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Supplementary Figure from Zenocutuzumab, a HER2xHER3 Bispecific Antibody, Is Effective Therapy for Tumors Driven by NRG1 Gene Rearrangements

NRG1 rearrangements are recurrent oncogenic drivers in solid tumors. NRG1 binds to HER3, leading to heterodimerization with other HER/ERBB kinases, increased downstream signaling, and tumorigenesis. Targeting ERBBs, therefore, represents a therapeutic strategy for these cancers. We investigated zenocutuzumab (Zeno; MCLA-128), an antibody-dependent cellular cytotoxicity–enhanced anti-HER2xHER3 bispecific antibody, in NRG1 fusion–positive isogenic and patient-derived cell lines and xenograft models. Zeno inhibited HER3 and AKT phosphorylation, induced expression of apoptosis markers, and inhibited growth. Three patients with chemotherapy-resistant NRG1 fusion–positive metastatic cancer were treated with Zeno. Two patients with ATP1B1–NRG1–positive pancreatic cancer achieved rapid symptomatic, biomarker, and radiographic responses and remained on treatment for over 12 months. A patient with CD74–NRG1-positive non–small cell lung cancer who had progressed on six prior lines of systemic therapy, including afatinib, responded rapidly to treatment with a partial response. Targeting HER2 and HER3 simultaneously with Zeno is a novel therapeutic paradigm for patients with NRG1 fusion–positive cancers.Significance:NRG1 rearrangements encode chimeric ligands that activate the ERBB receptor tyrosine kinase family. Here we show that targeting HER2 and HER3 simultaneously with the bispecific antibody Zeno leads to durable clinical responses in patients with NRG1 fusion–positive cancers and is thus an effective therapeutic strategy.This article is highlighted in the In This Issue feature, p. 1171

NRG1 rearrangements are recurrent oncogenic drivers in solid tumors. NRG1 binds to HER3, leading to heterodimerization with other HER/ERBB kinases, increased downstream signaling, and tumorigenesis. Targeting ERBBs, therefore, represents a therapeutic strategy for these cancers. We investigated zenocutuzumab (Zeno; MCLA-128), an antibody-dependent cellular cytotoxicity–enhanced anti-HER2xHER3 bispecific antibody, in NRG1 fusion–positive isogenic and patient-derived cell lines and xenograft models. Zeno inhibited HER3 and AKT phosphorylation, induced expression of apoptosis markers, and inhibited growth. Three patients with chemotherapy-resistant NRG1 fusion–positive metastatic cancer were treated with Zeno. Two patients with ATP1B1–NRG1–positive pancreatic cancer achieved rapid symptomatic, biomarker, and radiographic responses and remained on treatment for over 12 months. A patient with CD74–NRG1-positive non–small cell lung cancer who had progressed on six prior lines of systemic therapy, including afatinib, responded rapidly to treatment with a partial response. Targeting HER2 and HER3 simultaneously with Zeno is a novel therapeutic paradigm for patients with NRG1 fusion–positive cancers. Significance: NRG1 rearrangements encode chimeric ligands that activate the ERBB receptor tyrosine kinase family. Here we show that targeting HER2 and HER3 simultaneously with the bispecific antibody Zeno leads to durable clinical responses in patients with NRG1 fusion–positive cancers and is thus an effective therapeutic strategy. This article is highlighted in the In This Issue feature, p. 1171

ZVAD protects HNSCC cells from erlotinib-induced cell death.

Human HNSCC tumors display elevated expression of IL-1 pathway components.

SQ20B Network list.

PCR primer description table.

Knockdown of TRIF does not reduce erlotinib-induced IL-6.

Cal-27 Network list.

Transient siRNA Knockdown of IL1R1 reduces erlotinib-induced IL-6 secretion.

This is the figure legends for the supplementary figures.

Bispecific antibodies (BsAb) are a new generation of antibody-based therapy, conveying artificial specificity to polyclonal T cells or radiohaptens. These drugs have been successfully implemented to cure hematologic malignancies and are under clinical investigation for solid tumors including HRNB. BsAbs designed to engage T cells or increase the therapeutic index of radiotherapy hold the potential to significantly improve the long-term survival of HRNB patients by shrinking bulky tumors and more effectively eliminating micrometastases and preventing relapse. BsAbs can also be used to arm T cells, yielding a product analogous to CAR T cells, possibly with an improved safety profile. A thoughtful and realistic integration of these therapies into the standard of care should benefit more patients worldwide. Here we describe the history of development of BsAbs for HRNB, which dates back almost three decades. We discuss the merits and pitfalls of all relevant BsAbs, including T cell-engagers and agents used for radioimmunotherapy, highlighting the importance of structural design and interdomain spacing for anti-tumor efficacy.

BackgroundSuccess of T cell immunotherapy hinges on the tumor microenvironment (TME), and abnormal tumor vasculature is a hallmark of most solid tumors and associated with immune evasion. The efficacy of T cell engaging bispecific antibody (BsAb) treatment relies on the successful trafficking and cytolytic activity of T cells in solid tumors. Normalization of tumor vasculature using vascular endothelial growth factor (VEGF) blockades could improve efficacy of BsAb-based T cell immunotherapy.MethodsAnti-human VEGF (bevacizumab, BVZ) or anti-mouse VEGFR2 antibody (DC101) was used as VEGF blockade, and ex vivo armed T cells (EATs) carrying anti-GD2, anti-HER2, or anti-glypican3 (GPC3) IgG-(L)-scFv platformed BsAb were used. BsAb-driven intratumoral T cell infiltration and in vivo antitumor response were evaluated using cancer cell line-derived xenografts (CDXs) or patient-derived xenografts (PDXs) carried out in BALB-Rag2-/-IL-2R-γc-KO (BRG) mice. VEGF expression on human cancer cell lines was analyzed by flow cytometry, and VEGF levels in mouse serum were measured using VEGF Quantikine ELISA Kit. Tumor infiltrating lymphocytes (TILs) were evaluated using flow cytometry and by bioluminescence; both TILs and tumor vasculature were studied using immunohistochemistry.ResultsVEGF expression on cancer cell lines increased with seeding density in vitro. BVZ significantly reduced serum VEGF levels in mice. BVZ or DC101 increased high endothelial venules (HEVs) in the TME and substantially enhanced (2.1–8.1 fold) BsAb-driven T cell infiltration into neuroblastoma and osteosarcoma xenografts, which was preferential for CD8(+) TILs versus CD4(+) TILs, leading to superior antitumor effects in multiple CDX and PDX tumor models without added toxicities.ConclusionsVEGF blockade using specific antibodies against VEGF or VEGFR2 increased HEVs in the TME and cytotoxic CD8(+) TILs, significantly improving the therapeutic efficacy of EAT strategies in preclinical models, supporting the clinical investigation of VEGF blockades to further enhance BsAb-based T cell immunotherapies.

Desmoplastic small round cell tumor (DRSCT) is a highly aggressive primitive sarcoma that primarily affects adolescent and young adult males. The 5-year survival rate is 15-30% and few curative treatment options exist. Although there is no standard treatment for DSRCT, patients are most often treated with a combination of aggressive chemotherapy, radiation, and surgery. Targeted therapy inhibitors of PDGFA and IGF-1R, which are almost uniformly overexpressed in DSRCT, have largely failed in clinical trials. As in cancer in general, interest in immunotherapy to treat DSRCT has increased in recent years. To that end, several types of immunotherapy are now being tested clinically, including monoclonal antibodies, radionuclide-conjugated antibodies, chimeric antigen receptor T cells, checkpoint inhibitors, and bispecific antibodies (BsAbs). These types of therapies may be particularly useful in DSRCT, which is frequently characterized by widespread intraperitoneal implants, which are difficult to completely remove surgically and are the frequent cause of relapse. Successful treatment with immunotherapy or radioimmunotherapy following debulking surgery could eradiate these micrometasteses and prevent relapse. Although there has been limited success to date for immunotherapy in pediatric solid tumors, the significant improvements in survival seen in the treatment of other pediatric solid tumors, such as metastatic neuroblastoma and its CNS spread, suggest a potential of immunotherapy and specifically compartmental immunotherapy in DSRCT.

Background The epidermal growth factor receptor (EGFR) monoclonal IgG1 antibody cetuximab is approved for first-line treatment of recurrent and metastatic (R/M) HNSCC as a part of the standard of care EXTREME regimen (platinum/5-fluorouracil/cetuximab). This regimen has relatively high response and disease control rates but is generally not curative and many patients will experience recurrent disease and/or metastasis. Therefore, there is a great need to identify predictive biomarkers for recurrence and disease progression in cetuximab-treated HNSCC patients to facilitate patient management and allow for treatment modification. The goal of this work is to assess the potential of activating interleukin-1 (IL-1) ligands (IL-1 alpha [IL-1α], IL-1 beta [IL-1β]) as predictive biomarkers of survival outcomes in HNSCC patients treated with cetuximab-based chemotherapy. Methods Baseline gene, serum and tumor expression of interleukin-1 (IL-1) ligands were analyzed from The Cancer Genome Atlas (TCGA) database or clinical trials of cetuximab-based therapies and interrogated for associations with clinical outcome data. Results High tumor gene expression of IL-1β was associated with a more favorable overall survival in cetuximab-treated HNSCC patients but not in non-cetuximab-treated patients. In HNSCC patients treated with cetuximab-based chemotherapy, higher gene and circulating levels of IL-1α and IL-1β were correlated with a more favorable progression free survival compared to patients with low or undetectable levels of IL-1 ligands. Conclusions These findings suggest that IL-1 ligands may function as predictive biomarkers for tumor response to cetuximab-based chemotherapy in HNSCC patients and warrants further investigation and validation in larger clinical studies.

Fusions involving the neuregulin 1 gene (NRG1) occur at low frequency in pancreatic, lung, and other cancers. NRG1 fusion oncoproteins bind to HER3, leading to heterodimerization with HER2 and potent activation of downstream signaling mainly via the PI3K-AKT pathway. Zenocutuzumab (Zeno, MCLA-128), an ADCC-enhanced anti-HER2×HER3 bi-specific antibody, uniquely ‘docks' on HER2, to position the antibody and subsequently ‘block' NRG1 from interacting with HER3, effectively preventing HER2:HER3 heterodimerization and downstream signaling. Our goal in this study was to evaluate the efficacy of Zeno in preclinical models of NRG1 fusion-positive cancers. We tested Zeno in a panel of isogenic and patient-derived cell line and xenograft (PDX) models of lung, breast and pancreatic cancers. Cell lines either expressed an NRG1 fusion endogenously (MDA-MB-175-VII, DOC4-NRG1) or by lentiviral transfer of cDNAs (ATP1B1-NRG1 and SLC3A2-NRG1 in H6c7 pancreatic ductal cell line; CD74-NRG1 and VAMP2-NRG1 in immortalized human bronchial epithelial cells; and DOC4-NRG1 in MCF7 breast cancer cells). PDX models were generated from NSCLC samples harboring CD74-NRG1 (ST3204) or SLC3A2-NRG1 (LUAD-0061AS3) fusions and from a high grade serous ovarian cancer harboring a CLU-NRG1 fusion (OV-10-0050). Zeno treatment of NRG1 fusion-expressing breast, pancreatic, and lung cancer cell lines resulted in dose-dependent reduction of growth and abrogated phosphorylation of HER3, HER4, AKT, p70S6 kinase and STAT3 in all cell lines tested. Phosphorylation of HER2, EGFR and MEK/ERK was inhibited, albeit with some variation, in a cell line-specific manner. Growth of isogenic control cell lines without NRG1 fusion was not significantly altered. In breast and lung cancer cell lines, Zeno treatment down-regulated cyclin D1 expression and induced expression of the negative cell cycle regulators P21 or P27. Evidence of apoptosis activation (cleaved PARP, expression of BIM and PUMA) was also observed in cells exposed to Zeno. Treatment of mice bearing LUAD-0061AS3, ST3204 and OV-10-0050 PDX tumors (2.5, 8, 25 mg/kg, QW) caused a dose-dependent inhibition of tumor growth, with tumor shrinkage observed at higher doses. Finally, we assessed the ability of Zeno to induce antibody-dependent cellular cytotoxicity using a chromium release assay and peripheral blood mononuclear cells. Zeno induced significant cytotoxicity in MDA-MB-175-VII cells while a non-ADCC enhanced, non-specific IgG had no effect. Here we show that Zeno effectively blocks the growth of NRG1 fusion-positive cell line and xenograft models of tumors arising from lung, pancreas and other organs, and these results support the continued development of Zeno to treat patients with this molecularly defined subset of cancers. Citation Format: Igor Odintsov, Inna Khodos, Madelyn Espinosa-Cotton, Allan J. Lui, Marissa Mattar, Alison M. Schram, Ron C. Schackmann, Jeroen Lammerts van Bueren, Cecile A. Geuijen, Elisa de Stanchina, Marc Ladanyi, Romel Somwar. The HER2×HER3 bi-specific antibody Zenocutuzumab is effective at blocking growth of tumors driven by NRG1 gene fusions [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 956.

Background Despite the high prevalence of epidermal growth factor receptor (EGFR) overexpression in head and neck squamous cell carcinomas (HNSCCs), incorporation of the EGFR inhibitor cetuximab into the clinical management of HNSCC has not led to significant changes in long-term survival outcomes. Therefore, the identification of novel therapeutic approaches to enhance the clinical efficacy of cetuximab could lead to improved long-term survival for HNSCC patients. Our previous work suggests that EGFR inhibition activates the interleukin-1 (IL-1) pathway via tumor release of IL-1 alpha (IL-1α), although the clinical implications of activating this pathway are unclear in the context of cetuximab therapy. Given the role of IL-1 signaling in anti-tumor immune response, we hypothesized that increases in IL-1α levels would enhance tumor response to cetuximab. Methods Parental and stable myeloid differentiation primary response gene 88 (MyD88) and IL-1 receptor 1 (IL-1R1) knockdown HNSCC cell lines, an IL-1R antagonist (IL-1RA), neutralizing antibodies to IL-1α and IL-1β, and recombinant IL-1α and IL-1β were used to determine cytokine production (using ELISA) in response to cetuximab in vitro. IL-1 pathway modulation in mouse models was accomplished by administration of IL-1RA, stable overexpression of IL-1α in SQ20B cells, administration of rIL-1α, and administration of a polyanhydride nanoparticle formulation of IL-1α. CD4+ and CD8+ T cell-depleting antibodies were used to understand the contribution of T cell-dependent anti-tumor immune responses. Baseline serum levels of IL-1α were measured using ELISA from HNSCC patients treated with cetuximab-based therapy and analyzed for association with progression free survival (PFS). Results Cetuximab induced pro-inflammatory cytokine secretion from HNSCC cells in vitro which was mediated by an IL-1α/IL-1R1/MyD88-dependent signaling pathway. IL-1 signaling blockade did not affect the anti-tumor efficacy of cetuximab, while increased IL-1α expression using polyanhydride nanoparticles in combination with cetuximab safely and effectively induced a T cell-dependent anti-tumor immune response. Detectable baseline serum levels of IL-1α were associated with a favorable PFS in cetuximab-based therapy-treated HNSCC patients compared to HNSCC patients with undetectable levels. Conclusions Altogether, these results suggest that IL-1α in combination with cetuximab can induce a T cell-dependent anti-tumor immune response and may represent a novel immunotherapeutic strategy for EGFR-positive HNSCCs. Electronic supplementary material The online version of this article (10.1186/s40425-019-0550-z) contains supplementary material, which is available to authorized users.

Interleukin-1 alpha (IL-1α) is a pleiotropic cytokine involved in inflammation and immune response and is upregulated in many solid tumors including head and neck squamous cell carcinomas. Although IL-1αexpression is generally associated with poor prognosis, the implications of the subcellular localization of IL-1αexpression in patient outcomes are poorly understood. This study is aimed at investigating the prognostic value of nuclear and cytoplasmic immunohistochemical IL-1αexpression in oral squamous cell carcinomas (OSCCs). Tissue microarrays containing 146 OSCCs were analyzed for IL-1αand epidermal growth factor receptor (EGFR) expression by immunohistochemistry. IL-1αand EGFR expression scores were correlated with clinicopathological parameters and survival outcomes. IL-1αexpression was observed in the nuclear and/or cytoplasmic compartments in 98% of evaluable tumors and 78% of tumors expressed IL-1αin both compartments. There were no differences observed in overall survival or progression-free survival between high, moderate, or negative IL-1αnuclear/cytoplasmic expression scores. When IL-1αnuclear/cytoplasmic expression scores were stratified by positive or negative EGFR expression, tumors with a combined EGFR-positive and high nuclear IL-1αexpression profile were significantly more likely to possess perineural invasion and were significantly associated with a high risk of tumor recurrence and worse progression-free survival compared to all other EGFR and combined IL-1α/EGFR expression profiles. Altogether, nuclear IL-1αexpression may enhance the prognostic value of EGFR in OSCC and warrants further study as a prognostic biomarker for recurrence.

BackgroundAcute myeloid leukemia (AML) remains one of the most challenging hematological malignancies. Despite progress in therapeutics, majority of patients succumb to this neoplasm. CD33 is a proven therapeutic target, given its expression on most AML cells. Almost all anti-CD33 antibodies target the membrane distal immunoglobulin V (IgV) domain of the CD33 extracellular domain.MethodsIn this manuscript, we present data on three bispecific antibodies (BsAbs) against the CD33 IgV and membrane proximal immunoglobulin C (IgC) domains. We use in vitro binding and cytotoxicity assays to show the effect of these BsAbs on AML cell lines. We also use immunodeficient mice-bearing leukemias from cell lines and patient-derived xenografts to show the effect of these BsAbs in vivo.ResultsIn vitro, the IgV-targeting BsAb had higher binding to AML cell lines using flow cytometry and delivered more potent cytotoxicity in T-cell-dependent cytotoxicity assays; importantly, the IgC domain-targeting outperformed the IgV domain-targeting BsAb in medullary and extramedullary leukemia animal models.ConclusionsThese data support further clinical development of this BsAb for first-in-human phase I clinical trial.

EGFR is upregulated in the majority of head and neck squamous cell carcinomas (HNSCC). However, many patients with HNSCC respond poorly to the EGFR inhibitors (EGFRI) cetuximab and erlotinib, despite tumor expression of EGFR. Gene expression analysis of erlotinib-treated HNSCC cells revealed an upregulation of genes involved in MyD88-dependent signaling compared with their respective vehicle-treated cell lines. We therefore investigated whether MyD88-dependent signaling may reduce the antitumor efficacy of EGFRIs in HNSCC. Erlotinib significantly upregulated IL6 secretion in HNSCC cell lines, which our laboratory previously reported to result in reduced drug efficacy. Suppression of MyD88 expression blocked erlotinib-induced IL6 secretion in vitro and increased the antitumor activity of erlotinib in vivo. There was little evidence of Toll-like receptor or IL18 receptor involvement in erlotinib-induced IL6 secretion. However, suppression of IL1R signaling significantly reduced erlotinib-induced IL6 production. A time-dependent increase of IL1α but not IL1β was observed in response to erlotinib treatment, and IL1α blockade significantly increased the antitumor activity of erlotinib and cetuximab in vivo. A pan-caspase inhibitor reduced erlotinib-induced IL1α secretion, suggesting that IL1α was released because of cell death. Human HNSCC tumors showed higher IL1α mRNA levels compared with matched normal tissue, and IL1α was found to be negatively correlated with survival in patients with HNSCC. Overall, the IL1α/IL1R/MYD88/IL6 pathway may be responsible for the reduced antitumor efficacy of erlotinib and other EGFRIs, and blockade of IL1 signaling may improve the efficacy of EGFRIs in the treatment of HNSCC. Cancer Res; 75(8); 1657–67. ©2015 AACR.

Interleukin-1 alpha (IL-1α) is a pleiotropic cytokine involved in inflammation and immune response and is upregulated in many solid tumors including head and neck squamous cell carcinomas (HNSCCs). Although IL-1α expression is generally associated with poor prognosis, the implications of the subcellular localization of IL-1α expression in patient outcomes are poorly understood. This study is aimed at investigating the clinical relevance of immunohistochemical IL-1α expression in HNSCCs. Tissue microarrays (TMAs) containing human papillomavirus (HPV)-positive and HPV-negative HNSCCs were analyzed for IL-1α and epidermal growth factor receptor (EGFR) expression by immunohistochemistry. Nuclear and cytoplasmic IL-1α and EGFR expression scores were correlated with clinicopathological parameters and patient outcomes. IL-1α expression was observed in the nuclear and/or cytoplasm compartments in 98% of evaluable tumors and 78% of tumors expressed IL-1α in both compartments. A higher percentage of tumors with combined high nuclear and moderate cytoplasmic IL-1α expression were observed in HPV-negative tumors compared to HPV-positive tumors. In HPV-negative tumors a combined EGFR-negative and high nuclear IL-1α expression profile was associated with a low risk of tumor recurrence and favorable overall survival compared to all other EGFR/IL-1α expression profiles. Lastly, a higher frequency of tumors with combined high nuclear and moderate cytoplasmic IL-1α expression was observed in cetuximab monotherapy responders compared to non-responders. Altogether, IL-1α in combination with EGFR expression may be a strong indicator of risk of recurrence in HPV-negative HNSCCs and warrants further study as a tissue biomarker for predicting HNSCC patient outcomes. Citation Format: Andrean L. Simons, Anand Rajan, Katherine Gibson-Corley, Georgina Ofori-Amanfo, Patrick Ten Eyck, Madelyn Espinosa-Cotton, Sandra Schmitz, Joseph Coppock, Steven Sperry. Impact of interleukin-1 alpha and EGFR expression on recurrence and survival outcomes in head and neck squamous cell carcinomas [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 3150.

Despite the incorporation of the epidermal growth factor receptor (EGFR) inhibitor cetuximab into the clinical management of head and neck squamous cell carcinoma (HNSCC), limited to no long-term changes in overall survival are observed in HNSCC patients even though EGFR is expressed at high levels in these tumors. Therefore, the identification of novel therapeutic approaches to enhance the clinical efficacy of cetuximab could lead to improved long-term survival for HNSCC patients. Our previous work indicated that cetuximab triggers the release of pro-inflammatory cytokines directly from HNSCC tumor cells, which was mediated by interleukin-1 (IL-1) signaling. The IL-1 pathway plays a central role in immune response by regulating the expression of various inflammatory genes in immune cells. IL-1 signaling is also involved in antitumor immunity specifically via natural killer (NK)-cell mediated antibody-dependent cell-mediated cytotoxicity (ADCC), which is believed to be the major mechanism of action of cetuximab. Therefore we propose that IL-1 signaling is required for HNSCC tumor response to cetuximab. We found that blockade of IL-1 signaling using an IL-1-receptor antagonist (IL-1RA, anakinra) suppressed the anti-tumor efficacy of cetuximab, while IL-1 alpha (IL-1α) overexpression enhanced HNSCC tumor response to cetuximab in SQ20B xenograft mouse models. Blockade of IL-1 signaling also suppressed cetuximab-induced NK-cell-mediated ADCC by a mechanism involving decreased inducible nitric oxide synthase (iNOS) activity. These results support the idea that IL-1 signaling is necessary for HNSCC tumor response to cetuximab and that iNOS activity may be an important mediator in the mechanism of action of IL-1 signaling.

Despite the incorporation of the epidermal growth factor receptor (EGFR) inhibitor cetuximab into the clinical management of head and neck squamous cell carcinoma (HNSCC), limited to no long-term changes in overall survival are observed in HNSCC patients even though EGFR is expressed at high levels in these tumors. Therefore, the identification of novel therapeutic approaches to enhance the clinical efficacy of cetuximab could lead to improved long-term survival for HNSCC patients. Our previous work suggests that cetuximab activates the interleukin-1 (IL-1) pathway via tumor release of IL-1 alpha (IL-1α), although the implications of activating this pathway are unclear. The IL-1 pathway plays a central role in immune response and displays both pro-tumor and anti-tumor activities. IL-1 may promote tumor growth by upregulating the secretion of pro-inflammatory mediators involved in angiogenesis and metastasis. On the other hand, IL-1 signaling may promote antitumor immunity specifically via natural killer (NK)-cell mediated antibody-dependent cell-mediated cytotoxicity (ADCC), which is also an important mechanism of action of cetuximab. The goal of our work is to determine how modulation of the IL-1 pathway affects HNSCC tumor response to cetuximab. We found that blockade of IL-1 signaling using an IL-1-receptor antagonist (IL-1RA, anakinra), neutralizing IL-1α/IL-1β antibodies, and genetic knockdown of the IL-1R all suppressed the anti-tumor efficacy of cetuximab, while IL-1α overexpression and treatment with recombinant IL-1α enhanced HNSCC tumor response to cetuximab in immunodeficient and immunocompetent HNSCC mouse models. Mechanistically, these results appear to be due to modulation of ADCC, as we found that IL-1 blockade significantly reduced cetuximab-mediated ADCC in vitro. Additionally, we found that HNSCC patients with high baseline circulating levels of IL-1 ligands (IL-1α, IL-1β) were significantly more likely to respond favorably to cetuximab monotherapy compared to patients with low or no baseline circulating IL-1 ligands. Altogether, these results suggest that IL-1 signaling is necessary for HNSCC tumor response to cetuximab. Therefore, IL-1α warrants further study as a novel therapeutic to enhance response to cetuximab and as an immunologic biomarker for cetuximab response. Citation Format: Madelyn M. Espinosa-Cotton, Rachel A. Dahl, Elana Fertig, Isaac Jensen, Ayana J. McLaren, Kenley Miller, Samuel N. Rodman, Sandra Schmitz, Andrean L. Simons. Interleukin-1 signaling is required for HNSCC tumor response to cetuximab [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 5734.

Various types of cancers including head and neck squamous cell carcinomas (HNSCCs) are highly inflammatory, and the cytokine interleukin-6 (IL-6) is believed to be associated with this inflammatory phenotype. Unfortunately, global inhibition of IL-6 signaling has demonstrated little to no clinical efficacy in various types of cancers. Previous studies in the field have reported major differences between IL-6 classical signaling through the membrane bound IL-6R (mIL-6R) and IL-6 trans-signaling through the soluble IL-6R (sIL-6R), and the idea that these 2 pathways may have vastly different functions. Recent studies in models of inflammation have proposed that IL-6 classical signaling serves an anti-inflammatory/homeostatic role while IL-6 trans-signaling stimulates proinflammatory events. Therefore, IL-6 trans-signaling (and not classical signaling) may be associated with the proinflammatory phenotype observed in HNSCCs and may also be responsible for the increased tumor progression and treatment resistance frequently encountered with this disease. The goal of this work is to investigate if selective inhibition of IL-6 trans-signaling will demonstrate superior antitumor efficacy compared to global inhibition of IL-6 signaling in HNSCC cells and if selective inhibition of IL-6 trans-signaling will enhance the antitumor efficacy of chemo/radiotherapy in HNSCC cells. We observed that the HNSCC cell lines SQ20B, SCC-25, FaDu and Cal-27 all express mIL-6R and secrete IL-6, sIL-6R, and the natural inhibitor of sIL-6R signaling–soluble glycoprotein 130 (sgp130)–at varying levels. Simultaneous inhibition of IL-6 classical and trans-signaling using the IL-6R antagonist tocilizumab, and selective inhibition of IL-6 trans-signaling using the extracellular portion of gp130 fused to the Fc portion of human IgG1 (sgp130-Fc), showed no significant effects on cell survival in vitro. However, sgp130-Fc significantly suppressed SQ20B tumor growth in athymic nude mice while tocilizumab had no effect. Significant antitumor effects of sgp130-Fc were also observed in a murine SCCVII/C3H syngeneic mouse model. Cell lines treated with cisplatin and/or X-ray radiation (0-4 Gy) increased IL-6, sIL-6R and sgp130 in a dose-dependent manner and sgp130-Fc significantly increased SQ20B tumor response to chemo/radiotherapy in vivo. Altogether, this work suggests that selective inhibition of IL-6 trans-signaling using sgp130-Fc may be more effective than global IL-6 inhibition with respect to suppressing HNSCC tumor growth and warrants further study as an adjuvant to standard HNSCC therapy. Citation Format: Andrean L. Simons, Rachel A. Dahl, Madelyn Espinosa-Cotton, Samuel N. Rodman, Stefan Rose-John. Inhibition of IL-6 trans-signaling in HNSCC [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 5611.

Epidermal growth factor receptor (EGFR) tyrosine kinase inhibitors (TKIs) including erlotinib have demonstrated poor clinical response rates for head and neck squamous cell carcinoma (HNSCC) to date compared to the EGFR monoclonal antibody cetuximab. Therefore we seek to identify molecular mechanisms involved in tumor response to erlotinib and ultimately improve the anti-tumor efficacy of erlotinib for HNSCC patients. We have shown that erlotinib-treated HNSCC cell lines induced the expression and secretion of a wide variety of genes involved in inflammation and immune response including interleukin-1 (IL-1) and interleukin-6 (IL-6) compared to control-treated cells. The secretion of IL-6 from HNSCC cells in response to erlotinib was found to be triggered by MyD88-dependent IL-1 signaling and suppression of IL-1 signaling (using a neutralizing IL-1α antibody) significantly enhanced the anti-tumor efficacy of erlotinib in vivo. In erlotinib-resistant HNSCC cell lines, IL-1 signaling was significantly upregulated compared to their respective parental cell lines. Blockade of IL-1 signaling using a neutralizing IL-1α antibody or an IL-1 receptor (IL-1R) antagonist was able to significantly suppress the growth of erlotinib-resistant HNSCC xenografts as a single agent and in combination with erlotinib which was associated with reduced immune cell recruitment and angiogenesis. Altogether, blockade of the IL-1 pathway may represent a novel strategy to improve tumor response to erlotinib and other EGFR TKIs in HNSCC patients. Citation Format: Andrean Simons, Aditya Stanam, Adam Koch, Madelyn Espinosa-Cotton, Katherine Gibson-Corley. Targeting IL-1 signaling to improve tumor response to erlotinib in HNSCC [abstract]. In: Proceedings of the AACR-AHNS Head and Neck Cancer Conference: Optimizing Survival and Quality of Life through Basic, Clinical, and Translational Research; April 23-25, 2017; San Diego, CA. Philadelphia (PA): AACR; Clin Cancer Res 2017;23(23_Suppl):Abstract nr 48.

Treatment for head and neck squamous cell carcinoma (HNSCC) patients includes the epidermal growth factor receptor (EGFR) monoclonal antibody cetuximab. However, response to cetuximab is low and resistance remains a critical issue. Therefore, it is imperative to develop strategies to enhance tumor response to this drug. Inflammation plays an important role in tumor progression by increasing the release of cytokines that promote tumor cell survival. Our prior work has shown that a variety of pro-inflammatory cytokines involved in tumor survival are upregulated upon treatment with EGFR inhibitors in HNSCC cells, suggesting that poor tumor response to EGFR inhibitors may be due to increased tumor-associated inflammation. Our lab has shown that the increased proinflammatory tumor response due to EGFR inhibition may be activated by interleukin-1 (IL-1) signaling, suggesting that the IL-1 pathway may be an important target to enhance the efficacy of anti-EGFR therapy. Here we investigated the effect of IL-1 blockade on HNSCC tumor response to cetuximab treatment. We found that cetuximab induced IL-6 secretion from SQ20B HNSCC cells and knockdown of the adapter protein MyD88, which is essential for IL-1 signaling, suppressed IL-6 secretion at baseline and in response to cetuximab treatment. Similarly, knock down of the IL-1 receptor (IL-1R) decreased baseline and cetuximab-induced IL-6 secretion, although IL-1R knockdown did not improve response to cetuximab in SQ20B xenografts in vivo. We additionally blocked IL-1 signaling using a recombinant IL-1R antagonist (IL-1RA). IL-1RA pre-treatment reduced baseline and cetuximab-induced IL-6 secretion in vitro. Although IL-1RA did not significantly enhance SQ20B tumor response to cetuximab, IL-1RA as a single agent significantly suppressed tumor growth in female mice but not in male mice. Finally, treatment with a human-specific neutralizing IL-1 alpha (IL-1α) antibody (but not IL-1 beta (IL-1β)) reduced baseline and cetuximab-induced IL-6 secretion in vitro, and significantly increased SQ20B tumor response to cetuximab in vivo. Altogether we propose that targeting IL-1α specifically may be a viable strategy to improve HNSCC tumor response to cetuximab. Citation Format: Madelyn Espinosa-Cotton, Ayana J. McLaren, Adam T. Koch, Andrean L, Simons. Targeting interleukin-1 to increase cetuximab efficacy in head and neck cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 3705. doi:10.1158/1538-7445.AM2017-3705

Patients with advanced recurrent and/or metastatic head and neck squamous cell carcinoma (HNSCC) have few effective treatment options and a poor median survival of 6-8 months. Limited improvement in overall survival has been observed in HNSCC patients treated with EGFR inhibitors despite high expression of epidermal growth factor receptor (EGFR) in these tumors. Remarkable clinical results are emerging from immunotherapy targeting programmed death-1 (PD1) and its ligand programmed death ligand-1 (PDL1). Blockade of the PD1/PDL1 interaction is believed to enhance endogenous anti-tumor immunity by restoring T cell cytotoxic action. PDL1 is highly expressed in primary, recurrent, and metastatic HNSCC and phase 1 clinical trials are ongoing for monoclonal antibodies targeting the PD1/PDL1 axis in HNSCC. We found that activation of EGFR in HNSCC cells by epidermal growth factor (EGF) significantly increases PDL1 expression in a dose dependent manner. EGFR inhibition using tyrosine kinase inhibitors (TKI) or cetuximab suppresses this EGF-induced PDL1 expression in cells with both wild type and constitutively active EGFR. This is consistent with published data that constitutively active EGFR in lung cancer cells correlates with higher PDL1 expression, and this can be reversed by EGFR inhibition. These results suggest there may be interactions between EGFR-targeted therapy and immunotherapy involving the PD1/PDL1 axis. To evaluate potential therapeutic interactions, we used an immunocompetent, syngeneic mouse model of human EGFR-expressing TUBO cancer cells on a BALB/c background. The murine TUBO-EGFR cells are sensitive to human EGFR inhibitors both in vitro and in vivo. Initial studies show that treatment of tumors with EGFR inhibitor (erlotinib) results in increased intratumoral CD8+ T cell infiltration and tumor regression, suggesting that EGFR inhibition can impact the anti-tumor T cell response. We are currently evaluating the effects of EGFR inhibition on PDL1 expression and tumor response to PD1/PDL1 blockade using this model. Further, a clinical trial investigating the impact of cetuximab treatment on intratumoral expression of PDL1 in HNSCC patients is planned to open shortly. We conclude that signaling mediated by EGFR can impact expression of PDL1 by HNSCC. Understanding the interactions between EGFR inhibitors and immune checkpoint blockade could lead to new approaches to combining TKI and cancer immunotherapy. Citation Format: Laura M. Rogers, Madelyn M. Espinosa-Cotton, Andrean L. Simons, George J. Weiner. Impact of therapeutic EGFR inhibition on immune checkpoint blockade in head and neck squamous cell carcinoma. [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 546.