Andrés López-Albaitero

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.

Antitumor Activity of Human Papillomavirus Type 16 E7–Specific T Cells against Virally Infected Squamous Cell Carcinoma of the Head and Neck

Human papillomavirus (HPV)-associated squamous cell carcinoma of the head and neck (SCCHN) seems to be a suitable target for cancer vaccination. HPV-encoded oncogenic proteins, such as E7, are promising tumor-specific antigens and are obligatory for tumor growth. Because few immunologic studies have analyzed the endogenous HPV-specific immune response in this subset of SCCHN patients, we studied T-cell frequencies against HPV-16 E7(11-20) or E7(86-93) in tumor-bearing, human leukocyte antigen (HLA)-A*0201+ SCCHN patients, whose tumors were either HPV-16+ or HPV-16-. In HPV-16+ SCCHN patients, frequencies of T cells against either peptide were significantly elevated (P < 0.005) compared with HPV-16- patients or healthy volunteers. Tetramer+ T cells showed evidence of terminally differentiated phenotype (CD45RA+CCR7-) and an elevated level of CD107a staining for degranulation. Despite detectable expression of the restricting HLA class I allele, HLA-A*0201-E7(11-20)- or HLA-A*0201-E7(86-93)-specific CTL obtained by in vitro stimulation of healthy donor peripheral blood mononuclear cells only recognize a naturally HPV-16-transformed, HLA-A*0201+ SCCHN cell line after pretreatment with IFN-gamma. This cell line had little or no expression of LMP2, TAP1, and tapasin, critical components of the HLA class I antigen-processing machinery, which were up-regulated by IFN-gamma treatment. Immunohistochemistry of HPV-16+ SCCHN tumors showed that these antigen-processing machinery components are down-regulated in tumors in vivo compared with adjacent normal squamous epithelium. Thus, immunity to HPV-16 E7 is associated with the presence of HPV-16 infection and presentation of E7-derived peptides on SCCHN cells, which show evidence of immune escape. These findings support further development of E7-specific immunotherapy and strategies for up-regulation of antigen-processing machinery components in HPV-associated SCCHN.

Role of Antigen-Processing Machinery in the In Vitro Resistance of Squamous Cell Carcinoma of the Head and Neck Cells to Recognition by CTL

Squamous cell carcinoma of the head and neck (SCCHN) cells are poorly recognized in vitro by CTL despite expressing the restricting HLA class I allele and the targeted tumor Ag (TA). Several lines of evidence indicate that the lack of SCCHN cell recognition by CTL reflects defects in targeted TA peptide presentation by HLA class I Ag to CTL because of Ag-processing machinery (APM) dysfunction. First, lack of recognition of SCCHN cells by CTL is associated with marked down-regulation of the IFN-γ-inducible APM components low-m.w. protein 2, TAP1, TAP2, and tapasin. Second, SCCHN cell recognition by CTL is restored by pulsing cells with exogenous targeted TA peptide. Third, the restoration of CTL recognition following incubation of SCCHN cells with IFN-γ is associated with a significant (p = 0.001) up-regulation of the APM components TAP1, TAP2, and tapasin. Lastly, and most conclusively, SCCHN cell recognition by CTL is restored by transfection with wild-type TAP1 cDNA. Our findings may explain the association between APM component down-regulation and poor clinical course of the disease in SCCHN. Furthermore, the regulatory nature of the APM defects in SCCHN cells suggests that intralesional administration of IFN-γ may have a beneficial effect on the clinical course of the disease and on T cell-based immunotherapy of SCCHN by restoring SCCHN cell recognition by CTL.

Natural killer (NK): dendritic cell (DC) cross talk induced by therapeutic monoclonal antibody triggers tumor antigen-specific T cell immunity.

Tumor antigen (TA)-targeted monoclonal antibodies (mAb), trastuzumab, cetuximab, panitumumab, and rituximab, have been among the most successful new therapies in the present generation. Clinical activity is observed as a single agent, or in combination with radiotherapy or chemotherapy, against metastatic colorectal cancer, head and neck cancer, breast cancer, and follicular lymphoma. However, the activity is seen only in a minority of patients. Thus, an intense need exists to define the mechanism of action of these immunoactive mAb. Here, we discuss some of the likely immunological events that occur in treated patients: antibody-dependent cellular cytotoxicity (ADCC), cross talk among immune cells including NK cells and dendritic cells (DCs), and generation of TA-specific T lymphocyte responses. We present evidence supporting the induction of “NK:DC cross talk,” leading to priming of TA-specific cellular immunity. These observations show that mAb-mediated NK cell activation can be greatly enhanced by the action of stimulatory cytokines and surface molecules on maturing DC and that NK:DC interaction facilitates the recruitment of both NK cells and DC to the tumor site(s). The cooperative, reciprocal stimulatory activity of both NK cells and DC can modulate both the innate immune response in the local tumor microenvironment and the adaptive immune response in secondary lymphoid organs. These events likely contribute to clinical activity, as well as provide a potential biomarker of response to mAb therapy.

Novel Immunogenic HLA-A*0201-restricted Epidermal Growth Factor Receptor-specific T-cell Epitope in Head and Neck Cancer Patients

Therapeutic targeting of the epidermal growth factor receptor (EGFR), which is highly overexpressed and correlated with poor prognosis in colorectal and head and neck squamous cell carcinoma (SCCHN), has shown clinical efficacy using the blocking mAbs, cetuximab or panitumumab, but only in 10% to 20% of patients. Clinical responsiveness is correlated with certain Fcγ receptor genotypes, suggesting immune activity may contribute to therapeutic efficacy. In addition, cetuximab-resistant tumor cells exhibit ubiquitination and degradation of EGFR, which would increase its processing as a tumor antigen for cytotoxic T lymphocyte (CTL) lysis. Thus, T cell-based immunotherapy might enhance the antitumor efficacy of EGFR-specific mAbs, but CTL epitopes are poorly defined. To permit combinatorial EGFR-targeted immunotherapy, we identified a novel immunogenic wild-type sequence peptide, EGFR853−861 and modified its anchor sequence to enhance HLA-A*0201 binding and stimulation of cross-reactive anti-wild–type EGFR853−861-specific CTL. Cross-reactivity was also observed with HER2861−869. EGFR853−861-specific CTL recognition of SCCHN cells was increased by incubation of tumor cells with cetuximab, which led to EGFR degradation. In addition, EGFR853−861-specific CTLs were elevated in the circulation of SCCHN patients as compared with healthy control peripheral blood mononuclear cells. Thus, a novel, immunogenic EGFR-encoded CTL epitope may be incorporated into vaccines and would be useful for combinatorial immunotherapy with EGFR-specific mAbs in cancer patients.

Quantitative Expression and Immunogenicity of MAGE-3 and -6 in Upper Aerodigestive Tract Cancer

The MAGE antigens are frequently expressed cancer vaccine targets. However, quantitative analysis of MAGE expression in upper aerodigestive tract (UADT) tumor cells and its association with T‐cell recognition has not been performed, hindering the selection of appropriate candidates for MAGE‐specific immunotherapy. Using quantitative RT‐PCR (QRT‐PCR), we evaluated the expression of MAGE‐3/6 in 65 UADT cancers, 48 normal samples from tumor matched sites and 7 HLA‐A*0201+ squamous cell carcinoma of the head and neck (SCCHN) cell lines. Expression results were confirmed using Western blot. HLA‐A*0201:MAGE‐3‐ (271–279) specific cytotoxic T lymphocytes (MAGE‐CTL) from SCCHN patients and healthy donors showed that MAGE‐3/6 expression was highly associated with CTL recognition in vitro. On the basis of the MAGE‐3/6 expression, we could identify 31 (47%) of the 65 UADT tumors, which appeared to express MAGE‐3/6 at levels that correlated with efficient CTL recognition. To confirm that the level of MAGE‐3 expression was responsible for CTL recognition, 2 MAGE‐3/6 mRNAhigh SCCHN cell lines, PCI‐13 and PCI‐30, were subjected to MAGE‐3/6‐specific knockdown. RNAi‐transfected cells showed that MAGE expression and MAGE‐CTL recognition were significantly reduced. Furthermore, treatment of cells expressing low MAGE‐3/6 mRNA with a demethylating agent, 5‐aza‐2′‐deoxycytidine (DAC), increased the expression of MAGE‐3/6 and CTL recognition. Thus, using QRT‐PCR UADT cancers frequently express MAGE‐3/6 at levels sufficient for CTL recognition, supporting the use of a QRT‐PCR‐based assay for the selection of candidates likely to respond to MAGE‐3/6 immunotherapy. Demethylating agents could increase the number of patients amenable for targeting epigenetically modified tumor antigens in vaccine trials.

Maturation pathways of dendritic cells determine TAP1 and TAP2 levels and cross-presenting function.

Ability to cross-present exogenous antigens in the human leukocyte antigen class I pathway is key to the antigen presenting function of mature tumor cell-loaded dendritic cells (DC). Conditions of DC maturation have been shown to be important for DCs ability to produce proinflammatory cytokines and induce T cell effector functions. However, it remains unknown if the different pathways of maturation are associated with modulation of the ability of mature DCs to cross-present tumor antigens (TA). Here, we compare DC matured with 3 clinically relevant cytokine combinations including interleukin (IL)-1β, tumor necrosis factor-α, IL-6 (termed DC-0), DC-0 cells incubated with prostaglandin-2 (termed DC-0+prostaglandin-2), or DC treated with interferon-γ, interferon-α, tumor necrosis factor-α, Poly I:C, and IL1-β (termed DC-1). We found that these DC vary in their ability to cross-present TA to cytotoxic T lymphocytes (CTL), with the DC-1 cytokine combination being significantly more effective than the other 2. TA cross presentation and CTL priming were strongly correlated with level of expression of the antigen processing machinery components, TAP1 and TAP2, indicating that these components could be used as biomarkers to standardize DC preparations for optimal function. However, the up-regulation of TAP1/TAP2 was not sufficient to explain the enhanced cross-presentation ability of DC-1 cells, as the use of IFN-γ alone to up-regulate TAP1/TAP2 did not generate DC as effective at cross-presentation as the full DC-1 maturation cytokine combination. These data indicate for the first time that the pathways of DC maturation modulate antigen processing machinery component expression to different extents and that differently matured DC vary in the ability to cross-present TA to human leukocyte antigen class I-restricted CTL.

Immunotherapy for head and neck cancer

Overall survival for patients with squamous cell carcinoma of the head and neck (SCCHN) has not improved appreciably over the past few decades. Novel therapeutic approaches, such as immunotherapy, are under clinical investigation since the standard treatments are toxic and have not successfully controlled this disease with sufficiently high success rates. Cancer immunotherapy describes various techniques to expand and activate the immune system to control tumor growth in vivo, and clinical evaluation has so far demonstrated low toxicity. Immunotherapy appears to have the most applicability in settings of minimal residual disease and to reduce distant metastases after other therapeutic interventions, and its potential clinical value is now receiving intensive evaluation. Emerging forms of SCCHN immunotherapy involve both the use of monoclonal antibodies (mAb) that target growth factor receptors where immune activation appears to contribute to tumor cell lysis, as well as various forms of active vaccination strategies which activate and direct the patients cellular immunity against the tumor. This article reviews immunotherapeutic strategies currently in clinical trials or under development for patients with SCCHN.

R079: Transcutanous Immunotherapy in HPV-16+ Head and Neck Cancer

and CT staging score. NP patients were substratified into three subgroups: NP without BHR, NP with asymptomatic BHR, and NP with BHR and asthma according to their bronchial phenotype by the results of provocative testing. RESULTS: The mean CL level in polyp-tissues, but not in blood, was higher than in the control specimens. In NP patients, tissue-CL was associated with endoscopy score. Tissue-CL and endoscopy score were associated with BHR/ asthma phenotype. CONCLUSION: These results suggest an important role for oxidative stress in the pathophysiology of nasal polyposis. SIGNIFICANCE: Free radical levels in polyp-tissues associated with NP severity and with BHR/asthma phenotype in nonallergic NP patients. SUPPORT: This work was supported by a grant (Grant No.: NSC 92-2314-B-039-026) from the National Science Council (Republic of China) and was also supported in part by a research grant (Grant No. DMR-93-031) from the China Medical University Hospital.