Neu Antigen Research Articles

Induced tumor associated autoantibodies (iTAAs) by hapten plus drugs for targeting oncogenic nuclear antigens at sentinel lymph node of pancreatic cancer

Background: the abscopal is a hypothesis of effect on non-irradiated tumors after localized radiation therapy which associated with the products of tumor-associated gene as autoantibodies (aTAAs) in reaction to the tumor-associated antigens (TAAs). When TAAs interact with hapten within tumor after hapten plus chemotherapy drugs intratumoral injection to pancreatic cancer like tumor lysates vaccine, TAAs-hapten stimulates the immune system as a neu antigen and produces autologous tumor antibodies, it is called induced tumor-associated autoantibodies (iTAAs) which is lightly difference to aTAAs since epitope of antigen linked with hapten. Method: immunofluorescence (IF) was applied for detect the binding of the iTAAs in tumor cells at sentinel lymph node. Results: aTAAS naturally could not target and bind the TAAs in tumor since due to immune tolerance. The iTAAs can target and bind the TAAS, also the iTAAs targeted in the tumors of sentinel lymph node with complements C help, it was found Cmyc (P = 0.0017 at one week; P = 0.0001 at two weeks), p53 (P = 0.0373; 29.13 ± 6.91, and P = 0.0254), and Zeta (P = 0.1513, P = 0.0044) increased significantly in the tumor cells or perinuclear tumor cells in lymph node at one and two weeks after treatment. Conversely, IMP1 (P = 0.6154; P = 0.0138), Koc (P = 0.5684, P = 0.0103), Survivn (P = 0.1020; P = 0.0147) and Rala (P = 0.3226; P = 0.0249) increased significantly in the cell or perinuclear of tumor in lymph node at two weeks later following. Conclusions: it indicated all of iTAAs plays a function in the binding of original cellular tumor genes at sentinel lymph node while the aTAAs could not bind the cellular tumor genes at lymph node due to immune tolerance. We could make a hypothesis: TAAs inside the cancer cells’ nuclei can be targeted by the iTAAs which produced by hapten enhanced intratumoral chemotherapy. The iTAA may play a distinctive role in controlling tumor cell growth and resulting in an abscopal effect, which is one of hapten associated with TAA induced immune response.

Abstract P2-02-05: Dynamic circulating tumor cell changes in enumeration and HER2 expression during systemic therapy for metastatic breast cancer

Abstract Introduction: CTCs are tumor cells that circulate in the blood of patient with primary and MBC and, are responsible for seeing of metastasis. The monitoring of CTCs in MBC emerged as strong prognostic and possible predictive biomarker in oncology over the past couple of decades. Meanwhile, overexpression of HER2 protein has been associated with rapid cell division and worse prognosis of MBC. Here we report a significant correlation between dynamic CTCs enumeration and CTCs-HER2 expression during the systemic therapies, with potential implication to understand treatment resistance. Methods: A total of 298 whole blood samples (7.5ml/each) were collected from 149 patients with stage IV breast cancer (2016-2020) at the Northwestern University Robert H Lurie Comprehensive Cancer Center, before (Baseline) and 3 months after (Time point 2) initiation of systemic treatment. CTC enumerations were performed using the FDA approved CellSearch™ system (Menarini) which is specific for the intracellular protein cytokeratin (CK) in epithelial cells, DAPI stains the cell nucleus, anti-CD45-APC is specific for leukocytes, and anti-HER-2/neu-FLU is specific for HER-2/neu antigen. The CTCs were classified as CK+, EpCAM+, DAPI+ and CD45-. We developed a criteria for evaluation of HER2 expression by 4 different categories (0,1+,2+,3+) based on expression intensity in our lab (present in 2021 ASCO). In this study we included CTCs with all intensities of HER2 expression (1+ to 3+) which was standardized in our lab. Mann-Whitney U test was used for statistics. Results: Of the 149 baseline samples, CTC≥1 were found in 101 patients (67.8%). A change in CTCs between baseline and time point 2 for these 101 patients: Three groups were identified: Group 1: 33patients (33%) with increase CTCs; Group 2: 64 patients (63%) with decreased CTC; Group 3: 4 patients (4%) with no change The median increase of total CTCs and HER2+ CTCs in Group 1 were 7.0 and 2.0, respectively; the median decrease of total CTCs and HER2+ CTCs in Group 2 were 9.5 and 2.0, respectively. The change of HER2+ CTCs was significantly correlated with the change of total CTCs after systemic therapy, with the correlation coefficient as rs=0.662 (p<0.001) for these 101 patients. A significant positive correlation between HER2+ CTCs and total CTCs were found in both Group 1 and Group 2, rs=0.717 (p<0.001) and rs=0.604 (p<0.001) respectively. Furthermore, the ratio of HER2+ CTCs (HER2+ CTCs/total CTCs) in both Group 1 and Group 2 was also significantly correlated with total CTCs after therapy with the corresponding correlation coefficient as rs=0.536 (p<0.001) and rs=0.388 (p<0.001) in Group 1 and Group 2, respectively. Conclusion: Our study demonstrated that dynamic changes of CTCs after systemic therapies, are positively correlated with the HER2 expression in CTCs in different levels of baseline CTCs amounts. Observation of HER2 expression and ratio in CTCs during the course of systemic therapy is useful in monitoring therapy efficacy and potential disease progress. Citation Format: Qiang Zhang, Weijun Qin, Paolo D'Amico, Andrew A. Davis, Jianhua Jiao, Lorenzo Gerratana, Saya L. Jacob, Youbin Zhang, Jeannine Donahue, Wenan Qiang, Ami N Shah, Amir Behdad, Lisa Flaum, William Gradishar, Leonidas C Platanias, Massimo Cristofanilli. Dynamic circulating tumor cell changes in enumeration and HER2 expression during systemic therapy for metastatic breast cancer [abstract]. In: Proceedings of the 2021 San Antonio Breast Cancer Symposium; 2021 Dec 7-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2022;82(4 Suppl):Abstract nr P2-02-05.

Abstract P4-01-08: Anatomical staging and value of CTCs in locally advanced breast cancer (LABC) and metastatic breast cancer (MBC)

Abstract Introduction: The stage of a metastatic breast cancer (MBC) is determined by the cancer’s characteristics based on AJCC TNM system, including the size of the cancer tumor and invasion into nearby tissue (T), lymph nodes (N), and other parts of the body beyond the breast (M). Additional information including hormone-receptor status, HER2 status, and possibly Oncotype DX score has contributed to include biological characteristics and improve both, prognostic and predictive value of staging. Metastatic breast cancer (MBC) is a clinical and biologically heterogeneous condition poorly defined by current staging system. We recently report that CTCs enumeration can classify MBC in two distinct prognostic groups independently of clinical and molecular characteristics (Crit Rev Oncol Hematol. 2019). Moreover, our group reported that CTCs is associated with HER2 expression in MBC which may indicate more aggressive tumor (2019 AACR #1919). Here we compared CTCs enumeration of Stage III and Stage IV, and it would be helpful to evaluate the MBC metastasis capability and treatment in clinic. Methods: The study included 64 specimens prospectively collected under IRB-approved protocol from 36 patients with Stage III BCa, and 424 specimens from 203 patients with stage IV BCa who received standard systemic treatments based on disease subtypes at NMH (2016-2019). Duplicate whole blood samples (7.5ml/each) were collected in EDTA tubes from these patients who were longitudinally characterized for CTCs before therapy (baseline, BL), and 3 months (first evaluation, FE) or 6 months (second evaluation, SE) after systemic therapies respectively. CTCs enrichment and enumeration were performed in FDA approved semi-automated fluorescence CELLTRACKS ANALYZERII® System (Menarini) by using CELLSEARCH® CXC Kit contains antibodies targeting the Epithelial Cell Adhesion Molecule (EpCAM) antigen for capturing CTCs, anti-CK-PE which is specific for the intracellular protein cytokeratin in epithelial cells, DAPI for staining the cell nucleus, anti-CD45-APC is specific for leukocytes, and anti-HER-2/neu-FLU is specific for HER-2/neu antigen (2019 ASCO #1036). The CTCs were classified based on morphology and correct phenotype as CK+, EpCAM+, DAPI+ and CD45−. Kruskal-Wallis test was used for statistics. Results: There were median of 6.0 CTCs found in baseline level of Stage III patients, including average of 3. HER2− CTCs and 2.66 HER2+ CTCs. On the other hand, there were median of CTCs as 96.16 were detected at baseline level of Stage IV patients including average of 56.45 HER2− CTCs and 39.8 HER2+ CTCs respectively, which is about 15.9 folds higher than CTCs in Stage III patients (P<0.001). Moreover, median of 14.51 CTCs (including 7.92 HER+ CTCs) were found in all 64 Stage III specimens, which is significantly lower than average of 62.25 CTCs (including 24.8 HER2+ CTCs) in total 419 Stage IV specimens. Meanwhile, CTC-clusters were found in 8.33% (3 out of 36) patients with average of 10.66 clusters in each positive specimen in Stage III group, in compared to 13.33% (27 out of 203) patients with average of 11.44 clusters in each positive specimen respectively in Stage IV group. Furthermore, within the Stage IV group, there were 117 out of 203 patients with FE CTCs test, among which 60 had also SE CTC tests. The total average CTCs decreased from 96.16 to 33 from BL to FE level, and then decreased to 15.86 in SE level after systemic therapies. Conclusions: In this study, we showed that the total CTCs, HER2+ CTCs, and CTC-clusters is dramatically higher in Stage IV patients compared to Stage III patients, which provides an additional “staging tool” associated with a new measure of metastasis capability. Enumeration of CTCs can offer a strong supplemental criteria for TNM system for clinical decision-making. Citation Format: Qiang Zhang, Lorenzo Gerratana, Lisa Flaum Flaum, Ami Shah Shah, Andrew Davis, Amir Behdad, William Gradishar, Leon Platanias, Massimo Cristofanilli. Anatomical staging and value of CTCs in locally advanced breast cancer (LABC) and metastatic breast cancer (MBC) [abstract]. In: Proceedings of the 2019 San Antonio Breast Cancer Symposium; 2019 Dec 10-14; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2020;80(4 Suppl):Abstract nr P4-01-08.

Abstract 5195: Increased circulating tumor cell (CTC) clusters are associated with significantly higher levels of HER2 expression and metastasis in stage III/IV breast cancer

Abstract Introduction: Molecular and genomic characterization of CTCs may help to understand breast cancer prognosis and predict treatment benefit, especially for metastatic or recurrent disease. Overexpression of HER2 protein has been associated with rapid cell division and the metastasis and prognosis of advanced breast cancer in the clinic. Herein, we report a correlation between the detection of CTC clusters and HER2 overexpression in otherwise biomarker negative disease. Methods: 146 whole blood samples (7.5ml/each) were collected from 95 patients with stage III/IV breast cancer at baseline, before and after systemic therapy. All samples were transported and stored at 15-30 °C in EDTA tubes. CTC enrichment and enumeration were performed in FDA approved semi-automated fluorescence CELLTRACKS ANALYZERII® System (Janssen Diagnostics) by using CELLSEARCH® CXC Kit (Cell Search) contains ferrofluid-based capture reagent particles with a magnetic core surrounded by a polymeric layer coated with antibodies targeting the Epithelial Cell Adhesion Molecule (EpCAM) antigen for capturing CTCs. Furthermore, it includes immunofluorescent staining reagents including Anti-CK-PE which is specific for the intracellular protein cytokeratin in epithelial cells, DAPI stains the cell nucleus, anti-CD45-APC is specific for leukocytes, and anti-HER-2/neu-FLU is specific for HER-2/neu antigen. The CTCs were classified based on morphology and correct phenotype as CK+, EpCAM+, DAPI+ and CD45-. Database of CTCs was generated and linked with clinical database. Kruskal-Wallis test was used for statistics. Results: Of the 146 samples analyzed, 80 samples contained CTCs, among which 54 cases did not form clusters (Group 1), 11 samples had 1-5 clusters (Group 2), and 15 had >5 clusters (Group 3). The average ratio of CTCs/ total EpCAM+ was directly correlated with the number of clusters and were reported as 15.36%, 25.32% and 43.65% for the 3 cohorts respectively (P<0.01). The highest expression of HER-2 was found in Group 3 (66.67% samples are positive, and 120 positive cells/each sample). This value was significantly higher than Group 2 (27.27% samples are positive, 24.33 positive cells/each sample) and Group 1 (20.37% samples are positive, 6.18 positive cells/each sample) (P<0.001). The results indicated that high level of CTCs clusters strongly associated with over expression of HER2 in CTCs. Conclusion: We demonstrate that patients with detectable CTCs clusters in comparison with CTCs with no or low numbers of clusters, high levels of CTCs clusters (> 5 clusters/7.5ml blood) is a strong indicator for more aggressive of CTCs with high level of HER2 expression, which tend to be more aggressive in metastasis and worse prognosis than HER2-negative breast cancers. Along with and cancer stage and HER2 status, CTCs clusters helps determine your treatment options in clinic. Citation Format: Qiang Zhang, Youbin Zhang, Lisa Flaum, Lorenzo Gerratana, William Gradishar, Leonidas Platanias, Massimo Cristofanilli. Increased circulating tumor cell (CTC) clusters are associated with significantly higher levels of HER2 expression and metastasis in stage III/IV breast cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 5195.

A PI3K p110α-selective inhibitor enhances the efficacy of anti-HER2/neu antibody therapy against breast cancer in mice

ABSTRACTCombination therapies with phosphoinositide 3-kinase (PI3K) inhibitors and trastuzumab (anti-human epidermal growth factor receptor [HER]2/neu antibody) are effective against HER2+ breast cancer. Isoform-selective PI3K inhibitors elicit anti-tumor immune responses that are distinct from those induced by inhibitors of class I PI3K isoforms (pan-PI3K inhibitors). The present study investigated the therapeutic effect and potential for stimulating anti-tumor immunity of combined therapy with an anti-HER2/neu antibody and pan-PI3K inhibitor (GDC-0941) or a PI3K p110α isoform-selective inhibitor (A66) in mouse models of breast cancer. The anti-neu antibody inhibited tumor growth and enhanced anti-tumor immunity in HER2/neu+ breast cancer TUBO models, whereas GDC-0941 or A66 alone did not. Anti-neu antibody and PI3K inhibitor synergistically promoted anti-tumor immunity by increasing functional T cell production. In the presence of the anti-neu antibody, A66 was more effective than GDC-0941 at increasing the fraction of CD4+, CD8+, and IFN-γ+CD8+ T cells in the tumor-infiltrating lymphocyte population. Detection of IFN-γ levels by enzyme-linked immunospot assay showed that the numbers of tumor-specific T cells against neu and non-neu tumor antigens were increased by combined PI3K inhibitor plus anti-neu antibody treatment, with A66 exhibiting more potent effects than GDC-0941. In a TUBO (neu+) and TUBO-P2J (neu−) mixed tumor model representing immunohistochemistry 2+ tumors, A66 suppressed tumor growth and prolonged survival to a greater extent than GDC-0941 when combined with anti-neu antibody. These results demonstrate that a PI3K p110α-isoform-selective inhibitor is an effective adjunct to trastuzumab in the treatment of HER2-positive breast cancer.

Combination therapy of a PI3K p110α-isoform-selective inhibitor and anti-HER2/neu antibody enhances the anti-tumor immunity in breast cancer mouse model

Abstract Trastuzumab (Herceptin) is a humanized recombinant monoclonal antibody against HER2 that has shown the clinical benefit in HER2+ breast cancer patients. Because non-responders to trastuzumab have been observed, one strategy to overcome the resistance is combination therapy. Combination therapies with phosphoinositide 3-kinase (PI3K) inhibitors and trastuzumab (anti-HER2/neu antibody) are effective against HER2+ breast cancer mouse model. The therapeutic effect and anti-tumor immunity of combination therapy with an anti-HER2/neu antibody and a pan-PI3K inhibitor (GDC-0941) or a p110a isoform-selective inhibitor (A66) was evaluated in vivo breast cancer models. Combined the anti-neu and PI3Ki treatment synergistically occurred in anti-tumor immunity and HER2/neu-mediated memory T cell responses. In the presence of the anti-neu antibody, A66 was more effective in increasing the T cells and IFN-γ+ CD8+ T cells in TILs. IFN-γ ELISPOT data showed that tumor-specific T cells against neu and non-neu tumor antigens increased synergistically after combination PI3Ki and anti-neu treatment, and A66 was more potent. In a TUBO (neu+) and TUBO-P2J (neu-) mixed model representing IHC2+ tumors, A66 showed greater tumor mass control and survival prolongation than GDC-0941, when combined with the anti-neu antibody. Combined with the anti-neu antibody, A66 was more effective than GDC-0941, and A66 synergized with the anti-neu antibody in terms of anti-tumor immunity. We propose A66 plus anti-HER2/neu antibody as an effective strategy in trastuzumab-resistant breast cancers.

P2.03b-057 Diagnostic Value of Tumor Markers in Lung Adenocarcinoma-Associated Cytologically Positive and Negative Pleural Effusions: Topic: Biomarkers

Cytology fails to detect neoplastic cells in 40–50% of malignant pleural effusions (PEs), which commonly accompany lung adenocarcinomas. PE samples were collected from 74 lung adenocarcinoma patients with associated cytologically positive (41) and negative (33) effusions, and from 99 patients with benign conditions including tuberculosis, pneumonia, congestive heart failure, and liver cirrhosis. We evaluated the diagnostic sensitivity and optimal cutoff points for tumor markers Her-2/neu, Cyfra 21-1, and carcinoembryonic antigen (CEA) to distinguish lung adenocarcinoma-associated cytologically negative pleural effusions (LAC-CNPEs) from benign PEs. Mean levels of Her-2/neu, Cyfra 21-1, and CEA were significantly higher in LAC-CNPEs than in benign pleural effusions (P = 0.0050, = 0.0039, and < 0.0001, respectively). The cutoff points for Her-2/neu, Cyfra 21-1, and CEA were optimally set at 3.6 ng/mL, 60 ng/mL, and 6.0 ng/mL. Their sensitivities ranged from 12.1%, to 30.3%, to 63.6%, respectively. CEA combined with Cyfra 21-1 increased diagnostic sensitivity to 66.7%. Combining CEA with Cyfra 21-1 will provide the best differentiation between LAC-CNPEs and benign PEs with two tumor markers to date, and allows early diagnosis and early treatment for two-thirds of affected patients.

Abstract 2551: B cells induced during CD4+ T-cell mediated destruction of primary tumors in neuN mice produce mAbs that are capable of inhibiting spontaneous tumor growth

Abstract We previously identified an induced serologic response in FVB-neuN mice bearing primary breast tumors following adoptive therapy with CD4+ T cells. Because the CD4+ T cells were initially primed with breast tumor progenitor cells, we tested whether the B cells induced in treated mice also recognized antigens expressed specifically by breast tumor progenitor cells. We generated hybridomas and then screened supernatant for binding to target cells by FACS analysis. We isolated three clones that produced IgG (mAb1-2, mAb2, and mAb3) which recognized cell surface molecules on tumorsphere cultures from a NeuN breast tumor line but not the parental adherent culture from which they were derived. We used a Her-2/neu antigen loss variant to show that Her-2/neu was not itself the antigen. Interestingly, the three mAbs also recognized 4T1tumorspheres, an independently derived breast tumor from another mouse strain, but not adherent 4T1 cultures. Moreover, the mAbs were not reactive with normal spleen, liver, or kidney cells. To investigate whether any of these mAbs had anti-tumor function we used Her-2/neuN transgenic mice which develop mammary adenocarcinoma starting at 5 months of age. We selected mice with a single spontaneous tumor in situ when tumors became palpable and visible. The mice received mAb1-2, mAb2, mAb3 (150 μg/dose) or PBS on day 0, day 1 then every other day until day 23. There was no toxicity from the treatment and mice treated with PBS or mAb3 all showed progressive tumor growth. However, administration of mAb1-2 inhibited primary tumor progression in 5/8 mice and mAb2 inhibited tumor progression in 9/13 mice (p&amp;lt;0.05). Some mice with response to mAb had ongoing attenuation of tumor growth even after completion of mAb treatments. These data suggest that mice responding to CD4 T cell immunotherapy were able to stimulate endogenous B cells with reactivity against auto-antigens expressed by tumor progenitor cells. Our approach restricted our screen to cell surface antigens because we used FACS to identify hybridoma supernatant which specifically bound to tumor progenitor cells. This allowed us to focus on mAb that might have some direct therapeutic effect and two of the antibodies did inhibit tumor growth. At this point we do not know whether mAb1-2 or mAb2 inhibit tumor growth through blockade of signaling or through cytotoxic mechanisms but the antigen seems to be expressed by the majority of spontaneous breast tumors arising in FVB-neuN mice. Our current studies are attempting to identify the antigen recognized by each mAb. Citation Format: Li-Xin Wang, Mona Patel, Michael Berk, Gregory Plautz. B cells induced during CD4+ T-cell mediated destruction of primary tumors in neuN mice produce mAbs that are capable of inhibiting spontaneous tumor growth. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 2551. doi:10.1158/1538-7445.AM2014-2551

IFN-γ Rα Is a Key Determinant of CD8+ T Cell-Mediated Tumor Elimination or Tumor Escape and Relapse in FVB Mouse

During the past decade, the dual function of the immune system in tumor inhibition and tumor progression has become appreciated. We have previously reported that neu-specific T cells can induce rejection of neu positive mouse mammary carcinoma (MMC) and also facilitate tumor relapse by inducing neu antigen loss and epithelial to mesenchymal transition (EMT). Here, we sought to determine the mechanism by which CD8+ T cells either eliminate the tumor, or maintain tumor cells in a dormant state and eventually facilitate tumor relapse. We show that tumor cells that express high levels of IFN-γ Rα are eliminated by CD8+ T cells. In contrast, tumor cells that express low levels of IFN-γ Rα do not die but remain dormant and quiescent in the presence of IFN-γ producing CD8+ T cells until they hide themselves from the adaptive immune system by losing the tumor antigen, neu. Relapsed tumor cells show CD44+CD24- phenotype with higher rates of tumorigenesis, in vivo. Acquisition of CD44+CD24- phenotype in relapsed tumors was not solely due to Darwinian selection. Our data suggest that tumor cells control the outcome of tumor immune surveillance through modulation of the expression of IFN-γ Rα.

Isolation of scFv antibody fragments against HER2 and CEA tumor antigens from combinatorial antibody libraries derived from cancer patients

Tumor cells expressing HER-2/neu and CEA antigens are potentially ideal targets for antibody-targeted therapy. In this study, two large human combinatorial libraries have been generated from the lymph nodes of breast cancer patients that express HER2 and CEA antigens in their tumors. These 'immune' libraries have been constructed in two different formats of scFv, differing in the length of the peptide linker connecting the two variable VH and VL domains. Libraries derived from these patients may contain a larger pool of anti-tumor antigen antibodies and are useful repertoire for isolating scFvs against any tumor markers. The results of this study showed that we were successful in obtaining human scFvs against HER-2/neu and CEA. For HER-2, cell-panning strategy was performed and resulted in two scFv binders that detected the complete HER-2 receptor on the cell membrane and internalized to the cells. Also, preliminary ELISA data showed that several anti-CEA scFv binders were isolated by panning.

OP0218 Regulation of auto-antibody production by auto-immune complexes on follicular dendritic cells

BackgroundFollicular dendritic cells (FDCs) are characteristic components of organized tertiary lymphoid structures (TLS) containing germinal centres (GCs) in chronically inflamed tissues. However, the role of FDCs in initiation, maintenance, and...

Three-Dimensional Scaffolds to Evaluate Tumor Associated Fibroblast-Mediated Suppression of Breast Tumor Specific T Cells

In the tumor microenvironment, the signals from tumor-associated fibroblasts (TAF) that suppress antitumor immunity remain unclear. Here, we develop and investigate an in vitro three-dimensional (3D) scaffold model for the novel evaluation of TAF interaction with breast tumor cells and breast specific, neu antigen (p98) reactive T cells. Breast cancer cells seeded on 3D chitosan-alginate (CA) scaffolds showed productive growth and formed distinct tumor spheroids. Antigen specific p98 T cells, but not naïve T cells, bound significantly better to tumor cells on scaffolds. The p98 T cells induced potent tumor cell killing but T helper cell cytokine function was impaired in the presence of TAF coseeding on scaffolds. We found that the immunosuppression was mediated, in part, by transforming growth factor beta (TGF-b) and interleukin-10 (IL-10). Therefore, TAF appear capable of inducing potent T cell suppression. CA scaffolds can provide clinically relevant findings prior to preclinical testing of novel immunotherapies.

Oral Vaccination With Adeno-associated Virus Vectors Expressing the Neu Oncogene Inhibits the Growth of Murine Breast Cancer

Recombinant adeno-associated viruses (AAV) have been used for therapeutic gene transfer. These vectors offer a number of advantages including resistance to the effects of pH, a broad cellular tropism, efficient gene transfer, persistence of gene expression, and little toxicity. AAV vectors; however, at high doses can induce humoral and cellular immune responses. While potentially problematic for replacement gene therapy, this effect may be advantageous for antitumor vaccination. We examined the activity of an oral and intramuscular antitumor vaccination using AAV serotypes 5 and 6 expressing a truncated neu oncogene in a neu-positive murine TUBO breast cancer model. Mice receiving a single oral administration of AAV5-neu or AAV6-neu demonstrated improved survival. Oral vaccination significantly improved survivals compared with intramuscular vaccination. Mice vaccinated with AAV6-neu survived longer than those treated with AAV5-neu. Vaccination with AAV5-neu or AAV6-neu induced both humoral and cellular immune responses against the NEU antigen. These responses were more robust in the mice undergoing oral vaccination compared with mice receiving the intramuscular vaccination. Protection from tumor was long lasting with 80% of the animals treated with oral AAV6-neu surviving a re-challenge with TUBO cells at 120 and 320 days post-vaccination. Further evaluation of AAV-based vectors as tumor vaccines is warranted.

A Review on Aetio-Pathogenesis of Breast Cancer

This is a literature review of the aetilogy and pathogenesis of breast cancer, which is the most common cancer worldwide, and the second leading cause of cancer death, especially in Western countries. Several aetiological factors have been implicated in its pathogenesis, and include age, genetics, family history, diet, alcohol, obesity, lifestyle, physical inactivity, as well as endocrine factors. Although the roles of BRCA1, BRCA2 and HER-2/neu antigens is well established in the aetiology of breast cancer, recent evidence has shed more light on triple negative breast cancer (with absence of BRCA1, BRCA2 and HER-2/neu antigens), which has a worse prognosis and is associated with certain patient characteristics. Knowledge of these factors is important in identifying high risk groups and individuals, which will help in screening, early detection, and follow-up. This will help to decrease the morbidity and mortality from this life-threatening disease.

Targeting of Primary Breast Cancers and Metastases in a Transgenic Mouse Model Using Rationally Designed Multifunctional SPIONs

Breast cancer remains one of the most prevalent and lethal malignancies in women. The inability to diagnose small volume metastases early has limited effective treatment of stage 4 breast cancer. Here we report the rational development and use of a multifunctional superparamagnetic iron oxide nanoparticle (SPION) for targeting metastatic breast cancer in a transgenic mouse model and imaging with magnetic resonance (MR). SPIONs coated with a copolymer of chitosan and polyethylene glycol (PEG) were labeled with a fluorescent dye for optical detection and conjugated with a monoclonal antibody against the neu receptor (NP-neu). SPIONs labeled with mouse IgG were used as a nontargeting control (NP-IgG). These SPIONs had desirable physiochemical properties for in vivo applications such as near neutral zeta potential and hydrodynamic size around 40 nm and were highly stable in serum containing medium. Only NP-neu showed high uptake in neu expressing mouse mammary carcinoma (MMC) cells which was reversed by competing free neu antibody, indicating their specificity to the neu antigen. In vivo, NP-neu was able to tag primary breast tumors and significantly, only NP-neu bound to spontaneous liver, lung, and bone marrow metastases in a transgenic mouse model of metastatic breast cancer, highlighting the necessity of targeting for delivery to metastatic disease. The SPIONs provided significant contrast enhancement in MR images of primary breast tumors; thus, they have the potential for MRI detection of micrometastases and provide an excellent platform for further development of an efficient metastatic breast cancer therapy.

A vaccine approach for breast cancer immunotherapy by targeting HER2/neu antigen to B cells via CD19 molecule.

287 Background: Trastuzumab has been proven to be effective in the immunotherapy for Her-2/neu-positive breast cancer. However, this adoptively transferred therapeutic Ab must be continuously given to the patient with huge financial cost. Thus, it would be desirable if tumor vaccines could elicit long-lasting trastuzumab-like Ab. Methods: Constructing fusion protein of anti-mouse CD19 single chain variable fragment (anti-CD19-scFv) with human Her-2/neu extracellular domain P3-4 (P3-4) by extracting total RNA from Rat anti-mouse CD19 hybridoma (1D3) and first strand cDNA was synthesized accordingly. VH and VL were amplified using designed primers. Single chain Fv (VL-VH) was then synthesized by overlapping PCR. Then constructs were sequenced and cloned into pET-20b(+) vectors. Fragments P3-4 of extracellular domains of human Her-2/neu was cloned from pcDNA3.1-Her2 and then cloned into pET-20b(+)-scFv. Series of constructs were expressed and purified according to standard protocol and verified by Western Blot. Results: Both in vitro and in vivo studies demonstrated that fusion proteins anti-CD19-scFv and anti-CD19-scFv-P3-4 but not P3-4 could specifically bind to B cells with high affinity. Mice immunized with anti-CD19-scFv-P3-4 secreted higher titers of IgG and IgM than those from controls (p&lt;0.05). Studies also demonstrated that sera from anti-CD19-scFv-P3-4 but not anti-CD19-scFv or P3-4 immunized mice stained with Her-2/neu expressing SKOV-3 tumor cells. These Abs also competitively inhibited trastuzumab-mediated Ag binding, suggesting that trastuzumab-like Ab responses were elicited. WT mice immunized with anti-CD19-scFv-P3-4 fusion protein then challenged with D2/F2-Her-2 mammary tumor cells showed significantly reduced tumor burden compared to those immunized with control fusion proteins (p&lt;0.05) and had enhanced median overall survival to 45 days versus 34 days in WT mice immunized with either anti-CD19-scFv or P3-4. Conclusions: Targeting of Her-2/neu antigens to B cells stimulates Th-dependent humoral immune responses with anti tumor effect in mouse model. These findings provide a novel avenue for successful development of breast cancer vaccination strategy and help to fight for cancer.

Targeting HER2/neu antigen to B cells via CD19 molecule: A vaccine approach for breast cancer immunotherapy.

e13074 Background: Herceptin has been proven to be effective in the immunotherapy for Her-2/neu+ breast cancer. However, this adoptively transferred therapeutic Ab must be continuously given to the patient with huge financial cost. Thus, it would be desirable if tumor vaccines could elicit long-lasting Herceptin-like Ab. Methods: Constructing fusion protein of anti-mouse CD19 single chain variable fragment (anti-CD19-scFv) with human Her-2/neu extracellular domain P3-4 (P3-4) by extracting total RNA from Rat anti-mouse CD19 hybridoma (1D3) and first strand cDNA was synthesized accordingly. VH and VL were amplified using designed primers. Single chain Fv (VL-VH) was then synthesized by overlapping PCR. Then constructs were sequenced and cloned into pET-20b(+) vectors. Fragments P3-4 of extracellular domains of human Her-2/neu was cloned from pcDNA3.1-Her2 and then cloned into pET-20b(+)-scFv. Series of constructs were expressed and purified according to standard protocol and verified by Western Blot. Results: Both in vitro and in vivo studies demonstrated that fusion proteins anti-CD19-scFv and anti-CD19-scFv-P3-4 but not P3-4 could specifically bind to B cells with high affinity. Mice immunized with anti-CD19-scFv-P3-4 secreted higher titers of IgG and IgM than those from controls (p<0.05). Studies also demonstrated that sera from anti-CD19-scFv-P3-4 but not anti-CD19-scFv or P3-4 immunized mice stained with Her-2/neu expressing SKOV-3 tumor cells. These Abs also competitively inhibited Herceptin-mediated Ag binding, suggesting that Herceptin-like Ab responses were elicited. WT mice immunized with anti-CD19-scFv-P3-4 fusion protein then challenged with D2/F2-Her-2 mammary tumor cells showed significantly reduced tumor burden compared to those immunized with control fusion proteins (p<0.05) and had enhanced median overall survival to 45 days versus 34 days in WT mice immunized with either anti-CD19-scFv or P3-4. Conclusions: Targeting of Her-2/neu antigens to B cells stimulates Th-dependent humoral immune responses with anti tumor effect in mouse model. These findings provide a novel avenue for successful development of breast cancer vaccination strategy and help to fight for cancer.

Abstract 747: Tumor vaccine adjuvant effect of TLR7 agonist Imiquimod is limited by inducing immusuppressive cells

Abstract Toll-like receptor (TLR) agonists are immune stimulators and have been studied as potential vaccine adjuvants with great interest. We evaluated the adjuvant effects of the TLR7 agonist imiquimod as compared to GM-CSF, which is widely used as a vaccine adjuvant in clinical trials. We found that both topical imiquimod and intradermal GM-CSF effectively induced the accumulation and activation of dentritic cells in draining lymph nodes and equally enhanced OVA specific CD4+ and CD8+ T cell responses in an OVA-tg mouse model. We further evaluated the efficacy of tumor prevention of imiquimod and GM-CSF in a neu-transgenic (neu-tg) mouse model of human breast cancer which over-expresses both neu and IGFBP2 tumor associated antigens. The neu-tg mice were immunized with a vaccine composed of three immunogenic IGFBP2 peptides prior to being inoculated with syngeneic MMC tumor cells. The tumor growth was significantly inhibited in the groups which received GM-CSF as an adjuvant, but not in imiquimod-treated groups. We questioned whether imiquimod induces immune suppression in response to tumor associated antigens and assessed the frequencies of myeloid-derived suppressive cells (MDSC) and T regulatory cells (Treg) in the splenocytes after three immunizations. We found that both CD11c+GR1+ MDSC and CD4+FOXP3+Treg cells were significantly increased in imiquimod-treated mice, but did not increase in GM-CSF-treated group. Serum levels of IL-10 were also significantly increased in imiquimod-treated, but not in GM-CSF-treated group. IFN-gamma secretion in response to IGFBP2 antigen, on the contrary, was remarkably enhanced in the GM-CSF-treated mice, but not in the imiquimod-treated mice as tested by an IFN-gamma ELISPOT assay. Thus, the TLR7 agonist imiquimod, as a vaccine adjuvant did not achieve a protective effect on breast cancer growth. The limited anti-tumor effect observed is associated with elevated levels of immunosuppressive MDSC and Treg cells. These results may have significant implications in the future development of imiquimod as a vaccine adjuvant. 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 747. doi:10.1158/1538-7445.AM2011-747

Abstract 748: TLR3 agonist Ampligen® as an adjuvant inhibits the growth of breast cancer

Abstract A potent adjuvant is the key for a successful vaccine against tumor antigens. We evaluated the adjuvant effect of a TLR3 agonist, Ampligen® on the growth of breast cancer in a transgenic mouse model. Ampligen® [rintatolimod; poly(I)·poly(C12,U)] is a synthetic mismatched double strand RNA that specifically stimulates TLR3 (Hemispherx Biopharma, Philadelphia, PA). A neu-tg mice [strain name, FVB/N-TgN (MMTVneu) 202Mul] over-expressing both neu and IGFBP-2 tumor associated antigens (Park KH et al CR 2008) were used for this study. Three IGFBP2 peptides were used as immunogen. The mice were vaccinated with the peptides and Ampligen® three times, 2 weeks apart, with PBS, peptides alone, and Ampligen® alone as controls. Syngeneic MMC tumor cells were injected into the mice two weeks after the last vaccine, and the growth of tumors was monitored afterwards. Three other immune modulators (GM-CSF, TLR7 agonist Imiquimod and TLR9 agonist CpG ODN 2395) were also used to evaluate the efficacy of Ampligen® and combination adjuvant approaches. Our results indicate that peptides alone did not mediate an anti-tumor effect. Although Ampligen® alone at high concentration (100ug) had a weak anti-tumor effect as compared to the control with 23% tumor inhibition, vaccination with IGFBP2 antigen and Ampligen® remarkably inhibited tumor growth by 60%. A dose response of Ampligen® was observed with 20ug as the optimal dose. The analysis of the levels of multiplex cytokines (IL-12p70, IL-1a, IL-1b, IL-2, IL-5, IL-10, IL-17, IFNg and TNFa) in serum two days after last vaccine showed that the level of IL-12p70 was increased in peptides/Ampligen® treated mice as compared to control (5.9±0.9 vs. 3.1±0.8 pg/ml;n=5; p=0.041) or other adjuvants. The serum levels of other cytokines were not statistically different. We compared the adjuvant effect of Ampligen® with GM-CSF, Imiquimod and ODN 2395. Besides Imiquimod, all these adjuvants significantly inhibited tumor growth as compared with peptides alone (p&amp;lt;0.002). Ampligen® was more effective than ODN2394 (p=0.02). The combination of Ampligen® with these other immunostimulators didn't further enhance the antitumor effect. In conclusion, Ampligen® as an adjuvant inhibited the growth of breast cancer with an increased level of Th1 cytokines and is a potent tumor vaccine adjuvant with the potential for clinical use. 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 748. doi:10.1158/1538-7445.AM2011-748

Abstract 754: Heat shock protein vaccines for immunotherapy ofHer-2/neu positive breast cancers

Abstract Over-expression of Her-2/neu protein, a member of the epidermal growth factor receptor (EGFR) involved in cell growth results in cancer development. 20-30% of breast cancers over-express Her-2/neu protein and this has been correlated with poor prognosis, leading to recurrence after their initial treatment. Thus new strategies are necessary to combat this devastating disease. The selective expression of HER-2/neu in epithelial carcinogenesis makes HER-2/neu antigen an ideal target for immunotherapy. The objective of our research is to explore vaccine strategies that impacts both B and T cell responses in HER-2/neu breast cancer models. We are exploring heat shock proteins (HSP) vaccines that will chaperone tumor-associated antigens including our antigen of interest Her-2/neu protein to elicit innate and adaptive responses. We are utilizing the rat Her-2/neu as our model antigen and are administering these vaccines in transplantable and transgenic her-2/neu murine models after tumor implantation. Our first approach was to generate a Her-2/neu derived HSP gp96 in a secretable form. It has been reported that gp96 can ubiquitously bind to cell associated peptides and cross present these antigens to CD8+ T-cells to generate an effective cytotoxic T cell (CTL) response. Cancer cells expressing rat Her-2/neu were stably transfected with a mouse gp96IgG eukaryotic expression vector, where the endoplasmic retention signal sequence KDEL of gp96 was replaced with the hinge and CH2 and CH3 domains of murine IgG1. 2×104 stably transfected cells secreted within 24h 100- 200ng gp96IgG. Our goal is to generate Her-2/neu specific CD8 T cell responses that would result in vivo tumor delay. Our second approach was to test the adjuvanticity of a mixture of HSPs in combination with irradiated tumor cells. Administration of the vaccine in a therapeutic setting not only delayed tumor growth, but also provided survival benefit. Additionally, vaccinated animals were superior in eliciting Her-2/neu specific antibody response when compared to the HSP vaccine or the irradiated tumor cells alone. Our long-term goal is to combine both approaches to enhance cytotoxic T cell (CTL) and B cell specific response against Her-2/neu specific cancers. Funded by: American Cancer Society MRSG# 08-064-01-LIB and Women's Cancer Association 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 754. doi:10.1158/1538-7445.AM2011-754