Gangjun Lei

HER2 Overexpression Elicits a Proinflammatory IL-6 Autocrine Signaling Loop That Is Critical for Tumorigenesis

HER2 overexpression occurs in approximately 25% of breast cancers, where it correlates with poor prognosis. Likewise, systemic inflammation in breast cancer correlates with poor prognosis, although the process is not understood. In this study, we explored the relationship between HER2 and inflammation, comparing the effects of overexpressing wild-type or mutated inactive forms of HER2 in primary human breast cells. Wild-type HER2 elicited a profound transcriptional inflammatory profile, including marked elevation of interleukin-6 (IL-6) expression, which we established to be a critical determinant of HER2 oncogenesis. Mechanistic investigations revealed that IL-6 secretion induced by HER2 overexpression activated Stat3 and altered gene expression, enforcing an autocrine loop of IL-6/Stat3 expression. Both mouse and human in vivo models of HER2-amplified breast carcinoma relied critically on this HER2-IL-6-Stat3 signaling pathway. Our studies offer the first direct evidence linking HER2 to a systemic inflammatory mechanism that orchestrates HER2-mediated tumor growth. We suggest that the HER2-IL-6-STAT3 signaling axis we have defined in breast cancer could prompt new therapeutic or prevention strategies for treatment of HER2-amplified cancers.

Ligand-Independent Toll-like Receptor Signals Generated by Ectopic Overexpression of MyD88 Generate Local and Systemic Antitumor Immunity

Although critical for initiating and regulating immune responses, the therapeutic use of individual cytokines as anticancer immunotherapeutic agents has achieved only modest clinical success. Consequently, many current strategies have focused on the use of specific immunotherapeutic agonists that engage individual receptors of innate immune networks, such as the Toll-like receptor (TLR) system, each resulting in specific patterns of gene expression, cytokine production, and inflammatory outcome. However, these immunotherapeutics are constrained by variable cellular TLR expression and responsiveness to particular TLR agonists, as well as the specific cellular context of different tumors. We hypothesized that overexpression of MyD88, a pivotal regulator of multiple TLR signaling pathways, could circumvent these constraints and mimic coordinated TLR signaling across all cell types in a ligand-independent fashion. To explore this hypothesis, we generated an adenoviral vector expressing MyD88 and show that Ad-MyD88 infection elicits extensive Th1-specific transcriptional and secreted cytokine signatures in all murine and human cell types tested in vitro and in vivo. Importantly, in vivo intratumoral injection of Ad-MyD88 into established tumor masses enhanced adaptive immune responses and inhibited local tumor immunosuppression, resulting in significantly inhibited local and systemic growth of multiple tumor types. Finally, Ad-MyD88 infection of primary human dendritic cells, tumor-associated fibroblasts, and colorectal carcinoma cells elicited significant Th1-type cytokine responses, resulting in enhanced tumor cell lysis and expansion of human tumor antigen-specific T cells. Thus, Ad-MyD88 initiated robust antitumor activity in established murine tumor microenvironments and in human contexts, suggesting its potential effectiveness as a clinical immunotherapeutic strategy.

Synergism from combined immunologic and pharmacologic inhibition of HER2 in vivo

The monoclonal antibody trastuzumab and the EGFR/HER2 tyrosine kinase inhibitor lapatinib improve the clinical outcome of patients with HER2‐overexpressing breast cancer. However, the majority of metastatic cancers will eventually progress, suggesting the need for other therapies. Because HER2 overexpression persists, we hypothesized that the anti‐HER2 immune response induced by cancer vaccines would be an effective strategy for treating trastuzumab‐ and lapatinib‐refractory tumors. Furthermore, we hypothesized that the antibody response could synergize with lapatinib to enhance tumor inhibition. We developed a recombinant adenoviral vector expressing a kinase‐inactive HER2 (Ad‐HER2‐ki) to use as a cancer vaccine. Vaccine‐induced polyclonal HER2‐specific antiserum was analyzed for receptor internalization and signaling effects alone and in combination with lapatinib. Ad‐HER2‐ki vaccine‐induced potent T cell and antibody responses in mice and the vaccine‐induced polyclonal HER2‐specific antiserum mediated receptor internalization and degradation much more effectively than trastuzumab. Our in vitro studies demonstrated that HER2 vaccine‐induced antibodies effectively caused a decrease in HER2 expression, but when combined with lapatinib caused significant inhibition of HER2 signaling, decreased pERK and pAKT levels and reduced breast tumor cell proliferation. In addition, a known mechanism of resistance to lapatinib, induction of survivin, was inhibited. The combination of Ad‐HER2‐ki plus lapatinib also showed superior antitumor efficacy in vivo. Based on these results, we feel clinical studies using this approach to target HER2‐overexpressing breast cancer, including trastuzumab‐ and lapatinib‐resistant tumors is warranted.

An Adenoviral Vaccine Encoding Full-Length Inactivated Human HER2 Exhibits Potent Immunogenicty and Enhanced Therapeutic Efficacy Without Oncogenicity

Purpose: Overexpression of the breast cancer oncogene HER2 correlates with poor survival. Current HER2-directed therapies confer limited clinical benefits and most patients experience progressive disease. Because refractory tumors remain strongly HER2+, vaccine approaches targeting HER2 have therapeutic potential, but wild type (wt) HER2 cannot safely be delivered in imunogenic viral vectors because it is a potent oncogene. We designed and tested several HER2 vaccines devoid of oncogenic activity to develop a safe vaccine for clinical use. Experimental Design: We created recombinant adenoviral vectors expressing the extracellular domain of HER2 (Ad-HER2-ECD), ECD plus the transmembrane domain (Ad-HER2-ECD-TM), and full-length HER2 inactivated for kinase function (Ad-HER2-ki), and determined their immunogenicity and antitumor effect in wild type (WT) and HER2-tolerant mice. To assess their safety, we compared their effect on the cellular transcriptome, cell proliferation, anchorage-dependent growth, and transformation potential in vivo. Results: Ad-HER2-ki was the most immunogenic vector in WT animals, retained immunogenicity in HER2-transgenic tolerant animals, and showed strong therapeutic efficacy in treatment models. Despite being highly expressed, HER2-ki protein was not phosphorylated and did not produce an oncogenic gene signature in primary human cells. Moreover, in contrast to HER2-wt, cells overexpressing HER2-ki were less proliferative, displayed less anchorage-independent growth, and were not transformed in vivo. Conclusions: Vaccination with mutationally inactivated, nononcogenic Ad-HER2-ki results in robust polyclonal immune responses to HER2 in tolerant models, which translates into strong and effective antitumor responses in vivo. Ad-HER2-ki is thus a safe and promising vaccine for evaluation in clinical trials. Clin Cancer Res; 16(5); 1466–77

Polyclonal HER2-specific antibodies induced by vaccination mediate receptor internalization and degradation in tumor cells

IntroductionSustained HER2 signaling at the cell surface is an oncogenic mechanism in a significant proportion of breast cancers. While clinically effective therapies targeting HER2 such as mAbs and tyrosine kinase inhibitors exist, tumors overexpressing HER2 eventually progress despite treatment. Thus, abrogation of persistent HER2 expression at the plasma membrane to synergize with current approaches may represent a novel therapeutic strategy.MethodsWe generated polyclonal anti-HER2 antibodies (HER2-VIA) by vaccinating mice with an adenovirus expressing human HER2, and assessed their signaling effects in vitro and anti-tumor effects in a xenograft model. In addition, we studied the signaling effects of human HER2-specific antibodies induced by vaccinating breast cancer patients with a HER2 protein vaccine.ResultsHER2-VIA bound HER2 at the plasma membrane, initially activating the downstream kinases extracellular signal-regulated protein kinase 1/2 and Akt, but subsequently inducing receptor internalization in clathrin-coated pits in a HER2 kinase-independent manner, followed by ubiquitination and degradation of HER2 into a 130 kDa fragment phosphorylated at tyrosine residues 1,221/1,222 and 1,248. Following vaccination of breast cancer patients with the HER2 protein vaccine, HER2-specific antibodies were detectable and these antibodies bound to cell surface-expressed HER2 and inhibited HER2 signaling through blocking tyrosine 877 phosphorylation of HER2. In contrast to the murine antibodies, human anti-HER2 antibodies induced by protein vaccination did not mediate receptor internalization and degradation.ConclusionThese data provide new insight into HER2 trafficking at the plasma membrane and the changes induced by polyclonal HER2-specific antibodies. The reduction of HER2 membrane expression and HER2 signaling by polyclonal antibodies induced by adenoviral HER2 vaccines supports human clinical trials with this strategy for those breast cancer patients with HER2 therapy-resistant disease.

Polyfunctional anti-human epidermal growth factor receptor 3 (anti-HER3) antibodies induced by HER3 vaccines have multiple mechanisms of antitumor activity against therapy resistant and triple negative breast cancers

BackgroundUpregulation of human epidermal growth factor receptor 3 (HER3) is a major mechanism of acquired resistance to therapies targeting its heterodimerization partners epidermal growth factor receptor (EGFR) and human epidermal growth factor receptor 2 (HER2), but also exposes HER3 as a target for immune attack. We generated an adenovirus encoding full length human HER3 (Ad-HER3) to serve as a cancer vaccine. Previously we reported the anti-tumor efficacy and function of the T cell response to this vaccine. We now provide a detailed assessment of the antitumor efficacy and functional mechanisms of the HER3 vaccine-induced antibodies (HER3-VIAs) in serum from mice immunized with Ad-HER3.MethodsSerum containing HER3-VIA was tested in complement-dependent cytotoxicity (CDC) and antibody-dependent cellular cytotoxicity (ADCC) assays and for its effect on HER3 internalization and degradation, downstream signaling of HER3 heterodimers and growth of metastatic HER2+ (BT474M1), HER2 therapy-resistant (rBT474), and triple negative (MDA-MB-468) breast cancers.ResultsHER3-VIAs mediated CDC and ADCC, HER3 internalization, interruption of HER3 heterodimer-driven tumor signaling pathways, and anti-proliferative effects against HER2+ tumor cells in vitro and significant antitumor effects against metastatic HER2+ BT474M1, treatment refractory HER2+ rBT474 and triple negative MDA-MB-468 in vivo.ConclusionsIn addition to the T cell anti-tumor response induced by Ad-HER3, the HER3-VIAs provide additional functions to eliminate tumors in which HER3 signaling mediates aggressive behavior or acquired resistance to HER2-targeted therapy. These data support clinical studies of vaccination against HER3 prior to or concomitantly with other therapies to prevent outgrowth of therapy-resistant HER2+ and triple negative clones.

Complimentary mechanisms of dual checkpoint blockade expand unique T-cell repertoires and activate adaptive anti-tumor immunity in triple-negative breast tumors

ABSTRACT Triple-negative breast cancer (TNBC) is an aggressive and molecularly diverse breast cancer subtype typified by the presence of p53 mutations (∼80%), elevated immune gene signatures and neoantigen expression, as well as the presence of tumor infiltrating lymphocytes (TILs). As these factors are hypothesized to be strong immunologic prerequisites for the use of immune checkpoint blockade (ICB) antibodies, multiple clinical trials testing single ICBs have advanced to Phase III, with early indications of heterogeneous response rates of <20% to anti-PD1 and anti-PDL1 ICB. While promising, these modest response rates highlight the need for mechanistic studies to understand how different ICBs function, how their combination impacts functionality and efficacy, as well as what immunologic parameters predict efficacy to different ICBs regimens in TNBC. To address these issues, we tested anti-PD1 and anti-CTLA4 in multiple models of TNBC and found that their combination profoundly enhanced the efficacy of either treatment alone. We demonstrate that this efficacy is due to anti-CTLA4-driven expansion of an individually unique T-cell receptor (TCR) repertoire whose functionality is enhanced by both intratumoral Treg suppression and anti-PD1 blockade of tumor expressed PDL1. Notably, the individuality of the TCR repertoire was observed regardless of whether the tumor cells expressed a nonself antigen (ovalbumin) or if tumor-specific transgenic T-cells were transferred prior to sequencing. However, responsiveness was strongly correlated with systemic measures of tumor-specific T-cell and B-cell responses, which along with systemic assessment of TCR expansion, may serve as the most useful predictors for clinical responsiveness in future clinical trials of TNBC utilizing anti-PD1/anti-CTLA4 ICB.

Abstract A38: Checkpoint blockade elicits unique T cell expansion to promote tumor regression

While PD-1 and CTLA-4 immune checkpoint antibodies have led to durable clinical activity in certain cancers, only a fraction of patients exhibit responses. In these responsive tumors, PD-1 and CTLA-4 antibodies are thought to interfere with tumor immunosuppression of T-cells; however, the exact mechanisms of action and potential synergism between these therapies remains unclear. As triple-negative breast cancers (TNBCs) are characterized by elevated expression of inflammatory and immunosuppressive molecules, as well as high levels of immune infiltrating T cells (TILs), we hypothesized that they would be susceptible to treatment with PD-1 and CTLA-4 antibodies. To test this hypothesis and further define the mechanisms of action of checkpoint blockade, we generated a model of murine TNBC (E0771) utilizing ovalbumin (OVA) as a defined antigen that is tumor-specific and recognizable by transgenic T cells (OT-I cells). Consistent with human TNBCs, E0771 tumors exhibit robust T cell infiltration, with >60% of CD4+ T cells being T-regulatory cells (Tregs), and tumor cells express high levels of PDL1. We found that despite the generation of systemic anti-tumor responses and the addition of OT-I cells, TNBC immunosuppression shielded tumors from immune mediated regression. We then tested the efficacy of anti-PD1 and anti-CTLA4 targeting antibodies to inhibit this tumor immunosuppression and demonstrate that they had an anti-tumor effect by blocking PD-1 signaling in the tumor microenvironment and reducing intratumoral Tregs, respectively. When combined, these distinct mechanisms of action led to regression of ~80% of tumors and were significantly associated with anti-tumor adaptive responses. T cell receptor (TCR) sequencing of TILS in treated mice demonstrated a hyperexpansion of several clones, while also a broadening of the total number of unique clones present. Surprisingly, we found that despite using a homogenous tumor model and adoptively transferring OT-1 cells, TCR sequencing revealed clonal populations that were almost entirely unique for each tumor, with the OTI TCR not representing an expanded clone. As such, our study demonstrates that dual CTLA-4 and PD-1 checkpoint blockade inhibits immunosuppression of T cells in the tumor microenvironment through different and complementary mechanisms to expand and broaden unique intrinsic T cell repertoires in the tumor. Citation Format: Erika J. Crosby, Junping Wei, Xiao Yi Yang, Gangjun Lei, Tao Wang, Cong-Xiao Liu, Pankaj Agarwal, H. Kim Lyerly, Zachary C. Hartman. Checkpoint blockade elicits unique T cell expansion to promote tumor regression. [abstract]. In: Proceedings of the AACR Special Conference on Tumor Immunology and Immunotherapy; 2016 Oct 20-23; Boston, MA. Philadelphia (PA): AACR; Cancer Immunol Res 2017;5(3 Suppl):Abstract nr A38.

Abstract B63: The utility and mechanistic basis of CTLA-4 and PD-1 immunotherapeutic checkpoint inhibitors in enhancing tumor-specific adaptive responses to effectively treat triple-negative breast cancer

Abstracts: AACR Special Conference: Advances in Breast Cancer; October 17-20, 2015; Bellevue, WA While most breast cancers driven by estrogen-mediated signaling (ER+) or HER2 expression (HER2+) can be effectively treated by agents targeting these pathways, recent deep sequencing efforts have failed to identify widespread targetable drivers in Triple Negative Breast Cancers (TNBCs). Despite a lack of unifying drivers, our lab and others have uncovered that these cancers are highly inflammatory and associated with higher numbers of infiltrating immune cells (including CD8+ T-cells and Foxp3+ T-regulatory cells) , as well as expressing immuno-suppressive molecules (such as PD-L1). We hypothesized that despite TNBC molecular heterogeneity, the intrinsic inflammatory and immuno-suppressive features of these cancers could make them highly susceptible to checkpoint inhibitor-based immunotherapy. To test this hypothesis, we explored the utility and mechanistic basis of both PD-1 and CTLA-4 inhibition in generating tumor-specific immunity in an established murine model of TNBC. To test this hypothesis, we utilized a murine TNBC line (E0771) engineered with an OVA antigen to track and quantify tumor-specific immune responses. Consistent with patient samples, we found that TNBC tumors from this model contained significant numbers of T-cell infiltrates, a large proportion of which were CTLA-4+ PD-1+ Foxp3 T-regulatory cells (~66% of CD4+ TILs). We further found that TNBC tumor cells expressed surface PD-L1, which was significantly enhanced by INF-gamma stimulation. While these tumor-bearing mice exhibited systemic T-cell and B-cell immunity to tumor-specific antigens, established TNBC tumors were highly immuno-suppressive and resistant to antigen-specific T-cell attack in vivo. Having established a clinically applicable model of immuno-suppressive TNBC in an immuno-competent setting, we next tested the efficacy of CTLA-4 and PD-1 targeting antibodies, both alone and in combination. We found that delivery of CTLA-4 targeting antibodies elicited a significant anti-tumor response that was mediated by ADCC suppression of Foxp3+ T-regulatory cells in the tumor microenvironment; as well as partially by blockade of CTLA-4 function, which together allowed for more potent tumor-specific T-cell immunity in treated animals. Administration of PD-1 antibodies also inhibited TNBC tumor growth through blockade of PD-L1/PD-1 signaling in T-cells in the TNBC tumor microenvironment. When these therapies were combined, we found that they greatly enhanced anti-tumor responses through significant reduction in Foxp3+ T-regulatory cells, as well as through significantly induction of tumor-specific T-cell responses, due to tandem non-redundant CTLA-4 and PD-1 blockade. Surprisingly, we found that these responses were individually heterogeneous, despite the use of a single cell type in an inbred strain of mouse under identical conditions. Notably, these responses strongly correlated with the strength of the adaptive tumor-specific immune response suggesting that individual microenvironment differences impact the establishment of robust anti-tumor immunity in TNBC. Collectively, our study demonstrates the efficacy of a combined checkpoint inhibitor approach utilizing CTLA-4 and PD-1 in TNBC and suggests that the clinical use of these approved therapies may vary based on individual microenvironmental perturbations that influence tumor immunity. Citation Format: Erika Crosby, Jun-Ping Wei, Xiao-Yi Yang, Gangjun Lei, Tao Wang, H Kim Lyerly, Zachary C. Hartman. The utility and mechanistic basis of CTLA-4 and PD-1 immunotherapeutic checkpoint inhibitors in enhancing tumor-specific adaptive responses to effectively treat triple-negative breast cancer. [abstract]. In: Proceedings of the AACR Special Conference on Advances in Breast Cancer Research; Oct 17-20, 2015; Bellevue, WA. Philadelphia (PA): AACR; Mol Cancer Res 2016;14(2_Suppl):Abstract nr B63.

Abstract 2421: An adenoviral vaccine encoding full-length inactivated human HER2 exhibits potent immunogenicity and enhanced therapeutic efficacy without oncogenicity

While HER2 oncogene directed therapies such as lapatinib and trastuzumab confer clinical benefits, a majority of metastatic HER2+ patients eventually experience progressive disease; however, their tumors remain strongly HER2+. Thus, current therapeutic limitations combined with continued HER2 expression in patients makes immunotherapeutic vaccination against HER2 an attractive strategy. But as HER2 is an oncogene, it is imperative to develop HER2 vaccines with maximal immunologic potential, but minimal oncologic potential. To address these issues, we created recombinant adenoviral vectors expressing the extracellular domain of HER2 (Ad-HER2-ECD), ECD plus the transmembrane domain (Ad-HER2-ECD-TM) and full length HER2 inactivated for kinase function (Ad-HER2-ki) and determined their relative immunogenicity, anti-tumor effectiveness, as well as their oncogenic potential. As an immunotherapeutic vaccine, we found that that both Ad-HER2-ki and Ad-HER2-ECD-TM were highly effective in eliciting significant T-cell and antibody responses to HER2 in naive mouse models compared to plasmid vaccination with HER2-ki constructs, thus validating the immunologic efficacy of the adenoviral platform. In contrast, we did not observe any induction of HER2 specific T-cell or antibody responses in HER2-ECD vaccinated animals. While the strong immune responses from both Ad-HER2-ki and Ad-HER2-ECD-TM vectors translated into significantly retarded tumor growth in naive animals, our studies revealed that Ad-HER2ki vaccinated animals had the most significant HER2-specific T-cell responses as well as the most significant anti-tumor response. When a HER2+ tolerant mouse model was used, we observed that direct Ad-HER2-ki vaccination elicited only slightly diminished T-cell and antibody responses compared to HER2 naive mice. Most significantly, we were able to demonstrate that Ad-HER2-ki vaccination of HER2+ tolerant mice elicits significant anti-HER2+ tumor responses. Subsequent investigation into the oncogenicity associated with strong overexpression of HER2-ki revealed no evidence for its oncogenic functionality in terms of phosphorylation or transcriptional signature in primary human cells. We also found no evidence for its oncogenicity in enabling enhanced cellular proliferation, anchorage-independent growth, or transformation in vivo. In sum, we found that vaccination with mutationally inactivated, non-oncogenic Ad-HER2-ki results in robust polyclonal immune responses to HER2 in tolerant models, which translate into strong and effective anti-tumor responses in vivo. Ad-HER2-ki is thus a safe and promising vaccine for evaluation in clinical trials. 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 2421.