Knutson, Keith L.

Abstract Elements of the immune system act as intimate regulators of cancer progression, inhibiting early stages of tumor growth, through immunosurveillance while facilitating later stages of tumor progression. Recent findings have revealed that activated CD8 T cells can stimulate mammary epithelial tumor cells to undergo epithelial-mesenchymal transition (EMT) and to acquire the greatly increased tumorigenic capability and chemotherapeutic resistance of breast cancer stem cells (BCSC). These studies provide a window to understanding how BCSC arise and are maintained within tumors, and how to best target these processes for therapeutic benefit. Cancer Res; 70(8); 3005–8. ©2010 AACR.

AbstractPurpose: Over the past two decades, there has been significant interest in targeting HER-2/neu in immune-based approaches for the treatment of HER-2/neu+ cancers. For example, peptide vaccination using a CD8 T cell–activating HER-2/neu epitope (amino acids 369-377) is an approach that is being considered in advanced phase clinical trials. Studies have suggested that the persistence of HER-2/neu–specific CD8 T cells could be improved by incorporating human leukocyte antigen (HLA) class II epitopes in the vaccine. Our goal in this study was to identify broad coverage HLA-DR epitopes of HER-2/neu, an antigen that is highly expressed in a variety of carcinomas.Experimental Design: A combination of algorithms and HLA-DR–binding assays was used to identify HLA-DR epitopes of HER-2/neu antigen. Evidence of preexistent immunity in cancer patients against the identified epitopes was determined using IFN-γ enzyme-linked immunosorbent spot (ELIspot) assay.Results: Eighty-four HLA-DR epitopes of HER-2/neu were predicted, 15 of which had high binding affinity for ≥11 common HLA-DR molecules. A degenerate pool of four HLA-DR–restricted 15-amino acid epitopes (p59, p88, p422, and p885) was identified, against which >58% of breast and ovarian cancer patients had preexistent T-cell immunity. All four epitopes are naturally processed by antigen-presenting cells. Hardy-Weinberg analysis showed that the pool is useful in ∼84% of population. Lastly, in this degenerate pool, we identified a novel in vivo immunodominant HLA-DR epitope, HER-2/neu88-102 (p88).Conclusion: The broad coverage and natural immunity to this epitope pool suggests potential usefulness in HER-2/neu–targeting, immune-based therapies such as vaccines. Clin Cancer Res; 16(3); 825–34

Abstract The breast cancer stem cell (BCSC) hypotheses suggest that breast cancer is derived from a single tumor-initiating cell with stem-like properties, but the source of these cells is unclear. We previously observed that induction of an immune response against an epithelial breast cancer led in vivo to the T-cell–dependent outgrowth of a tumor, the cells of which had undergone epithelial to mesenchymal transition (EMT). The resulting mesenchymal tumor cells had a CD24−/loCD44+ phenotype, consistent with BCSCs. In the present study, we found that EMT was induced by CD8 T cells and the resulting tumors had characteristics of BCSCs, including potent tumorigenicity, ability to reestablish an epithelial tumor, and enhanced resistance to drugs and radiation. In contrast to the hierarchal cancer stem cell hypothesis, which suggests that breast cancer arises from the transformation of a resident tissue stem cell, our results show that EMT can produce the BCSC phenotype. These findings have several important implications related to disease progression and relapse. [Cancer Res 2009;69(7):2887–95]

AbstractRecent studies have shown the importance of helper CD4 T cells in initiating and sustaining tumor-specific CD8 T-cell immunity. This has paved the way for identifying MHC class II epitopes that could be incorporated into class I–based vaccines. In this study, the goal was to identify an HLA-DR–degenerate epitope pool derived from insulin-like growth factor binding protein 2 (IGFBP-2). IGFBP-2, a regulator of insulin-like growth factor action, is overexpressed in the majority of breast and ovarian cancers. Using algorithms, we predicted 29 HLA-DR1–binding epitopes. Binding assays targeting 15 different HLA-DRs revealed that 10 epitopes were degenerate, binding to at least four different HLA-DR variants. An IFN-γ enzyme-linked immunosorbent spot assay was used to assess immunity to these 10 epitopes in 48 patients with either breast or ovarian cancer and 18 controls. Elevated T-cell immunity in patients was detected in 4 of the 10 epitopes (IGFBP2.17, IGFBP2.22, IGFBP2.249, and IGFBP2.293). The cumulative T-cell frequency of these four epitopes was elevated in patients relative to controls. All four peptides are naturally processed and presented to CD4 T-cells. The degenerate pool of peptides covers nearly 80% of patients and may be useful for augmenting CD4 T-cell immunity in patients undergoing immunization. [Cancer Res 2008;68(12):4893–901]

Abstract We have previously shown T-cell–mediated rejection of the neu-overexpressing mammary carcinoma cells (MMC) in wild-type FVB mice. However, following rejection of primary tumors, a fraction of animals experienced a recurrence of a neu antigen-negative variant (ANV) of MMC (tumor evasion model) after a long latency period. In the present study, we determined that T cells derived from wild-type FVB mice can specifically recognize MMC by secreting IFN-γ and can induce apoptosis of MMC in vitro. Neu transgenic (FVBN202) mice develop spontaneous tumors and cannot reject it (tumor tolerance model). To dissect the mechanisms associated with rejection or tolerance of MMC tumors, we compared transcriptional patterns within the tumor microenvironment of MMC undergoing rejection with those that resisted it either because of tumor evasion/antigen loss recurrence (ANV tumors) or because of intrinsic tolerance mechanisms displayed by the transgenic mice. Gene profiling confirmed that immune rejection is primarily mediated through activation of IFN-stimulated genes and T-cell effector mechanisms. The tumor evasion model showed combined activation of Th1 and Th2 with a deviation toward Th2 and humoral immune responses that failed to achieve rejection likely because of lack of target antigen. Interestingly, the tumor tolerance model instead displayed immune suppression pathways through activation of regulatory mechanisms that included in particular the overexpression of interleukin-10 (IL-10), IL-10 receptor, and suppressor of cytokine signaling (SOCS)-1 and SOCS-3. These data provide a road map for the identification of novel biomarkers of immune responsiveness in clinical trials. [Cancer Res 2008;68(7):2436–46]

Abstract Studies have shown that the immune system can recognize self-antigens under conditions (eg, cell injury) in which the self-tissue might elaborate immune-activating endogenous danger signals. Uric acid (UA) is an endogenous danger signal recently identified to be released from dying cells. Prior work has shown that UA activates immune effectors of both the innate and adaptive immune system, including neutrophils and cytotoxic T-cell immunity. However, it was unclear whether UA could enhance antibody immunity, which was examined in this study. When added to dying tumor cells or with whole protein antigen, UA increased IgG1-based humoral immunity. Further, UA blocked growth of tumor in subsequent tumor challenge experiments, which depended on CD4, but not CD8, T cells. Sera derived from UA-treated animals enhanced tumor growth, suggesting it had little role in the antitumor response. UA did not signal for T-cell expansion or altered tumor-infiltrating leukocyte populations. Consistent with the lack of T-cell expansion, when applied to dendritic cells, UA suppressed T-cell growth factors but up-regulated B cell–activating cytokines. Understanding the nature of endogenous danger signals released from dying cells may aid in a better understanding of mechanisms of immune recognition of self.

AbstractPurpose: Passive immunotherapy with antitumor antibodies has the potential to induce active tumor immunity via the opsonic enhancement of immunogenicity of tumor antigen. We have assessed whether immune sensitization to the HER-2/neu tumor antigen occurs during treatment with the anti-HER-2/neu monoclonal antibody trastuzumab.Experimental Design: Twenty-seven patients treated with trastuzumab and chemotherapy were assessed for the induction of HER-2/neu–specific immunity. Sera and peripheral blood mononuclear cells obtained before and after trastuzumab therapy were compared for the presence of anti-HER-2/neu endogenous Igλ antibodies and HER-2/neu–specific CD4 responses by ELISA and enzyme-linked immunospot, respectively.Results: Anti-HER-2/neu antibodies were detectable in 8 of 27 (29%) patients before trastuzumab treatment and in 15 of 27 (56%) patients during trastuzumab treatment. In the overall study population, anti-HER-2/neu humoral responses significantly increased during therapy (P < 0.001) and were not associated with development of an anti-idiotypic response. In 10 evaluable individuals, 6 showed augmented HER-2/neu–specific CD4 T-cell responses during therapy. Of the 22 individuals treated for metastatic disease, those patients showing objective clinical responses exhibited more frequent (P = 0.004) and larger (P = 0.006) treatment-associated anti-HER-2/neu humoral responses.Conclusion: Humoral immune sensitization occurs during treatment with chemotherapy and trastuzumab. Further studies are warranted to investigate whether augmented anti-HER-2/neu humoral and cellular immunity contributes mechanistically to clinical outcome.

AbstractPurpose: Regulatory T cells (Tregs) have been implicated as inhibitors of antitumoral immunity, and evidence suggests that elimination of Tregs may augment natural and pharmacologic immunity. We tested for the presence of putative Tregs within renal cell carcinoma (RCC) tumors.Experimental Design: We identified 170 patients who underwent radical or partial nephrectomy for clear cell RCC between 2000 and 2002. Specimens were stained with anti-CD4, anti-CD25, and anti-Foxp3 antibodies and examined using confocal microscopy. Associations of CD4+CD25+Foxp3− and CD4+CD25+Foxp3+ T cells with death from RCC were evaluated using Cox proportional hazards regression models.Results: At last follow-up, 46 of 170 patients had died; of these, 37 died from RCC at a median of 1.4 years following nephrectomy (range, 0-4.4). Among the 124 remaining patients, median follow-up was 3.7 years (range, 0-5.7). Forty-three (25.3%) tumors harbored CD4+CD25+Foxp3+ T cells. The presence of Foxp3+ T cells was not significantly associated with RCC death univariately. One hundred forty-three (84.1%) tumors harbored CD4+CD25+Foxp3− T cells. The indicator for ≥10% CD4+CD25+Foxp3− T cells was significantly associated with RCC death univariately [risk ratio (RR), 2.60; 95% confidence interval (95% CI), 1.35-4.98; P = 0.004], after adjusting for tumor B7-H1 expression (RR, 2.53; 95% CI, 1.32-4.85; P = 0.005) and lymphocytic infiltration (RR, 2.53; 95% CI, 1.32-4.87; P = 0.005).Conclusions: Increased presence of CD4+CD25+Foxp3+ T cells was not significantly associated with RCC death. In contrast, CD4+CD25+Foxp3− T cells, which may represent a unique set of Tregs or activated helper T cells, was significantly associated with outcome.

AbstractOur goal is to develop peptide vaccines that stimulate tumor antigen–specific T-cell responses against frequently found cancers. Previous work has shown that to generate effective T-cell responses, peptides have to be administered in combination with strong adjuvants such as Toll-like receptor agonists. However, most animal tumor model systems used to study peptide vaccines were not truly representative of malignant diseases in humans because they solely used transplantable tumor lines, and instead of true tumor antigens, they used highly immunogenic foreign proteins. Here, we describe a peptide vaccination strategy, which is highly effective in delaying or preventing the occurrence of spontaneous breast tumors. Transgenic female BALB-neuT mice that carry the activated rat HER-2/neu oncogene were vaccinated with a synthetic peptide from the rat HER-2/neu gene product, which represents an epitope for CTLs in combination with a Toll-like receptor agonist adjuvant. Our results show that to obtain tumor antigen-specific CTL responses and antitumor effects, the vaccine had to be administered repetitively, or the function of CD4/CD25 T regulatory cells had to be blocked with anti-CD25 antibody therapy. Mice that were vaccinated with this approach remained tumor-free or were able to control spontaneous tumor growth and exhibited long-lasting CTL responses, not only against the immunizing peptide but also against other peptides derived from rat HER-2/neu product (i.e., epitope spreading). These results suggest that similar strategies should be followed for conducting clinical studies in patients. [Cancer Res 2007;67(3):1326–34]

PurposeStudies have demonstrated that the generation of immunity to tumor antigens is associated with improved prognosis for many cancers. A candidate antigen is the folate receptor alpha (FRα), which is overexpressed in breast and ovarian cancers. Our goal in this study was to attain a better understanding of the extent of endogenous FRα immunity.MethodsUsing a CD4+T cell epitope prediction algorithm, we predicted promiscuous epitopes of FRα, and tested for immunity in 30 breast (n = 17) or ovarian (n = 13) cancer patients and 18 healthy donors using enzyme-linked immunospot analysis.ResultsFourteen peptides were predicted, seven each from the carboxy- and amino-terminus halves of the protein. More than 70% of patients demonstrated immunity to at least one FRα peptide. Patients responded to an average of 3 ± 0.5 peptides, whereas healthy donors responded to 1 ± 0.4 peptides (P = .004). Five peptides were recognized by more than 25% of patients. Responses to three peptides were higher (P < .05) in patients than in healthy donors, suggesting augmented immunity. Compared with healthy individuals, patients developed higher immunity to the amino-terminus half of the receptor (P = .03). There was no difference between each group in the responses to nonspecific (P = .2) and viral stimuli (P = .5). Lastly, patients demonstrated elevated levels of FRα antibodies consistent with a coordinated immune response.ConclusionThese findings demonstrate that the FRα is a target of the immune system in breast and ovarian cancer patients. Understanding which antigens are targeted by the immune system may be important for prognosis or immune-based therapies.