Endogenous anti-HER2 antibodies block HER2 phosphorylation and signaling through extracellular signal-regulated kinase.

Immunologic targeting of the oncoprotein HER2/neu with monoclonal antibodies is an important component of current therapeutic strategies for patients with locally and systemically advanced breast cancer. Engineered antibodies targeting HER2 may have agonist or antagonist effects on HER2, but little is known about whether endogenous antibodies modulate HER2 activity. Vaccination of patients with HER2 peptides successfully induced antibodies in a minority of patients with HER2-expressing malignancy. A subset of antibodies specifically suppressed phosphorylation of HER2 on tyrosine Y1248, a residue critical for HER2 signaling through extracellular signal-regulated kinase. These antibodies also suppressed extracellular signal-regulated kinase phosphorylation and inhibited colony formation in soft agar. The majority of the antibodies that suppressed HER2 phosphorylation displayed specificity for amino acids 328 to 345 and 369 to 384. The isotype of anti-HER2 antibodies was predominantly IgG3 of low avidity, suggesting a Th1 response to peptide vaccine. Endogenous anti-HER2 antibodies can effectively suppress HER2 kinase activity and downstream signaling to inhibit the transformed phenotype of HER2-expressing tumor cells.

Generation of T-cell immunity to the HER-2/neu protein after active immunization with HER-2/neu peptide-based vaccines.

PURPOSE: The HER-2/neu protein is a nonmutated tumor antigen that is overexpressed in a variety of human malignancies, including breast and ovarian cancer. Many tumor antigens, such as MAGE and gp100, are self-proteins; therefore, effective vaccine strategies must circumvent tolerance. We hypothesized that immunizing patients with subdominant peptide epitopes derived from HER-2/neu, using an adjuvant known to recruit professional antigen-presenting cells, granulocyte-macrophage colony-stimulating factor, would result in the generation of T-cell immunity specific for the HER-2/neu protein. PATIENTS AND METHODS: Sixty-four patients with HER-2/neu-overexpressing breast, ovarian, or non-small-cell lung cancers were enrolled. Vaccines were composed of peptides derived from potential T-helper epitopes of the HER-2/neu protein admixed with granulocyte-macrophage colony-stimulating factor and administered intradermally. Peripheral-blood mononuclear cells were evaluated at baseline, before vaccination, and after vaccination for antigen-specific T-cell immunity. Immunologic response data are presented on the 38 subjects who completed six vaccinations. Toxicity data are presented on all 64 patients enrolled. RESULTS: Ninety-two percent of patients developed T-cell immunity to HER-2/neu peptides (stimulation index, 2.1 to 59) and 68% to a HER-2/neu protein domain (stimulation index range, 2 to 31). Epitope spreading was observed in 84% of patients and significantly correlated with the generation of a HER-2/neu protein-specific T-cell immunity (P =.03). At 1-year follow-up, immunity to the HER-2/neu protein persisted in 38% of patients. CONCLUSION: The majority of patients with HER-2/neu-overexpressing cancers can develop immunity to both HER-2/neu peptides and protein. In addition, the generation of protein-specific immunity, after peptide immunization, was associated with epitope spreading, reflecting the initiation of an endogenous immune response. Finally, immunity can persist after active immunizations have ended.

Effect of dose on immune response in patients vaccinated with an her-2/neu intracellular domain protein--based vaccine.

PURPOSE: To evaluate the safety of an HER-2/neu intracellular domain (ICD) protein vaccine and to estimate whether vaccine dose impacts immunogenicity. PATIENTS AND METHODS: Twenty-nine patients with HER-2/neu-overexpressing breast or ovarian cancer and with no evidence of disease after standard therapy received a low- (25 microg), intermediate- (150 microg), or high-dose (900 microg) HER-2/neu ICD protein vaccine. The vaccine was administered intradermally, monthly for 6 months, with granulocyte-macrophage colony-stimulating factor as an adjuvant. Toxicity and both cellular and humoral HER-2/neu-specific immunity was evaluated. RESULTS: The vaccine was well tolerated. The majority of patients (89%) developed HER-2/neu ICD-specific T-cell immunity. The dose of vaccine did not predict the magnitude of the T-cell response. The majority of patients (82%) also developed HER-2/neu-specific immunoglobulin G antibody immunity. Vaccine dose did not predict magnitude or avidity of the HER-2/neu-specific humoral immune response. Time to development of detectable HER-2/neu-specific immunity, however, was significantly earlier for the high- versus low-dose vaccine group (P =.003). Over half the patients retained HER-2/neu-specific T-cell immunity 9 to 12 months after immunizations had ended. CONCLUSION: The HER-2/neu ICD protein vaccine was well tolerated and effective in eliciting HER-2/neu-specific T-cell and antibody immunity in the majority of breast and ovarian cancer patients who completed the vaccine regimen. Although the dose of vaccine did not impact the magnitude of T-cell or antibody immunity elicited, patients receiving the highest dose developed HER-2/neu-specific immunity more rapidly than those who received the lowest dose.

Hormone receptor status does not affect the clinical benefit of trastuzumab therapy for patients with metastatic breast cancer.

BACKGROUND: Hormone receptor (HR) and HER2 signaling pathways are involved in the regulation of breast cancer proliferation. The impact of HR status on the clinical outcome of patients with HER2-overexpressing disease treated with the monoclonal antibody trastuzumab is unknown. PATIENTS AND METHODS: To evaluate this, we conducted a retrospective analysis of 805 patients with metastatic breast cancer enrolled in 3 clinical trials comparing trastuzumab in combination with chemotherapy versus chemotherapy alone or trastuzumab monotherapy as first-, second-, or third-line treatment. Patients whose tumor samples overexpressed HER2 by fluorescence in situ hybridization (FISH) were stratified based on HR status, and clinical outcomes were compared. RESULTS: Tumor samples from 596 of 805 patients were HER2overexpressing by FISH; 45% of these were HR-positive and 43% were HR-negative (HR status was unknown in 12%). Overall response rate (ORR) and time to progression (TTP) were significantly higher in patients treated with chemotherapy plus trastuzumab than in those treated with chemotherapy alone, irrespective of HR status. Median survival was longer for patients with HR-positive tumors receiving combination therapy compared with those with HR-negative tumors. In the trastuzumab monotherapy trials, ORR and TTP were similar for patients with HR-positive and HR-negative tumors. Median survival was longer for patients with HR-positive tumors compared with those with HR-negative tumors. CONCLUSION: Hormone receptor status did not affect the clinical benefit of trastuzumab given as a single agent or combined with chemotherapy. The addition of trastuzumab to chemotherapy provides an improved clinical benefit compared with chemotherapy alone, regardless of HR status.

Immunization of cancer patients with a HER-2/neu, HLA-A2 peptide, p369-377, results in short-lived peptide-specific immunity.

Ideally, vaccines should be designed to elicit long-lived immunity. The goal of this study was to determine whether HER-2/neu peptide-specific CD8+ T-cell immunity could be elicited using an immunodominant HER-2/neu-derived HLA-A2 peptide alone in the absence of exogenous help. Granulocyte macrophage colony-stimulating factor (GM-CSF) was used as adjuvant. Six HLA-A2 patients with HER-2/neu-overexpressing cancers received 6 monthly vaccinations with a vaccine preparation consisting of 500 microg of HER-2/neu peptide, p369-377, admixed with 100 microg of GM-CSF. The patients had either stage III or IV breast or ovarian cancer. Immune responses to the p369-377 were examined using an IFN-gamma enzyme-linked immunosorbent spot assay. Before vaccination, the median precursor frequency (range), defined as precursors per 10(6) peripheral blood mononuclear cell, to p369-377 was 0 (no range). After vaccination, the median precursor frequency to p369-377 in four evaluable patients was 0 (0-116). Overall, HER-2/neu peptide-specific precursors developed to p369-377 in two of four evaluable subjects. The responses were short-lived and not detectable at 5 months after the final vaccination. Immunocompetence was evident, because patients had detectable enzyme-linked immunosorbent spot responses to tetanus toxoid and influenza. These results demonstrate that HER-2/neu MHC class I epitopes can induce HER-2/neu peptide-specific IFN-gamma-producing CD8+ T cells. However, the magnitude of the responses were low, as well as short-lived, suggesting that CD4+ T-cell help is required for lasting immunity to this epitope.

Immunization of cancer patients with HER-2/neu-derived peptides demonstrating high-affinity binding to multiple class II alleles.

PURPOSE: The purpose of this study was to immunize patients with HER-2/neu-overexpressing cancer with a multipeptide vaccine comprised of four class II HER-2/neu peptides that had been identified as the most immunogenic in a previous clinical trial. Furthermore, we questioned whether MHC binding affinity could predict the in vivo immunogenicity of the HER-2/neu helper peptides. EXPERIMENTAL DESIGN: Four putative class II HER-2/neu peptides, which were found to generate detectable specific T-cell responses (stimulation index > 2) in a majority of patients in a previous study, were used to formulate a single vaccine. The multipeptide vaccine was administered intradermally with granulocyte macrophage colony-stimulating factor as an adjuvant. Ten patients with HER-2/neu overexpressing breast or lung cancer were enrolled. HER-2/neu peptide-and protein-specific T cell and antibody immune responses were measured. Competitive inhibition assays were used to analyze the class II HER-2/neu peptides for their binding affinity to 14 common HLA-DR alleles. RESULTS: Twenty-five percent of patients developed HER-2/neu peptide-specific T-cell immunity, and 50% developed HER-2/neu peptide-specific antibody immunity. No patient developed HER-2/neu protein-specific T cell or antibody immunity. The majority of peptides exhibited high binding affinity, in vitro, to >/==" BORDER="0">3 of the 14 DR alleles analyzed. CONCLUSION: The group of peptides used in this study demonstrated high binding affinity to multiple DR alleles suggesting that in vitro binding affinity may be able to predict the in vivo immunogenicity of class II peptides. However, only a minority of patients immunized with the multipeptide vaccine developed HER-2/neu peptide-specific T cell or antibody immunity, and none developed HER-2/neu protein-specific immunity.

Adoptive T-cell therapy for the treatment of solid tumours.

Solid tumours can be eradicated by infusion of large amounts of tumour-specific T-cells in animal models. The successes seen in preclinical models, however, have not been adequately translated to human disease due, in part, to the inability to expand tumour antigen-specific T-cells ex vivo. Polyclonality and retention of antigen-specificity are two important properties of infused T-cells that are necessary for successful eradication of tumours. Investigators are beginning to evaluate the impact of attempting to reconstitute full T-cell immunity representing both major T-cell subsets, cytolytic T-cells and T-helper (Th) cells. One of the more important and often overlooked steps of successful adoptive T-cell therapy is the ex vivo expansion conditions, which can dramatically alter the phenotype of the T-cell. A number of cytokines and other soluble activation factors that have been characterised over the last decade are now available to supplement in vitro antigen presentation and IL-2. Newer molecular techniques have been developed and are aimed at genetically altering the characteristics of T-cells including their antigen-specificity and growth in vivo. In addition, advanced imaging techniques, such as positron emission tomography (PET), are being implemented in order to better define the in vivo function of ex vivo expanded tumour-specific T-cells.

Kinetics of tumor-specific T-cell response development after active immunization in patients with HER-2/neu overexpressing cancers.

The ability of a cancer vaccine to elicit a specific measurable T-cell response is increasingly being used to prioritize immunization strategies for therapeutic development. Knowing the optimal time during a vaccine regimen to measure the development of tumor-specific immunity would greatly facilitate the assessment of T-cell responses. The purpose of this study was to overview the kinetics of HER-2/neu-specific T-cell immunity evolution during and after the administration of HER-2/neu peptide-based vaccination in the adjuvant setting. Furthermore, we questioned whether the presence of preexistent HER-2/neu T-cell immunity or the timing of immunity development over the course of active immunization influenced the intensity of the elicited HER-2/neu-specific T-cell immunity. Our findings demonstrate that maximal tumor-specific immune responses may occur toward the end of the vaccination regimen or even after the scheduled vaccines have been completed. Additionally, the presence of tumor antigen-specific immunity prior to vaccination is associated with greater magnitude immune responses.

Delayed type hypersensitivity response to recall antigens does not accurately reflect immune competence in advanced stage breast cancer patients.

The development of delayed-type hypersensitivity (DTH) response to recall antigens has long been utilized as a measure of immune competence. It is assumed that because patients with advanced stage cancers exhibit multiple immune system defects they may not be responsive to immunization. We pre-selected patients with advanced HER-2/neu (HER2) overexpressing breast and ovarian cancers for enrolment into a phase I trial designed to evaluate the immunogenicity of a HER2 peptide vaccine based on the patient's immune competence as assessed by DTH skin testing to common recall antigens (Multitest CMI, Institut Merieux, Lyon, France). At the time of a positive DTH response to tetanus toxoid (tt) peripheral blood was obtained to measure T cell responses to tt. Of 53 patients evaluated, 38 (72%) were not anergic. Among the 15 (28%) who were, seven patients with advanced stage breast cancer were re-tested a median of 26 days (range 12-150 days) after receiving a tt bopster vaccination. Five of the seven had positive DTH responses when re-challenged with tt and six had peripheral blood tetanus specific T cell response with stimulation index >2.0. Thus, the majority of patients studied with advanced stage breast or ovarian cancer were able to mount a DTH response to common recall antigens. Moreover, a negative response by DTH testing to a battery of common recall antigens was not a reflection of the breast cancer patient's ability to mount a cell-mediated immune response to a vaccinated antigen, tt.

Humoral epitope-spreading following immunization with a HER-2/neu peptide based vaccine in cancer patients.

HER-2/neu is a tumor antigen in patients with breast and ovarian cancer. Multiple varieties of vaccine strategies are being developed to immunize patients against HER-2/neu. Studies in animal models have demonstrated both T cell and antibody immunity are needed to mediate an antitumor response. Thirty-five patients, immunized with HER-2/neu peptide based vaccines, were evaluated for the generation of HER-2/neu-specific antibody immunity. Sixty percent of patients developed HER-2/neu IgG specific antibody responses to at least one peptide included in their vaccine. Twenty-nine percent of patients developed IgG immunity to the native HER-2/neu protein after peptide immunization. Humoral intramolecular epitope-spreading within the HER-2/neu protein occurred in 49% of immunized patients. Intermolecular epitope-spreading to p53 was evident in 20% of vaccinated patients. Of those patients who developed new immunity to p53, 71% had demonstrated antibody epitope-spreading within HER-2/neu.

Immunotherapy of glioblastoma multiforme.

Glioblastoma multiforme is immunogenic and several glioblastoma multiforme-related antigens have now been identified. In addition, the immunologic characteristics of the tumor microenvironment that may affect tumor growth are becoming increasingly understood. The type of immune-based approach selected to treat glioblastoma multiforme will depend on the tumor burden. For minimal disease states, active vaccination may be useful for generating adequate protection from relapse. However, for more advanced stage disease states, more rigorous strategies may need to be applied, such as adoptive T-cell therapy, antibody therapy or a combination of different techniques. The immunosuppressive environment observed during advanced malignancy may need to be reversed for improved efficacy of immune-based therapies.

Flt3 ligand as a vaccine adjuvant in association with HER-2/neu peptide-based vaccines in patients with HER-2/neu-overexpressing cancers.

Dendritic cells (DCs) are potent antigen-presenting cells and have shown promise to function as "natural" vaccine adjuvants. Currently, most cancer vaccine trials using DCs generate autologous DCs ex vivo for each patient. Systemic treatment with Flt3 ligand (FL) results in a marked increase of DCs in tissues such as spleen and lymph nodes in mice and in the peripheral blood and skin of humans. In light of these observations, we questioned whether FL could be used systemically as a vaccine adjuvant to stimulate DC mobilization in vivo, circumventing the need to generate DCs ex vivo. Ten patients with HER-2/neu-overexpressing cancer were enrolled in a phase 1 study to receive a HER-2/neu peptide-based vaccine targeting the intracellular domain of the HER-2/neu protein. All patients received 20 microg/kg FL per day subcutaneously for 14 days. Five patients received the HER-2/neu peptide-based vaccine alone on day 7 of the 14-day cycle, and 5 patients received the vaccine admixed with 150 microg granulocyte macrophage-colony-stimulating factor (GM-CSF) on day 7 of the FL cycle. T-cell proliferative responses to HER-2/neu peptides and intracellular domain protein suggest that vaccine regimens including FL as an adjuvant were not effective in eliciting a significant HER-2/neu protein-specific T-cell proliferative response. However, including FL as a vaccine adjuvant was effective in boosting the precursor frequency of interferon-gamma-secreting HER-2/neu-specific T cells. The small sample size of each group, however, did not allow a statistically significant comparison of immune responses between the FL alone and FL with GM-CSF arms. Finally, vaccine regimens including FL as a vaccine adjuvant were associated with the development of apparent autoimmune phenomena in some patients.

Pilot study of an HLA-A2 peptide vaccine using flt3 ligand as a systemic vaccine adjuvant.

A pilot vaccine study was conducted to test the safety and immunological efficacy of four monthly immunizations of an MHC class I peptide vaccine, the E75 HLA-A2 epitope from HER-2/neu, using flt3 ligand as a systemic vaccine adjuvant. Twenty HLA-A2-expressing subjects with advanced stage prostate cancer were randomly assigned to one of four immunization or treatment schedules: (a) Flt3 ligand (20 microg/kg per day) administered subcutaneously daily for 14 days on a 28-day cycle, monthly for four months; (b) flt3 ligand course as above with the E75 peptide vaccine administered on day 7 of each flt3 ligand cycle; (c) flt3 ligand course as above with the E75 peptide vaccine administered on day 14 of each flt3 ligand cycle; or (d) E75 peptide admixed with granulocyte-macrophage colony-stimulating factor and administered intradermally once every 28 days, as has previously been reported. The primary endpoints of the study were the determination of safety and immunological efficacy in generating E75-specific T cells as determined by peptide-specific interferon-gamma ELIspot. Adverse events included one grade 3 skin reaction and the development of grade 2 autoimmune hypothyroidism in two subjects with preexisting subclinical autoimmune hypothyroidism. Dendritic cells were markedly increased in the peripheral blood of subjects receiving flt3 ligand with each repetitive cycle, but augmentation of antigen-presenting cells within the dermis was not observed. Apart from a single subject, no significant peptide-specific T-cell responses were detected by ELIspot, whereas delayed-type hypersensitivity responses were detectable in control subjects and in subjects receiving peptide vaccine early in the course of flt3 ligand administration. The absence of robust peripheral immune responses in the current study may be attributable to the small numbers of subjects or differences in the subject population. In addition, the inability of fit3 ligand to augment the number of peripheral skin antigen-presenting cells may have contributed to the absence of robust peptide-specific immunity detectable in the peripheral blood of immunized subjects treated with flt3 ligand.