Novel engineered trastuzumab conformational epitopes demonstrate in vitro and in vivo antitumor properties against HER-2/neu

Trastuzumab is a growth-inhibitory humanized Ab targeting the oncogenic protein HER-2/neu. Although trastuzumab is approved for treatment of advanced breast cancer, a number of concerns exist with passive immunotherapy. Treatment is expensive and has a limited duration of action, necessitating repeated administrations of the mAb. Active immunotherapy with conformational B cell epitopes affords the possibility of generating an enduring immune response, eliciting protein-reactive high-affinity anti-peptide Abs. The three-dimensional structure of human HER-2 in complex with trastuzumab reveals that the Ag-binding region of HER-2 spans residues 563-626 that comprises an extensive disulfide-bonding pattern. To delineate the binding region of HER-2, we have designed four synthetic peptides with different levels of conformational flexibility. Chimeric peptides incorporating the measles virus fusion "promiscuous" T cell epitope via a four-residue linker sequence were synthesized, purified, and characterized. All conformational peptides were recognized by trastuzumab and prevented the function of trastuzumab inhibiting tumor cell proliferation, with 563-598 and 597-626 showing greater reactivity. All epitopes were immunogenic in FVB/N mice with Abs against 597-626 and 613-626 recognizing HER-2. The 597-626 epitope was immunogenic in outbred rabbits eliciting Abs which recognized HER-2, competed with trastuzumab for the same epitope, inhibited proliferation of HER-2-expressing breast cancer cells in vitro and caused their Ab-dependent cell-mediated cytotoxicity. Moreover, immunization with the 597-626 epitope significantly reduced tumor burden in transgenic BALB-neuT mice. These results suggest the peptide B cell immunogen is appropriate as a vaccine for HER-2-overexpressing cancers because the resulting Abs show analogous biological properties to trastuzumab.     

High affinity mimotope of the polysaccharide capsule of Cryptococcus neoformans identified from an evolutionary phage peptide library

Cryptococcus neoformans causes a life-threatening meningoencephalitis in a significant percentage of AIDS patients. Mice immunized with a glycoconjugate vaccine composed of the glucuronoxylomannan (GXM) component of the cryptococcal capsular polysaccharide conjugated to tetanus toxoid (TT) produce Abs that, based on the epitope recognized, can be either protective or nonprotective. Since nonprotective Abs block the efficacy of protective Abs, we are interested in developing a vaccine that would focus the immune response specifically to protective epitopes. Previously, we screened a phage display library with 2H1, a protective anti-GXM mAb, and isolated PA1, a representative peptide that had a K(d) of 295 nM for 2H1. Mice immunized with PA1 conjugated to keyhole limpet hemocyanin developed high anti-peptide (1/13,000), but low anti-GXM (maximum, 1/200) titers. We now report our efforts to improve this vaccine by screening a sublibrary with six random amino acids added to either end of the PA1 motif to identify higher affinity peptides. P206.1, a peptide isolated from this sublibrary, had 80-fold higher affinity for 2H1 (K(d) = 3.7 nM) than PA1. P206.1 bound protective, but not nonprotective, anti-GXM Abs. Mice immunized with P206.1 conjugated to various carriers did not mount an Ab response to GXM despite developing high anti-peptide titers. However, mice primed with GXM-TT and boosted with P206.1-TT developed significant anti-GXM titers (maximum, 1/180,000). This latter immunization scheme focused the immune response on protective epitopes, since only 2-5% of these titers were directed against nonprotective de-O-acetylated GXM epitopes compared with 20-60% in animals primed and boosted with GXM-TT.     

Substitution analog peptide derived from HER-2 can efficiently induce HER-2-specific, HLA-A24 restricted CTLs

In order to broaden the possibility for anti-HER-2/neu (HER-2) immune targeting, it is important to identify HLA-A24 restricted peptide epitopes derived from HER-2, since HLA-A24 is one of the most common alleles in Japanese and Asian people. In the present study, we have screened HER-2-derived, HLA-A24 binding peptides for cytotoxic T lymphocyte (CTL) epitopes. A panel of HER-2-derived peptides with HLA-A24 binding motifs and the corresponding analogs designed to enhance HLA-A24 binding affinity were selected. Identification of HER-2-reactive and HLA-A24 restricted CTL epitopes were performed by a reverse immunology approach. To induce HER-2-reactive and HLA-A24 restricted CTLs, PBMCs from healthy donors were repeatedly stimulated with monocytes-derived, mature DCs pulsed with HER-2 peptide. Subsequent peptide-induced T cells were tested for the specificity by enzyme linked immunospot, cytotoxicity and tetramer assays. CTL clones were then obtained from the CTL lines by limiting dilution. Of the peptides containing HLA-A24 binding motifs, 16 peptides (nine mers) including wild type peptides (IC₅₀<1,000 nM) and substituted analog peptides (IC₅₀<50 nM) were selected for the present study. Our studies show that an analog peptide, HER-2(905AA), derived from HER-2(905) could efficiently induce HER-2-reactive and HLA-A24 restricted CTLs. The reactivity of the HER-2(905AA)-induced CTL (CTL905AA) was confirmed by different CTL assays. The CTL905AA clones also were able to lyse HER-2(+), HLA-A24(+) tumor cells and cytotoxicity could be significantly reduced in cold target inhibition assays using cold targets pulsed with the HER-2(905) wild type peptide as well as the inducing HER-2(905AA) analog peptide. A newly identified HER-2(905) peptide epitope is naturally processed and presented as a CTL epitope on HER-2 overexpressing tumor cells, and an MHC anchor-substituted analog, HER-2(905AA), can efficiently induce HER-2-specific, HLA-A24 restricted CTLs.     

A chimeric multi-human epidermal growth factor receptor-2 B cell epitope peptide vaccine mediates superior antitumor responses

Immunotherapeutic approaches to cancer should focus on novel undertakings that modulate immune responses by synergistic enhancement of antitumor immunological parameters. Cancer vaccines should preferably be composed of multiple defined tumor Ag-specific B and T cell epitopes. To develop a multiepitope vaccine, 12 high ranking B cell epitopes were identified from the extracellular domain of the human epidermal growth factor receptor-2 (HER-2) oncoprotein by computer-aided analysis. Four novel HER-2 B cell epitopes were synthesized as chimeras with a promiscuous T cell epitope (aa 288-302) from the measles virus fusion protein (MVF). Two chimeric peptide vaccines, MVF HER-2(316-339) and MVF HER-2(485-503) induced high levels of Abs in outbred rabbits, which inhibited tumor cell growth. In addition, Abs induced by a combination of two vaccines, MVF HER-2(316-339) and MVF HER-2(628-647) down-modulated receptor expression and activated IFN-gamma release better than the individual vaccines. Furthermore, this multiepitope vaccine in combination with IL-12 caused a significant reduction (p = 0.004) in the number of pulmonary metastases induced by challenge with syngeneic tumor cells overexpressing HER-2. Peptide Abs targeting specific sites in the extracellular domain may be used for exploring the oncoprotein's functions. The multiepitope vaccine may have potential application in the treatment of HER-2-associated cancers.     

Generation of Peptide mimics of the epitope recognized by trastuzumab on the oncogenic protein Her-2/neu

Immunizations with the oncogenic protein Her-2/neu elicit Abs exerting diverse biological effects--depending on epitope specificity, tumor growth may be inhibited or enhanced. Trastuzumab (herceptin) is a growth-inhibitory humanized monoclonal anti-Her-2/neu Ab, currently used for passive immunotherapy in the treatment of breast cancer. However, Ab therapies are expensive and have to be repeatedly administered for long periods of time. In contrast, active immunizations produce ongoing immune responses. Therefore, the study aims to generate peptide mimics of the epitope recognized by trastuzumab for vaccine formulation, ensuring the subsequent induction of tumor growth inhibitory Abs. We used the phage display technique to generate epitope mimics, mimotopes, complementing the screening Ab trastuzumab. Five candidate mimotopes were isolated from a constrained 10 mer library. These peptides were specifically recognized by trastuzumab, and showed distinctive mimicry with Her-2/neu in two experimental setups. Subsequently, immunogenicity of a selected mimotope was examined in BALB/c mice. Immunizations with a synthetic mimotope conjugated to tetanus toxoid resulted in Abs recognizing Her-2/neu in a blotted cell lysate as well as on the SK-BR-3 cell surface. Analogous to trastuzumab, the induced Abs caused internalization of the receptor from the cell surface to endosomal vesicles. These results indicate that the selected mimotopes are suitable for formulation of a breast cancer vaccine because the resulting Abs show similar biological features as trastuzumab.     

Breast cancer: the upgraded role of HER-3 and HER-4

Breast cancer is the most common cancer in women and the ErbB receptor family holds crucial role in its pathogenesis. Among them, epidermal growth factor receptor and HER-2 are the most studied members and their overexpression has been associated with aggressive clinical behaviour. These data were further strengthened by the clinical success of trastuzumab, a monoclonal antibody against HER-2 in breast cancer patients with HER-2 overexpression and/or amplification. However, trastuzumab failure in some patients may partly be attributed to co-expression of other ErbB receptors. Herein, we provide updated views regarding the role of HER-3 and HER-4 in breast cancer. Accumulated evidence implies that these receptors should be considered more than heterodimerisation partners. Their expression profile might be useful in predicting responsiveness to current treatment options, while new strategies targeting their ligands and downstream effectors are being developed.     

Enhanced mucosal immunoglobulin A response and solid protection against foot-and-mouth disease virus challenge induced by a novel dendrimeric peptide

The successful use of a dendrimeric peptide to protect pigs against challenge with foot-and-mouth disease virus (FMDV), which causes the most devastating animal disease worldwide, is described. Animals were immunized intramuscularly with a peptide containing one copy of a FMDV T-cell epitope and branching out into four copies of a B-cell epitope. The four immunized pigs did not develop significant clinical signs upon FMDV challenge, neither systemic nor mucosal FMDV replication, nor was its transmission to contact control pigs observed. The dendrimeric construction specifically induced high titers of FMDV-neutralizing antibodies and activated FMDV-specific T cells. Interestingly, a potent anti-FMDV immunoglobulin A response (local and systemic) was observed, despite the parenteral administration of the peptide. On the other hand, peptide-immunized animals showed no antibodies specific of FMDV infection, which qualifies the peptide as a potential marker vaccine. Overall, the dendrimeric peptide used elicited an immune response comparable to that found for control FMDV-infected pigs that correlated with a solid protection against FMDV challenge. Dendrimeric designs of this type may hold substantial promise for peptide subunit vaccine development.     

DNA fusion vaccine designed to induce cytotoxic T cell responses against defined peptide motifs: implications for cancer vaccines

DNA vaccination offers a strategy to induce immune attack on cancer cells, but tumor Ags are often weak. Inclusion of a "foreign" protein increases immunogenicity, and we found previously that fusion of the fragment C (FrC) of tetanus toxin to the tumor Ag sequence promotes Ab and CD4(+) responses against B cell tumors. For CTL responses, use of the full two-domain FrC may be less helpful, because known immunogenic MHC class I-binding peptides in the second domain could compete with attached tumor-derived epitopes. Therefore, we removed the second domain, retaining the N-terminal domain, which contains a "universal" helper epitope. We investigated the ability to induce CTL responses of candidate peptides placed at the C terminus of this domain. As test peptides, we repositioned the two known CTL motifs from the second domain to this site. Strong CTL responses to each peptide were induced by the engineered construct, as compared with the native FrC construct. Induced CTLs were able to specifically kill tumor cells transfected with FrC as a surrogate tumor Ag both in vitro and in vivo. Further reduction of the domain to a short helper epitope generated only weak CTL responses against fused peptides, and synthetic peptides mixed with the plasmid containing the first domain were ineffective. The single FrC domain-peptide vaccine design also was able to induce high levels of CTLs against a known epitope from carcinoembryonic Ag. Response to peptide was suppressed if two FrC domains were present, consistent with immunodominance. These principles and designs may have relevance for cancer vaccines delivered via DNA.     

Structural basis of enhanced binding of extended and helically constrained peptide epitopes of the broadly neutralizing HIV-1 antibody 4E10

Potent, broadly HIV-1 neutralizing antibodies (nAbs) may be invaluable for the design of an AIDS vaccine. 4E10 is the broadest HIV-1 nAb known to date and recognizes a contiguous and highly conserved helical epitope in the membrane-proximal region of gp41. The 4E10 epitope is thus an excellent target for vaccine design as it is also highly amenable to peptide engineering to enhance its helical character. To investigate the structural effect of both increasing the peptide length and of introducing helix-promoting constraints in the 4E10 epitope, we have determined crystal structures of Fab 4E10 bound to an optimized peptide epitope (NWFDITNWLWYIKKKK-NH(2)), an Aib-constrained peptide epitope (NWFDITNAibLWRR-NH(2)), and a thioether-linked peptide (NWFCITOWLWKKKK-NH(2)) to resolutions of 1.7 A, 2.1 A, and 2.2 A, respectively. The thioether-linked peptide is the first reported structure of a cyclic tethered helical peptide bound to an antibody. The introduced helix constraints limit the conformational flexibility of the peptides without affecting interactions with 4E10. The substantial increase in affinity (10 nM versus 10(4) nM of the IC(50) of the original KGND peptide template) is largely realized by 4E10 interaction with an additional helical turn at the peptide C terminus that includes Leu679 and Trp680. Thus, the core 4E10 epitope was extended and modified to a WFX(I/L)(T/S)XX(L/I)W motif, where X does not play a major role in 4E10 binding and can be used to introduce helical-promoting constraints in the peptide epitope.     

A contraceptive peptide vaccine targeting sulfated glycoprotein ZP2 of the mouse zona pellucida

In this study, we have mapped and characterized a B cell epitope of sulfated glycoprotein ZP2 (ZP2) as a step toward the development of a multi-epitope zona pellucida (ZP) vaccine. Recombinant polypeptides expressed by random deoxyribonuclease-digested fragments of ZP2 cDNA were screened for binding to IE-3, a monoclonal antibody to murine ZP2. Positive clones contained cDNA inserts encoding polypeptide corresponding to ZP2(103-134). When normal or ovariectomized female mice were immunized with three overlapping peptides that span this region of ZP2 (101-120, 111-130, 121-140), only ZP2(121-140) elicited IgG antibodies that reacted with mouse ovarian ZP, indicative of the presence of native B epitope and helper T cell epitope in ZP2(121-140). To more finely map the ZP2 B cell epitope, a random peptide display library was screened with the IE-3 antibody, and a consensus tetramer sequence VxYK that matched the ZP2(123-126) sequence VRYK was located. Competitive immunofluorescence analysis with single alanine-substituted VxYK peptides ranked the relative contribution of the three critical B cell epitope residues as Y > V > K. A chimeric peptide was constructed that contained the YRYK motif of ZP2 and a bovine RNase T cell epitope. Although (C57BL/6xA/J) F1 (B6AF1) female mice immunized with the chimeric peptide developed ZP antibody response, this peptide elicited antibody only in mice of the histocompatibility complex (MHC) H-2(k or b) haplotype. In contrast, ZP2(121-140) peptide elicited antibody in inbred mice with three additional mouse MHC haplotypes. Moreover, although ZP2(121-140) contained a T cell epitope, no oophoritis was observed after immunization of B6AF1 mice with ZP2(121-140) in complete Freund's adjuvant (CFA). In a preliminary trial, female B6AF1 mice immunized with ZP2(121-140) in CFA had reduced litter sizes as compared with mice injected with CFA alone.     

Immunological features and efficacy of the reconstructed epitope vaccine CtUBE against Helicobacter pylori infection in BALB/c mice model

Urease is an essential virulence factor and colonization factor for Helicobacter pylori, of which the urease B subunit (UreB) is considered as an excellent vaccine candidate antigen. In previous study, an epitope vaccine with cholera toxin B subunit (CTB) and an epitope (UreB_321–339) named CtUBE was constructed and the mice were protected significantly after intragastric vaccination with the CtUBE liposome vaccine. However, the fusion protein CtUBE was expressed as inclusion bodies and was difficultly purified. Besides, the immunogenicity and specificity of the CtUBE vaccine was not investigated in a fairly wide and detailed way. In this study, the fusion peptide CtUBE was reconstructed and expressed as a soluble protein with pectinase signal peptide at the N terminus and the 6-his tag at its C-terminal, and then the immunogenicity, specificity, prophylactic, and therapeutic efficacy of the reconstructed CtUBE (rCtUBE) vaccine were evaluated in BALB/c mice model after purification. The experimental results indicated that mice immunized with rCtUBE could produce comparatively high level of specific antibodies which could respond to natural H. pylori urease, UreB, or the minimal epitope UreB_327–334 involved with the active site of urease, and showed effectively inhibitory effect on the enzymatic activity of urease. Besides, oral prophylactic or therapeutic immunization with rCtUBE significantly decreased H. pylori colonization compared with oral immunization with rCTB or PBS, and the protection was correlated with antigen-specific IgG, IgA, and mucosal sIgA antibody responses, and a Th2 cells response. This rCtUBE vaccine may be a promising vaccine candidate for the control of H. pylori infection.     

A HER-2/neu peptide admixed with PLA microspheres induces a Th1-biased immune response in mice

The elimination of cancer cells requires strong cellular immune responses, and these responses are induced by the activation of Th1 lymphocytes. In this work, the possibility of inducing a Th1 type of immune response in vivo by mixing a HER-2/neu synthetic CTL (cytotoxic T lymphocyte) peptide [HER-2/neu (789-797)], with poly-lactide (PLA) microspheres was investigated. Various formulations of the peptide were administered to HLA-A2.1 transgenic (HHD) mice. Cellular experiments, assessing proliferation and cytokine determination in splenocyte culture supernatants, were carried out in order to evaluate the type of immune response to the antigen. The in vivo administration of the peptide antigen admixed with the PLA microspheres induced a potent immune response which was comparable to that induced by the combination of the antigen in complete Freund's adjuvant (CFA). Furthermore, the cytokine profile produced by the T lymphocytes of the immunized animals indicated that the combination of the peptide antigen with the PLA microspheres induced a strong Th1 biased immune response to the antigen. The time of peptide incubation with the microspheres prior to administration did not affect the immune response, which further simplifies the preparation of this type of vaccine. The results justify further investigation of the possibility of inducing effective cellular immune responses against cancer cells overexpressing HER-2/neu molecules by simply mixing appropriate HER-2/neu peptide antigens with PLA microspheres.     

Identification and characterization of Ch806 mimotopes

The chimeric antibody 806 (Ch806) is a promising antitumor agent that recognizes both the epidermal growth factor receptor variant III (EGFRvIII) and the overexpressed epidermal growth factor receptor (EGFR) in cancer tissues but does not recognize the wild type EGFR in normal tissues. However, passive antibody immunization could not produce effective antitumor titers unless the immunization was administered repeatedly over long periods. To overcome this limitation, we generated epitope mimics that bind to Ch806 and tested whether the peptide mimics could induce the production of similar antibodies when actively immunizing mice with the peptides. We used the PH.D-12 phage display peptide library to identify peptides that bind to the monoclonal antibody (mAb) 12H23, which also recognizes similar epitopes of Ch806. Two mimotopes (WHTEILKSYPHE and LPAFFVTNQTQD) were shown to mimic the mAb 12H23 and Ch806 epitope using immunoassays. The mimotopes were conjugated to immunogenic carrier proteins and used to intraperitoneally immunize BALB/c mice. Interestingly, sera from the mice immunized with the isolated mimotopes not only recognize the recombinant or synthetic 806 eptitope, but can also recognize EGFR that is overexpressed in A431 cells and EGFRvIII expressed in Huh7-EGFRvIII cells, whereas sera from mice immunized with the control peptide-KLH (keyhole limpet hemocyanin) and carrier KLH alone failed to show a similar reactivity. Furthermore, in an antibody-dependent cellular cytotoxicity assay (ADCC), the mimotope-induced antibodies specifically lysed human Huh-7-EGFRvIII cells. Our data indicate that the isolated mimotopes reported here may potentially be used as new alternative agents for treating cancer with EGFRvIII expression or EGFR overexpression.     

Establishment and characterization of a cell line (BTIC) including HER-2-positive cells derived from pleural effusion of recurrent breast invasive ductal carcinoma, scirrhous type

Human epidermal growth factor receptor-2 (HER-2) overexpression in breast cancer occurs in 20% to 30% of patients with breast cancer. Trastuzumab (Herceptin) targets HER-2 tyrosine kinase receptors expressed on tumor cells and mediates anti-proliferative effects against HER-2-positive tumor cells. Adjuvant chemotherapy with trastuzumab has improved the prognosis of patients with HER-2 positive high-grade breast cancer. However, patients often experience appearance and proliferation of recurrent tumor cells after trastuzumab treatment. In this study, we report the successful establishment and characterization of a cell line (BTIC) derived from a patient with recurrent breast cancer after adjuvant chemotherapy with trastuzumab. Characteristics of the BTIC cell line were investigated by phase contrast or electron microscopic observations, chromosome analysis, xenotransplantation, immunohistochemistry and radioimmunoassay for tumor markers. We confirmed that the BTIC cell line grown as multilayered culture in culture dishes, has a poorly developed endoplasmic reticulum in the cytoplasm and some desmosomes. The population doubling time was approximately 44 hr. A graft in nude mouse after xenotransplantation was diagnosed as scirrhous carcinoma. Immunohistochemistry on cultured BTIC cells revealed that the BTIC cells were negative for estrogen receptor and progesterone receptor, and 30% positive for HER-2. Radioimmunoassay indicated secretion of HER-2 protein, NCC-ST-439 and CA15-3.     

Combination treatment with HER-2 and VEGF peptide mimics induces potent anti-tumor and anti-angiogenic responses in vitro and in vivo

HER-2 is a member of the EGF receptor family and is overexpressed in 20-30% of breast cancers. HER-2 overexpression causes increased expression of VEGF at both the RNA and protein levels. HER-2 and VEGF are therefore considered good targets for cancer treatment, which has led to the development of two humanized monoclonal antibodies (mAb) pertuzumab and bevacizumab. Although passive immunotherapy with these Abs are approved for treatment of advanced breast cancer, a number of concerns exist. Treatment is expensive, has a limited duration of action, and is usually accompanied by serious side effects. We hypothesized that therapy with conformational peptide mimics aimed at blocking receptor-ligand interaction is potentially safer with little toxicity, cheaper with a longer half-life, and has greater penetrating abilities than mAbs. We designed and synthesized peptides based on the binding of HER-2 with pertuzumab and VEGF with VEGFR2. We show that treatment with the peptide mimics induces potent anti-tumor responses in vitro as determined by cell viability, proliferation, and HER2 phosphorylation assays. We also demonstrate in a transplantable BALB/c mouse tumor model that treatment with the peptide mimics resulted in a greater delay in tumor growth and development. Similarly, treatment with the peptide mimics inhibited angiogenesis in vivo as assessed by a Matrigel plug assay. To address the problem of degradability of L-amino acid peptides in vivo, we synthesized the retro-inverso D-peptide mimics that resulted in higher efficacy in treatment. Our study shows that combination treatment with HER-2 and VEGF peptide mimics provides greater efficacy than individual treatments.     

HER-2/neu Cancer Vaccines: Present Status and Future Prospects

Immunotherapeutic approaches to cancer should focus on novel undertakings that modulate immune responses by synergistic enhancement of anti-tumor immunological parameters. Cancer vaccines should preferably be composed of multiple defined tumor antigen specific B- and T-cell epitopes. The main focus of this article is to briefly review the present status of Her-2/neu vaccine strategies and to describe the innovative strategies developed in my laboratory for a vaccine against HER-2/neu (ErbB-2) with emphasis on the humoral arm of the immune response. Elucidating the underlining mechanisms of anti-tumor effects elicited by peptide vaccines against a self-protein is a requirement for developing an immunotherapeutic strategy that might be effective in human cancer vaccines. Our approach entails the identification of biologically relevant epitopes, establishing relevant in vitro assays for monitoring vaccine efficacy, devising strategies to engineer conformationally dependent sequences, developing highly immunogenic vaccines for an outbred population and delivering the immunogen/vaccine in a safe and efficacious vehicle, utilizing transgenic animal models for assessing tumor development, and developing challenge models using transplantable tumors to study efficacy of vaccine constructs. We have developed a multi-HER-2/neu B-cell epitope approach and shown in preclinical studies that immunization with a combination of two B-cell epitope was more effective in preventing mammary tumors than a single epitope. We have translated that work to the clinic (OSU 0105) in an FDA approved, NCI sponsored “Phase 1 Active Immunotherapy trial with Chimeric and Multi-epitope based peptide vaccine targeting HER-2 oncoprotein and nor-MDP adjuvant in patients with metastatic and/or recurrent solid tumors” at the James Cancer Hospital at the Ohio State University. The correlation between overexpression of HER-2/neu and up-regulation of VEGF has been demonstrated in breast cancer patients. Thus, blocking angiogenesis is an attractive strategy to inhibit tumor growth, invasion, and metastasis. The hypothesis that combination of anti-angiogenic therapy and tumor immunotherapy of cancer may be synergistic is an important future goal. In this review, I will discuss insights into our preclinical studies that might aid in the design of the next generation of cancer vaccines and become an integrated component of prophylactic/preventive and therapeutic approach.     

Targeting high affinity and epitope-distinct oligoclonal nanobodies to HER2 over-expressing tumor cells

Modern anti-HER2 antibody therapy tends to exploit a panel of different antibodies against different epitopes on the antigen. For this aim, nanobodies are very striking targeting agents and can be easily produced against any cell-specific membrane antigen. The oligoclonal nanobodies can be used to block more than one functional epitope on a target antigen and inhibit the generation of escape variants associated with cancer therapy. In this study, 12 nanobody clones selected from an immune camel library were examined for their ability to differ between tumor markers. These oligoclonal nanobodies targeted breast cancer cells better than each individual nanobody. In epitope mapping, several nanobodies overlapped in the epitope recognized by trastuzumab and some of the non-overlapping nanobodies could affect the binding of trastuzumab to HER2. This study demonstrates that the oligoclonal nanobodies are potential therapeutic tools that can be used instead of, or in combination with trastuzumab to assess tumor viability during treatment.     

Candidate epitope identification using peptide property models: application to cancer immunotherapy

Peptides derived from pathogens or tumors are selectively presented by the major histocompatibility complex proteins (MHC) to the T lymphocytes. Antigenic peptide-MHC complexes on the cell surface are specifically recognized by T cells and, in conjunction with co-factor interactions, can activate the T cells to initiate the necessary immune response against the target cells. Peptides that are capable of binding to multiple MHC molecules are potential T cell epitopes for diverse human populations that may be useful in vaccine design. Bioinformatical approaches to predict MHC binding peptides can facilitate the resource-consuming effort of T cell epitope identification. We describe a new method for predicting MHC binding based on peptide property models constructed using biophysical parameters of the constituent amino acids and a training set of known binders. The models can be applied to development of anti-tumor vaccines by scanning proteins over-expressed in cancer cells for peptides that bind to a variety of MHC molecules. The complete algorithm is described and illustrated in the context of identifying candidate T cell epitopes for melanomas and breast cancers. We analyzed MART-1, S-100, MBP, and CD63 for melanoma and p53, MUC1, cyclin B1, HER-2/neu, and CEA for breast cancer. In general, proteins over-expressed in cancer cells may be identified using DNA microarray expression profiling. Comparisons of model predictions with available experimental data were assessed. The candidate epitopes identified by such a computational approach must be evaluated experimentally but the approach can provide an efficient and focused strategy for anti-cancer immunotherapy development.     

Ii-Key/HER-2/<Emphasis Type="Italic">neu</Emphasis>(776-90) hybrid peptides induce more effective immunological responses over the native peptide in lymphocyte cultures from patients with HER-2/<Emphasis Type="Italic">neu</Emphasis>+ tumors

We have demonstrated that coupling an immunoregulatory segment of the MHC class II-associated invariant chain (Ii), the Ii-Key peptide, to a promiscuous MHC class II epitope significantly enhances its presentation to CD4+ T cells. Here, a series of homologous Ii-Key/HER-2/neu(776-790) hybrid peptides, varying systematically in the length of the epitope(s)-containing segment, are significantly more potent than the native peptide in assays using T cells from patients with various types of tumors overexpressing HER-2/neu. In particular, priming normal donor and patient PBMCs with Ii-Key hybrid peptides enhances recognition of the native peptide either pulsed onto autologous dendritic cells (DCs) or naturally presented by IFN-gamma-treated autologous tumor cells. Moreover, patient-derived CD4+ T cells primed with the hybrid peptides provide a significantly stronger helper effect to autologous CD8+ T cells specific for the HER-2/neu(435-443) CTL epitope, as illustrated by either IFN-gamma ELISPOT assays or specific autologous tumor cell lysis. Hybrid peptide-specific CD4+ T cells strongly enhanced the antitumor efficacy of HER-2/neu(435-443) peptide-specific CTL in the therapy of xenografted SCID mice inoculated with HER-2/neu overexpressing human tumor cell lines. Our data indicate that the promiscuously presented vaccine peptide HER-2/neu(776-790) is amenable to Ii-Key-enhancing effects and supports the therapeutic potential of vaccinating patients with HER-2/neu+ tumors with such Ii-Key/HER-2/neu(776-790) hybrid peptides.