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Supplementary Figures 1-3, Tables 1-3 from Synthetic MicroRNA Designed to Target Glioma-Associated Antigen 1 Transcription Factor Inhibits Division and Induces Late Apoptosis in Pancreatic Tumor Cells

Purpose: To determine whether the synthetic microRNAs (miRNA) could effectively target tumor cells we designed several miRNA complementary to glioma-associated antigen-1 (Gli-1) mRNA and investigated their ability to inhibit tumor cell proliferation. The sonic hedgehog pathway is an early and late mediator of tumorigenesis in epithelial cancers. Activation of sonic hedgehog signaling seems to precede transformation of tissue stem cells to cancerous stem cells, with the Gli-1 transcription factor functioning as a mediator of environmental signals. Inhibiting cancer cell proliferation by targeting the Gli-1 effector pathway is difficult to achieve by chemotherapeutic agents or short interfering RNA.Experimental Design: We hypothesized that targeting the 3′-untranslated region of Gli-1 mRNA would effectively inhibit tumor cell proliferation. To test this hypothesis, we used synthetic miRNAs of our own design and corresponding duplex/small temporal RNAs by introducing three-nucleotide loops in the 3′-untranslated region Gli-1 sequence of high GU content.Results: We found that miRNA (Gli-1-miRNA-3548) and its corresponding duplex (Duplex-3548) significantly inhibited proliferation of Gli-1+ ovarian (SK-OV-3) and pancreatic (MiaPaCa-2) tumor cells. The miRNAs mediated delayed cell division and activation of late apoptosis in MiaPaCa-2 cells. This is the first demonstration of inhibition of pancreatic tumor cell division by designed miRNA.Conclusions: Gli-1 miRNAs should significantly add to the general understanding of the mechanisms of metastasis and contribute toward the design of better treatments for epithelial cancers.

Supplementary Figures 1-3 from Taxol Increases the Amount and T Cell–Activating Ability of Self-Immune Stimulatory Multimolecular Complexes Found in Ovarian Cancer Cells

HLA-A2-restricted CTLs, which lysed high molecular weight (HMW)-melanoma-associated antigen (MAA)+ melanoma cells, were induced in patients with melanoma immunized with MELIMMUNE, a combination of the murine anti-idiotypic (anti-id) monoclonal antibodies (mAb) MEL-2 and MF11–30 (MW Pride et al., Clin Cancer Res 1998;4:2363.). In the present study we investigated whether CTL epitopes are present in anti-id mAb MF-11–30 and activate T cells to recognize HMW-MAA on melanoma cells. One candidate epitope in the mAb MF11–30 VH chain, VH (3–11), was selected based on the presence of HLA-A2 anchor residues and partial homology with the HMW-MAA epitope, HMW-MAA (76–84). Lymphocytes from HLA-A2+-immunized patients proliferated to VH (3–11) peptide and to a variant HMW-MAA peptide to a significantly greater extent than autologous lymphocytes stimulated with an irrelevant peptide and lymphocytes from nonimmunized patients. No proliferative response was detected to the wild-type HMW-MAA peptide (76–84). Significant increase in IFN-γ production but not in interleukin 10 production in response to VH (3–11) and to variant HMW-MAA peptide (76–84) was observed in lymphocytes from the immunized patients. Stimulation of lymphocytes from HLA-A2+ patients with the two peptides induced CTL, which lysed HMW-MAA+/HLA-A2+ A375SM melanoma cells. This is the first report documenting the presence of immunogenic peptides in a murine anti-id mAb for a defined epitope expressed by a human melanoma-associated antigen. These results may be relevant for development of novel vaccines based on homology between anti-id mAb and tumor-associated antigen amino acid sequences.

It has been proposed that chemotherapy enhances tumor antigen (TA)–specific immunity. The molecular form of TA from ovarian tumor that activates cellular immunity is unknown. We report here identification of a novel molecular form of immunogenic TA for CD8+ cells named self-immune stimulatory multimolecular complexes (ISMMC). ISMMC consist of a molecular complex of polyosome/ribosome-bound ubiquitinated nascent HER-2 polypeptides. This complex is chaperoned by heat shock protein Gp96, which mediates ISMMC uptake by antigen-presenting cells through the scavenger receptor CD91. RNAs in ISMMC stimulate immature dendritic cells to secrete interleukin 12 and induce IFN-γ in peripheral blood mononuclear cells. ISMMC dissociate, retrotranslocate from the lysosome to cytoplasm, and are processed to peptides by the proteasome. At subpharmacologic doses, Taxol increased the amount of ISMMC by three to four times and modified their composition by inducing the attachment of cochaperones of HSP70, such as the mitotic-phase phosphoprotein 11J. On a total protein basis, Taxol induced ISMMC, expanded more CD8+ cells, activated more CD56+ NKG2D+ cells to produce IFN-γ, and were more potent inducers of high T-cell receptor density Perforin+ cells than native ISMMC and peptide E75. Elucidation of the composition of ISMMC and identification of adducts formed by Taxol should be important for developing molecular cancer vaccines. [Cancer Res 2007;67(17):8378–87]

Supplementary Tables 1-5, Figure Legends 1-3 from Taxol Increases the Amount and T Cell–Activating Ability of Self-Immune Stimulatory Multimolecular Complexes Found in Ovarian Cancer Cells

BRAK/CXCL14 is a CXC chemokine constitutively expressed at the mRNA level in certain normal tissues but absent from many established tumor cell lines and human cancers. Although multiple investigators cloned BRAK, little is known regarding the physiologic function of BRAK or the reason for decreased expression in cancer. To understand the possible significance associated with loss of BRAK mRNA in tumors, we examined the pattern of BRAK protein expression in normal and tumor specimens from patients with squamous cell carcinoma (SCC) of the tongue and used recombinant BRAK (rBRAK) to investigate potential biological functions. Using a peptide-specific antiserum, abundant expression of BRAK protein was found in suprabasal layers of normal tongue mucosa but consistently was absent in tongue SCC. Consistent with previous in situ mRNA studies, BRAK protein also was expressed strongly by stromal cells adjacent to tumors. In the rat corneal micropocket assay, BRAK was a potent inhibitor of in vivo angiogenesis stimulated by multiple angiogenic factors, including interleukin 8, basic fibroblast growth factor, and vascular endothelial growth factor. In vitro, rBRAK blocked endothelial cell chemotaxis at concentrations as low as 1 nmol/L, suggesting this was a major mechanism for angiogenesis inhibition. Although only low affinity receptors for BRAK could be found on endothelial cells, human immature monocyte-derived dendritic cells (iDCs) bound rBRAK with high affinity (i.e., Kd, ∼2 nmol/L). Furthermore, rBRAK was chemotactic for iDCs at concentrations ranging from 1 to 10 nmol/L. Our findings support a hypothesis that loss of BRAK expression from tumors may facilitate neovascularization and possibly contributes to immunologic escape.

Supplementary Methods from Fine Specificity of High Molecular Weight-Melanoma-Associated Antigen-Specific Cytotoxic T Lymphocytes Elicited by Anti-Idiotypic Monoclonal Antibodies in Patients with Melanoma

Supplementary Figure 1 from BRAK/CXCL14 Is a Potent Inhibitor of Angiogenesis and a Chemotactic Factor for Immature Dendritic Cells

Bacterial cell wall polysaccharides, such as PGN, bind and activate TLR-2, NOD2 and PGRP on monocytes/macrophages and activate inflammation. We found that the peptides containing basic amino acids (cations) at N -terminus and tyrosine at C-terminus interfered with activating ability of PGN. This finding is significant because the ECD of TLR-2 in vivo encounters a large number of proteins or peptides. Some should bind ECD and "pre-form" TLR-2 to respond or not to its activators, although they cannot activate TLR-2 alone. TLR-2 is receptor for a large number of ligands, including lipopeptides and bacterial cell wall glycoproteins. A binding site for lipopeptides has been identified; however, a binding site for soluble or multimeric PGN has not been proposed. To identify the candidate binding sites of peptides and PGN on TLR-2, we modeled docking of peptides and of the PGN monomer (PGN-S-monomer) to extracellular domain (ECD-TLR-2) of the unbound TLR-2. Quantification, in silico, of free energy of binding (DG) identified 2 close sites for peptides and PGN. The PGN-S-monomer binding site is between amino acids TLR-2, 404-430 or more closely TLR-2, 417-428. The peptide-binding site is between amino acids TLR-2, 434-455. Molecular models show PGN-S-monomer inserts its N -acetyl-glucosamine (NAG) deep in the TLR-2 coil, while its terminal lysine interacts with inside (Glu(403)) and outside pocket (Tyr(378)). Peptides insert their two N -terminal arginines or their C-terminal tyrosines in the TLR-2 coil. PGN did not bind the lipopeptide-binding site in the TLR-2. It can bind the C-terminus, 572-586 (DG = 0.026 kcal), of "lipopeptide-bound" TLR-2. An additional, low-affinity PGN-binding site is TLR-2 (227-237). MTP, MDP, and lysine-less PGN bind to TLR-2, 87-113. This is the first report identifying candidate binding sites of monomer PGN and peptides on TLR-2. Experimental verification of our findings is needed to create synthetic adjuvant for vaccines. Such synthetic PGN can direct both adjuvant and cancer antigen to TLR-2.

Many viral oncolytic approaches against cancer are based on the ability of specific viruses to replicate in tumors expressing components of the constitutively activated Ras/mitogen-activated protein kinase (MAPK) pathways and/or inhibited or dysregulated alpha/beta interferon (IFN-α/β) response pathways. A major issue when considering these approaches is their applicability to tumors that lack activated Ras. To identify the effector mechanisms activated by oncolytic viruses, we investigated inhibition of proliferation of the prostate cancer line LNCap by the recombinant TR-NS1 influenza A virus, a genetically attenuated influenza A/PR8/34 virus expressing a truncated nonstructural protein (NS1) of 126 amino acids. LNCap cells lack constitutively activated MAPK, extracellular signal-regulated kinase (ERK), and p38 and are resistant to death by IFN-α. Truncation of the NS1 protein of influenza viruses is known to result in viral attenuation due to a reduced ability of the NS1 to inhibit the IFN-α/β response. Infection with TR-NS1 virus rapidly activated ERK-1 more than ERK-2 in LNCap cells. Importantly, TR-NS1 virus infection transiently inhibited cell proliferation and induced apoptosis in LNCap cells. Addition of peripheral blood mononuclear cells (PBMC) and interleukin 12 (IL-12) to TR-NS1 virus-infected LNCap cells (TR-NS1-LNCap) resulted in faster elimination of TR-NS1-LNCap cells compared with LNCap cells. Moreover, TR-NS1-LNCap cells induced IFN-γ in PBMC. The levels of IFN-γ were amplified by IL-12. TR-NS1-LNCap cells also induced tumor-lytic cytotoxic T lymphocytes (CTL). These CTL lysed noninfected LNCap cells in a CD8-dependent manner. Activation of cellular immunity to tumor cells by viruses is an intriguing effector pathway, which should be especially significant for elimination of human tumors that lack activated Ras.

Purpose E75 is an immunogenic peptide from the HER2/neu protein that is highly expressed in breast cancer. We are conducting a clinical trial of an E75 + granulocyte-macrophage colony-stimulating factor vaccine to assess safety, immunologic response, and the prevention of clinical recurrences in patients with disease-free, node-positive breast cancer (NPBC). Patients and Methods Fifty-three patients with NPBC were enrolled and HLA typed. HLA-A2+ patients (n = 24) were vaccinated, and HLA-A2− patients (n = 29) are observed prospectively as clinical controls. Local/systemic toxicities, immunologic responses, and time to recurrence are being measured. Results Only minor toxicities have occurred (one grade 3 [4%]). All patients have demonstrated clonal expansion of E75-specific CD8+T cells that lysed HER2/neu-expressing tumor cells. An optimal dosage and schedule have been established. Patients have developed delayed-type hypersensitivity reactions to E75 postvaccination compared with controls (33 v 7 mm; P < .01). HLA-A2+ patients have been found to have larger, more poorly differentiated, and more hormonally insensitive tumors compared to HLA-A2− patients. Despite this, the only two deaths have occurred in the control group. The disease-free survival in the vaccinated group is 85.7% compared to 59.8% in the controls at 22 months' median follow-up with a recurrence rate of 8% compared to 21%, respectively (P < .19). Median time to recurrence in the vaccinated patients was prolonged (11 v 8 months), and recurrence correlated with a weak delayed-type hypersensitivity response. Conclusion This HER2/neu (E75) vaccine is safe and effective in eliciting a peptide-specific immune response in vivo. Induced HER2/neu immunity seems to reduce the recurrence rate in patients with NPBC.

Antiidiotypic antibodies have been and are being used for cancer immunotherapy based on the rationale that Ab2 carrying an “internal image” of the corresponding tumor antigen can induce tumor antigen–specific antibodies (i.e., Ab3 and inhibit tumor growth). Recent evidence indicates that Ab2 also induces cellular responses by CD4+ and CD8+ T cells. This finding has raised the question of where the short peptides, which express CD8+ T-cell–defined epitopes, are located and their relationship with the tumor antigen. We found that two of the four known Ab2 associated with tumor antigen, with known amino acid sequence, express unique NH2-terminal VH sequences which precede the framework regions. Both the unique and the shared NH2-terminal VH sequences are nested MHC class I antigen–binding peptides. These peptides were highly homologous with peptides from corresponding tumor antigen (carcinoembryonic antigen, CD55, and human high molecular weight melanoma–associated antigen) but differed from the tumor antigen peptides by the presence of the side chain known to mediate stronger forces of interaction with other atoms. The presence of candidate CTL epitopes in NH2-terminal VH of Ab2 homologous with tumor antigen may be important for the development of novel immunotherapeutic strategies for cancer.

HLA-A2-restricted CTLs, which lysed high molecular weight (HMW)-melanoma-associated antigen (MAA)+ melanoma cells, were induced in patients with melanoma immunized with MELIMMUNE, a combination of the murine anti-idiotypic (anti-id) monoclonal antibodies (mAb) MEL-2 and MF11–30 (MW Pride et al., Clin Cancer Res 1998;4:2363.). In the present study we investigated whether CTL epitopes are present in anti-id mAb MF-11–30 and activate T cells to recognize HMW-MAA on melanoma cells. One candidate epitope in the mAb MF11–30 VH chain, VH (3–11), was selected based on the presence of HLA-A2 anchor residues and partial homology with the HMW-MAA epitope, HMW-MAA (76–84). Lymphocytes from HLA-A2+-immunized patients proliferated to VH (3–11) peptide and to a variant HMW-MAA peptide to a significantly greater extent than autologous lymphocytes stimulated with an irrelevant peptide and lymphocytes from nonimmunized patients. No proliferative response was detected to the wild-type HMW-MAA peptide (76–84). Significant increase in IFN-γ production but not in interleukin 10 production in response to VH (3–11) and to variant HMW-MAA peptide (76–84) was observed in lymphocytes from the immunized patients. Stimulation of lymphocytes from HLA-A2+ patients with the two peptides induced CTL, which lysed HMW-MAA+/HLA-A2+ A375SM melanoma cells. This is the first report documenting the presence of immunogenic peptides in a murine anti-id mAb for a defined epitope expressed by a human melanoma-associated antigen. These results may be relevant for development of novel vaccines based on homology between anti-id mAb and tumor-associated antigen amino acid sequences.

The development of cancer vaccines requires approaches to induce expansion and functional differentiation of tumor antigen-specific cytotoxic T lymphocyte (CTL) effectors which posses cytolytic capability and produce cytokines. Efficient induction of such cells is hindered by the poor immunogenicity of tumor antigens and by the poor transduction efficiency of dendritic cells (DCs) with current nonreplicating vectors. We have investigated the use of influenza A virus, a potent viral inducer of CTLs, as a vector expressing the immunodominant HER-2 CTL epitope KIF (E75). For this purpose, an attenuated influenza A/PR8/34 virus with a truncated nonstructural (NS1) gene was generated containing the E75 epitope in its neuraminidase protein (KIF-NS virus). Stimulation of peripheral blood mononuclear cells from healthy donors and of tumor-associated lymphocytes from ovarian and breast cancer patients with DCs infected with KIF-NS virus (KIF-NS DC) induced CTLs that specifically recognized the peptide KIF and HER-2-expressing tumors in cytotoxicity assays and secreted gamma interferon (IFN-γ) and interleukin-2 at recall with peptide. Priming with KIF-NS DCs increased the number of E75 + CD45RO + cells by more than 10-fold compared to nonstimulated cells. In addition, KIF-NS virus induced high levels of IFN-α in DCs. This is the first report demonstrating induction of human epitope-specific CTLs against a tumor-associated antigen with a live attenuated recombinant influenza virus vector. Such vectors may provide a novel approach for tumor antigen delivery, lymphocyte activation, and differentiation in human cancer vaccine development.

To design side chain variants for modulation of immunogenicity, we modeled the complex of the HLA-A2 molecule with an immunodominant peptide, E75, from the HER-2/neu protooncogene protein recognized by CTL. We identified the side chain orientation of E75. We modified E75 at the central Ser5 (E75 wild-type), which points upward, by removing successively the HO (variant S5A) and the CH2-OH (variant S5G). Replacement of the OH with an aminopropyl (CH2)3-NH3 (variant S5K) maintained a similar upward orientation of the side chain. S5A and S5G were stronger stimulators while S5K was a weaker stimulator than E75 for induction of lytic function, indicating that the OH group and its extension hindered TCR activation. S5K-CTL survived longer than did CTL induced by E75 and the variants S5A and S5G, which became apoptotic after restimulation with the inducer. S5K-CTL also recognized E75 endogenously presented by the tumor by IFN-γ production and specific cytolysis. S5K-CTL expanded at stimulation with E75 or with E75 plus agonistic anti-Fas mAb. Compared with S5K-CTL that had been restimulated with the inducer S5K, S5K-CTL stimulated with wild-type E75 expressed higher levels of E75+ TCR and BCL-2. Activation of human tumor-reactive CTL by weaker agonists than the nominal Ag, followed by expansion with the nominal Ag, is a novel approach to antitumor CTL development. Fine tuning of activation of tumor-reactive CTL by weak agonists, designed by molecular modeling, may circumvent cell death or tolerization induced by tumor Ag, and thus, may provide a novel approach to the rational design of human cancer vaccines.

HER-2/neu peptides have recently been shown to induce a proliferative response by peripheral CD4(+) T cells in breast cancer patients. To investigate potential differences in the local cellular immune response between breast cancer patients with and without nodal metastases, lymphocytes were isolated from axillary lymph nodes from patients with breast cancer, and proliferative and cytokine responses to HER-2/neu peptides were determined. Freshly isolated lymphocytes from lymph nodes of 7 women undergoing surgery for invasive breast cancer were plated at 20 x 10(5) cells per well in triplicate. Cells were stimulated with HER-2/neu peptides at 50 microg/ml and with control antigens. Incorporation of tritium-labeled thymidine was determined 4 days later. The levels of the cytokines interferon-gamma (IFN-gamma), interleukin-4 (IL-4), and IL-10 were determined at priming and at restimulation with HER-2/neu peptides using a cytokine-specific, double-sandwich, enzyme-linked immunosorbent assay (ELISA). Lymphocytes isolated from the axillary lymph nodes of the patients mounted significant cellular immune response to HER-2/neu peptides, manifested by proliferation and specific cytokine elaboration. Proliferative responses to HER-2/neu peptides were seen in lymphocytes of patients with and without overexpression of HER-2/neu in the primary tumor. In some patients, the proliferative response to HER-2/neu peptides in lymphocytes from lymph nodes with metastases was absent or blunted compared with the response in lymphocytes from lymph nodes without metastases from the same patient (p0.05). HER-2/neu peptides induced a predominantly T helper type 1 (Th1) pattern of cytokine response in nodal lymphocytes isolated from breast cancer patients. A Th1-specific cytokine production pattern was maintained at priming and restimulation with HER-2/neu peptides and was amplified with IL-12 costimulation. These results indicate that HER-2/neu peptides can activate T cells in draining lymph nodes from women with invasive breast cancer. This activation is associated with a predominantly Th1 cytokine response, which suggests that conditioning with HER-2/neu peptides may be of value in the development of breast cancer vaccines.

We investigated the ubiquitination and degradation of a tumor antigen, the HER-2/neu (HER-2) protooncogene product which is overexpressed in epithelial cancers. HER-2 degradation was investigated in the ovarian tumor line, SKOV3.A2, that constitutively overexpressed long-life HER-2. We used as agonist geldanamycin (GA), which initiated downmodulation of HER-2 from the cell surface. HER-2 was polyubiquitinated and degraded faster in the presence than in the absence of GA. GA did not decrease HLA-A2 expression. Presentation of the immunodominant cytotoxic T lymphocyte (CTL) epitope, E75 (369–377) from SKOV.A2 was inhibited by proteasome inhibitors, such as LLnL but was enhanced by cysteine protease inhibitors such as E64, indicating that both the proteasome and cysteine proteases are involved in epitope formation but have different effects. Enhanced tumor recognition was not an immediate or early effect of GA treatment, but was evident after 20 h of GA treatment. In contrast, 20 h GA treatment did not increase tumor sensitivity to LAK cell lysis. Twenty hour GA-treated SKOV3.A2 cells expressed an unstable HER-2 protein synthesized in the presence of GA, of faster electrophoretic mobility than control HER-2. This suggested that the newly synthesized HER-2 in the presence of GA was the main source of epitopes recognized by CTL. Twenty hour GA-treated SKOV3.A2 cells were better inducers of CTL activity directed to a number of HER-2 CTL epitopes, in peripheral blood mononuclear cells compared with control untreated SKOV3.A2 cells. Thus, induction of HER-2 protein instability enhanced the sensitivity of tumor for CTL lysis. Increased HER-2 CTL epitopes presentation may have implications for overcoming the poor immuno-genicity of human tumors, and design of epitope precursors for cancer vaccination.

CXC chemokines play an important role in recruitment of T cells to the site of activation and regulation of angiogenesis. CXC chemokines are secreted by T cells stimulated with cytokines or by established cytotoxic T lymphocyte (CTL) lines at recognition of conventional antigen (Ag), but the activation requirements and the relationship of interferon-gamma (IFN-gamma) inducible protein (IP-10) secretion with IFN-gamma induction in lymphocytes are still unclear. We studied the induction of IP-10 from nonadherent peripheral blood mononuclear cells (PBMC) by IFN-gamma, interleukin-12 (IL-12), and the HER-2 peptide E75, which forms a CTL-defined antigen. We found that IFN-gamma alone was a weak inducer of IP-10 in these cells, whereas IL-12 was a significantly stronger inducer of IP-10. In the presence of IL-12, the tumor peptide E75 (HER-2, 369-377) was a stronger inducer of IP-10 than was IL-12 alone. E75 and its variants mutated at position 5 could also induce IP-10 in the absence of exogenous IL-12 or IFN-gamma. IP-10 induction by E75 required HLA-A2 presentation and B7-CD28 interactions and was partially inhibited by blocking of CD40-CD40L interactions. These results indicate that presentation of tumor peptides to peripheral T cells can induce a fast chemokine response, which in its early phase may be higher than the IFN-gamma response. This shows that the IP-10 response was independent of any early-phase IFN-gamma response in peripheral T cells. This may be important for understanding the regulation of the balance between chemoattractant chemokines (CC) and CXC chemokines by tumor Ag and may have implications for understanding the mechanisms of polarization of T cells and conditioning of antigen-presenting cells (APC) by tumor antigens.

Summary: Class I expression in context with T-cell receptor expression is crucial for peptide presentation and induction of CD8+ cytotoxic T lymphocytes (CTL). Presentation of class I bound peptides is dependent on transporter-associated proteins (TAP) expression and function. Tumor infiltrating lymphocytes from a patient with melanoma were isolated, expanded in vitro in the presence of interleukin-2, and tested for cytotoxicity against HLA-A2 positive, MART-1 positive autologous tumor cells, an HLA-A2-positive, MART-1 positive melanoma cell line (Mel-501), and HLA-A2-negative melanoma cells. Significant killing occurred against both A2-positive cell lines (63% and 65%, respectively), but not against the A2-negative line (18%) or A2-positive autologous tumor (1.5%). These CTL preferentially recognized the MART-1 peptide F119, 27–35, and gp100 peptide F125, 280–288, resulting in a 30% to 60% enhancement of lysis when autologous tumor or major histocompatibility complex class I “empty” T2 cells were pulsed with either peptide. To address whether the deficiency in autologous tumor recognition might be related to a deficiency in Ag presentation, we screened for the presence of TAP1 and TAP2 transcripts by polymerase chain reaction, Southern blotting, and scanning densitometry using sequence-specific primers and probes. Both TAP1 and TAP2 expression levels in the autologous tumor were minimal, yet were upregulated 7-to 18-fold, respectively, by interferon-γ. Despite this increase, a similar increase in cytotoxicity did not occur. In short, deficiencies in TAP presentation may have functional significance for tumor escape from immunosurveillance and with respect to impending vaccine trials.

Although natural killer (NK) cells have been described as non-MHC-restricted, new evidence suggests that NK activity can be either up- or down-regulated after interaction with the peptide-MHC-class-I complex expressed on target cells. However, the epitope(s) recognized by NK cells have remained ill-defined. We investigated NK cell recognition of synthetic peptides representing a portion of a self-protein encoded by the HER-2/neu (HER-2) proto-oncogene and presented by HLA-A2. HER-2 nonapeptides C85, E89, and E75 were found partially to protect T2 targets from lysis by freshly isolated and interleukin-2(IL-2)-activated NK cells (either HLA-A2(+) or A2(-)). This inhibition was not solely due to changes in the level of HLA-A2 expression or conformation of serological HLA-A2 epitopes. Using single-amino-acid variants at position 1 (P1) of two HER-2 peptides, we observed that protection of targets was dependent on the sequence and the side-chain. These results suggest similarities in the mechanism of target recognition by NK and T cells. This information may be important for understanding the mechanisms of tumor escape from immunosurveillance and could help explain the aggressiveness of HER-2-overexpressing tumor cells.

Resveratrol, a polyphenolic natural product abundantly present in grape skins, is a candidate cancer chemopreventive agent that antagonizes each stage of carcinogenesis and inhibits protein kinase C (PKC), a key mediator of tumor promotion. While resveratrol has been shown to antagonize both isolated and cellular forms of PKC, the weak inhibitory potency observed against isolated PKC cannot account for the reported efficacy of the polyphenol against PKC in cells. In this report, we analyze the mechanism of PKC inhibition by resveratrol. Our results indicate that resveratrol has a broad range of inhibitory potencies against purified PKC that depend on the nature of the substrate and the cofactor dependence of the phosphotransferase reaction. Resveratrol weakly inhibited the Ca2+/phosphatidylserine-stimulated activity of a purified rat brain PKC isozyme mixture (IC(50) = 90 microM) by competition with ATP (K(i) = 55 microM). Consistent with the kinetic evidence for a catalytic domain-directed mechanism, resveratrol inhibited the lipid-dependent activity of PKC isozymes with divergent regulatory domains similarly, and it was even more effective in inhibiting a cofactor-independent catalytic domain fragment (CDF) of PKC generated by limited proteolysis. This suggested that regulatory features of PKC might impede resveratrol inhibition of the enzyme. To explore this, we examined the effects of resveratrol on PKC-catalyzed phosphorylation of the cofactor-independent substrate protamine sulfate, which is a polybasic protein that activates PKC by a novel mechanism. Resveratrol potently inhibited protamine sulfate phosphorylation (IC(50) = 10 microM) by a mechanism that entailed antagonism of the activation of PKC by protamine sulfate and did not involve competition with either substrate. On the basis of the presence of PKC isozymes at subcellular sites rich in polybasic proteins, it has been proposed that certain endogenous polybasic PKC substrates may activate PKC in cells by the same mechanism as protamine sulfate. Our results suggest that antagonism by resveratrol of the phosphorylation of cellular PKC substrates that resemble protamine sulfate in their interactions with PKC may contribute to the efficacy of resveratrol against PKC in cells.

Synthetic peptide analogues of sequences in the HER-2 protooncogene (HER-2) were selected based on the presence of HLA-A2.1 anchor motifs to identify the epitopes on HER-2 recognized by ovarian tumor-reactive CTL. 19 synthetic peptides were evaluated for recognition by four HLA-A2 ovarian-specific cytotoxic T lymphocyte (CTL) lines obtained from leukocytes associated with ovarian tumors. The nonapeptide E75 (HER-2, 369-377:KIFGSLAFL) was efficient in sensitizing T2 cells for lysis by all four CTL lines. This peptide was specifically recognized by cloned CD8+ CTL isolated from one of the ovarian-specific CTL lines. E75-pulsed T2 cells inhibited lysis by the same CTL clone of both an HLA-A2+ HER-2high ovarian tumor and a HER-2high cloned ovarian tumor line transfected with HLA-A2, suggesting that this or a structurally similar epitope may be specifically recognized by these CTL on ovarian tumors. Several other HER-2 peptides were recognized preferentially by one or two CTL lines, suggesting that both common and private HER-2 epitopes may be immunogenic in patients with ovarian tumors. Since HER-2 is a self-antigen, these peptides may be useful for understanding mechanisms of tumor recognition by T cells, immunological tolerance to tumor, and structural characterization of tumor antigens.

Novel therapeutic approaches to eliminate cancer stem cells (CSCs) are being developed. This development is imperative as CSCs are resistant to drugs; they divide activated by ligands on the epithelium or on neighboring cancer cells. Specific commands for division originate from Notch-1 ligands. Notch-1 cleavage inhibitors can have opposite effects from the ones expected when the levels of Notch ligands are high on neighboring cancer cells. High levels of Jagged-1 are a common feature of ovarian tumors. Some gene pathways enhance, others repress transcription of Notch-1, while Notch-1 itself activates Myc and HIF-1α. RNA-based therapies need effector RNAs (eRNAs) with broad and focused specificity. eRNAs are short RNAs (20-30 nt long) which mediate biological effects. Two to three inhibitory RNAs with high net folding/hybridization/binding (and thereafter folding), and free energy (Net-ΔG) with multiple mRNAs can replace many miRs as eRNAs and overcome the complexity of identification of specific targets for each miR and competitive inhibition on delivery of small amounts of many miRs at the same time. To discover candidate eRNAs with multiple high affinity target sites or sequences (and thereafter targets), we searched for sequences containing more than randomly probable G and C. G and C bind with more hydrogen bonds than the pair A:T. We identified the sequence, Notch-1,33-56 in the ORF of Notch-1 mRNA. Notch-1,33-56 has a GC frame of 2 asymmetrical halves in 24 nucleotides. Each GC group has a different third nucleotide. Since GC is repeated, the third nucleotide defines the specificity as a 'bar code'. The complementary strand to Notch-1,33-56, binds in silico nt at 5'-UTR, ORF and 3'-UTR of mRNA. For simplification, the sequence of Notch-1,33-56 was designated HHN1 and its complementary strand, anti-HHB. We introduced novel quantitative parameters: Net-ΔG and mean Net-ΔG/bond. We quantified the Net-ΔG of folding, in silico, of anti-HHB with additional targets in Notch-1,1-404. The targets of anti-HHB contained 11-12 complementary nucleotides and formed small loops with anti-HHB upon folding. Anti-HHB folded with 3-4 distinct targets in each mRNA from 50 mRNAs. Targets were in 5'-UTR (40%), ORF (50%) and 3'-UTR (10%). Anti-HHB also folded with high Net-ΔG with Notch-1 targets, c-Myc and HIF-1α, suggesting it can inhibit EMT. Human embryonal stem cell (hESC) miRs, 1909 and 1915, folded with Notch-1,8-29 and Notch-1,33-56, respectively with a similar Net-ΔG as anti-HHB. This finding suggested a natural feedback mechanism aiming to inhibit Notch-1 translation which is activated in stem cells by miRs with a similar sequence as anti-HHB, and anti-HHB can be used when the miRs 1915 and 1909 are absent. The consensus sequence of 18 targets folded with HHB with the highest Net-ΔG (range -10.20 to 24.00 Kcal/mol) similar to that of two Drosophila transposons. Targeting 'domesticated transposons' carried by humans with eRNAs may become a universal approach to treat cancer. Anti-HHB is the first candidate eRNA to fold, in silico, with multiple targets in 5'-UTR and ORF of Notch-1 partners with at least 2-times higher ΔG than natural miRs with 3'-UTR Notch-1.

Notch and Hedgehog activate cell-cycle progression of adult and cancer stem cells. Notch is activated by DLL and Jag presents on neighboring cells. We investigated the effects of density of the Notch-activating ligand, Jag-1, and targeting Gli-1, in activation of division of paclitaxel/taxol-resistant, (PTX Res) ovarian cancer cells SKOV3 (SKOV3). We used the specific gamma-presenilin inhibitor, DAPT, to identify the specificity of activating signals for Notch-1 and created 'butterfly-duplex-3548-Gli-1-inhibitory RNA' (i-Gli-1.RNA) to inhibit cell division. To accurately quantify kinetics of division, the expression of CD44 and CD24 was determined in each gated population of divided cells. CD44 High proliferated when activated by Jag-1 Low and poorly when activated by Jag-1 High. DAPT inhibited proliferation of cells activated by Jag-1 Low, and increased proliferation of cells activated by Jag-1 High. Only 5-10% of cells activated by Jag-1 High and Jag-1 Low divided fast, polynomial, and symmetric. i-Gli-1.RNA eliminated more than 50% of the small CD44 High/CD24 Neg cells in divisions 3 and 4. This effect appeared specific compared with cells transfected with negative control siRNA. i-Gli-1.RNA had no effect on large CD44 High/CD24 Neg cells, but inhibited the population of CD44 High/CD24 Low cells. Expansion of CD44 High inversely correlated with Jag-1 density on activating autologous tumor and fibrosarcoma cells. Created i-RNAs may decrease the resting CSC pool. Notch and Gli-1 signals play an important role in proliferation/division and survival of cancer stem cells. Targeting Notch-1 through its enhancer Gl-1, should be significant for novel treatments to eliminate taxol-resistant cancer stem cells (CSC). i.Gli-1 RNA should be more effective if used together with Taxol.

The expression of gamma/delta T cell antigen receptors (TcR) in T cell lines or clones derived from tumor-infiltrating lymphocytes (TIL) from patients with solid tumors was investigated. gamma/delta TcR T cell lines were derived from TIL from patients with Wilms tumor, sarcoma or metastatic melanoma by stimulation with autologous tumor cells alone and recombinant interleukin 2 and they exhibited nonspecific cytotoxicity against autologous and allogeneic tumor cells, or cells of the K562 or the MEL21 tumor cell lines. Two T cell lines were derived from a patient with Wilms tumor. One of them expressed a non-disulfide-linked gamma/delta TcR using the 60-kDa gamma chain, whereas, the other expressed a disulfide-linked gamma/delta TcR. A T cell line was derived from a patient with sarcoma and expressed a disulfide-linked gamma/delta TcR, whereas, a T cell line derived from a patient with melanoma expressed a non-disulfide-linked gamma chain of 62 kDa. Several T cell clones were developed from patients with metastatic melanoma or Wilms tumor and expressed either disulfide- or non-disulfide-linked gamma/delta TcR. Northern analysis of RNA from certain of these clones revealed a full-length gamma chain transcript, whereas, the alpha or beta chain transcripts were either absent or truncated. These T cell clones exhibited nonspecific cytotoxicity. Both disulfide- and non-disulfide-linked TIL T cell lines and clones expressed the delta TCS1 determinant. gamma/delta TcR+ cells in freshly prepared TIL from these patients were present in low proportions (less than 5%) and their delta TCS1/delta 1 ratios were within the range observed in the peripheral blood of normal donors. These results demonstrate that both disulfide- and non-disulfide-linked gamma/delta TcR are expressed on T cell lines and clones derived from TIL from solid tumors. Non-disulfide-linked gamma/delta TcR using the 56-66-kDa gamma chain are frequently found on TIL-derived T cell lines and clones. These 56-66-kDa gamma chains are rarely expressed on T cell lines or clones derived from peripheral blood lymphocytes of normal donors.

The objective of this study was to discover whether the peptides LRMK and LRMK-Ava linked to the N-terminus of peptides HER-2 (774-788) and HER-2 (776-788), respectively, help differentiation of E75-TCR(+)CD8(+) cells. Activation was quantified in terms of proliferation of E75-TCR(+)CD8(+) cells expressing high, medium and low density amounts of the specific TCR. Differentiation to functional CD8(+) cells was quantified as induction of Perforin (Perf), the lytic-enzyme which mediates the effector function of CD8(+) cells, in E75-TCR(+)CD8(+) cells. Peripheral blood mononuclear cells (PBMCs) of 3 patients activated with E75(+)AE-37 and E75(+)AE-47 more greatly increased the number of E75-TCR(Hi) CD8(+)Perf(+) cells than PBMCs activated by AE-47 alone or AE-47(+) E75. E75 plus cytokines and cytokines alone activated more E75-TCR(Low) cells than did AE-37 and AE-47. E75(+) AE-37 and AE-37 also induced differentiation of small- and medium-size activated CD8(+) cells from BRC ascites, in allogeneic activation, to Perf(+) cells. Preferential differentiation of E75-TCR(+)CD8(+)Perf(+) cells in distinct patients by AE-37 and AE-47 indicates that cancer vaccines will benefit from such correct individual and disease-associated help. Additional studies using the natural peptides p776 and F7 are needed to understand whether the LRMK-(Ava) tetra-, or pentamer augments or inhibits differentiation of CD8(+) cells, compared with native, natural HER-2 peptides and/or protects CD8(+) cells activated by E75 and by other HLA-I bound peptides from death. Our findings also develop a model for uniform quantification of differentiated CD8(+) effectors.

The objective of this study was to find whether the peptide LRMK linked to the N-terminus of HER-2, 774-788 and shorter peptides create a T helper antigen, which can replace all functional HER-2 peptides in a cancer vaccine. Of the 6 LRMK-HER-2 peptides tested in the presence of IL-12, AE-37, AE-38 and AE-39 induced higher IFN-gamma in PBMCs from 4 healthy donors than the other peptides. AE-37 and AE-39 contained the immunogenic HER-2 peptide, p776 (774-788), while AE-38 contained the truncated HER-2 peptide G89 (777-788). The free, unprotected HER-2 peptide G89 (777-789) activated IFN-gamma production in PBMCs from another BRC patient. Responses to free p776 and F7 could not be tested. Three out of 11 patients responded strongly only to AE-37, while 3 of 10 patients responded strongly only to G89. One responded only to AE-39. All 3 patients diagnosed with DICS of mixed HER(hi), ER+ PR+ type responded to AE-37. Three out of 4 patients diagnosed with luminal cancer and one HERhi, ER- PR- BRC patient responded only to G89. AE-37, at low concentration, helped to expand more E75-TCR(Hi+Med) cells than the negative control AE-47, for IFN-gamma induction AE-47, but was a weaker helper than AE-47 at higher concentrations, and eliminated E75-TCR(Hi) cells. The strongest and most consistent effect of AE-37 was the elimination of CD4(Hi) CD25(Hi) cells. When LRMK is linked to AE-37, the side-chains of L(P-10) and R(P-9) are positioned away from MHC; LRMK forms a bi-strophynx (rotating-double-hook-like) structure when attached to AE-37 and the minimum HER-2 peptide in the peptide-binding groove (PBG). K(P-8) anchors the bi-strophynx to HLA-DR outside the PBG in a novel site DRalpha, 49-52. The HER-2 amino acids, Y(P-1) and R(P3) point towards TCR; R(P-9) and M(P-8) can contact the TCRValpha1. The positions of K(P-8) in AE-37 and Ava (epsilonV)(P-8) in AE-39 modulated immunogenicity of p776. It is unknown whether LRMK adds TCR contact points to the minimal HLA-DR-bound HER-2 peptide (780-788) or activates T-cells of other specificities to produce cytokines and die. Preferential activation of Th1 cells in distinct individuals by AE-37, G89 and AE-39 indicates that cancer vaccines will benefit from correct individual and disease-associated help. Emergence of distinct daughters of luminal and myoepithelial BRC stem cells during metastasis through "de-differentiation" and "reverse differentiation" of drug-resistant cancer requires personalized vaccines, which use an optimal-helper antigen.

The transcription factor glioma-associated antigen-1 (Gli-1) mediates activation of the sonic hedgehog (Shh) pathway, a process that precedes the transformation of tissue stem cells into cancerous stem cells and that is involved in early and late epithelial tumorigenesis. Hypothesizing that targeting the 3'-untranslated region (3'-UTR) of Gli-1 mRNA would effectively inhibit epithelial tumor cell proliferation, we evaluated several complementary miRNA molecules for their ability to do so. The synthetic miRNAs and corresponding duplex/small temporal RNAs were introduced as 3-nucleotide (nt) loops into GU-rich portions of the 3'UTR Gli-1 sequence. One particular miRNA (miRNA Gli-1-3548) and its corresponding duplex (Duplex 3548) significantly inhibited proliferation of Gli-1+ ovarian (SK-OV-3) and pancreatic (MiaPaCa-2) tumor cells by delaying cell division and activating late apoptosis in MiaPaCa-2 cells. Here, we describe the design of effective miRNA sequences and their applications as anti-gene agents.

A human monoclonal antibody designated AC6C3 was developed by fusing regional lymph node lymphocytes from a patient with epithelial ovarian carcinoma with cells of the hybrid myeloma SPAZ 4. This monoclonal antibody recognized a determinant expressed on the cell surface of ovarian tumor cell lines. The AC6C3 hybridoma has been maintained for more than 24 months by repeated cloning and secretes IgM at concentrations of 2-8 micrograms/10(6) cells/24h. The AC6C3 monoclonal antibody reacted with a cell surface component of ovarian tumor cell lines, as determined by cell surface immunofluorescence staining using the fluorescent activated cell sorter (FACS). In contrast, nylon wool nonadherent peripheral blood lymphocytes or red blood cells from normal donors were negative (less than 5% of the cells were stained). Immunoperoxidase staining with the AC6C3 monoclonal antibody of nonpermeabilized cryostat sections of freshly obtained or cryopreserved ovarian carcinoma specimens and human ovarian tumor xenografts demonstrated strong reactivity of these specimens. Most normal tissues including brain, liver, heart, kidney and peritoneum demonstrated negative or weak reactions with AC6C3. Other carcinomas including breast, colon and some malignancies of neuroectodermal origin were strongly reactive with AC6C3. AC6C3 mediated complement-dependent cytotoxicity and identified a 32 Kd band in Western blotting and immunoprecipitation experiments conducted on surface labelled SKOV3 cells. The association constant for AC6C3 was determined at 2.3 x 10(10) M-1.

The transcription factor glioma-associated antigen-1 (Gli-1) mediates activation of the sonic hedgehog (Shh) pathway, a process that precedes the transformation of tissue stem cells into cancerous stem cells and that is involved in early and late epithelial tumorigenesis. Hypothesizing that targeting the 3'-untranslated region (3'-UTR) of Gli-1 mRNA would effectively inhibit epithelial tumor cell proliferation, we evaluated several complementary miRNA molecules for their ability to do so. The synthetic miRNAs and corresponding duplex/small temporal RNAs were introduced as 3-nucleotide (nt) loops into GU-rich portions of the 3'UTR Gli-1 sequence. One particular miRNA (miRNA Gli-1-3548) and its corresponding duplex (Duplex 3548) significantly inhibited proliferation of Gli-1+ ovarian (SK-OV-3) and pancreatic (MiaPaCa-2) tumor cells by delaying cell division and activating late apoptosis in MiaPaCa-2 cells. Here, we describe the design of effective miRNA sequences and their applications as anti-gene agents.

The objective of this study was to determine whether peptides consisting of the Ii-Key peptide LRMK linked to the N-terminal ends of HER-2 peptides would stimulate the expansion of antigen-specific E75-TCR+CD8+ cells. The peptides tested were N-acetylated and linked to an alpha-amide at the C-terminus; some of the peptides contained epsilon-aminovaleric acid (Ava) between the LRMK and the HER-2 peptide. Of the seven LRMK-HER-2 peptides tested to date, three effectively induced IFN-gamma production by peripheral blood mononuclear cells (PBMCs) from healthy donors and women with ductal carcinoma in situ. A fusion peptide, LRMK-Ava-HER-2(777-789), was more immunogenic than the natural HER-2(777-789) antigen, G89, with regard to IFN-gamma production. In combination with the CD8-activating peptide E75 [HER-2(369-377)] LRMK-p776 and LRMK-Ava-F7 induced the proliferation of E75-TCR(Med+Hi) CD8+ cells to a greater extent than did 1,000 or 5,000 nM of E75 alone, respectively. The induction effects were strongest at 600 nM for LRMK-p776 and 3,000 nM for LRMK-Ava-F7. At 3,000 nM, LRMK-p776 was cytotoxic to PBMCs. LRMK-p776 and F7 had a similar specificity and preferences for binding HLA-DR molecules. The molecular modeling of HLA-DR:LRMK-p776 and HLA-DR:LRMK-Ava-F7 complexes revealed the side chains of the peptides, which pointed towards the T-cell receptor. Differences in side chain orientation introduced by various N-terminal extensions of MHC class II-bound peptides should be important for directing CD4+ cells to stimulate CD8+ cells or for eliminating regulatory T cells in cancer immunotherapy.

Purpose: E75 is an immunogenic peptide from the HER2/neu protein, which is overexpressed in many breast cancer patients. We have conducted two overlapping E75 vaccine trials to prevent recurrence in node-positive (NP) and node-negative (NN) breast cancer patients.Experimental Design: E75 (HER2/neu 369-377) + granulocyte macrophage colony-stimulating factor was given intradermally to previously treated, disease-free NP breast cancer patients in a dose escalation safety trial and to NN breast cancer patients in a dose optimization study. Local and systemic toxicity was monitored. Immunologic responses were assessed using in vitro assays and in vivo delayed-type hypersensitivity responses. Clinical recurrences were documented.Results: One hundred and eighty-six patients were enrolled in the two studies (NP, 95; NN, 91). Human leucocyte antigen A2 (HLA-A2) and HLA-A3 patients were vaccinated (n = 101), whereas all others (n = 85) were followed prospectively as controls. Toxicities were minimal, and a dose-dependent immunologic response to the vaccine was shown. Planned primary analysis revealed a recurrence rate of 5.6% in vaccinated patients compared with 14.2% in the controls (P = 0.04) at a median of 20 months follow-up. As vaccine-specific immunity waned over time, the difference in recurrence lost significance at 26 months median follow-up (8.3% versus 14.8%); however, a significant difference in the pattern of recurrence persisted.Conclusions: E75 is safe and effective in raising a dose-dependent HER2/neu immunity in HLA-A2 and HLA-A3 NP and NN breast cancer patients. More importantly, E75 may reduce recurrences in disease-free, conventionally treated, high-risk breast cancer patients. These findings warrant a prospective, randomized phase III trial of the E75 vaccine with periodic booster to prevent breast cancer recurrences.

We recently reported that the myristoylated peptide N-myristoyl-Lys-Arg-Thr-Leu-Arg (N-m-KRTLR) is a novel protein kinase C inhibitor. In this study, we investigated the biological effects of N-m-KRTLR using as an in vitro model the induction of the IL-2 receptor and IL-2 secretion by Jurkat cells in response to stimulation with 12-O tetradecanoylphorbol-13-acetate (TPA) plus phytohemagglutinin (PHA) and TPA plus OKT3 mAb. N-m-KRTLR significantly suppressed induction of the IL-2 receptor on the surface of the Jurkat cells by TPA plus either PHA or OKT3 mAb. Furthermore, N-m-KRTLR inhibited the production and release of IL-2 from cultured Jurkat cells stimulated with TPA plus either PHA or OKT3 mAb. Similarly, this peptide significantly inhibited the IL-2 production in normal human peripheral blood mononuclear cells in response to stimulation by TPA and PHA. In contrast, this peptide did not affect expression of the CD3 complex on the surface of the Jurkat cells either alone or in the presence of TPA or PHA. Furthermore, N-m-KRTLR did not interfere with the spontaneous proliferation of the Jurkat cells, and its effects on IL-2 secretion and IL-2 receptor expression in the Jurkat cells were evident without loss of cell viability. These results suggest that the novel protein kinase C inhibitor N-m-KRTLR may selectively inhibit certain activation pathways of Jurkat cells and indicate the usefulness of N-m-KRTLR in the analysis of discrete events in T cell activation.

It has been proposed that chemotherapy enhances tumor antigen (TA)–specific immunity. The molecular form of TA from ovarian tumor that activates cellular immunity is unknown. We report here identification of a novel molecular form of immunogenic TA for CD8+ cells named self-immune stimulatory multimolecular complexes (ISMMC). ISMMC consist of a molecular complex of polyosome/ribosome-bound ubiquitinated nascent HER-2 polypeptides. This complex is chaperoned by heat shock protein Gp96, which mediates ISMMC uptake by antigen-presenting cells through the scavenger receptor CD91. RNAs in ISMMC stimulate immature dendritic cells to secrete interleukin 12 and induce IFN-γ in peripheral blood mononuclear cells. ISMMC dissociate, retrotranslocate from the lysosome to cytoplasm, and are processed to peptides by the proteasome. At subpharmacologic doses, Taxol increased the amount of ISMMC by three to four times and modified their composition by inducing the attachment of cochaperones of HSP70, such as the mitotic-phase phosphoprotein 11J. On a total protein basis, Taxol induced ISMMC, expanded more CD8+ cells, activated more CD56+ NKG2D+ cells to produce IFN-γ, and were more potent inducers of high T-cell receptor density Perforin+ cells than native ISMMC and peptide E75. Elucidation of the composition of ISMMC and identification of adducts formed by Taxol should be important for developing molecular cancer vaccines. [Cancer Res 2007;67(17):8378–87]

It has been brought to our attention that one of the folate binding protein (FBP) peptides, which we reported first as antigenic and immunogenic in cancer patients, the FBP 191-199, is "off by one amino acid from the amino acid sequence that is listed in GenBank". We searched the published information on FBP and found that the FBP 191-199, which we reported contains threonine 197 instead of the GenBank tyrosine 197. In addition, we found mutations in the FBP (191-199) in other positions, as well as in the flanking residues which direct processing. The potential significance of these changes for cancer vaccines is discussed. It is highly recommended that future human studies with FBP will analyze both GenBank and published sequences in the literature. The large number of mutations in immunogenic FBP-tumor antigens, reported more recently, should be considered during preclinical testing for vaccine and gene therapy in human cancers.

Notch is a plasma membrane receptor involved in the control of cell fate specification and in the maintenance of the balance between proliferation and differentiation in many cell lineages. Disruption of Notch has been implicated in a variety of hematological and solid cancers. Numb is also expressed in many adult mammalian cells. Adult cells divide symmetrically, and Numb is symmetrically partitioned at mitosis. The Numb-mediated regulation of Notch is believed to play a causative role in naturally occurring breast cancers. Reduction of Numb levels in breast tumors is regulated by proteasomal degradation. We reasoned that if the disregulated negative control of Notch by Numb protein is the consequence of Numb proteasomal degradation, then degradation of Numb can generate peptides which are transported, presented by MHC-I molecules. Surprisingly we found few candidate naturally processed peptides from Notch 1, Notch2, and Numb1. CD8 + T cells expressing TCRs which specifically recognized peptides Notch1 (2112-2120) and Numb1 (87-95) were presented in the ascites of ovarian cancer patients. Many of these cells were differentiated and expressed high levels of Perforin. The natural immunogenicity of Notch1 and particularly of Numb1 suggests a mechanism of immunosurveillance which is overcome during tumor progression. Immunotherapy with tumor antigens from Notch and Numb should be important for treatment of cancer patients.

Notch is a plasma membrane receptor involved in the control of cell fate specification and in the maintenance of the balance between proliferation and differentiation in many cell lineages. Disruption of Notch has been implicated in a variety of hematological and solid cancers. Numb is also expressed in many adult mammalian cells. Adult cells divide symmetrically, and Numb is symmetrically partitioned at mitosis. The Numb-mediated regulation of Notch is believed to play a causative role in naturally occurring breast cancers. Reduction of Numb levels in breast tumors is regulated by proteasomal degradation. We reasoned that if the disregulated negative control of Notch by Numb protein is the consequence of Numb proteasomal degradation, then degradation of Numb can generate peptides which are transported, presented by MHC-I molecules. Surprisingly we found few candidate naturally processed peptides from Notch1, Notch2, and Numb1. CD8+ T cells expressing TCRs which specifically recognized peptides Notch1 (2112-2120) and Numb1 (87-95) were presented in the ascites of ovarian cancer patients. Many of these cells were differentiated and expressed high levels of Perforin. The natural immunogenicity of Notch1 and particularly of Numb1 suggests a mechanism of immunosurveillance which is overcome during tumor progression. Immunotherapy with tumor antigens from Notch and Numb should be important for treatment of cancer patients.

Mobilization of tumor-reactive CD8 + T cells remains the major challenge of cancer immunotherapy. Knowing how and when the T cell response expands and differentiates after antigen stimulation would make a significant contribution to the development of tumor vaccines. In the current study, we used CFSE-based cell sorting and cDNA microarray to identify the gene expression profile of adjacent generations of T cells after PHA stimulation. Early-divided generations of T cells responded to stimulation by activating cell cycle and surviving gene pathways, while late generations of T cells had more dramatic changes in transcription of cytokine genes. Reconstruction of biochemical pathways, activated in both early and late generations of T cells, also confirmed the impact of division in focal-adhesion kinases. Because most tumors are infiltrated by lymphocytes, our studies indicate a novel approach to identify 'systemic biological responses' of T cells, which could determine the design, and optimization of effective tumor vaccines.

Mobilization of tumor-reactive CD8+ T cells remains the major challenge of cancer immunotherapy. Knowing how and when the T cell response expands and differentiates after antigen stimulation would make a significant contribution to the development of tumor vaccines. In the current study, we used CFSE-based cell sorting and cDNA microarray to identify the gene expression profile of adjacent generations of T cells after PHA stimulation. Early-divided generations of T cells responded to stimulation by activating cell cycle and surviving gene pathways, while late generations of T cells had more dramatic changes in transcription of cytokine genes. Reconstruction of biochemical pathways, activated in both early and late generations of T cells, also confirmed the impact of division in focal-adhesion kinases. Because most tumors are infiltrated by lymphocytes, our studies indicate a novel approach to identify 'systemic biological responses' of T cells, which could determine the design, and optimization of effective tumor vaccines.

Purpose: To determine whether the synthetic microRNAs (miRNA) could effectively target tumor cells we designed several miRNA complementary to glioma-associated antigen-1 (Gli-1) mRNA and investigated their ability to inhibit tumor cell proliferation. The sonic hedgehog pathway is an early and late mediator of tumorigenesis in epithelial cancers. Activation of sonic hedgehog signaling seems to precede transformation of tissue stem cells to cancerous stem cells, with the Gli-1 transcription factor functioning as a mediator of environmental signals. Inhibiting cancer cell proliferation by targeting the Gli-1 effector pathway is difficult to achieve by chemotherapeutic agents or short interfering RNA. Experimental Design: We hypothesized that targeting the 3′-untranslated region of Gli-1 mRNA would effectively inhibit tumor cell proliferation. To test this hypothesis, we used synthetic miRNAs of our own design and corresponding duplex/small temporal RNAs by introducing three-nucleotide loops in the 3′-untranslated region Gli-1 sequence of high GU content. Results: We found that miRNA (Gli-1-miRNA-3548) and its corresponding duplex (Duplex-3548) significantly inhibited proliferation of Gli-1+ ovarian (SK-OV-3) and pancreatic (MiaPaCa-2) tumor cells. The miRNAs mediated delayed cell division and activation of late apoptosis in MiaPaCa-2 cells. This is the first demonstration of inhibition of pancreatic tumor cell division by designed miRNA. Conclusions: Gli-1 miRNAs should significantly add to the general understanding of the mechanisms of metastasis and contribute toward the design of better treatments for epithelial cancers.

MicroRNA (miRNA) are a class of non-coding RNAs found both in normal tissues and cancer cells. The natural miRNA, which target cancer genes for transcriptional repression are still unknown. Approaches for synthetic miRNA design targeting cancer genes have not yet been established. We designed miRNAs targeting the 3' UTR (nt 4350-4372) of HER-2 proto-oncogene. One miRNA (miR-14U) was designed by introducing a mutation in the nt 14 counting from the 5' end of anti-sense RNA. Two others (miR4350-10GGA and miR4350-11AAGCU) were designed by introducing either loop-forming nucleotides in position 10 or a part of the complementary sequence of the Brd-box consensus sequence, in position 11. miR4350-10GGA was more effective than the anti-sense strand in decreasing the numbers of ovarian tumor SKOV3 cells, which over-expressed HER-2 protein. Its inhibitory effects were lower than that of corresponding double-stranded (ds) RNA4350-4372. Inhibition of HER-2 expression mediated by miRNAs was higher in cells expressing higher levels of HER-2 protein than in cells expressing lower levels of HER-2 protein. This is the first demonstration of inhibition of expression of a constitutively over-expressed tumor protein by designed synthetic miRNA and its cor-responding dsRNA targeting 3' untranslated regions in mRNA. Our results also show that measuring the effects of miRNA and dsRNA in pooled populations of tumor cells, which express various levels of target oncogenes, may reflect the survival responses of siRNA resistant tumors rather than the growth inhibitory responses of siRNA-sensitive tumors.

MicroRNA (miRNA) are a class of non-coding RNAs found both in normal tissues and cancer cells. The natural miRNA, which target cancer genes for transcriptional repression are still unknown. Approaches for synthetic miRNA design targeting cancer genes have not yet been established. We designed miRNAs targeting the 3' UTR (nt 4350-4372) of HER-2 proto-oncogene. One miRNA (miR-14U) was designed by introducing a mutation in the nt 14 counting from the 5' end of anti-sense RNA. Two others (miR4350-10GGA and miR4350-11AAGCU) were designed by introducing either loop-forming nucleotides in position 10 or a part of the complementary sequence of the Brd-box consensus sequence, in position 11. miR4350-10GGA was more effective than the anti-sense strand in decreasing the numbers of ovarian tumor SKOV3 cells, which over-expressed HER-2 protein. Its inhibitory effects were lower than that of corresponding double-stranded (ds) RNA4350-4372. Inhibition of HER-2 expression mediated by miRNAs was higher in cells expressing higher levels of HER-2 protein than in cells expressing lower levels of HER-2 protein. This is the first demonstration of inhibition of expression of a constitutively over-expressed tumor protein by designed synthetic miRNA and its cor-responding dsRNA targeting 3' untranslated regions in mRNA. Our results also show that measuring the effects of miRNA and dsRNA in pooled populations of tumor cells, which express various levels of target oncogenes, may reflect the survival responses of siRNA resistant tumors rather than the growth inhibitory responses of siRNA-sensitive tumors.

Development of cancer vaccines requires approaches to induce expansion and functional differentiation of tumor antigen-specific effector and memory cells. The later are particularly relevant for prevention of disease relapse. Efficient induction of memory cells is hindered by the lack of information about the relationship between TCR stimulation and the cytokines required for Ag-specific memory CD8+ cells and proliferation and survival. Since viruses are known to induce memory T cells, an attenuated influenza A/PR8/34 virus with a truncated nonstructural (NS1) gene was generated containing the HER-2 CTL E75 epitope in its neuraminidase protein (KIF-NS virus). Stimulation of PBMC from healthy donors and of tumor-associated lymphocytes (TAL) from ovarian cancer patients with dendritic cells (DC) infected with KIF-NS (KIF-NS-DC), induced higher numbers of immediate memory effector CD8+ CD44hi CD122hi cells, expressing TCR specific for E75 (E75-TCR) than stimulation with peptide E75. Survival of CD44hi CD122hi cells was dependent on the levels of TCR; cells expressing lower levels of E75-TCR (MFI: 10(2)-10(3)) survived better in IL-2 while cells expressing high levels of TCR (MFI: 10(3)-10(4)) survived better in IL-15. This is the first report demonstrating induction of human Ag-specific memory CD8+ cells against a human tumor-antigen using a live attenuated recombinant influenza virus vector. Such vectors may provide a novel approach for preventive immunity in human cancer vaccine development.

CD8+ cells expressing high numbers of TCR per cell (TCRhi) are considered important mediators of antitumor effects. To understand the relationship between TCR density and antigen affinity for TCR in the outcome of stimulation with antigen and differentiation of CTL recognizing tumor antigen, we analyzed perforin induction in ovarian tumor-associated lymphocytes in response to the smallest possible changes in the atomic forces of interaction between antigen and TCR. Stimulating undifferentiated, apoptosis-resistant CD8+ cells expressing high levels of E75-TCR (TCRhi) with variants of the CTL epitope E75, HER-2 (369-377), induced their stepwise differentiation, first to IFN-γ+ Perf− and to TCRhi IFN-γ+ Perf+ cells. Blocking caspase-9 activation at antigen stimulation also enhanced the generation of TCRhi Perfhi cells, demonstrating that TCR density dictated the pathway of death activated by stimulation with the same agonist. Expansion and differentiation of TCRhi Perf+ CTL required an agonist of optimal CH2 side chain length, which in this study was equal to two CH2 groups appended to E75 at the Gly4 position. Side chains one CH2 shorter or longer than optimal were either less stimulatory or induced death of TCRhi Perf+ cells. Differentiation of TCRhi CD8+ cells can be finely tuned by synthetic amino acids in the peptide, whose side chains induce small increments in the affinity of the antigen for TCR below the affinity which induce apoptosis.

The HER2/neu protein is over-expressed in multiple epithelial tumors and the source of immunogenic peptides currently under investigation in vaccine trials in ovarian and breast cancers. We sought to define the correlation between HER2/neu expression and risk for prostate cancer recurrence and then determine the potential efficacy of anti-HER2/neu vaccination in prostate cancer patients at risk for recurrence. The risk for prostate-specific antigen (PSA) recurrence in 95 patients undergoing prostatectomy at the Walter Reed Army Medical Center (WRAMC) was calculated and correlated to HER2/neu expression, as determined by immunohistochemical staining. Peripheral blood lymphocytes (PBL) were then isolated from six consecutive human leukocyte antigen (HLA) A2+ patients with HER2/neu+ prostate tumors. These PBL were grown in parallel cultures and stimulated either with no peptide, HER2/neu E75 peptide, or control peptide. The cultures were compared for stimulated proliferation, induced peptide-specific cytotoxicity and tumor-specific cytotoxicity. When assessed by risk group, 69% of the high risk patients' tumors over-expressed HER2/neu compared to 47% of the intermediate risk group (p

BRAK/CXCL14 is a CXC chemokine constitutively expressed at the mRNA level in certain normal tissues but absent from many established tumor cell lines and human cancers. Although multiple investigators cloned BRAK, little is known regarding the physiologic function of BRAK or the reason for decreased expression in cancer. To understand the possible significance associated with loss of BRAK mRNA in tumors, we examined the pattern of BRAK protein expression in normal and tumor specimens from patients with squamous cell carcinoma (SCC) of the tongue and used recombinant BRAK (rBRAK) to investigate potential biological functions. Using a peptide-specific antiserum, abundant expression of BRAK protein was found in suprabasal layers of normal tongue mucosa but consistently was absent in tongue SCC. Consistent with previous in situ mRNA studies, BRAK protein also was expressed strongly by stromal cells adjacent to tumors. In the rat corneal micropocket assay, BRAK was a potent inhibitor of in vivo angiogenesis stimulated by multiple angiogenic factors, including interleukin 8, basic fibroblast growth factor, and vascular endothelial growth factor. In vitro, rBRAK blocked endothelial cell chemotaxis at concentrations as low as 1 nmol/L, suggesting this was a major mechanism for angiogenesis inhibition. Although only low affinity receptors for BRAK could be found on endothelial cells, human immature monocyte-derived dendritic cells (iDCs) bound rBRAK with high affinity (i.e., Kd, ∼2 nmol/L). Furthermore, rBRAK was chemotactic for iDCs at concentrations ranging from 1 to 10 nmol/L. Our findings support a hypothesis that loss of BRAK expression from tumors may facilitate neovascularization and possibly contributes to immunologic escape.

The HER2/neu protein is over-expressed in multiple epithelial tumors and the source of immunogenic peptides currently under investigation in vaccine trials in ovarian and breast cancers. We sought to define the correlation between HER2/neu expression and risk for prostate cancer recurrence and then determine the potential efficacy of anti-HER2/neu vaccination in prostate cancer patients at risk for recurrence. The risk for prostate-specific antigen (PSA) recurrence in 95 patients undergoing prostatectomy at the Walter Reed Army Medical Center (WRAMC) was calculated and correlated to HER2/neu expression, as determined by immunohistochemical staining. Peripheral blood lymphocytes (PBL) were then isolated from six consecutive human leukocyte antigen (HLA) A2+ patients with HER2/neu+ prostate tumors. These PBL were grown in parallel cultures and stimulated either with no peptide, HER2/neu E75 peptide, or control peptide. The cultures were compared for stimulated proliferation, induced peptide-specific cytotoxicity and tumor-specific cytotoxicity. When assessed by risk group, 69% of the high risk patients' tumors over-expressed HER2/neu compared to 47% of the intermediate risk group (p0.05). Evaluation of the in vitro immune response of PBL isolated from six consecutive prostate cancer patients revealed a statistically significant increase in E75-stimulated lymphocytic proliferation. E75-stimulated lymphocytes demonstrated an E75-specific cytolytic response in 6/6 prostate cancer patients that increased with successive stimulations. Moreover, these E75-specific lymphocytes also demonstrated tumor-specific lysis against HER2/neu-expressing prostate cancer cell lines. The majority of prostate cancer patients at high risk for recurrence have HER2/neu expressing tumors. Hence, HER2/neu is a viable target for immunotherapeutics such as preventative immunization strategies with HER2/neu peptide vaccines.

CD8+ cells expressing high numbers of TCR per cell (TCRhi) are important mediators of anti-tumor effects. To understand the relationship between TCR density and Ag affinity for TCR in the outcome of differentiation of CTL recognizing tumor Ag, we analyzed perforin induction in ovarian tumor-associated lymphocytes in response to the smallest possible changes in the atomic forces of interaction between Ag and TCR. Stimulating undifferentiated, apoptosis-resistant CD8+ cells expressing high levels of E75-TCR (TCRhi) with variants of the CTL epitope E75, HER-2 (369-377), induced their stepwise differentiation, first to IFN-gamma+ Perf-, and then to TCRhi IFN-gamma+ Perf+ cells. Blocking caspase-9 activation at Ag stimulation also enhanced the generation of TCRhi Perf+ cells, demonstrating that TCR density dictated the pathway of death activated by stimulation with the same agonist. Expansion and differentiation of TCRhi Perf+ CTL required an agonist of optimal CH2 side chain length. Side chains one CH2 shorter or longer than optimal were either less stimulatory or induced death of TCRhi Perf+ cells. Differentation of TCRhi CD8+ cells can be finely tuned by side chains which induce small increments in the affinity of the Ag for TCR below the affinity which induce apoptosis.