Murine HER-2 Research Articles

AYVM to AYMM Transition on HER2 Exon 20 Insertion Induces Tyrosine Kinase Inhibitor Resistance in NSCLC.

Pyrotinib, a novel pan-HER tyrosine kinase inhibitor, has demonstrated substantial anti-tumor activity in patients with NSCLC harboring HER2 mutations. Nevertheless, the inevitable resistance to pyrotinib necessitates an in-depth understanding of the underlying mechanisms. Resistance-associated mutations were identified through genomic sequencing of paired baseline and post-resistance samples from 40 patients. Integrated computational and experimental approach were utilized to validate the resistance mechanisms and explore strategies for overcoming resistance in vitro and in vivo. Analysis of novel mutations upon the development of resistance did not identify any predominant secondary HER2 mutations. Nevertheless, 12 secondary HER2 mutations (38.7%) occurred either as single nucleotide variations (75%) or insertions-deletions (25%), on the basis of HER2 p.Y772_P775dup mutation. Only two mutations led to HER2 autophosphorylation and IL3-independent proliferation of Ba/F3 cells from the invitro experiments, implying that the remaining 10 secondary mutations were passenger mutations. Further invivo and invitro validation showed that the HER2 p.E770_A771insAYMM mutation diminished the sensitivity of murine HER2 mutant lung adenocarcinoma cell line to pyrotinib, with ineffective inhibition of HER2 and its downstream pathways. Drug screening indicated that mobocertinib and dacomitinib could effectively restrain the growth of tumors bearing the HER2 p.E770_A771insAYMM mutation. Our findings unveil a new form of resistance-a secondary mutation superimposed on the original mutation-and offer insights into a potentially sequential strategy for overcoming resistance to pyrotinib.

Abstract 726: The retinoid X receptor agonist MSU42011 enhances efficacy of anti-PD1 therapy in a HER2+ breast cancer model through a CD4-IL12 axis

Abstract Immunotherapy is rarely effective in breast cancer and most current therapeutic options fail to target the stromal compartment. We have developed a new small molecule, MSU42011, that induces tumor regression in experimental models of Kras-driven lung cancer and HER2+ breast cancer. This tumor regression is dependent on a functional immune system. Here, we dissected the tumor stroma, revealing a CD4 and IL12a dependency for the anti-tumor activity of MSU42011. Cancer immunotherapy responses are mostly driven by CD8 T cells. Using a murine HER2+ tumor model, anti-CD8 antibodies (200ug/mouse) were injected 24 hours before treatment with MSU42011 (100MG/KG in power diet) was started. Mice were maintained on diet for 10 days. Anti-CD8 antibodies were also administered on days 3, 7 and 10. The combination did not affect the inhibition of tumor growth by MSU42011, suggesting that its anti-tumor efficacy is not dependent on CD8 T cells. In contrast, tumors treated with MSU42011 and anti-CD4 antibodies grew (p=0.0016 versus MSU42011 alone) at the same rate as tumors treated with control diet (p=0.0139 versus MS42011). Furthermore, anti-CD4 reversed inhibition of tumor growth by MSU42011 in a carcinogen-induced lung tumor model. Interestingly, MSU42011 increased the levels of IL12 by 4x (p=0.01) and IFN by 8x (p=0.03) compared to the controls. These data suggest that MSU42011 increased Th1 CD4 T cell polarization; the increase in IL12 was lost following CD4 T cell depletion. Following depletion of IL12a with antibodies (loading dose of 1 mg, followed by 0.5mg per mouse IP), tumors treated with the combination of MSU42011 and anti-IL12a antibodies grew at the same rate as the controls. T cell engagement with MSU42011 suggests that this small molecule is a good candidate for combination with immunotherapy. HER2+ MMTV-Neu mice with established tumors were treated with the combination of MSU42011 and anti-PD1 antibodies. Treatment with MSU42011 at 100mg/Kg of diet for 10 days, followed by anti-PD1 antibodies (200ug) twice weekly failed to increase survival. In contrast, 2 doses of anti-PD1 antibodies (300ug), followed by treatment with MSU42011 increased survival by 10 days compared to controls. The tumors in the MSU42011+anti-PD1 group initially grew rapidly, with increased redness and heat, suggesting an enhanced inflammatory response. When anti-PD1 antibodies were given at a lower loading dose (200ug and then 100ug twice weekly) followed by a higher dose of MSU42011 (300 mg/Kg of diet), survival markedly increased (p=0.052). These findings demonstrate that activation of RXR in the stroma can be used to increase the effectiveness of immunotherapy. Citation Format: Ana Sofia Leal, Karen T. Liby. The retinoid X receptor agonist MSU42011 enhances efficacy of anti-PD1 therapy in a HER2+ breast cancer model through a CD4-IL12 axis [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 726.

Systemically administered low-affinity HER2 CAR T cells mediate antitumor efficacy without toxicity

BackgroundThe paucity of tumor-specific targets for chimeric antigen receptor (CAR) T-cell therapy of solid tumors necessitates careful preclinical evaluation of the therapeutic window for candidate antigens. Human epidermal growth factor...

Carbon ion irradiation plus CTLA4 blockade elicits therapeutic immune responses in a murine tumor model

Radiotherapy can act as an in situ vaccine, activating preventive tumor-specific immune responses in patients. Although carbon ion radiotherapy has superior biophysical properties over conventional photon irradiation, the immunological effects induced by this radiation type are poorly understood. Multiple strategies combining radiotherapy with immune checkpoint inhibition (radioimmunotherapy) to enhance antitumor immunity have been described; however, immune cell composition in tumors following radioimmunotherapy with carbon ions remains poorly explored. We developed a bilateral tumor model based on time-shifted subcutaneous injection of murine Her2+ EO771 tumor cells into immune-competent mice followed by selective irradiation of the primary tumor. αCTLA4-, but not αPD-L1-based radioimmunotherapy, induced complete tumor rejection and mediated the eradication of even non-irradiated, distant tumors. Cured mice were protected against the EO771 rechallenge, indicating long-lasting, tumor-specific immunological memory. Single-cell RNA sequencing and flow cytometric analyses of irradiated tumors revealed activation of NK cells and distinct tumor-associated macrophage clusters with upregulated expression of TNF and IL1 responsive genes. Distant tumors in the irradiated mice showed higher frequencies of naïve T cells activated upon the combination with CTLA4 blockade. Thus, radioimmunotherapy with carbon ions plus CTLA4 inhibition reshapes the tumor-infiltrating immune cell composition and can induce complete rejection even of non-irradiated tumors. Our data suggest combining radiotherapy approaches with CTLA4 blockade to achieve durable antitumor immunity. Evaluation of future radioimmunotherapy approaches should not be restricted to immunological impact at the irradiation site but should also consider systemic immunological effects on non-irradiated tumors.

Abstract P4-14-11: Prolonged survival of mice bearing HER2 positive breast cancer with a therapeutic cancer vaccine via intra-reconstructed abdominal flap administration

Abstract Background: Previously we reported a porous silicon micro-particle based cancer vaccine, which stimulated robust CD8+ T cell-dependent anti-tumor immunity in mice bearing HER2-positive breast cancer. In this study we evaluated the efficacy of the vaccine via single injection into reconstructed abdominal flap after primary tumor mastectomy. Method: To generate murine model of HER2 positive breast cancer murine, 1 × 105 TUBO/luciferase cells were injectedinto both mammary gland fat pad and left ventricle of each 6-8 weeks old female Balb/c mice. Primary and systemic metastatic tumor growth were monitored with a IVIS-200 imaging system. Four days after tumor inoculation, mammary gland fat pad with primary tumor was resected by mastectomy, and superficial inferior epigastric(SIE) vessels based abdominal flap was used for abdominal reconstruction. During the surgery, mice also received one of the three treatments below: 1) negative control without treatments; 2) single intra-flap vaccination of cancer vaccine; and 3) single injection of cancer vaccine via footpad injection. Therapeutic efficacy were monitored with IVIS system weekly. To analyze immune response, popliteal and inguinal lymph nodes were collected one day and seven days post vaccination, and activation of dendritic cells and T cells was evaluated with a flow cytometry. HER2 specific T cells in splenocytes which highly expressed interferon-gamma (IFNγ) were counted by ELISpot. CD3+ T cell infiltration in brain metastatic nodules were analyzed by immunohistochemical assay. Result: Prolonged survival of mice bearing HER2-positive breast cancer was achieved by both intra-flap and intra-dermal vaccination of cancer vaccine. Mice vaccinated via intra-flap injection extended median survival by 9 days (p=0.025), although not as long as intra-dermal treatment (15 days, p=0.034), comparing to control group. In lymph nodes, activated dendritic cells increased in both two vaccinated groups. Interestingly, intra-flap vaccination induced higher response in inguinal lymph node, which considered as the major draining lymph node of this route. Meanwhile, intra-dermal vaccination raised higher immune response in popliteal lymph node. In ELISpot assay, IFNγ secreted T cells were found abundant in splenocytes from both two vaccination groups. Furthermore, efficient infiltration of T cells inside the brain metastatic nodules was found in intra-dermal injection group, which provided potential protection from distal recurrence after mastectomy. Conclusion: Cancer vaccine administrated via intra-flap injection effectively stimulated systemic immune response, and slowed down tumor progression in murine HER2 positive breast cancer model. Citation Format: Xiaoling Liu, Junhua Mai, Chaoyang Meng, Wei Wei, Haifa Shen. Prolonged survival of mice bearing HER2 positive breast cancer with a therapeutic cancer vaccine via intra-reconstructed abdominal flap administration [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 P4-14-11.

Abstract 3955: Tumor macrophage-mediated antibody dependent cell phagocytosis (ADCP) is theprimary mechanism mediating HER2 mAb (Trastuzumab) anti-tumor responses which can be synergistically enhanced by CD47 innate immune checkpoint blockade

Abstract Approximately 20% of breast cancers (BC) are defined by HER2 overexpression and treated using HER2-specific monoclonal antibodies (mAb), such as Trastuzumab. Previous studies have demonstrated Trastuzumab can limit signaling, elicit antibody dependent cell cytotoxicity (ADCC), antibody dependent phagocytosis (ADCP), and adaptive immune responses to HER2, however its primary therapeutic mechanism of action (MOA) remains unclear. As HER2 mAb efficacy is subverted in advanced cancers, understanding and boosting their MOA is of critical clinical interest and scientific significance for HER2+ BC as well as for other targeted mAbs. Using a series of fully murine Trastuzumab mAbs of different isotypes, we found that the IgG2A (high A/I) isotype was essential for tumor growth suppression, suggesting an innate immune MOA. In contrast to previous reports, our studies revealed that NK cells, neutrophils, T-cell, or B-cell populations were unnecessary for this anti-tumor MOA. However, we found that a murine IgG2A Trastuzumab (mTras) altered the immune microenvironment in vivo through neutrophil suppression (~4 fold, p<.001) but stimulated a significant expansion (~7 fold, p<.001) and activation of resident tumor associated-macrophages (TAMs). Using DiD dye-labelled tumors, we confirmed that mTras elicited striking HER2-specific ADCP in ~50% of resident TAMs (versus ~16% in control), while in vitro studies confirmed that mTras-mediated macrophage anti-tumor efficacy was dependent upon ADCP, but not ADCC, through activation of FCGR4. Critically, we also found BC CD47 expression suppressed ADCP and resulted in suboptimal mTras anti-tumor efficacy. To explore this innate resistance pathway, we utilized CD47 KO BC lines and CD47 mAbs and found that both significantly enhanced mTras-mediated ADCP in vitro (>4 fold, p<.01) without altering ADCC activity. Moreover, we also found that this combination significantly enhanced TAM expansion and activation in vivo, which significantly enhanced human HER2+ BC anti-tumor efficacy (88% complete regression vs. 0% for single agent) and prolonged survival in multiple murine HER2+ BC models, including an endogenous treatment-resistant HER2+ BC mouse model. Thus, our study demonstrates the primary MOA of a clinically relevant HER2 mAb requires engagement with TAMs to stimulate their expansion and elicit ADCP, which could be significantly enhanced by CD47-SIRPa blockade. This suggests that the strategic use of CD47-SIRPa innate blockade may allow for significant enhancement of anti-tumor immunity in combination with other cancer antigen targeting mAb therapies (targeting EGFR, CD20, etc) as a more effective strategy to stimulate anti-tumor immune responses in advanced immunosuppressive cancers. Citation Format: Li-Chung Tsao, Jun-Ping Wei, Gang-jun Lei, Tao Wang, Xiao-Yi Yang, Cong-Xiao Liu, H. Kim Lyerly, Zachary C. Hartman. Tumor macrophage-mediated antibody dependent cell phagocytosis (ADCP) is theprimary mechanism mediating HER2 mAb (Trastuzumab) anti-tumor responses which can be synergistically enhanced by CD47 innate immune checkpoint blockade [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 3955.

Sorafenib combined with HER-2 targeted vaccination can promote effective T cell immunity in vivo

The tumor microenvironment (TME) is established and maintained through complex interactions between tumor cells and host stromal elements. Therefore, therapies that target multiple cellular components of the tumor may be most effective. Sorafenib, a multi-kinase inhibitor, alters signaling pathways in both tumor cells and host stromal cells. Thus, we explored the potential immune-modulating effects of sorafenib in a murine HER-2-(neu) overexpressing breast tumor model alone and in combination with a HER-2 targeted granulocyte-macrophage colony-stimulating factor (GM-CSF)-secreting vaccine (3T3neuGM). In vitro, sorafenib inhibited the growth of HER-2 overexpressing NT2.5 tumor cells, inducing apoptosis. Sorafenib also interfered with ERK MAPK, p38 MAPK, and STAT3 signaling, as well as cyclin D expression, but did not affect HER-2 or AKT signaling. In vivo, single agent sorafenib disrupted the tumor-associated vasculature and induced tumor cell apoptosis, effectively inducing the regression of established NT2.5 tumors in immune competent FVB/N mice. Immune depletion studies demonstrated that both CD4+ and CD8+ T cells were required for tumor regression. Sorafenib treatment did not impact the rate of tumor clearance induced by vaccination with 3T3neuGM in tumor-bearing FVB/N mice relative to either sorafenib treatment or vaccination alone. In vivo studies further demonstrated that sorafenib enhanced the accumulation of both CD4+ and CD8+ T cells into the TME of vaccinated mice. Together, these findings suggest that GM-CSF-secreting cellular immunotherapy may be integrated with sorafenib without impairing vaccine-based immune responses.

LEAPS Vaccine Incorporating HER-2/neu Epitope Elicits Protection That Prevents and Limits Tumor Growth and Spread of Breast Cancer in a Mouse Model.

The prototype J-LEAPS T cell vaccine for HER-2/neu breast cancer (J-HER) consists of the murine HER-2/neu66–74 H-2d CD8 T cell epitope covalently attached through a triglycine linker to the J-immune cell binding ligand (ICBL) (human β2 microglobulin38–50 peptide). The J-ICBL was chosen for its potential to promote Th1/Tc1 responses. In this proof-of-concept study, the ability of J-HER to prevent or treat cancer was tested in the TUBO cell-challenged BALB/c mouse model for HER-2/neu-expressing tumors. The J-HER vaccine was administered as an emulsion in Montanide ISA-51 without the need for a more potent adjuvant. When administered as a prophylactic vaccination before tumor challenge, J-HER protected against tumor development for at least 48 days. Despite eliciting protection, antibody production in J-HER-immunized, TUBO-challenged mice was less than that in unimmunized mice. More importantly, therapeutic administration of J-HER one week after challenge with TUBO breast cancer cells limited the spread of the tumors and the morbidity and the mortality in the challenged mice. The ability to elicit responses that prevent spread of the TUBO tumor by J-HER suggests its utility as a neoimmunoadjuvant therapy to surgery. Individual or mixtures of J-LEAPS vaccines can be readily prepared to include different CD8 T cell epitopes to optimize tumor therapy and customize treatment for individuals with different HLA types.

Abstract 320: Inhibition of adenosine A2A receptor (A2AR) by CPI-444 enhances CD8+ T cell killing of a HER-2/neu expressing murine tumor

Abstract Immunotherapies such as checkpoint inhibitors that block immune suppressive mechanisms are transforming the way some cancers are treated. The identification of additional mechanisms that suppress immune cell function is stimulating the search for other immune modulating agents that are additive or synergistic with current immune checkpoint inhibitors or are capable of overcoming mechanisms of resistance and tolerance. Extracellular adenosine and its signaling through A2AR increases the activity of regulatory T cells (Tregs), and attenuates tumor-specific CD4+/CD8+ T cells. In this study we evaluated the role of a small-molecule inhibitor of A2AR in enhancing antitumor immunity against murine Her-2/neu (neu)-expressing breast cancer. We examined whether an A2AR inhibitor would enhance the activity of cancer specific CD8+ T cells when given with a T cell inducing vaccine. To test this, neu-expressing mammary tumor bearing HER-2/neu transgenic (Neu-N) mice were treated with low dose cyclophosphamide (Cy) to deplete Tregs followed one day later with a granulocyte-macrophage colony-stimulating factor (GM-CSF) and neu-expressing whole cell vaccine (GVAX). Mice were treated with the A2AR inhibitor, CPI-444, by oral gavage daily for twelve days. Mice also received adoptively transferred high-avidity naïve neu-specific CD8+ T cells intravenously one day after vaccination. Mice were monitored for tumor progression, and tumor-infiltrating lymphocytes (TIL) were harvested from additional mice at multiple time points and analyzed for T cell activation by flow cytometry. Inflammatory changes in the tumor microenvironment were assessed by immunohistochemistry. We found that T cells adoptively transferred to mice together with Cy, a neu-targeted vaccine and CPI-444 are more effective at reducing established tumors and delaying tumor progression when compared with mice treated with Cy, vaccine, adoptively transferred T cells and vehicle control. Of particular note, mice receiving the same treatment but with CPI-444 in lieu of vehicle had a significant mean difference in tumor area of 6.446mm2 (P<0.05) when compared to the vaccine and vehicle control cohort. When compared to the tumor alone cohort, mice receiving the full therapeutic regimen had a significant mean difference in tumor area of 13.59mm2 (P<0.001). Mechanistic studies are underway to assess the activity of the adoptively transferred T cells in the TIL. These studies may provide the rationale to test an A2AR inhibitor in combination with adoptively transferred T cells and/or vaccines in patients with cancer. Citation Format: Blake A. Scott, Todd Armstrong, Elizabeth M. Jaffee. Inhibition of adenosine A2A receptor (A2AR) by CPI-444 enhances CD8+ T cell killing of a HER-2/neu expressing murine tumor. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 320.

In Vitro and In Vivo Assessment of Nonionic Iodinated Radiographic Molecules as Chemical Exchange Saturation Transfer Magnetic Resonance Imaging Tumor Perfusion Agents.

The aim of this study was to evaluate 4 nonionic x-ray iodinated contrast agents (CAs), commonly used in radiographic procedures, as novel chemical exchange saturation transfer (CEST) magnetic resonance imaging (MRI) agents by assessing their in vitro exchange properties and preliminary in vivo use as tumor enhancing agents. The CEST properties, as function of pH (range, 5.5-7.9) and of radio frequency conditions (irradiation field strength range of 1-9 μT and time of 1-9 seconds), have been determined at 7 T and 310 K for 4 x-ray CAs commonly used in clinical settings, namely, iomeprol, iohexol, ioversol, and iodixanol. Their in vivo properties have been investigated upon intravenous injection in a murine HER2+ breast tumor model (n = 4 mice for each CA) using both computed tomography (CT) and MRI modalities. The prototropic exchange rates measured for the 4 investigated iodinated molecules showed strong pH dependence with base catalyzed exchange rate that was faster for monomeric compounds (20-4000 Hz in the pH range of 5.5-7.9). Computed tomography quantification showed marked (up to 2 mg I/mL concentration) and prolonged accumulation (up to 30 minutes postinjection) inside tumor regions. Among the 4 agents we tested, iohexol and ioversol display good CEST contrast properties at 7 T, and in vivo results confirmed strong and prolonged contrast enhancement of the tumors, with elevated extravasation fractions (74%-91%). A strong and significant correlation was found between CT and CEST-MRI tumor-enhanced images (R = 0.70, P < 0.01). The obtained results demonstrate that iohexol and ioversol, 2 commonly used radiographic compounds, can be used as MRI perfusion agents, particularly useful when serial images acquisitions are needed to complement CT information.

Immunotargeting of Antigen xCT Attenuates Stem-like Cell Behavior and Metastatic Progression in Breast Cancer

Resistance to therapy and lack of curative treatments for metastatic breast cancer suggest that current therapies may be missing the subpopulation of chemoresistant and radioresistant cancer stem cells (CSC). The ultimate success of any treatment may well rest on CSC eradication, but specific anti-CSC therapies are still limited. A comparison of the transcriptional profiles of murine Her2(+) breast tumor TUBO cells and their derived CSC-enriched tumorspheres has identified xCT, the functional subunit of the cystine/glutamate antiporter system xc(-), as a surface protein that is upregulated specifically in tumorspheres. We validated this finding by cytofluorimetric analysis and immunofluorescence in TUBO-derived tumorspheres and in a panel of mouse and human triple negative breast cancer cell-derived tumorspheres. We further show that downregulation of xCT impaired tumorsphere generation and altered CSC intracellular redox balance in vitro, suggesting that xCT plays a functional role in CSC biology. DNA vaccination based immunotargeting of xCT in mice challenged with syngeneic tumorsphere-derived cells delayed established subcutaneous tumor growth and strongly impaired pulmonary metastasis formation by generating anti-xCT antibodies able to alter CSC self-renewal and redox balance. Finally, anti-xCT vaccination increased CSC chemosensitivity to doxorubicin in vivo, indicating that xCT immunotargeting may be an effective adjuvant to chemotherapy.

Targeting Her-2/neu breast cancers with a plant derived natural compound, Withaferin A (53.15)

Abstract Over expression of oncogenic Her-2/neu protein in breast cancer cells correlates with poor prognosis and patients initially responsive to Her-2/neu targeted treatments often experience recurrence within a year of treatment. Thus, new strategies to combat these cancers should be explored. Our research focuses on the development of Withaferin A (WA), a steroidal lactone derived from the Indian ayurvedic medicinal plant Withania somnifera as an immunotherapeutic agent. The benefit of using natural plant derived products is their low toxicity, multimodal action and potential ability to enhance the immune system. Our goal is to establish WA as a chemoimmunotherapeutic agent in the treatment of Her-2/neu breast cancers. We provide evidence that WA is effective in vitro against both human and murine Her-2/neu breast cancer cells. WA induces cell death by apoptosis and increases expression of immunogenic stress proteins. Furthermore, WA up-regulated costimualtory molecules, cytokine production and antigen presentation in cultured bone marrow derived murine dendritic cells (BMDC). Additionally, in vivo administration of WA in a therapeutic setting significantly delayed tumor growth with a 10% survival benefit. Our long-term goal is to elucidate the anti-tumor immunomodulatory mechanisms of WA. Such agents that can deliver multimodal action towards cancer at both a chemotherapeutic and immunotherapeutic level might prove to be the best alternative to current treatment strategies.

CD40-Targeted Recombinant Adenovirus Significantly Enhances the Efficacy of Antitumor Vaccines Based on Dendritic Cells and B Cells

Despite the advantages of using adenoviral vectors for specific antigenic gene delivery in the development of antigen-presenting cell (APC)-based vaccines, the lack of the coxsackievirus-adenovirus receptor (CAR) on APCs limits the use of adenoviral vectors for in vitro gene delivery. In this study, we used a recombinant adapter protein, CFm40L, which consists of the ectodomain of CAR genetically fused to the ectodomain of CD40 ligand (CD40L) via a trimerization motif, to target Her-2/neu- or human papillomavirus 16 (HPV16) E6/E7-encoding adenoviruses to CD40 on dendritic cells (DCs) and B cells. Targeting CD40 enabled the enhancement of tumor antigen delivery and simultaneous activation of APCs via the CD40-CD40L interaction. We found that these transduced DCs and B cells substantially enhanced the CTL response against human Her-2/neu- and HPV16 E6/E7-expressing tumors, resulting in significant inhibition of tumor growth in a murine tumor model. In addition, the use of the CFm40L adapter protein in combination with gemcitabine treatment allowed for a successful immune response against a self-tumor antigen, murine Her-2/neu. Our results suggest that targeting adenovirus to APCs via CD40, using CFm40L, represents a great improvement in anticancer cellular vaccines.

Abstract 2412: Sorafenib can be effectively combined with GM-CSF secreting cellular immunotherapy in a mouse model of HER-2+ breast cancer

Abstract Due to the complexities of the tumor microenvironment and the frequent emergence of drug resistance with treatment, it is increasingly evident that cancer therapy will have to hit multiple host and tumor targets simultaneously. We previously reported that DC101, an antiangiogenic monoclonal antibody specific for the vascular endothelial growth factor receptor-2 (VEGFR-2), can enhance tumor immunity by a T cell-dependent mechanism. Sorafenib, a small molecule that also disrupts angiogenesis, blocks signaling through multiple kinases that control tumor growth and progression. It has also been reported to inhibit both dendritic cell (DC) and T cell function. To further explore the intrinsic tumor- and host-based effects of sorafenib, we tested it against murine HER-2+ breast tumor cells (NT) in vitro, and in NT-tumor-bearing FVB/N (immune-competent) and neu-N (immune tolerant to HER-2) mice in vivo. Sorafenib inhibited the growth of NT tumor cells in vitro, inducing apoptosis. Western blot analysis revealed that Sorafenib interfered with ERK/MAPK, but not AKT, signaling pathways, both of which are constitutively activated when HER-2 is over-expressed. Single agent sorafenib effectively controlled the growth of established tumors in immune competent FVB/N mice, but did not result in complete tumor clearance. In contrast, sorafenib alone did not significantly impact the outgrowth of established tumors in neu-N mice, where tumor-specific immune tolerance is in play. Consistent with this observation, depletion studies conducted in immune competent FVB/N mice revealed that the control of tumor growth is in part dependent on T cells, but not macrophages or NK cells. While sorafenib treatment alone did not induce immunity specific for RNEU420–429 (the immunodominant HER-2 epitope), it did accelerate tumor clearance induced by vaccination with a granulocyte-macrophage colony-simulating factor (GM-CSF)-secreting, HER-2+ cellular vaccine in tumor-bearing FVB/N mice compared to either drug treatment or vaccination alone. Finally, sorafenib neither augmented nor inhibited the vaccine-induced T cell response specific for RNEU420–429 as measured by ELISPOT and intracellular cytokine staining. Overall, these findings suggest that vaccine-based immunotherapy can interact with kinase inhibitors by distinct mechanisms for enhanced therapeutic response. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 2412.

Interferon-gamma renders tumors that express low levels of Her-2/neu sensitive to cytotoxic T cells.

Her-2/neu is a tumor-associated antigen that has been targeted with both antibodies and cytotoxic T lymphocytes (CTL). Despite the isolation of Her-2/neu-reactive CTL in vaccinated patients, their therapeutic use has been limited by the observation that they often do not robustly recognize Her-2/neu(+) tumors. We sought to determine the mechanism for this escape using Ag201P and Ag201M cells, which are murine osteosarcoma tumor lines that express a functional HLA-A2/K(b) molecule. We now demonstrate that Ag201P and Ag201M express low levels of murine Her-2/neu, and that Ag201M was modestly and inconsistently recognized by an HLA-A2-restricted, Her-2/neu-reactive human CTL clone. In order to determine whether inefficient antigen processing might account for the weak recognition, COS-A2 cells were transfected with a short Her-2/neu minigene coding for the immunodominant Her-2/neu:369 epitope that did not require antigen processing or a long Her-2/neu minigene that did require antigen processing. Her-2/neu-reactive CTL clones only recognized COS-A2 cells transfected with the short minigene, indicating that lack of proper antigen processing could be responsible for the poor recognition of target cells. To confirm these results, it was demonstrated that following treatment with interferon-gamma, both Ag201P and Ag201M robustly and consistently stimulated the CTL clones. Furthermore, CTL clone recognition was enhanced following interferon-gamma treatment using another murine tumor line that expressed low levels of Her-2/neu (B16-A2/K(b)). The enhanced recognition of Ag201P and Ag201M in the presence of interferon-gamma was not due to an upregulation of Her-2/neu protein expression. Collectively, these results suggest that inefficient antigen processing of Her-2/neu can contribute to the lack of tumor recognition by CTL. These results also suggest that even tissues that express low levels of Her-2/neu might become CTL targets under conditions in which antigen processing is enhanced.

Gene Expression Analysis of Immune-Mediated Arrest of Tumorigenesis in a Transgenic Mouse Model of HER-2/neu-Positive Basal-Like Mammary Carcinoma

We previously showed that a vaccine combining interleukin 12 and allogeneic p185(neu)-positive mammary carcinoma cells completely prevented multifocal mammary carcinogenesis in HER-2/neu transgenic mice. To identify the molecular events responsible for effective tumor prevention and to define the tumor gene expression signature, we used microarrays to analyze the expression profile of mammary tissue of untreated transgenic mice and of vaccine-treated, tumor-free mice at different time points. Mammary tissue from vaccinated mice displayed a gene expression profile different from that of untreated, tumor-bearing mice but similar to that of normal/hyperplastic mammary gland. Comparison of treated and untreated mice at 15 weeks of age revealed up-regulation of genes encoding antibodies, chemokines, gamma-interferon-induced genes and inflammatory molecules, and down-regulation of early genes induced by tumor development. The gene expression signature of HER-2/neu-transformed tumor cells showed modulation of genes promoting proliferation, angiogenesis, migration, invasion, and metastasis and inhibiting apoptosis and immune response. Meta-analysis of microarray data on human breast cancer showed that the signature of tumors arising in murine HER-2/neu transgenic model correctly classified human HER-2/neu-expressing tumors and normal breast tissue. Moreover murine and human HER-2/neu-positive tumors share the signature of basal-like breast cancers. This gene expression analysis reveals the immune events associated with prevention of tumor development and shows that HER-2/neu transgenic mice represent a good model of a poor-prognosis group of human breast tumors.

Concordant morphologic and gene expression data show that a vaccine halts HER-2/neu preneoplastic lesions

While much experimental data shows that vaccination efficiently inhibits a subsequent challenge by a transplantable tumor, its ability to inhibit the progress of autochthonous preneoplastic lesions is virtually unknown. In this article, we show that a combined DNA and cell vaccine persistently inhibits such lesions in a murine HER-2/neu mammary carcinogenesis model. At 10 weeks of age, all of the ten mammary gland samples from HER-2/neu–transgenic mice displayed foci of hyperplasia that progressed to invasive tumors. Vaccination with plasmids coding for the transmembrane and extracellular domain of rat p185neu followed by a boost with rp185neu+ allogeneic cells secreting IFN-γ kept 48% of mice tumor free. At 22 weeks, their mammary glands were indistinguishable from those of 10-week-old untreated mice. Furthermore, the transcription patterns of the two sets of glands coincided. Of the 12,000 genes analyzed, 17 were differentially expressed and related to the antibody response. The use of B cell knockout mice as well as the concordance of morphologic and gene expression data demonstrated that the Ab response is the main mechanism facilitating tumor growth arrest. This finding suggests that a new way can be found to secure the immunologic control of the progression of HER-2/neu preneoplastic lesions.

Concordant morphologic and gene expression data show that a vaccine halts HER-2/neu preneoplastic lesions.

While much experimental data shows that vaccination efficiently inhibits a subsequent challenge by a transplantable tumor, its ability to inhibit the progress of autochthonous preneoplastic lesions is virtually unknown. In this article, we show that a combined DNA and cell vaccine persistently inhibits such lesions in a murine HER-2/neu mammary carcinogenesis model. At 10 weeks of age, all of the ten mammary gland samples from HER-2/neu-transgenic mice displayed foci of hyperplasia that progressed to invasive tumors. Vaccination with plasmids coding for the transmembrane and extracellular domain of rat p185neu followed by a boost with rp185neu+ allogeneic cells secreting IFN-gamma kept 48% of mice tumor free. At 22 weeks, their mammary glands were indistinguishable from those of 10-week-old untreated mice. Furthermore, the transcription patterns of the two sets of glands coincided. Of the 12,000 genes analyzed, 17 were differentially expressed and related to the antibody response. The use of B cell knockout mice as well as the concordance of morphologic and gene expression data demonstrated that the Ab response is the main mechanism facilitating tumor growth arrest. This finding suggests that a new way can be found to secure the immunologic control of the progression of HER-2/neu preneoplastic lesions.

Development of a cancer vaccine: peptides, proteins, and DNA.

Genetic changes leading to protooncogene activation qualitatively and/or quantitatively alter their gene products and are exclusively or largely restricted to transforming cells and their precursors. The overexpression of HER2 is among those changes and is often detected in adenocarcinomas such as breast, ovarian, lung, and gastric cancer. This provides a rationale for exploring the possibility that HER2 is a target of host immune responses against cancer cells. We have recently demonstrated that HER2 can be a target for tumor-rejecting immune responses against syngeneic murine HER2+ tumor cells. We defined two different peptides, HER2p63-71 and HER2p780-788, with a Kd anchor motif that can induce CD8+ cytotoxic T lymphocytes (CTLs). The growth of HER2+ syngeneic tumors was suppressed in mice immunized with HER2p63-71 or p780-788. Since murine Kd and human HLA-A24 share a similar anchor motif for peptides, HER2p63 71 and HER2p780-788 were examined for induction of CTLs in HLA-A24+ individuals. CD8+ CTL clones specific for these peptides were established and they lysed HER2+ tumor cells in a human leukocyte antigen (HLA)-A24-restricted manner. To elicit specific CD8+ T cell immune responses against cancer, the development of efficient devices to deliver tumor antigen peptides to the major histocompatibility complex (MHC) class I pathway constitutes a central issue. We have developed a novel formula of hydrophobized polysaccharide nanoparticles which can deliver a HER2 oncoprotein containing an epitope peptide to the MHC class I pathway. We designed a simple protein delivery system: cholesteryl group-bearing polysaccharides, mannan or pullulan (CHM or CHP, respectively), complexed with the truncated HER2 protein containing the 147 N-terminal amino acids. These complexes were able to induce CD8+ CTLs against HER2+ tumors. CTLs were MHC class I restricted and specifically recognized HER2p63-71, a part of a truncated HER2 protein used as an immunogen. The complete rejection of tumors also occurred when CHM-HER2 was applied early after tumor implantation. In the effector phase of in vivo tumor rejection, CD8+ T cells played a major role. The results suggest that this unique hydrophobized polysaccharide may help soluble proteins to induce cellular immunity. Such a novel vaccine may be of potential benefit in cancer prevention and cancer therapy.