Specific inhibition of the reaction between a tumor-inhibitory antibody and the ErbB-2 receptor by a mimotope derived from a phage display library.

Overexpression of ErbB-2, a coreceptor for stroma-derived growth factors, is involved in malignancies of epithelial tissues, and a humanized antibody to ErbB-2 was shown to be therapeutic in a clinical setting. In an effort to understand and enhance immunotherapy, the laboratory has raised several tumor inhibitory monoclonal antibodies (mAb), including mAb L26 that blocks inter-receptor interactions. Here the application of the phage display methodology for the isolation of a phage clone that specifically recognizes mAb L26 is described. The isolated mimetic peptide (mimotope) specifically inhibited the binding of mAb L26 to ErbB-2 overexpressing cells. No sequence homology was found between the mimotope and ErbB-2. implying that it mimics a conformational structure of the receptor. Preliminary studies showed that the lead peptide can be truncated by removal of two to three amino acids from either the N- or C-terminal end without drastically affecting the inhibitory properties of the mimotope. A tryptophan'glycine residue at the C-terminus and a lysine at the N-terminus of the peptide seemed to play a role in its ability to compete with L26 antibody for binding to ErbB-2 overexpressing cells. These results highlight the potential of active immunization with conformation mimicking peptides in ErbB-2 overexpressing tumors.

Immunogenicity of H-2Kb-low affinity, high affinity, and covalently-bound peptides in anti-tumor vaccination.

CTL induction by immunization with synthetic peptide epitopes has been shown to inhibit tumor growth and its metastatic spread. Ex vivo pulsing of peptides on MHC class I-bearing cells such as RMA-S cells or professional APCs elicits an effective CTL response. Since the stability of the MHC-peptide complex is strongly correlated with the overall immunogenecity, we compared the effect of immunization with low affinity, high affinity, and irreversibly bound MHC peptides in the context of immunotherapy of metastasis. MUT1, a tumor-associated antigen peptide that was isolated from 3LL Lewis lung carcinoma, is a low H-2Kb binder. MUT1 was modified into a high binder by changing positions 3, 5, and 8 to the favorable anchor residues. In addition, we introduced a photo-active chemical moiety, which can bind irreversibly to MHC upon illumination. These peptides, loaded onto RMA-S, were used to immunize mice against the 3LL tumor. Vaccination via the covalent conjugation of the low binder peptide was found to increase the CTL response measured against MUT1 loaded cells and against H-2Kb transfected D122 cells relative to the native MUT1 peptide. However, the photo cross-linking of the high affinity peptide to the MHC did not significantly improve the induction of specific CTL. The level of CTL activity was elevated to the same extent by either cross-linking the peptide to the MHC or by modifying it into a high-binder peptide. The protective capacity of all the peptide-based vaccines against D122 metastatic spread to the lungs was found to be comparable. These results indicate that augmentation of the affinity of a TAA peptide to the RMA-S surface MHC molecules, by conversion to a high-affinity mimotope or by photo-conjugation, can significantly enhance the immune response. There seems to be, however, a ceiling beyond which increase in the peptide-binding affinity does not lead to a corresponding enhancement of the overall immunogenicity of the peptide.

Migration of adhesive and nonadhesive particles in the rat intestine under altered mucus secretion conditions.

The migration rate of adhesive (polycarbophil) and nonadhesive (Eudragit RL-100) particles was studied in the small and large intestine of the anesthetized rat under altered mucus secretion conditions accomplished by cholinergic stimulation (a previously developed in situ model which distinctly accounts for the effect of regional changes in mucus turnover rate on mucoadhesion in the digestive tube of the rat). It was found that in the proximal jejunum the relative recovery time (RRT) of adhesive particles, but not nonadhesive particles, was decreased by carbachol stimulation. However, adhesive particles agglomerated a short while after their administration into this organ. In the colon RRT of both adhesive and nonadhesive particles decreased in a similar manner as the mucus secretion increased. It is concluded that, in the rat, interactions between intestinal mucus layer and adhesive and nonadhesive particles are similar. The corresponding similarity in the intestinal transit time for both types of particles raises doubts about the advantage of nonspecific adherence in the design of oral prolonged-release dosage forms.

'1-8 interferon inducible gene family': putative colon carcinoma-associated antigens.

D(b-/-)xbeta2 microglobulin (beta2m) null mice transgenic for a chimeric HLA-A2.1/D(b)-beta2m single chain (HHD mice) are an effective biological tool to evaluate the antitumour cytotoxic T-lymphocyte response of known major histocompatibility-restricted peptide tumour-associated antigens, and to screen for putative unknown novel peptides. We utilised HHD lymphocytes to identify immunodominant epitopes of colon carcinoma overexpressed genes. We screened with HHD-derived lymphocytes over 500 HLA-A2.1-restricted peptides derived from colon carcinoma overexpressed genes. This procedure culminated in the identification of seven immunogenic peptides, three of these were derived from the 'human 1-8D gene from interferon inducible gene' (1-8D). The 1-8D gene was shown to be overexpressed in fresh tumour samples. The three 1-8D peptides were both antigenic and immunogenic in the HHD mice. The peptides induce cytotoxic T lymphocytes that were able to kill a colon carcinoma cell line HCT/HHD, in vitro and retard its growth in vivo. One of the peptides shared by all the 1-8 gene family primed efficiently normal human cytotoxic T lymphocyte precursors. These results highlight the 1-8D gene and its homologues as putative immunodominant tumour-associated antigens of colon carcinoma.

Mucus gel thickness and turnover in the gastrointestinal tract of the rat: response to cholinergic stimulus and implication for mucoadhesion.

The thickness of the mucus gel and its turnover rate were measured in the stomach, proximal jejunum, cecum and proximal colon of the rat, using microscopy and staining techniques. The specific mucus-secretory responses to carbachol-induced cholinergic stimulus in these locations were also studied. The mucus gel was found to be the thinnest (18 +/- 1 microns) in the cecum, and the thickest in the stomach (39 +/- 14 microns). The effect of carbachol on mucus secretion was profound and dose dependent in the stomach, and less profound, although still dose dependent, in the proximal jejunum. The least responsive organs were the cecum and the proximal colon, where no effect was observed after three doses of carbachol. Mucus secretion rate was significantly higher in the jejunum (1.1 +/- 0.5 microgram glucose equivalent min-1 cm-2) than in the colon (0.5 +/- 0.2 microgram glucose equivalent min-1 cm-2). Also, the proximal jejunum was more responsive to the carbachol stimulus (mucus secretion rate of 5.4 +/- 2.2 micrograms glucose equivalent min-1 cm-2 after carbachol treatment) than the colon (mucus secretion rate of 1.0 +/- 0.4 micrograms glucose equivalent min-1 cm-2 after carbachol treatment). In vitro mucoadhesion studies with Polycarbophil disks were performed in the mucosal tissues of the stomach, jejunum, cecum and proximal colon of the rat with and without cholinergic (carbachol) stimulus. The adhesion force in the cecum and the colon was significantly stronger than in the stomach and proximal jejunum when the studies were performed at pH 2.(ABSTRACT TRUNCATED AT 250 WORDS)

Antigenicity and immunogenicity of an intracellular delivery system of major histocompatibility complex class I epitopes that bypasses proteasome processing.

The development of a cell-free synthetic vaccine to induce an effective cytotoxic T lymphocyte response is an important challenge in T-cell--mediated immunity. Because standard vaccinations with nominal epitopes were found to be only partially effective in vivo, the authors suggest an alternative strategy: the delivery of epitopes directly to the cell cytosol in a proteasome bypass mechanism of processing. Two model peptides, the presentation level on the cell surface of which can be directly assessed, were conjugated via a cross-linker to an internalization peptide derived from an antennapedia homeobox protein. The linker was designed to undergo spontaneous hydrolysis, after which the epitope is subsequently released. The conjugates were shown to enter RMA and P815 cells, where the epitopes were released mainly in cytosol and endogenously loaded on the major histocompatibility complex class I molecules to be presented on the cell surface. Concomitant inhibition of proteasome activity by MG132 significantly increased the presentation level of both model peptides, indicating proteasome-independent processing. This phenomenon was exploited to enhance the immunogenicity of the conjugates. Conjugates were emulsified with MG132 in incomplete Freund's adjuvant and injected into mouse footpads. Analysis of the draining lymph nodes indicated an increase in the percentage of both CD4+ and CD8+ lymphocytes. In vitro cytolytic assays implied significant, albeit moderate, priming only when the proteasome inhibitor was administered with the conjugate. This approach may be useful for the development of efficient synthetic cell-free vaccines.

Induction of antitumor immunity by proteasome-inhibited syngeneic fibroblasts pulsed with a modified TAA peptide.

CTLs specific for tumor antigens play a major role in immunity against cancer. Improved binding affinity of putative TAA peptides could enhance the in vivo immunogenicity of these self-altered self- tumor antigens. We examined here the efficacy of tumor vaccines composed of an altered peptide ligand of MUT-1, designated MUT-D, which exhibited significantly higher class-I allele K(b) binding affinity than its native counterpart MUT-1. The peptide was loaded on antigen presenting cells composed of the C57BL/6-syngeneic fibroblast cell line BLK.CL4. These cells were treated with proteasome inhibitor in order to shut off the degradation of proteins and the subsequent loading of endogenous peptides onto MHC class-I molecules, thus allowing for the pulsing of these cells with the modified peptide MUT-D. Proteasome-inhibited and modified peptide-loaded fibroblasts induced a peptide-specific CTL that significantly delayed primary tumor progression and protected the pre-immunized mice against the development of lung metastasis following the surgical removal of the primary tumor. Genetic modification of the fibroblasts to express the immunostimulatory cytokine IL-2 did not improve the APC function of the modified cells, nor did it result in augmentation of the potency of the vaccine. Our results suggest that the proteasome-inhibited fibroblasts pulsed with modified, high binder tumor-associated antigen peptide are good antigen-presenting cells and represent an effective form of tumor vaccine.

MHC class I-restricted epitope spreading in the context of tumor rejection following vaccination with a single immunodominant CTL epitope.

Epitope spreading is a process whereby epitopes distinct from and non-cross-reactive with an inducing epitope become targets of an evolving immune response. This phenomenon has been associated most notably with the progression of naturally occurring or experimentally induced chronic autoimmune diseases. We have investigated the potential occurrence of epitope spreading in the context of antitumor cytotoxic T cell (CTL) responses using chicken ovalbumin (OVA) as a model antigen. Our results indicate that following rejection of OVA-expressing EG.7 tumor cells effectuated by a CTL response which is induced against the MHC class I-restricted immunodominant epitope OVA257-264, there occurs intramolecular diversification of the CTL response to two additional OVA-derived epitopes, OVA176-183 and OVA55-62, as well as intermolecular spreading to other endogenous tumor-derived determinants. It seems that CTL-mediated tumor cell destruction in vivo favors cross-presentation of additional epitopes with the consequent activation of additional tumor-reactive lymphocytes. The process of epitope spreading in that context has obvious important implications for the design of antigen-specific antitumor immunotherapies.

Novel breast-tumor-associated MUC1-derived peptides: characterization in Db-/- x beta2 microglobulin (beta2m) null mice transgenic for a chimeric HLA-A2.1/Db-beta2 microglobulin single chain.

The MUC1 protein was found to be up-regulated in a spectrum of malignant tumors. T-cell responses to the MUC1 extracellular tandem repeat array (TRA) were observed in murine models as well as in breast-carcinoma patients. In the present study, we evaluated the anti-tumor potential of HLA-A2.1-motif-selected peptides from non-TRA domains of the molecule. Peptide immunogenicity was examined in the Db-/- x beta2 microglobulin (beta2m) null mice transgenic for a modified HLA-A2.1/Db-beta2 microglobulin single chain (HHD mice). Our results show the existence of 3 novel HLA-A2.1-restricted MUC1-derived cytotoxic T-lymphocyte (CTL) epitopes. These peptides are processed and presented by the HHD-transfected breast-tumor cell line MDA-MB-157. Moreover, CTL induced by these 3 peptides show higher lysis of target cells pulsed with breast-carcinoma-derived peptides than of targets pulsed with normal breast-tissue-derived peptides. These data suggest an important role for non-TRA MUC1-derived peptides as inducers of a MHC-restricted CTL reaction to a breast-carcinoma cell line and patient-derived tumor extracts.

Anti-tumor vaccination in heterozygous congenic F1 mice: presentation of tumor-associated antigen by the two parental class I alleles.

Peptide vaccination of homozygous mice against syngeneic tumors using single major histocompatibility complex (MHC) class I-restricted cytotoxic T lymphocyte (CTL) epitopes elicits effective immune responses against metastatic growth. So far, single-peptide vaccination of patients against their autologous tumors seems to elicit less satisfactory results. In this study, the authors tried to determine whether effective anti-metastatic immunity requires the presentation of peptides restricted by the two parental class I major histocompatibility complex alleles in heterozygous hosts. The immune response against the H-2b-derived 3LL Lewis lung carcinoma was evaluated in heterozygous recombinant congenic F1 mice (Kk x K(b)) and (Kd x K(b)). Vaccination of such heterozygous animals with dendritic cells expressing the two parental H-2K alleles, pulsed with total tumor extract, elicited a potent anti-metastatic response. A comparable response was obtained after vaccination with tumor cells genetically modified to express the two class I alleles. In contrast, vaccination of the heterozygous mice with dendritic cells expressing only one of the parental F1 H-2K alleles or with tumors expressing only one H-2K allele failed to elicit effective immunity against tumor metastasis in recombinant congenic F1 mice. It appears, therefore, that to achieve effective anti-metastatic immunotherapy in heterozygous organisms, presentation of cytotoxic T lymphocyte epitopes restricted by the two parental class I alleles is required.

MAGE-A8 overexpression in transitional cell carcinoma of the bladder: identification of two tumour-associated antigen peptides.

Bladder carcinoma is the fourth most common cancer in men and the eighth most common cancer among women. Our study is aimed to characterise tumour-associated antigen peptides of transitional cell carcinoma of the bladder (TCC). A DNA micro-array-based differential display analysis of 10 000 genes was carried out, and MAGE-A8 gene expression was detected in the tumour, and not in the normal bladder. High occurrence of MAGE-A8 expression was observed in fresh tumour samples (17 out of 23) and TCC lines (four of eight). The MAGE-A8 protein sequence was screened for HLA-A2.1-binding motifs, six potential peptides were synthesised, and peptides binding to HLA-A2.1 were assured. Immunogenicity and antigenicity of the MAGE-A8 peptides were examined in the HHD system, murine class I MHC knockout mice, transgenic for HLA-A2.1. The MAGE-A8 peptide immunogenicity was examined in three modes of vaccination, delivered intranasally with cholera toxin, injected into the tail base with complete Freund's adjuvant (CFA), or presented directly as loaded onto cell surface HLA-A2.1 molecules. Two peptides, 8.1 and 8.3, induce CTL that kills the T24 TCC line in vitro, and prime human lymphocyte response of healthy donors. These results demonstrate the potential use of the MAGE-A8 peptides for specific immunotherapy of TCC.