Neu Peptide Research Articles

Frequencies of an Immunogenic HER-2/neu Epitope of CD8+ T Lymphocytes Predict Favorable Clinical Outcomes in Prostate Cancer

HER-2/neu is the human epidermal growth factor receptor 2, which is associated with the progression of prostate cancer (PCa). HER-2/neu-specific T cell immunity has been shown to predict immunologic and clinical responses in PCa patients treated with HER-2/neu peptide vaccines. However, its prognostic role in PCa patients receiving conventional treatment is unknown, and this was addressed in this study. The densities of CD8+ T cells specific for the HER-2/neu(780-788) peptide in the peripheral blood of PCa patients under standard treatments were correlated with TGF-β/IL-8 levels and clinical outcomes. We demonstrated that PCa patients with high frequencies of HER-2/neu(780-788)-specific CD8+ T lymphocytes had better progression-free survival (PFS) as compared with PCa patients with low frequencies. Increased frequencies of HER-2/neu(780-788)-specific CD8+ T lymphocytes were also associated with lower levels of TGF-β and IL-8. Our data provide the first evidence of the predictive role of HER-2/neu-specific T cell immunity in PCa.

Ferritin Conjugates With Multiple Clickable Amino Acids Encoded by C-Terminal Engineered Pyrrolysyl-tRNA Synthetase.

This study reports the application of expanding genetic codes in developing protein cage-based delivery systems. The evolved Methanosarcina mazei pyrrolysyl-tRNA synthetase (PylRS)•tRNAPyl pairs derived from directed evolution are examined to probe their recognition for para-substituted phenylalanine analogs. The evolved MmPylRS, AzFRS, harboring a wide range of substrates, is further engineered at the C-terminal region into another variant, AzFRS-MS. AzFRS-MS shows suppression of the elevated sfGFP protein amount up to 10 TAG stop codons when charging p-azido-l-phenylalanine (AzF, 4), which allows the occurrence of click chemistry. Since protein nanocages used as drug delivery systems that encompass multiple drugs through a site-specific loading approach remain largely unexplored, as a proof of concept, the application of AzFRS-MS for the site-specific incorporation of AzF on human heavy chain ferritin (Ftn) is developed. The Ftn-4 conjugate is shown to be able to load multiple fluorescence dyes or a therapeutic agent, doxorubicin (Dox), through the strain-promoted azide-alkyne cycloaddition (SPAAC) click reaction. Aiming to selectively target Her2+ breast cancer cells, Ftn-4-DOX conjugates fused with a HER2 receptor recognition peptide, anti-Her2/neu peptide (AHNP), is developed and demonstrated to be able to deliver Dox into the cell and to prolong the drug release. This work presents another application of evolved MmPylRS systems, whose potential in developing a variety of protein conjugates is noteworthy.

Targeting MCF-7 Cell Line by Listeriolysin O Pore Forming Toxin Fusion with AHNP Targeted Peptide.

Background:Tumor-targeting peptides are attracting subjects in cancer therapy. These peptides, which are widely studied, deliver therapeutic agents to the specific sites of tumors. In this study, we produced a new form of recombinant listeriolysin O (LLO) with genetically fused Anti-HER2/neu peptide (AHNP) sequence adding to its C-terminal end. The aim of the study was to engineer this pore-forming toxin to make it much more specific to tumor cells.Materials and Method and Results:Two forms of the toxin (with and without peptide) were subcloned into a bacterial expression plasmid. Subcloning was performed using a polymerase chain reaction (PCR) product as a megaprimer in a quick-change PCR to introduce the whole insert gene into the expression plasmid. After expression of two recombinant forms of LLO in BL21 DE3 cells, purification was performed using Ni-NTA affinity column. MDA-MB-231 and MCF-7 cell lines (as negative and positive controls, respectively) were treated with both LLO toxins to evaluate their cytotoxicity and specificity. The IC50 of LLO on MDA-MB-231 and MCF-7 cells was 21 and 5 ng/ml, respectively. In addition, IC50 for the fusion AHNP-LLO toxin was 140 and 60 ng/ml, respectively. It was found that the cytotoxicity of the new engineered AHNP-LLO toxin has decreased by about 9x compared to the wild-type toxin and the specificity of the AHNP-LLO toxin has been also reduced.Conclusions:Results show that the C-terminal of the LLO should not be modified and it seems that N-terminal of the toxin should be preferred for engineering and adding peptide modules.

Polyethylene glycol-conjugated HER2-targeted peptides as a nuclear imaging probe for HER2-overexpressed gastric cancer detection in vivo

BackgroundThe human epidermal growth factor receptor 2 (HER2) involved proliferation, angiogenesis, and reduced apoptosis in gastric cancer (GC), which is a common target for tumor therapy. HER2 is usually overexpressed in more than 15% GC patients, developing a reliable diagnostic tool for tumor HER2 detection is important. In this study, we attend to use polyethylene glycol (PEG) linked anti-HER2/neu peptide (AHNP-PEG) as a nuclear imaging agent probe for HER2 detection in GC xenograft animal model.MethodsThe HER2 expression of human sera and tissues were detected in GC patients and normal subjects. GC cell lines NCI-N87 (high HER2 levels) and MKN45 (low HER2 levels) were treated with AHNP-PEG to assess the cell viability and HER2 binding ability. The NCI-N87 was treated with AHNP-PEG to observe the level and phosphorylation of HER2. The MKN45 and NCI-N87-induced xenograft mice were intravenous injection with fluorescence labeled AHNP-PEG for detecting in vivo fluorescence imaging properties and biodistribution. The AHNP-PEG was conjugated with diethylenetriaminopentaacetic acid (DTPA) for indium-111 labeling (111In-DTPA-AHNP-PEG). The stability of was assessed in vitro. The imaging properties and biodistribution of 111In-DTPA-AHNP-PEG were observed in NCI-N87-induced xenograft mice.ResultsThe serum HER2 (sHER2) levels in GC patients were significantly higher than the normal subjects. The sHER2 levels were correlated with the tumor HER2 levels in different stages of GC patients. The AHNP-PEG inhibited the cell growth and down-regulated HER2 phosphorylation in HER2-overexpressed human GC cells (NCI-N87) via specific HER2 interaction of cell surface. In addition, the GC tumor tissues from HER2-postive xenograft mice presented higher HER2 fluorescence imaging as compared to HER2-negative group. The HER2 levels in the tumor tissues were also higher than other organs in NCI-N87-induced xenograft mice. Finally, we further observed that the 111In-DTPA-AHNP-PEG was significantly enhanced in tumor tissues of NCI-N87-induced xenograft mice compared to control.ConclusionsThese findings suggest that the sHER2 measurement may be as a potential tool for detecting HER2 expressions in GC patients. The radioisotope-labeled AHNP-PEG may be useful to apply in GC patients for HER2 nuclear medicine imaging.

Targeted Ultrasound-Triggered Phase Transition Nanodroplets for Her2-Overexpressing Breast Cancer Diagnosis and Gene Transfection.

For successful gene therapy, it is imperative to accumulate therapeutic gene in tumor tissues followed by efficiently delivering gene into targeted cells. Ultrasound irradiation, as a noninvasive and cost-effective external stimulus, has been proved to be one of the most potential external-stimulating gene delivery strategies recently in further improving gene transfection. In this study, we developed tumor-targeting ultrasound-triggered phase-transition nanodroplets AHNP-PFP-TNDs comprising a perfluorinated poly(amino acid) C11F17-PAsp (DET) as a core for simultaneously loading perfluoropentane (PFP) and nucleic acids, and a polyanionic polymer PGA-g-PEG-AHNP as the shell for not only modifying the surface of nanodroplets but also introducing an anti-Her2/neu peptide (AHNP) aiming to targeted treatment of Her2-overexpressing breast cancer. The results showed the average diameter of AHNP-PFP-TNDs was below 400 nm, nearly spherical in shape. The modification of PGA-g-PEG-AHNP not only increased the serum stability of the nanodroplets but also improved the affinity between nanodroplets and Her2-overexpressing breast cells. Both intratumor and intravenous injection of AHNP-PFP-TNDs into nude mice bearing HGC-27 xenografts showed that the gene transfection efficiency and the ultrasound contrast effect were significantly enhanced after exposed to the ultrasound irradiation with optimized ultrasound parameters. Therefore, this targeting nanodroplets system could be served as a potential theranostic vector for tumor targeting ultrasound diagnosis and gene therapy.

Improved drug delivery and therapeutic efficacy of PEgylated liposomal doxorubicin by targeting anti-HER2 peptide in murine breast tumor model

Targeted cancer therapy is a powerful therapeutic strategy to management of cancer. HER2 as an anticancer target has long been studied. Its overexpression plays an important role in the pathogenesis and progressiveness of breast and other cancers. To establish efficient and reliable drug delivery to HER2-overexpressing cells, the authors of this study have developed anti-HER2 (ErbB2) peptide-liposomal formulations of doxorubicin (DOX) by an engineered breast tumor-targeting peptide ligand, AHNP, Anti-HER2/neu peptide, (FCDGFYACYADV) with three glycine amino acids as spacer before its original sequencing. Towards this goal, PEGylated liposome doxorubicin (PLD) bearing different ligand densities of AHNP was prepared and characterized for their size, zeta potential and peptide conjugation. The AHNP functionalization and density effects on breast tumor cell uptake, selective cytotoxicity, prevention of tumor growth and the tissue biodistribution of encapsulated DOX were studied in mice bearing TUBO breast cancer tumor model. The findings demonstrated that increasing the ligand density of AHNP increases cytotoxicity and cell-uptake in SKBR3 and TUBO cells which overexpress HER2 but not in MDA-MB-231with low HER2 expression profile. The anticancer activity was also superior for targeted liposomal DOX with more AHNP densities. Overall, the results showed that optimum AHNP density functionalization of PLD can significantly improve selectivity and the therapeutic index of liposomal DOX in the treatment of HER2 positive breast cancer and merits further investigation.

Dual-Ligand Modified Polymer-Lipid Hybrid Nanoparticles for Docetaxel Targeting Delivery to Her2/neu Overexpressed Human Breast Cancer Cells.

In this study, a dual-ligand polymer-lipid hybrid nanoparticle drug delivery vehicle comprised of an anti-HER2/neu peptide (AHNP) mimic with a modified HIV-1 Tat (mTAT) was established for the targeted treatment of Her2/neu-overexpressing cells. The resultant dual-ligand hybrid nanoparticles (NPs) consisted of a poly(lactide-co-glycolide) core, a near 90% surface coverage of the lipid monolayer, and a 5.7 nm hydrated polyethylene glycol shell. Ligand density optimization study revealed that cellular uptake efficiency of the hybrid NPs could be manipulated by controlling the surface-ligand densities. Furthermore, the cell uptake kinetics and mechanism studies showed that the dual-ligand modifications of hybrid NPs altered the cellular uptake pathway from caveolae-mediated endocytosis (CvME) to the multiple endocytic pathways, which would significantly enhance the NP internalization. Upon the systemic investigation of the cellular uptake behavior of dual-ligand hybrid NPs, docetaxel (DTX), a hydrophobic anticancer drug, was successfully encapsulated into dual-ligand hybrid NPs with high drug loading for Her2/neu-overexpressing SK-BR-3 breast cancer cell treatment. The DTX-loaded dual-ligand hybrid NPs showed a decreased burst release and a more gradual sustained drug release property. Because of the synergistic effect of dual-ligand modification, DTX-loaded dual-ligand hybrid NPs exerted substantially better therapeutic potency against SK-BR-3 cancer cells than other NP formulations and free DTX drugs. These results demonstrate that the dual-ligand hybrid NPs could be a promising vehicle for targeted drug delivery to treat breast cancer.

Anti-HER2/neu peptide-conjugated iron oxide nanoparticles for targeted delivery of paclitaxel to breast cancer cells.

Nanoparticles (NPs) for targeted therapy are required to have appropriate size, stability, drug loading and release profiles, and efficient targeting ligands. However, many of the existing NPs such as albumin, liposomes, polymers, gold NPs, etc. encounter size limit, toxicity and stability issues when loaded with drugs, fluorophores, and targeting ligands. Furthermore, antibodies are bulky and this can greatly affect the physicochemical properties of the NPs, whereas many small molecule-based targeting ligands lack specificity. Here, we report the utilization of biocompatible, biodegradable, small (∼30 nm) and stable iron oxide NPs (IONPs) for targeted delivery of paclitaxel (PTX) to HER2/neu positive breast cancer cells using an anti-HER2/neu peptide (AHNP) targeting ligand. We demonstrate the uniform size and high stability of these NPs in biological medium, their effective tumour targeting in live mice, as well as their efficient cellular targeting and selective killing in human HER2/neu-positive breast cancer cells.

Prothymosin α and a prothymosin α-derived peptide enhance TH1-type immune responses against defined HER-2/neu epitopes

BackgroundActive cancer immunotherapies are beginning to yield clinical benefit, especially those using peptide-pulsed dendritic cells (DCs). Different adjuvants, including Toll-like receptor (TLR) agonists, commonly co-administered to cancer patients as part of a DC-based vaccine, are being widely tested in the clinical setting. However, endogenous DCs in tumor-bearing individuals are often dysfunctional, suggesting that ex vivo educated DCs might be superior inducers of anti-tumor immune responses. We have previously shown that prothymosin alpha (proTα) and its immunoreactive decapeptide proTα(100–109) induce the maturation of human DCs in vitro. The aim of this study was to investigate whether proTα- or proTα(100–109)-matured DCs are functionally competent and to provide preliminary evidence for the mode of action of these agents.ResultsMonocyte-derived DCs matured in vitro with proTα or proTα(100–109) express co-stimulatory molecules and secrete pro-inflammatory cytokines. ProTα- and proTα(100–109)-matured DCs pulsed with HER-2/neu peptides induce TH1-type immune responses, prime autologous naïve CD8-positive (+) T cells to lyse targets expressing the HER-2/neu epitopes and to express a polyfunctional profile, and stimulate CD4+ T cell proliferation in an HER-2/neu peptide-dependent manner. DC maturation induced by proTα and proTα(100–109) is likely mediated via TLR-4, as shown by assessing TLR-4 surface expression and the levels of the intracellular adaptor molecules TIRAP, MyD88 and TRIF.ConclusionsOur results suggest that proTα and proTα(100–109) induce both the maturation and the T cell stimulatory capacity of DCs. Although further studies are needed, evidence for a possible proTα and proTα(100–109) interaction with TLR-4 is provided. The initial hypothesis that proTα and the proTα-derived immunoactive decapeptide act as “alarmins”, provides a rationale for their eventual use as adjuvants in DC-based anti-cancer immunotherapy.

Vaccination for the prevention and treatment of breast cancer with special focus on Her-2/neu peptide vaccines

Immunologic interventions in a subset of breast cancer patients represent a well-established therapeutic approach reflecting individualized treatment modalities. Thus, the therapeutic administration of monoclonal antibodies targeting tumor-associated antigens (TAA), such as Her-2/neu, represents a milestone in cancer treatment. However, passive antibody administration suffers from several drawbacks, including frequency and long duration of treatment. These undesirables may be avoidable in an approach based on generating active immune responses against these same targets. Only recently has the significance of tumors in relation to their microenvironments been understood as essential for creating an effective cancer vaccine. In particular, the immune system plays an important role in suppressing or promoting tumor formation and growth. Therefore, activation of appropriate triggers (such as induction of Th1 cells, CD8+ T cells, and suppression of regulatory cells in combination with generation of antibodies with anti-tumor activity) is a desirable goal. Current vaccination approaches have concentrated on therapeutic vaccines using certain TAA. Many cancer antigens, including breast cancer antigens, have been described and also given priority ranking for use as vaccine antigens by the US National Cancer Institute. One of the TAA antigens which has been thoroughly examined in numerous trials is Her-2/neu. This review will discuss delivery systems for this antigen with special focus on T and B cell peptide vaccines. Attention will be given to their advantages and limitations, as well as the use of certain adjuvants to improve anti-cancer responses.

Enzymatic Discovery of a HER-2/neu Epitope That Generates Cross-Reactive T Cells

Patients with HER-2/neu-expressing breast cancer remain at risk for relapse following standard therapy. Vaccines targeting HER-2/neu to prevent relapse are in various phases of clinical testing. Many vaccines incorporate the HER-2/neu HLA-A2-binding peptide p369-377 (KIFGSLAFL), because it has been shown that CTLs specific for this epitope can directly kill HER-2/neu-overexpressing breast cancer cells. Thus, understanding how tumors process this epitope may be important for identifying those patients who would benefit from immunization. Proteasome preparations were used to determine if p369-377 was processed from larger HER-2/neu-derived fragments. HPLC, mass spectrometry, cytotoxicity assays, IFN-γ ELISPOT, and human breast cancer cell lines were used to assess the proteolytic fragments. Processing of p369-377 was not detected by purified 20S proteasome and immunoproteasome, indicating that tumor cells may not be capable of processing this Ag from the HER-2/neu protein and presenting it in the context of HLA class I. Instead, we show that other extracellular domain HER-2/neu peptide sequences are consistently processed by the proteasomes. One of these sequences, p373-382 (SLAFLPESFD), bound HLA-A2 stronger than did p369-377. CTLs specific for p373-382 recognized both p373-382 and p369-377 complexed with HLA-A2. CTLs specific for p373-382 also killed human breast cancer cell lines at higher levels than did CTLs specific for p369-377. Conversely, CTLs specific for p369-377 recognized p373-382. Peptide p373-382 is a candidate epitope for breast cancer vaccines, as it is processed by proteasomes and binds HLA-A2.

Identification of HER-2/neu peptide p373-382 as a naturally processed immunodominant HLA-A2 binding epitope (46.11)

Abstract HER2 is a prime target for cancer therapeutics due to its high overexpression in tumors. Advanced clinical trials, in breast and prostate cancer, investigating the use of the HLA-A2-binding HER2 peptide p369-377 (E75) as a vaccine are ongoing. In the present study, we investigated the proteasomal processing of p369-377 and other epitopes from HER2. We did not detect processing of p369-377 by either the proteasome or immunoproteasome in vitro, suggesting that tumor cells do not naturally process this epitope from HER2 for presentation on HLA-A2. However, other HER2 peptides were found to be consistently processed by the proteasome and immunoproteasome. One newly identified processed peptide, HER2 p373-382, was found to bind HLA-A2 stronger than p369-377, despite algorithms that predicted p369-377 as a stronger binder. Cytotoxic T lymphocytes (CTL) specific for p373-382 recognized target cells pulsed with either p373-382 or p369-377. CTL specific for p373-382 also lysed HER2+HLA-A2+ human breast cancer cells more efficiently than p369-377-specific CTL. These results suggest that cancer cells are naturally processing and presenting p373-382. Furthermore, our results suggest that T cells generated against p369-377 in previous studies may have been cross-reacting with p373-382. Thus, the newly identified p373-382 peptide may be a more appropriate candidate for vaccines with this new evidence that it is processed and presented by HLA-A2 for recognition by HER2-specific CTL.

A Novel Dendritic Cell-based Immunization Approach for the Induction of Durable Th1-polarized Anti-HER-2/neu Responses in Women With Early Breast Cancer

Twenty-seven patients with HER-2/neu overexpressing ductal carcinoma in situ of the breast were enrolled in a neoadjuvant immunization trial for safety and immunogenicity of DC1-polarized dendritic cells (DC1) pulsed with 6 HER-2/neu promiscuous major histocompatibility complex class II-binding peptides and 2 additional human leukocyte antigen (HLA)-A2.1 class I-binding peptides. DC1 were generated with interferon-γ and a special clinical-grade bacterial endotoxin (lipopolysaccharide) and administered directly into groin lymph nodes 4 times at weekly intervals before scheduled surgical resection of ductal carcinoma in situ. Patients were monitored for the induction of new or enhanced antipeptide reactivity by interferon-γ ELISPOT and enzyme-linked immunosorbentassays performed on Th cells obtained from peripheral blood or excised sentinel lymph nodes. Responses by cytotoxic T lymphocyte against HLA-A2.1-binding peptides were measured using peptide-pulsed T2 target cells or HER-2/neu-expressing or nonexpressing tumor cell lines. DC1 showed surface phenotype indistinct from "gold standard" inflammatory cocktail-activated DC, but displayed a number of distinguishing functional characteristics including the secretion of soluble factors and enhanced "killer DC" capacity against tumor cells in vitro. Postimmunization, we observed sensitization of Th cells to at least 1 class II peptide in 22 of 25 (88%; 95% exact confidence interval, 68.8%-97.5%) evaluable patients, whereas 11 of 13 (84.6%; 95% exact confidence interval, 64%-99.8%) HLA-A2.1 patients were successfully sensitized to class I peptides. Perhaps most importantly, anti-HER-2/neu peptide responses were observed up to 52-month postimmunization. These data show that even in the presence of early breast cancer such DC1 are potent inducers of durable type I-polarized immunity, suggesting potential clinical value for development of cancer immunotherapy.

Clinical trial results of the HER‐2/neu (E75) vaccine to prevent breast cancer recurrence in high‐risk patients

The authors conducted exploratory phase 1-2 clinical trials vaccinating breast cancer patients with E75, a human leukocyte antigen (HLA) A2/A3-restricted HER-2/neu (HER2) peptide, and granulocyte-macrophage colony-stimulating factor. The vaccine is given as adjuvant therapy to prevent disease recurrence. They previously reported that the vaccine is safe and effective in stimulating expansion of E75-specific cytotoxic T cells. Here, they report 24-month landmark analyses of disease-free survival (DFS). These dose escalation/schedule optimization trials enrolled lymph node-positive and high-risk lymph node-negative patients with HER2 (immunohistochemistry [IHC] 1-3(+) ) expressing tumors. HLA-A2/A3(+) patients were vaccinated; others were followed prospectively as controls for recurrence. DFS was analyzed by Kaplan-Meier curves; groups were compared using log-rank tests. Of 195 enrolled patients, 182 were evaluable: 106 (58.2%) in the vaccinated group and 76 (41.8%) in the control group. The 24-month landmark analysis DFS was 94.3% in the vaccinated group and 86.8% in the control group (P = .08). Importantly, because of trial design, 65% of patients received a lower than optimal vaccine dose. In subset analyses, patients who benefited most from vaccination (vaccinated group vs control group) had lymph node-positive (DFS, 90.2% vs 79.1%; P = .13), HER2 IHC 1+-2+ (DFS, 94.0% vs 79.4%; P = .04), or grade 1 or 2 (DFS, 98.4% vs 86.0%; P = .01) tumors and were optimally dosed (DFS, 97.3% vs 86.8%; P = .08). A booster program has been initiated; no patients receiving booster inoculations have recurred. The E75 vaccine has clinical efficacy that is more prominent in certain patients. A phase 3 trial enrolling lymph node-positive patients with HER2 low-expressing tumors is warranted.

The relationship between immunologic response and recurrence in a phase II trial evaluating the HER-2/neu peptide vaccines AE37 and GP2 in breast cancer patients in the adjuvant setting

The relationship between immunologic response and recurrence in a phase II trial evaluating the HER-2/neu peptide vaccines AE37 and GP2 in breast cancer patients in the adjuvant setting

MUC1/HER2/neu peptide-based immunotherapeutic vaccines for breast adenocarcinomas.

e13046 Background: Vaccination of resected breast cancer patients has shown promise in prolonging disease-free survival, but immune responses to vaccine antigens have been inconsistent. We hypothesized that addition of the toll-like receptor 9 agonist CpG-7909 and granulocyte-macrophage colony stimulating factor (GM-CSF—sargramostim) to a peptide vaccine for resected breast cancer patients would increase the frequency of vaccine-specific cytotoxic T lymphocytes generated by patients in response to vaccination. Methods: Patients with resected MUC1-positive breast adenocarcinoma who had completed all standard adjuvant therapy were randomized to receive vaccines consisting of three MUC1- and HER-2/neu-derived peptides emulsified in Montanide ISA-51 with varying immune adjuvants: GM-CSF (Arm A); CpG-7909 (Arm B); or both GM-CSF and CpG-7909 (Arm C). Vaccines were given every four weeks for six treatment cycles; patients were then observed for an additional 18 months. Vaccine immune responses were monitored by staining peripheral blood samples with MHC tetramers containing the peptides of interest. Responses were defined as doubling of antigen-specific T cells from pre-treatment levels or conversion from undetectable (< 0.05%) to detectable frequencies of antigen-specific T cells. Results: Four patients experienced ≥ grade 3 toxicities: 1/16 in Arm A (skin infection), 2/11 in Arm B (injection site reaction, endocrinopathy), and 1/12 in Arm C (syncope). The most common toxicities of any grade were injection site reactions, arthralgias, myalgias, and fatigue. HER-2/neu-specific immune responses were induced in 10 of 15 patients tested (3 of 5 in Arm A, 3 of 5 in Arm B, and 4 of 5 in Arm C), while MUC1-specific immune responses were induced in 9 of 15 patients (1 of 5 in Arm A, 4 of 5 in Arm B, and 4 of 5 in Arm C). Conclusions: Vaccines consisting of MUC1 and HER-2/neu peptides with GM-CSF and/or CpG-7909 are generally well-tolerated and lead to T cell immune responses in the majority of vaccinated patients. Funded by the DOD Breast Cancer Research Program DAMD17-01-1-0318.

Sequential administration of trastuzumab and a CD8 T-cell-eliciting HER2/neu peptide vaccine in patients with breast cancer compared to trastuzumab alone.

564 Background: We are conducting multiple, prospective, phase II trials evaluating different HER-2/neu(HER2) peptide vaccines in the adjuvant setting to prevent breast cancer (BrCa) recurrence. E75 (HER2: 369-377) and another vaccine (X) are HLA-A2/3+-restricted peptides capable of stimulating CD8+ cytotoxic T cells with anti-HER2 tumor activity. Trastuzumab (Tz), a recombinant monoclonal anti-HER2 antibody, has been shown to reduce BrCa recurrence by 50%. We examined the sequential use of Tz and a CD8 T cell-eliciting vaccine to compare the efficacy of sequential passive and active immunization to passive immunotherapy alone. Methods: We examined patients from our E75 and vaccine X clinical trials. HLA-A2/3+ node positive or high-risk node negative BrCa patients with any level of HER2 expression, disease-free after standard treatments were vaccinated with E75 or X+GM-CSF (immunoadjuvant) vs. nothing (E75 trial) or GM-CSF alone (X trial). Vaccinations were given as 6 monthly intradermal inoculations. Patients receiving adjuvant Tz followed by vaccination were compared to those receiving Tz without vaccination (or GM-CSF alone). p-values were calculated using Fisher’s exact test. Results: Of 283 patients (E75=187; 108 vaccine, 79 control) (X=96; 41 vaccine, 55 control), 62 (22%) received adjuvant Tz (E75=15, X=47). Of the 62 Tz-treated patients, 32 received no vaccine, and their recurrence rate is 12.5% (4/32)—comparable with reported rates of similarly staged and treated patients. In contrast, 30 patients received either E75 (12) or X (18) + GM-CSF after completing adjuvant Tz, with a recurrence rate of 0% (0/30) (p=0.065). Overall median length of follow-up is 48 months (E75=57 mo, X=19 mo). Conclusions: BrCa patients enrolled in our phase II trials of the E75 or X peptide vaccines who received adjuvant Tz followed by vaccination with a CD8-eliciting peptide vaccine appear to have a lower recurrence rate than adjuvant Tz therapy alone. This finding suggests that the combination of passive and active HER-2/neu immunization may have better efficacy than passive immunotherapy alone and has prompted the initiation of combination clinical trials.

Abstract LB-413: Circulating regulatory T cells (CD4+CD25high CD127low) decrease in breast cancer patients after vaccination with a modified HER-2/neu HLA class II peptide (AE37) vaccine

Abstract BACKGROUND: Regulatory T cells (TReg) are elevated in breast cancer patients with the number of TReg cells being inversely associated with clinical outcome. We are conducting a prospective, randomized phase II trial of the HER-2/neu peptide vaccines GP2 and AE37. GP2 is an HLA-A2+ -restricted peptide (HER-2/neu aa: 654–662) capable of eliciting a CD8+ cytotoxic T cell response directed against HER-2/neu expressing tumors. AE37 is the Ii-Key hybrid of the HER-2/neu peptide AE36 (HER-2/neu aa:776–790) and is capable of stimulating CD4+ helper T-cells with anti-tumor activity. Given the ability of AE37 to activate CD4+ helper T cells, concern exists that unwanted stimulation of TReg cells may occur with vaccination; although, this was not seen in our phase I trial. We have sought to confirm this finding by evaluating peripheral TReg cell levels in patients from our larger phase II breast cancer vaccine trial. METHODS: Node positive or high-risk node negative breast cancer patients with any level of HER-2/neu expression rendered disease-free by standard treatments were enrolled. HLA-A2+ patients were placed in the GP2 arm and randomized to either GP2 + GM-CSF (immunoadjuvant) or GM-CSF alone. HLA-A2− patients were placed in the AE37 arm and randomized to either AE37 + GM-CSF or GM-CSF alone. Vaccinations were given as six monthly intradermal inoculations. CD4+CD25high CD127low (CD127low) TReg cell levels were measured before (R0), mid-series (R3), upon completion (R6), and at six (RC6) and twelve (RC12) months after completion of the vaccine series using antibody staining and flow cytometry analysis. p values were calculated using the Student's t-test. RESULTS: TReg cells were measured in 65 patients in the AE37 arm (34 vaccine, 31 adjuvant) and 33 patients in the GP2 arm (17 vaccine, 16 adjuvant). Patients receiving the AE37 vaccine exhibited a decrease in CD127low TReg cells from baseline at all measured timepoints (R0=4.2, R3=3.7, p&amp;lt;0.01; R6=3.7, p=0.01; RC6=3.5, p&amp;lt;0.01; RC12=3.7, p&amp;lt;0.00001) while the randomized adjuvant patients demonstrated no decrease at any time point except from R0 to R3 (R0=4.3, R3=3.6, p=0.02; R6=4.1, p=0.58; RC6=3.9, p=0.06; RC12=3.9, p=0.21). In the GP2 arm, CD127low TReg cell levels did not change for either GP2 vaccinated patients (R0=3.6, R3=3.7, p=0.47; R6=4.1, p=0.67; RC6=4.3, p=0.56; RC12=4.3, p=0.16) or in randomized adjuvant patients (R0=4.4, R3=4.0, p=0.58; R6=4.4, p=0.95; RC6=3.8, p=0.25; RC12, p=0.59). CONCLUSIONS: Administration of a CD4 T cell-eliciting HER-2/neu peptide (AE37) vaccine to breast cancer patients induced a sustained decrease in circulating CD127low TReg cells. No such decrease was seen in randomized adjuvant-treated patients. Use of the CD8-eliciting, GP2 vaccine did not change circulating TReg cell levels. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr LB-413. doi:10.1158/1538-7445.AM2011-LB-413

Abstract LB-414: Comparison of in vitro and in vivo immunologic responses in a prospective, randomized, single-blinded phase II trial evaluating the HER-2/neu peptide vaccines GP2 and AE37 in breast cancer patients

Abstract BACKGROUND: We are conducting a prospective, randomized, single-blinded phase II trial evaluating the HER-2/neu peptide vaccines AE37 and GP2 in the adjuvant setting to prevent breast cancer recurrence. AE37 is the Ii-Key hybrid of the HER-2/neu peptide AE36 (HER-2/neu aa:776–790) and is capable of stimulating CD4+ helper T-cells with anti-tumor activity. GP2 is an HLA-A2+ -restricted immunogenic peptide from the HER-2/neu protein (HER-2/neu aa: 654–662) which is capable of stimulating CD8+ cytotoxic T-lymphocytes with the ability to recognize and destroy HER-2/neu-expressing tumor cells. Here we present data comparing the immunological profiles between these CD4 and CD8 eliciting vaccines METHODS: Node positive or high-risk node negative breast cancer patients with any level of HER-2/neu expression rendered disease-free by standard treatments were enrolled. Patients were HLA-typed at enrollment. HLA-A2+ patients were placed in the GP2 arm and randomized to either GP2 + GM-CSF (immunoadjuvant) or GM-CSF alone. HLA-A2− patients were placed in the AE37 arm and randomized to either AE37 + GM-CSF or GM-CSF alone. Vaccinations were given in six monthly intradermal inoculations. In vitro (3H-thymidine proliferation for AE37, HLA-A2:immunoglobulin dimer for GP2) and in vivo (delayed-type hypersensitivity (DTH)) immune responses were measured before (R0), mid-series (R3), upon completion (R6), and at six (RC6) and twelve (RC12) months after completion of the vaccine series. RESULTS: We have enrolled 247 patients (AE37=171, 87 vaccine and 84 control) (GP2=76, 41 vaccine and 35 control). Proliferative responses to both AE37 and AE36 were increased from baseline in the vaccine group at all time points (AE37: R0=0.98, R3=3.29, R6=3.38, RC6=3.64, RC12=3.68, p&amp;lt;0.001; AE36: R0=0.95, R3=1.92, R6=2.12, RC6=2.18, RC12=1.80, p&amp;lt;0.001) while they did not change for the control group. The population of circulating GP2-specific CD8+ T-cells measured in the dimer assay increased from baseline in the vaccine group at all time points (R0=1.00, R3=1.61, R6=1.85, RC6=1.95, RC12=1.93, p&amp;lt;0.05) with no change in the control group. DTH responses increased in both AE37 and GP2 vaccinated patients from pre- to post-vaccination (AE37: 2.7 vs 30.2 mm, p&amp;lt;0.001; AE36: 2.2 vs 16.3 mm, p&amp;lt;0.001; GP2: 3.5 vs 29.4 mm, p&amp;lt;0.001) while there was no significant change in the control group. CONCLUSIONS: In a novel prospective, randomized, single-blinded phase II vaccine trial of the AE37 and GP2 peptide vaccines with a GM-CSF only control group, breast cancer patients vaccinated with either a CD4 or CD8 eliciting peptide vaccine exhibit high levels of HER-2/neu-specific in vitro and in vivo immune responses. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr LB-414. doi:10.1158/1538-7445.AM2011-LB-414

Investigation of Transmembrane Helix Hetero-Dimerization by Combinatorial Peptide Libraries and High-Throughput Measurement of FÖRSTER Resonance Energy Transfer in Liposomes

Receptor tyrosine kinases (RTKs) are membrane proteins containing an N-terminal extracellular ligand-binding domain, a single helical transmembrane (TM) domain, and an intracellular catalytic kinase domain. Lateral dimerization driven in part by the TM domain is proposed to be a crucial intermediate step in signal transduction across the plasma membrane. Defects in this process such as over-dimerization are closely linked to unregulated signaling and disease. Therefore, dimerization inhibitors developed using the chemical-physical basis of RTK TM domain dimerization could be promising therapeutic agents. Here we present a first step towards this goal. We designed a rational combinatorial peptide library based on the TM domain of Neu, an RTK from the Erb-B/HER epidermal growth factor receptor family (EGFR) with a pathogenic V to E mutation in the TM domain. Förster Resonance Energy Transfer (FRET) in lipid vesicles was used as a method to probe the dimerization between the library members and mutant Neu sequence. To enable high-throughput screening in liposomes, we developed a novel multi-well FRET assay. FRET donor-labeled library members and acceptor-labeled Neu peptides were incorporated into lipid vesicles in a rapid high-throughput 96-well plate format. We are using this high throughput screen to identify inhibitors of Neu dimerization. Our findings will be discussed in a context of throughput and sensitivity along with a statistical analysis that takes into account the FRET that arises from random proximity of donors and acceptors in the bilayer.