Vivian Goodell

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.

Humoral Immunity Directed against Tumor-Associated Antigens As Potential Biomarkers for the Early Diagnosis of Cancer

Over the past decade, it has been demonstrated that cancer is immunogenic, and multiple tumor antigens have been identified in cancer patients. It is now possible to potentially harness the immune response elicited by cancer growth as a potential diagnostic tool. Humoral immunity, or the development of autoantibodies against tumor-associated proteins, may be used as a marker for cancer exposure. Unlike circulating proteins that are shed by bulky tumors, serum autoantibodies are detectable even when antigen expression is minimal. This paper will review the methods used for tumor antigen discovery and overview what is known about autoantibodies targeting common cancer antigens with a focus on breast cancer. Data will be presented modeling the use of tumor antigen associated autoantibodies as a breast cancer diagnostic. The endogenous humoral immune response present in cancer patients may allow the identification of individuals exposed to the malignant transformation of somatic cells.

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.

Antibody Immunity to the p53 Oncogenic Protein Is a Prognostic Indicator in Ovarian Cancer

PURPOSE Presence of intratumoral T-cell infiltration has been linked to improved survival in ovarian cancer patients. We questioned whether antibody immunity specific for ovarian cancer tumor antigens would predict disease outcome. We evaluated humoral immune responses against ovarian cancer antigens p53, HER-2/neu, and topoisomerase IIalpha. PATIENTS AND METHODS Serum was collected from 104 women (median age, 59 years; range, 34 to 89 years) at the time of their initial definitive surgery for ovarian cancer. Serum was analyzed by enzyme-linked immunosorbent assay for antibodies to p53, HER-2/neu, and topoisomerase IIalpha proteins. Antibody immunity to tetanus toxoid was assessed as a control. The incidence of humoral immunity at the time of diagnosis to any of these three antigens was tabulated. For patients with advanced-stage disease (III/IV), correlation was made between the presence of tumor-specific immunity at the time of diagnosis and overall survival. Patients were followed for a median of 1.8 years. RESULTS Multivariate analysis showed the presence of p53 antibodies to be an independent variable for prediction of overall survival in advanced-stage patients. Overall survival was significantly higher for patients with antibodies to p53 when compared with patients without p53 antibodies (P = .01). The median survival for p53 antibody-positive patients was 51 months (95% CI, 23.5 to 60.5 months) compared with 24 months (95% CI, 19.4 to 28.6 months) for patients without antibodies to p53. CONCLUSION Data presented here demonstrate that advanced stage ovarian cancer patients can have detectable tumor-specific antibody immunity and that immunity to p53 may predict improved overall survival in patients with advanced-stage disease.

Direct detection of carcinoembryonic antigen autoantibodies in clinical human serum samples using a surface plasmon resonance sensor

The ability to detect biomarkers in human serum is important for cancer diagnostics. The work presented focuses on the establishment of a surface plasmon resonance (SPR) biosensor as a means for detecting varying levels of autoantibody biomarkers in human serum samples. Carcinoembryonic antigen (CEA) is a biomarker that is present in human serum. It is thought that CEA levels become elevated in patients with colon and ovarian cancer, causing a corresponding increase in the autoantibody level in human serum. Detection of this CEA autoantibody increase could be used to diagnose cancer in patients. Using a SPR biosensor, human serum samples were screened directly for CEA antibody levels. Results using a sandwich assay with a SPR sensor demonstrated the same linear trend as seen from an established enzyme-linked immunosorbent assay (ELISA) method. Serum samples from five healthy individuals were used to establish a threshold value for differentiating a cancerous serum sample from a negative sample with a 95% confidence. Three serum samples from cancer patients with positive CEA antibody levels as evaluated by ELISA were used to test the criterion.

Evaluation of Known Oncoantibodies, HER2, p53, and Cyclin B1, in Prediagnostic Breast Cancer Sera

Serum autoantibodies, directed against oncogenic proteins, have been frequently detected in the sera of patients with breast cancer. It is unknown whether serum antibodies that are identified in patients with established disease could also be detected in patients with newly diagnosed disease or even predate the diagnosis of breast cancer. Using sera collected at the time of treatment, at the time of diagnosis, or before the time of diagnosis, the current study aimed to address the temporal relationship between breast cancer development and serum antibody response. Starting from serum antibodies to eight known breast cancer antigens, we first identified four serum antibodies, HER2/neu, p53, carcinoembryonic antigen (CEA), and cyclin B1, which are significantly increased in the sera collected from patients with breast cancer at the time of treatment. These antibodies were also elevated in breast cancer sera collected at the time of diagnosis. Finally, comparison of antibody responses in prediagnostic samples from women before the development of breast cancer and in controls showed that antibodies to the HER2/neu and p53 can be detected in sera that were collected on average more than 150 days before a breast cancer diagnosis. These results showed that serum autoantibodies commonly reported in sera from patients with established disease can also be detected in prediagnostic sera and may be useful for the early detection of breast cancer. Cancer Prev Res; 5(8); 1036–43. ©2012 AACR.

Level of HER-2/neu protein expression in breast cancer may affect the development of endogenous HER-2/neu-specific immunity

We questioned whether the incidence or magnitude of the humoral or cellular immune response to the self-tumor antigen HER-2/neu is influenced by the level of HER-2/neu protein overexpression as defined by immunohistochemical staining of tumors in breast cancer patients. We obtained peripheral blood from 104 women with stage II, III, and IV pathologically confirmed HER-2/neu-overexpressing breast cancer. Patients were categorized with +1 (n = 14), +2 (n = 20), or +3 (n = 70) HER-2/neu overexpression by institutional pathologic report. Circulating antibodies to HER-2/neu were evaluated using ELISA. T-cell responses to HER-2/neu were measured using an antigen-specific tritiated thymidine incorporation assay. Eighty-two percent of subjects with HER-2/neu antibodies were +3 overexpressors compared with 18% +2 overexpressors and 0% +1 overexpressors, a highly significant difference (P < 0.001), and there were significant differences in the magnitude of the HER-2/neu-specific antibodies between groups with varying HER-2/neu protein expression (P = 0.022). In addition, 65% of subjects with HER-2/neu-specific T cells were +3 overexpressors compared with 16% +2 overexpressors and 19% +1 overexpressors (P = 0.001). Data presented here indicate that endogenous HER-2/neu-specific humoral and T-cell immunity is greater in patients with +3 protein overexpression in their tumors than in patients with lower levels of HER-2/neu expression. Overexpression of a self-tumor-associated protein is a potential mechanism by which immunogenicity is enhanced and may aid in the identification of biologically relevant proteins to target for immune-based molecular cancer therapies. [Mol Cancer Ther 2008;7(3):449–54]

Targeting serum antibody for cancer diagnosis: a focus on colorectal cancer.

The ability of the immune system to magnify the appearance of disease by generating relatively large amounts of antibody in response to small amounts of disease makes it a natural biosensor, and serum antibodies have emerged as promising biomarkers for the detection of cancer. This review summarizes recent progress in targeting serum antibodies for cancer diagnosis, with a particular focus on colorectal cancer (CRC). Several serum antibodies have been detected at increased levels in CRC patients, including p53, carcinoembryonic antigen, Ras, topoisomerase II-α, histone deacetylase 3 and 5, ubiquitin C-terminal hydrolase L3, tropomyosin and cyclin B1. As each antibody is only present in a limited proportion of patients (usually < 40%), a combination of serum antibodies that defines the ‘immunological signature’ of cancer needs to be developed. High-throughput methods to identify new serum antibodies for cancer diagnosis are also reviewed.

Insulin-like growth factor binding protein 2 is a target for the immunomodulation of breast cancer

Breast cancer is immunogenic and well suited to treatment via immunomodulation. The disease is often treated to remission and time to relapse is generally measured in years in many cases. Immune-based therapeutics, such as cancer vaccines, may be able to affect the clinical progression of micrometastatic disease. Immune targets must be identified that have the potential to inhibit tumor growth. Insulin-like growth factor-binding protein-2 (IGFBP-2) has direct effects on breast cancer proliferation via stimulation of critical signaling pathways. We questioned whether IGFBP-2 was an immune target in breast cancer. IGFBP-2-specific IgG antibody immunity was preferentially detected in breast cancer patients compared with controls (P = 0.0008). To evaluate for the presence of T-cell immunity, we identified potential pan-HLA-DR binding epitopes derived from IGFBP-2 and tested the peptides for immunogenicity. The majority of epitopes elicited peptide-specific T cells in both patients and controls and had high sequence homology to bacterial pathogens. IGFBP-2 peptide-specific T cells could respond to naturally processed and presented IGFBP-2 protein, indicating that these peptides were native epitopes of IGFBP-2. Finally, both immunization with IGFBP-2 peptides as well as adoptive transfer of IGFBP-2-competent T cells mediated an antitumor effect in a transgenic mouse model of breast cancer. This is the first report of IGFBP-2 as a human tumor antigen that may be a functional therapeutic target in breast cancer.

Tumor Antigen ^ Specific T-Cell Expansion Is Greatly Facilitated by In vivo Priming

Purpose: Adoptive T-cell therapy is a promising strategy for the treatment of patients with established tumors but is often limited to specific cancers where tumor-infiltrating lymphocytes, the source of T cells for ex vivo culture, can be obtained. In this study, we evaluated the feasibility of expanding HER-2/neu–specific T cells derived from peripheral blood ex vivo following in vivo priming with a HER-2/neu peptide vaccine. Experimental Design: Peripheral blood mononuclear cells from cytomegalovirus (CMV)–seronegative and CMV-seropositive donors as well as HER-2/neu–positive cancer patients who had or had not been vaccinated with a HER-2/neu peptide–based vaccine was used as a source of T lymphocytes. Antigen-specific T-cell lines were generated by in vitro stimulation with antigen followed by nonspecific expansion on CD3/CD28 beads. The ability to expand antigen-specific T cells was assessed using IFN-γ and granzyme B enzyme-linked immunosorbent spot. The phenotype of the resultant T-cell lines was evaluated by flow cytometry, including the presence of FOXP3-expressing CD4+ T cells. Results: The frequencies of CMV-specific T cells generated from CMV+ donors were >11-fold higher than the frequencies from CMV− donors (P = 0.001), with 22-fold increase of total number of CD3+ T cells. The frequencies of HER-2/neu–specific T cells generated from the primed patients were >25-fold higher than the frequencies from unvaccinated patients (P = 0.006), with an average of a 19-fold increase of total number of CD3+ T cells. Using peripheral blood as the source of T cells did not result in concurrent expansion of FOXP3+CD4+ regulatory T cells despite the use of interleukin-2 in in vitro culture. Both CD4+ and CD8+ HER-2/neu–specific T cells could be expanded. The extent of ex vivo expansion correlated with the magnitude of immunity achieved during immunization (P = 0.008). Conclusion: Tumor-specific T cells can be efficiently expanded from the peripheral blood ex vivo following in vivo priming with a vaccine. This approach provides an effective method to generate tumor-specific polyclonal T cells for therapeutic use that could be applied to cancer patients with any tumor type.