random phage library (CL10), expressing cysteine-flanked decapeptides, circularized by disulfide bridging, fused to pIII of the filamentous phage M13 (30). Biopannings were performed (outlined in Ref. 31) with some modifications. In short, ELISA plates (Nunc, Rosklide, Denmark) were coated with 40 µg/ml trastuzumab in bicarbonate buffer, pH 8.5. Wells were blocked with PBS/1% dry milk and incubated with an aliquot of the phage library (5 x 1010 phage particles) in PBS/1% dry milk/0.1% Tween 20, at room temperature for 2 h. Unbound phage particles were removed by extensive washing with PBS/0.5% Tween 20. Bound phage was eluted with 0.1 M glycine, pH 2.2, and immediately neutralized. Phage was amplified in Escherichia coli K91, precipitated from the bacterial culture supernatant with polyethylene glycol, and either immediately used for the next round of biopanning or stored at –20°C. For titer determination, aliquots of the eluate or the amplificate were plated in serial dilutions on Luria broth agar plates containing 75 µg/ml kanamycin.
Colony screening assay
After each round of biopanning, colony screenings for selection of specific phage clones were done according to Barbas (32). Immunoscreenings were performed with trastuzumab and isotype control rituximab (2.5 µg/ml in PBS/casein). Bound Ab was detected by 125I-labeled anti-human IgG (IBL, Hamburg, Germany). Filters were washed, dried, and exposed to Biomax-MS films (Kodak, Rochester, NY) at –70°C. Positive clones were amplified as previously described.
DNA sequencing
Single strand phage DNA was prepared using a QiaPrep Spin M13 kit (Qiagen, Hilden, Germany). The amount of prepared DNA was checked with an EtBr2/0.7% agarose gel under UV illumination. DNA sequencing was done by IBL (Vienna, Austria).
Specificity ELISA
ELISA plates (Nunc) were coated with trastuzumab or rituximab, 10 µg/ml in PBS by overnight incubation at 4°C. Plates were then washed with PBS/0.1% Tween 20, and nonspecific binding was blocked by incubation with PBS/0.1% Tween 20/1% BSA. Phage particles were added in a concentration of 1 x 1010 CFU/ml in PBS/0.1% Tween 20/0.1% BSA. Bound phage particles were detected with a peroxidase-conjugated rabbit anti-phage Ab (Amersham Pharmacia Biotech, Little Chalfont, U.K.). The reaction was developed with ABTS (Sigma-Aldrich, St. Louis, MO) as substrate. OD405–490 was measured in an ELISA reader (Dynatech, Denkendorf, Germany).
Mimicry ELISAs
ELISA plates (Nunc) were coated with trastuzumab (10 µg/ml) in PBS, and blocked as previously described. Phage particles (108 CFU/well) and cell extracts (100 µg protein/well) were added simultaneously and incubated for 1 h at 37°C and for 1 h at 4°C. After washing, bound phage was detected as described for specificity ELISA. Percentage of specific inhibition was calculated relative to unspecific (sterical) inhibition incurred by the control cell extract.
A second setup consisted of Costar cell culture cluster plates (Corning, Corning, NY) coated with a suboptimal concentration of SK-BR-3 cells (1.5 x 105 cells/ml). Trastuzumab aliquots (0.1 µg/ml) were preincubated with specific phage clones or wild-type phage at 1 x 109–1010 CFU/ml, then added onto the ELISA plates. Rituximab (0.1 µg/ml) was used as negative control. Bound Ab was detected with a peroxidase-conjugated goat anti-human IgG Ab (Jackson ImmunoResearch Laboratories, West Grove, PA). The reaction was developed as previously discussed. Percentage of inhibition was calculated relative to the wild-type phage control.
All ELISA experiments were conducted at least three times. To confirm statistical significance of findings, 
2 tests were performed.
Synthesis of vaccine constructs
As a paradigm, the peptide C-QMWAPQWGPD-C was manufactured synthetically (piChem, Graz, Austria). Conformational accuracy was verified with trastuzumab in a DotBlot assay. In short, the peptide was solubilized in PBS/20% dimethylformamide, and dotted onto nitrocellulose membrane at 1 mg/ml. Blot strips were incubated with trastuzumab or rituximab, respectively, and bound Ab detected with a peroxidase-conjugated goat anti-human IgG Ab (Jackson ImmunoResearch Laboratories), using the ECL detection protocol (Amersham Pharmacia Biotech). Subsequently, the peptide was coupled via its C terminus to the immunogenic carrier, tetanus toxoid (TT), by the m-maleinimidobenzoyl-N-hydrosuccinimide ester method, and the conjugate again checked for trastuzumab binding capability in a DotBlot assay (as previously described).
Immunization of BALB/c mice
Two groups of BALB/c mice (n = 7) from the Institute for Laboratory Animal Science and Genetics (University of Vienna, Vienna, Austria) were immunized i.p. with 10 µg of the mimotope conjugate (group A), or the carrier protein alone (group B), respectively, on days 1, 15, and 29. Aluminum hydroxide was used as an adjuvant in all groups. Blood was taken from the tail vein on days 0 (preimmune serum), 22, and 36. Mice were treated according to European Union Rules of Animal Care, with permission 66009/147/PR-4-2001 from the Austrian Ministry of Science.
Immunoblotting
Total cell lysates were separated by SDS-PAGE and immunoblots incubated with trastuzumab or serum pools as detecting Abs. Bound trastuzumab was detected by 125I-labeled anti-human IgG (IBL), bound mouse Abs by 125I-labeled anti-mouse Ig (Amersham Pharmacia Biotech).
Titers of sera were determined by incubation of serial dilutions with dotted TT, mimotope peptide, or blotted Her-2/neu, and bound Abs detected as previously described.
Fluorescence microscopy
SK-BR-3 cells were plated at low density on four-well Lab-Tek tissue culture chamber slides (Miles Laboratories, Naperville, IL), and grown overnight until half-confluent. They were then cooled to 4°C, washed with ice-cold PBS, fixed with 4% paraformaldehyde in PBS for 30 min, quenched with 50 mM NH4Cl in PBS, and blocked with 1% BSA/PBS. Membranes were stained with rabbit anti-placenta alkaline phosphatase Ab, followed by Alexa 568-conjugated goat anti-rabbit IgG (Molecular Probes, Eugene, OR). Her-2/neu was detected with mAb 4D5 (kindly provided by Genentech, South San Francisco, CA), or with purified IgG (prepared with PROSEP-A spin columns; Millipore, Bedford, MA) from second immune sera (pooled from all mice in group A), followed by FITC-conjugated goat anti-mouse IgG (Caltag Laboratories, Burlingame, CA). Purified IgG of group B and preimmune sera were used as controls. Nuclei were stained with 0.1 µg/ml Hoechst dye (Sigma-Aldrich) in PBS for 10 min. Cells were mounted in Mowiol mounting medium and viewed with a Zeiss Axioplan 2 (Carl Zeiss, Jena, Germany).
Transfection and internalization assay
SK-BR-3 cells were plated into chamber slides as previously described, and transfected with a Her-1/GFP construct (33), kindly provided by Dr. P. Bastiaens (EMBL, Heidelberg, Germany) using SuperFect Transfection Reagent (Qiagen), according to the manufacturer’s instructions. After a 2-day expression period, cells were incubated for 15 min with 10 µg/ml purified IgG from second immune sera of the mimotope-immunized and the control group, with trastuzumab, or with medium alone. Cells were fixed, mounted, and viewed as previously described.
|
Results |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
A constrained 10 mer random peptide phage display library was screened with trastuzumab. As a first indicator of successful biopanning, an increase of phage titers during rounds of panning was observed. The phage titer increased from 8 x 105 CFU/ml (first round) to 6.3 x 106 CFU/ml (second round) and finally to 4.3 x 107 CFU/ml (third round).
Colony screenings were performed after each round of panning. Phage clones reactive with trastuzumab, but not with the isotype-matched control Ab rituximab, were amplified and DNA sequenced. Five insert sequences were deduced from 94 positive phage clones (Table I). The insert C-QMWAPQWGPD-C was found most frequently, indicating high reactivity of the corresponding peptide with the trastuzumab paratope. The second most frequent sequence was C-KLYWADGEFT-C, followed by C-VDYHYEGAIT-C, C-VDYHYEGTIT-C, and C-KLYWADGELT-C.
|
Specificity ELISA
An ELISA was performed to confirm the colony screening results in a separate test system. All five mimotope candidates were specifically recognized by the selecting Ab trastuzumab, but not by the isotype-matched control Ab rituximab. Moreover, trastuzumab neither reacted with a circular 10 mer control peptide nor with wild-type phage (Fig. 1). Again, the peptide C-QMWAPQWGPD-C exhibited the highest reactivity with the Ab, corresponding to the colony screening result analysis. The other peptides did not parallel the colony screening results as much, but showed comparable binding intensities.
|
In a first setup, we tested three selected phage clones for mimicry with the Her-2/neu Ag. Phage particles and a cell extract containing Her-2/neu from the human breast cancer cell line SK-BR-3, or a Her-2/neu-negative control extract from MDA-MB-468 cells, were competing for ELISA plate-fixed trastuzumab. Results were reproducible with both cell lysate preparations. Only the SK-BR-3 cell extract was able to specifically displace bound phage from the Ab (Fig. 2). The three selected mimics, C-QMWAPQWGPD-C, C-KLYWADGEFT-C, and C-VDYHYEGTIT-C, were competitively removed by 59.4, 40.6, and 79.4%, respectively. Percentages were calculated relative to the nonspecific, possibly sterical, inhibition incurred by the control cell extract.
|
|
Based on 1) the highest frequency of detection in colony screenings, 2) the highest intensity of recognition by trastuzumab in the specificity ELISA, and 3) the satisfying mimicry test results, the peptide C-QMWAPQWGPD-C was considered to be the most promising candidate for immunogenicity evaluation, and therefore manufactured synthetically. As a fourth criterium to proceed to immunizations, 4) a synthetic peptide must be specifically recognized by the selecting Ab. Indeed, synthetic C-QMWAPQWGPD-C was recognized by trastuzumab, but not by the isotype control (Fig. 4). Also conjugated to the immunogenic carrier, TT, mimotope conformation was preserved (data not shown). The immunogenicity of the mimotope conjugate was evaluated in BALB/c mice. All mice developed high anti-TT titers, indicating successful immunization in both groups. All seven mice in group A, immunized with the C-QMWAPQWGPD-C conjugate, showed a humoral response toward this peptide, and more importantly, also developed Abs recognizing Her-2/neu in a blotted SK-BR-3 cell lysate (Table II and Fig. 5).
|
|
|
To demonstrate that the induced Abs also recognize Her-2/neu on the cell surface, we performed immunofluorescence staining. Monoclonal Ab 4D5, the murine archetype of herceptin, was used as a positive control (Fig. 6A). Purified IgG from sera of mice of group A showed intense staining of SK-BR-3 breast cancer cells (Fig. 6B), whereas purified IgG from mice of control group B, and preimmune sera, only caused background staining (Fig. 6, C and D, respectively). For comparability, membranes and nuclei were stained in the same way.
|
The antitumor activity of trastuzumab and other tumor-inhibitory anti-Her2/neu Abs was shown to be the result of internalization of the receptor-Ab complex and movement into endocytic vesicles (3, 15, 19). Consequently, we performed an internalization assay to assess whether the Abs induced by the mimotope have a similar biological activity. To visualize the internalization of the receptor, we transfected SK-BR-3 cells with GFP-labeled Her-1. Her-1 was chosen because it is known to form heterodimers with Her-2/neu, which are internalized together (3). SK-BR-3 cells have very low constitutive levels of the Her-1 receptor, so no unlabeled receptors could interfere in the assay, as would have been the case with a GFP-labeled Her-2/neu. Furthermore, transfection of SK-BR-3 cells with Her-1 also ensures a high rate of Her-1-Her-2/neu heterodimer formation.
After a 15 min incubation with either trastuzumab or purified IgG of mice from group A, a reduction of surface staining and the appearance of vesicles was observed (Fig. 7, top panels). The perinuclear distribution of the vesicular staining suggests late endosomes/lysosomes and thus entering of a degradation pathway. The control IgG, purified from mice immunized with the carrier alone, showed no internalization, but only surface staining, as did the cells treated with no Ab at all (Fig. 7, bottom panel).
|
|
Discussion |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
As the tertiary structure of Her-2/neu was described only recently (41), B cell epitopes for the design of inhibitory vaccines could only be predicted by applying complicated algorithms. But even so, these peptide regions are only estimates of possible conformational epitopes, and may still induce growth-enhancing Abs (42). Moreover, the Her family receptors are known to be glycosylated, and glycosylation has been shown to play a decisive role in the immunogenicity of tumor-associated Ags. This possibly explains the difficulties met by computer-aided epitope searches.
In this situation, the phage display technique is an interesting tool. It is a relatively new technique to define peptides mimicking natural epitopes, including conformational B cell epitopes (43), as is the case with the epitope recognized by trastuzumab (34). The Ag to be mimicked can even be a carbohydrate structure (44). Phage display peptide libraries consist of filamentous phage particles displaying random peptides of defined length on their surface, which are either fused to the phage minor coat protein pIII, or at a higher copy number, to the major coat protein pVIII. By biopanning (31) of phage display libraries, mimotopes can be selected. These are peptides from 6 to 28 amino acids in length, assembled either in linear or in constrained (circular) form.
In this study, we generated mimotopes of a known inhibitory epitope on Her-2/neu, the epitope recognized by trastuzumab. For this purpose, we chose a constrained 10mer phage library because of the favorable stability characteristics of these short circular inserts. Trastuzumab was used to select matching peptides from a repertoire of >109 possible ligands. The surface characteristics of these mimetic peptides are equivalent to the epitope of the selecting Ab, although their amino acid sequence may differ (45), as seen with the five mimotopes presented in this study. This finding is expected when selecting mimotopes for a conformational epitope, as only a small number of amino acids mimic a region composed of several polypeptide chains in the original Ag.
Mimicry tests are mandatory to designate a peptide as a mimotope. After the reactivity of the candidate peptides to the selecting Ab is confirmed in colony screenings and specificity tests, the structural equivalence to the original Ag has to be ascertained. All five candidate peptides in this study showed significant mimicry with Her-2/neu in two experimental setups. Only a cell extract containing Her-2/neu was able to displace phage displayed mimotopes from bound trastuzumab. In the second setup, selected peptides inhibited the Ab binding to Her-2/neu. Thus, we demonstrate that the described mimotopes represent true mimics of the trastuzumab epitope.
Mimotopes are suitable for the induction of an epitope-specific humoral immune response as, for instance, previously reported in the field of type I allergies (46) and also for possible vaccinations against malignant melanoma (47, 48). Both are areas in which epitope-specific induction of Abs is crucial, as otherwise symptoms can be aggravated or malignant growth enhanced. As sequences are easily obtained, mimotope peptides can be synthesized custom-made. Synthetic peptide vaccines are cost-effective, simple to produce and can easily be quality-controlled during the manufacturing process. They are chemically stable and contain no oncogenic, toxic, or infectious material. As active immunogens, synthetic mimotopes can induce continuously available tumor-targeting Abs. We demonstrate in this study that our mimotope vaccine is immunogenic and elicits Igs recognizing the original Ag, Her-2/neu. This was shown in Western blot and also in immunofluorescence experiments. In the latter assay, the induced anti-mimotope Abs caused cell membrane staining of the same pattern and intensity as the positive control Ab 4D5, the murine precursor of trastuzumab. As tumor growth inhibitory anti-Her-2/neu Abs are described to cause receptor internalization and accelerated degradation (3, 15, 19), we performed an internalization study. Indeed, the induced Abs showed the same vesicular staining pattern as trastuzumab, indicating similar biological activity.
Undoubtedly, the effects of induced Abs by mimotope vaccination will have to be closely monitored, even though the danger of tumor growth stimulating Abs was ruled out by deliberate epitope selection with the phage display technique. Although no toxicities could be observed in mimotope-immunized animals so far, the potential occurrence of cardiotoxicity, which is known to occur as a complication of trastuzumab administration (49), must be assessed. Nevertheless, the benefits of a narrowly focused active immune response against a major cancer Ag as Her-2/neu are expected to outweigh possible disadvantages by far.
In conclusion, we demonstrate trastuzumab mimotopes to be novel vaccine candidates. They are immunogenic and elicit Abs recognizing the original Ag, Her-2/neu. Moreover, these Abs also showed a trastuzumab-like activity in receptor internalization induction. Their major advantage is to focus a long lasting humoral immune response to a therapeutically relevant epitope. Sustained availability of growth-inhibitory anti-Her-2/neu Abs may complement previous trials, which aimed at other tumor characteristics. The active induction of Abs with qualities like trastuzumab may lead to the mimotopes’ use in disease prevention, adjuvant treatment, and therapy of advanced disease.
|
Acknowledgments |
|---|
|
Footnotes |
|---|
2 Address correspondence and reprint requests to Dr. Erika Jensen-Jarolim, Department of Pathophysiology, University Hospital of Vienna, Waehringer Guertel 18-20, A–1090 Vienna, Austria. E-mail address: erika.jensen-jarolim{at}meduniwien.ac.at
3 Abbreviations used in this paper: EGF, epidermal growth factor; TT, tetanus toxoid.
Received for publication November 17, 2003. Accepted for publication April 20, 2004.
|
References |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
. Cell 110:763.[Medline]
This article has been cited by other articles:
|
|
 |
|
  F. Perosa, E. Favoino, C. Vicenti, A. Guarnera, V. Racanelli, V. De Pinto, and F. Dammacco Two Structurally Different Rituximab-Specific CD20 Mimotope Peptides Reveal That Rituximab Recognizes Two Different CD20-Associated Epitopes J. Immunol., January 1, 2009; 182(1): 416 - 423. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 S. Wagner, C. Krepler, D. Allwardt, J. Latzka, S. Strommer, O. Scheiner, H. Pehamberger, U. Wiedermann, C. Hafner, and H. Breiteneder Reduction of Human Melanoma Tumor Growth in Severe Combined Immunodeficient Mice by Passive Transfer of Antibodies Induced by a High Molecular Weight Melanoma-Associated Antigen Mimotope Vaccine Clin. Cancer Res., December 15, 2008; 14(24): 8178 - 8183. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
K. H. Bramswig, R. Knittelfelder, S. Gruber, E. Untersmayr, A. B. Riemer, K. Szalai, R. Horvat, R. Kammerer, W. Zimmermann, C. C. Zielinski, et al. Immunization with Mimotopes Prevents Growth of Carcinoembryonic Antigen Positive Tumors in BALB/c Mice Clin. Cancer Res., November 1, 2007; 13(21): 6501 - 6508. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
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. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 M. Binder, F.-N. Vogtle, S. Michelfelder, F. Muller, G. Illerhaus, S. Sundararajan, R. Mertelsmann, and M. Trepel Identification of Their Epitope Reveals the Structural Basis for the Mechanism of Action of the Immunosuppressive Antibodies Basiliximab and Daclizumab Cancer Res., April 15, 2007; 67(8): 3518 - 3523. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
A. B. Riemer, E. Untersmayr, R. Knittelfelder, A. Duschl, H. Pehamberger, C. C. Zielinski, O. Scheiner, and E. Jensen-Jarolim Active Induction of Tumor-Specific IgE Antibodies by Oral Mimotope Vaccination Cancer Res., April 1, 2007; 67(7): 3406 - 3411. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 E. Jensen-Jarolim and A. B. Riemer Small mimotopes are big in identifying B-cell epitopes Blood, September 15, 2006; 108(6): 1794 - 1795. [Full Text] [PDF]
|
|
|
|
|
|
F. Perosa, E. Favoino, M. A. Caragnano, and F. Dammacco Generation of biologically active linear and cyclic peptides has revealed a unique fine specificity of rituximab and its possible cross-reactivity with acid sphingomyelinase-like phosphodiesterase 3b precursor Blood, February 1, 2006; 107(3): 1070 - 1077. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 A. B. Riemer, H. Kurz, M. Klinger, O. Scheiner, C. C. Zielinski, and E. Jensen-Jarolim Vaccination With Cetuximab Mimotopes and Biological Properties of Induced Anti-Epidermal Growth Factor Receptor Antibodies J Natl Cancer Inst, November 16, 2005; 97(22): 1663 - 1670. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
W. Luo, J. C.-f. Hsu, C.-Y. Tsao, E. Ko, X. Wang, and S. Ferrone Differential Immunogenicity of Two Peptides Isolated by High Molecular Weight-Melanoma-Associated Antigen-Specific Monoclonal Antibodies with Different Affinities J. Immunol., June 1, 2005; 174(11): 7104 - 7110. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 B. Jiang, W. Liu, H. Qu, L. Meng, S. Song, T. Ouyang, and C. Shou A Novel Peptide Isolated from a Phage Display Peptide Library with Trastuzumab Can Mimic Antigen Epitope of HER-2 J. Biol. Chem., February 11, 2005; 280(6): 4656 - 4662. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
N. K. Dakappagari, K. D. Lute, S. Rawale, J. T. Steele, S. D. Allen, G. Phillips, R. T. Reilly, and P. T. P. Kaumaya Conformational HER-2/neu B-cell Epitope Peptide Vaccine Designed to Incorporate Two Native Disulfide Bonds Enhances Tumor Cell Binding and Antitumor Activities J. Biol. Chem., January 7, 2005; 280(1): 54 - 63. [Abstract] [Full Text] [PDF]
|
|
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
) displaying circular peptide inserts with sequences as given on the x-axis, of a phage clone displaying an irrelevant control peptide (control phage) or of wild-type phage, respectively. Isotype-matched control Ab (
) rituximab does not interact with trastuzumab mimotopes.
) in SK-BR-3 breast cancer cell extract competes with phage-displayed peptides for binding to coated trastuzumab, thereby reducing the number of bound phage particles. An extract (
