Hyun-Bae Jie

Cetuximab-Activated Natural Killer and Dendritic Cells Collaborate to Trigger Tumor Antigen–Specific T-cell Immunity in Head and Neck Cancer Patients

Purpose: Tumor antigen–specific monoclonal antibodies (mAb) block oncogenic signaling and induce Fcγ receptor (FcγR)–mediated cytotoxicity. However, the role of CD8+ CTL and FcγR in initiating innate and adaptive immune responses in mAb-treated human patients with cancer is still emerging. Experimental Design: FcγRIIIa codon 158 polymorphism was correlated with survival in 107 cetuximab-treated patients with head and neck cancer (HNC). Flow cytometry was carried out to quantify EGF receptor (EGFR)–specific T cells in cetuximab-treated patients with HNC. The effect of cetuximab on natural killer (NK) cell, dendritic cell (DC), and T-cell activation was measured using IFN-γ release assays and flow cytometry. Results: FcγRIIIa polymorphism did not predict clinical outcome in cetuximab-treated patients with HNC; however, elevated circulating EGFR853–861–specific CD8+ T cells were found in cetuximab-treated patients with HNC (P < 0.005). Cetuximab promoted EGFR-specific cellular immunity through the interaction of EGFR+ tumor cells and FcγRIIIa on NK cells but not on the polymorphism per se. Cetuximab-activated NK cells induced IFN-γ–dependent expression of DC maturation markers, antigen processing machinery components such as TAP-1/2 and T-helper cell (TH1) chemokines through NKG2D/MICA binding. Cetuximab initiated adaptive immune responses via NK cell–induced DC maturation, which enhanced cross-presentation to CTL specific for EGFR as well as another tumor antigen, MAGE-3. Conclusion: Cetuximab-activated NK cells promote DC maturation and CD8+ T-cell priming, leading to tumor antigen spreading and TH1 cytokine release through “NK–DC cross-talk.” FcγRIIIa polymorphism did not predict clinical response to cetuximab but was necessary for NK–DC interaction and mAb-induced cross-presentation. EGFR-specific T cells in cetuximab-treated patients with HNC may contribute to clinical response. Clin Cancer Res; 19(7); 1858–72. ©2013 AACR.

CTLA-4+ Regulatory T Cells Increased in Cetuximab-Treated Head and Neck Cancer Patients Suppress NK Cell Cytotoxicity and Correlate with Poor Prognosis

The EGFR-targeted antibody cetuximab is effective against head and neck cancer (HNSCC), but in only 15% to 20% of patients, and the variability and extent of cetuximab-mediated cellular immunity is not fully understood. We hypothesized that regulatory T cells (Treg) may exert a functional and clinical impact on antitumor immunity in cetuximab-treated individuals. The frequency, immunosuppressive phenotype, and activation status of Treg and natural killer (NK) cells were analyzed in the circulation and tumor microenvironment of cetuximab-treated patients with HNSCC enrolled in a novel neoadjuvant, single-agent cetuximab clinical trial. Notably, cetuximab treatment increased the frequency of CD4(+)FOXP3(+) intratumoral Treg expressing CTLA-4, CD39, and TGFβ. These Treg suppressed cetuximab-mediated antibody-dependent cellular cytotoxicity (ADCC) and their presence correlated with poor clinical outcome in two prospective clinical trial cohorts. Cetuximab expanded CTLA-4(+)FOXP3(+) Treg in vitro, in part, by inducing dendritic cell maturation, in combination with TGFβ and T-cell receptor triggering. Importantly, cetuximab-activated NK cells selectively eliminated intratumoral Treg but preserved effector T cells. In ex vivo assays, ipilimumab targeted CTLA-4(+) Treg and restored cytolytic functions of NK cells mediating ADCC. Taken together, our results argue that differences in Treg-mediated suppression contribute to the clinical response to cetuximab treatment, suggesting its improvement by adding ipilimumab or other strategies of Treg ablation to promote antitumor immunity.

Immune biomarkers of anti-EGFR monoclonal antibody therapy

The tumor antigen (TA)-targeted monoclonal antibodies (mAb) cetuximab and panitumumab target the human epidermal growth factor receptor and have been integrated into treatment regimens for advanced squamous cell carcinoma of the head and neck (SCCHN). The therapeutic efficacy of these mAbs has been found to be enhanced when combined with radiotherapy and chemotherapy. However, clinical trials indicate that these findings are limited to fewer than 20% of treated patients. Therefore, identifying patients who are likely to benefit from these agents is crucial to improving therapeutic strategies. Interestingly, it has been noted that TA-targeted mAbs mediate their effects by contributing to cell-mediated cytotoxicity in addition to inhibition of downstream signaling pathways. Here, we describe the potential immunogenic mechanisms underlying these clinical findings, their role in the varied clinical response and identify the putative biomarkers of antitumor activity. We review potential immunological biomarkers that affect mAb therapy in SCCHN patients, the implications of these findings and how they translate to the clinical scenario, which are critical to improving patient selection and ultimately outcomes for patients undergoing therapy.The tumor antigen (TA)-targeted monoclonal antibodies (mAb) cetuximab and panitumumab target the human epidermal growth factor receptor and have been integrated into treatment regimens for advanced squamous cell carcinoma of the head and neck (SCCHN). The therapeutic efficacy of these mAbs has been found to be enhanced when combined with radiotherapy and chemotherapy. However, clinical trials indicate that these findings are limited to fewer than 20% of treated patients. Therefore, identifying patients who are likely to benefit from these agents is crucial to improving therapeutic strategies. Interestingly, it has been noted that TA-targeted mAbs mediate their effects by contributing to cell-mediated cytotoxicity in addition to inhibition of downstream signaling pathways. Here, we describe the potential immunogenic mechanisms underlying these clinical findings, their role in the varied clinical response and identify the putative biomarkers of antitumor activity. We review potential immunological biomarkers that affect mAb therapy in SCCHN patients, the implications of these findings and how they translate to the clinical scenario, which are critical to improving patient selection and ultimately outcomes for patients undergoing therapy.

Tumor Antigen-Specific Monoclonal Antibodies and Induction of T-Cell Immunity

For decades the primary available cancer therapies were relatively nonspecific cytotoxic agents which, while effective in some patients, were limited by narrow therapeutic indices, extensive toxicity and development of resistance, likely due to tumor heterogeneity. Although these chemotherapies remain common tools of conventional treatment, the approval of a growing number of tumor antigen (TA)-specific monoclonal antibodies (mAbs) by the US Food and Drug Administration has driven a shift in the paradigm of cancer therapy. For a subset of patients with lymphoma, colorectal, head and neck, and breast cancers, the inclusion of rituximab (anti-CD20), cetuximab (anti-human epidermal growth factor 1), and trastuzumab (anti-human epidermal growth factor 2) has resulted in overall improved clinical response rates and survival advantages. The mechanisms that contribute to these effects are limited not only to inhibition of signaling pathways but also include cell-mediated cytotoxicity by innate immune cells and priming of effector cells of adoptive immunity triggered by the TA-specific mAb. However, as the use of these therapeutic mAbs has become more widespread, it has been observed that there is significant variability of response in patients treated with these agents. Thus, the factors that mediate this variability in clinical responses must be elucidated to optimize the use of TA-specific mAbs.

Co-targeting of delta-like ligand 4 (DLL4) and vascular endothelial growth factor a (VEGF) with programmed death 1 (PD1) blockade inhibits tumor growth and facilitates anti-tumor immune responses

Blocking DLL4, a Notch ligand, effectively inhibits tumor growth by increasing non-functional angiogenesis and decreasing the cancer stem cell (CSC) population. Preclinical studies have demonstrated inhibition of tumor growth by anti-DLL4 treatment and have led us to enter demcizumab, an anti-DLL4 mAb, into ongoing clinical trials. Vascular endothelial growth factor A (VEGF A) also plays a central role in inducing tumor angiogenesis. VEGF signaling is also involved in recruiting immune suppressive myeloid cells. Therefore, targeting VEGF could induce favorable immune responses against cancer. We developed a bispecific monoclonal antibody that blocks both DLL4 and VEGF which is in Phase I clinical trials. In the present study we compare the impact of anti-DLL4 in combination with anti-VEGF and anti-DLL4 in combination with anti-VEGF and anti-Programmed Cell Death Protein 1 (PD1) on anti-tumor immune responses. Our data demonstrate that the triple blockade of DLL4-VEGF-PD1 significantly inhibited tumor growth with more pronounced tumor regression. Anti-DLL4 treatment reduced IL17 production, an effect not observed with anti-PD1, blockade of DLL4-VEGF or DLL4-VEGF-PD1, suggesting that blocking DLL4 alone and together with VEGF or VEGF and PD1 might have different mechanisms for regulating immune responses. Anti-PD1 increased specific CD8+ T cell-mediated IFN-gamma production while decreasing IL6. Interestingly, IL2 was increased at the tumor site by blockade of DLL4-VEGF-PD1 compared to controls. Since IL2 is required for secondary population expansion of CD8+ memory T cells, increased IL2 in the triple combination group suggests potential for increased T cell activation, maintenance and memory T cell function, as compared to single agent anti-DLL4 and anti-PD1. Memory CD8+ T cell frequencies were increased within the total CD8+ T cell population by DLL4-VEGF-PD1 triple blockade. Therefore, these results show that co-targeting of DLL4 and VEGF with PD1 might be an effective and durable anti-cancer therapy in part by promoting anti-tumor immune responses and inhibiting pro-tumor immune responses.

Abstract 2612: Anti-Tigit induces T cell mediated anti-tumor immune response and combines with immune checkpoint inhibitors to enhance strong and long term anti-tumor immunity

TIGIT (T cell immunoreceptor with Ig and ITIM domains) has been recently described as an inhibitory receptor which blocks CD8 T cell-mediated anti-tumor immune responses. We have generated an anti-mouse TIGIT antibody (313R12) to evaluate drug efficacy and mechanism of action in pre-clinical tumor models. Anti-TIGIT as a single agent promoted an anti-tumor immune response in multiple syngeneic mouse tumor models. Anti-TIGIT enhanced tumor specific T cell responses, particularly of the Th1 type and reduced Th2 type responses and also increased the function of cytotoxic T cells. Furthermore, anti-TIGIT displayed combination activity with immune checkpoint inhibitors anti-PD1 and anti-PDL1 in inhibiting tumor growth, promoting complete tumor rejection and significantly increasing mouse survival in the murine CT26 colon carcinoma model as compared to controls and single agents alone. Mice “cured” with anti-TIGIT/anti-PDL1 or anti-TIGIT/anti-PD1 combination treatments did not form tumors upon subsequent re-challenges with increasing number of CT26 tumor cells, suggesting the existence of immunologic memory. IL2 and tumor-specific IFN-γ production by splenic T cells were increased in mice who responded to combination treatment compared to controls. Additionally, both effector and memory CD8+ T cell frequencies were increased within the total CD8+ T cell population in responding mice. We also demonstrated a systemic increase in tumor-specific CD8 T cells after anti-TIGIT/anti-PDL1 combination treatment compared to controls. Therefore, these results suggest that co-targeting of TIGIT and PD1 or PDL1 may be an effective and durable cancer therapy by increasing T cell-mediated anti-tumor immune responses and promoting long-term immunological memory. Citation Format: Minu K. Srivastava, Rui Yun, Erin Mayes, Janice Yu, Hyun-Bae Jie, Fumiko Axelrod, Ming-Hong Xie, Jorge Monteon, Andrew Lam, May Ji, Yuwang Liu, John Lewicki, Tim Hoey, Austin Gurney, Angie Inkyung Park. Anti-Tigit induces T cell mediated anti-tumor immune response and combines with immune checkpoint inhibitors to enhance strong and long term anti-tumor immunity [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 2612. doi:10.1158/1538-7445.AM2017-2612

Abstract 2003: Antibody against TIGIT (T cell immunoreceptor with Ig and ITIM domains) induces anti-tumor immune response and generates long-term immune memory

T cell immunoreceptor with Ig and ITIM domains (TIGIT) is a co-inhibitory molecule containing an immunoreceptor tyrosine-based inhibition motif (ITIM) within its cytoplasmic tail, and is highly expressed on regulatory T cells and activated CD4+ T, CD8+ T, and NK cells. TIGIT competes with CD226, which contains an immunoreceptor tyrosine-based activation motif (ITAM) within its cytoplasmic tail for ligands poliovirus receptor (PVR) and poliovirus receptor-related 2 (PVRL2), with higher affinity to PVR. The ligands are expressed on the surface of antigen presenting cells and at high levels on most tumors. Therefore, when TIGIT is present, the ligands preferentially engage TIGIT rather than CD226, leading to cell suppression. We have generated antibodies against TIGIT that blocks ligand binding and inhibits TIGIT signaling. The clinical candidate, OMP-313M32 binds human TIGIT but not rodent and non-human primate TIGIT. Therefore, a surrogate antibody was generated for pre-clinical assessments in mice. Antibody 313R12 is an anti-mouse TIGIT antibody that can block mouse PVR ligand binding and inhibit TIGIT signaling in a manner similar to the clinical candidate OMP-313M32. 313R12 inhibited the growth of syngeneic colon and kidney tumors in immune competent mice. In some cases, anti-TIGIT antibody 313R12 caused complete tumor regression and a potent anti-tumor immune memory response as demonstrated by the lack of tumor growth upon re-challenge of mice that remained tumor-free after prior anti-TIGIT treatment. Mechanistically, anti-TIGIT antibody 313R12 was shown to induce a Th1 response and increase cytotoxic T lymphocyte (CTL) activity. By in vivo depletion of T cell populations, we have shown that CD8 T cell depletion completely abrogated the anti-TIGIT therapeutic effect, whereas CD4 T cell depletion led to partial reversal of efficacy of anti-TIGIT. Therefore, both CD4+ and CD8+ T cells are critical for anti-TIGIT-mediated immune responses. Using mice reconstituted with human hematopoietic stem cells, we also demonstrated that the clinical candidate OMP-313M32 inhibits patient-derived melanoma tumor growth. Taken together, these data demonstrate that anti-TIGIT therapy suppresses tumor growth and generates long-term immunological memory against multiple tumors. Citation Format: Angie Inkyung Park, Minu Srivastava, Erin Mayes, Hyun-Bae Jie, Rui Yun, Christopher Murriel, Ming-hong Xie, Andrew Lam, May Ji, Fumiko Axelrod, Jorge Monteon, John Lewicki, Tim Hoey, Austin Gurney. Antibody against TIGIT (T cell immunoreceptor with Ig and ITIM domains) induces anti-tumor immune response and generates long-term immune memory [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 2003. doi:10.1158/1538-7445.AM2017-2003

Abstract A058: GITRL-Fc, an immunotherapeutic agent that stimulates T-cell-mediated antitumor immune response

GITRL, (Glucocorticoid-Induced Tumor Necrosis Factor Receptor Ligand, TNFSF18) is a member of the TNF family of ligands and naturally exists as a membrane-anchored type II protein that self assembles as a trimer. GITRL activates the co-stimulatory receptor GITR. A novel single-gene linkerless GITRL trimer was shown to be functional when fused to either the N- or C-terminus of an immunoglobulin Fc domain, offering a flexible strategy that may also be amenable to the production of bispecific agents. GITRL-Fc activated GITR signaling more effectively than prototype GITR agonist antibody DTA-1. GITRL-Fc promoted a robust anti-tumor immune response in several murine tumor graft models, including the apparent total regression of some treated tumors. GITRL-Fc potentiated tumor specific T-cell responses, particularly of the Th1 type, increased antigen-specific CD8 response, and promoted a reduction in Treg-mediated immune-suppressive activity. Citation Format: Fumiko Axelrod, Hyun-Bae Jie, Erin Mayes, Jorge Monteon, Minu Srivastava, Rui Yun, Inkyung Angie Park, Austin Gurney. GITRL-Fc, an immunotherapeutic agent that stimulates T-cell-mediated antitumor immune response. [abstract]. In: Proceedings of the CRI-CIMT-EATI-AACR Inaugural International Cancer Immunotherapy Conference: Translating Science into Survival; September 16-19, 2015; New York, NY. Philadelphia (PA): AACR; Cancer Immunol Res 2016;4(1 Suppl):Abstract nr A058.

Anti-EGFR mAb cetuximab therapy increases T cell receptor (TCR) diversity in the peripheral blood and focuses TCR richness in the tumor microenvironment

T cell receptor (TCR) recognition of tumor antigen is essential for effective antitumor immunity. The immune system`s ability to respond to a broad spectrum of antigens requires a sophisticated selection of various TCR. So far, little is known about the role of TCR richness and clonality in the immune response to the EGFR-specific mAb, cetuximab. Therefore, we investigated differences in TCR sequences between HPV+ and HPV- patients, as well as differences in sequence characteristics between T cells of peripheral blood mononuclear cells (PBMC) and tumor infiltrating lymphocytes (TIL). Additionally, we were able to investigate the TCR richness and clonality in samples pre- and post-treatment in a clinical single agent cetuximab trial.

Level of PD-1 expression on CD8+ T cells influence prognosis and respond to PD-1 therapy in a murine model

Prognosis varies dramatically in head and neck squamous cell carcinoma (HNSCC) based on HPV status. In HPV+ patients, programmed death (PD)-1 expression has been linked to a better clinical outcome. We hypothesized that extent of PD-1 expression may differentially impact T cell phenotype, patient prognosis and response to anti-PD-1 immunotherapy in a murine HPV+ cancer model.