Head and neck squamous cell carcinoma (HNSCC), the sixth leading incident cancer worldwide, has been recognized as an immunosuppressive disease. HNSCC induces a tumor-permissive cytokine profile,[2, 3] qualitative and quantitative lymphocyte deficiencies,[4-6] anergy in major immune effector cells,[7-10] and poor antigen presentation.[11, 12] An increasing proportion of HNSCC in North America and Europe is caused by oral infection with human papillomavirus (HPV),[13-15] rather than the classic exposures of tobacco and alcohol. Whether caused by environmental carcinogenesis or transformation by HPV oncogenes, HNSCC thwarts immune surveillance, recognition and destruction, which must be reversed to maximize therapeutic efficacy.
Early clinical trials in HNSCC exploited available immunostimulatory cytokines, which faltered clinically due to disinterest in local delivery or prohibitive systemic toxicity.[16-18] Three parallel advancements have reawakened enthusiasm for the development of novel immunotherapies in HNSCC: 1) realization of the contribution of immune mechanisms to the clinical activity of cetuximab,[19, 20] the monoclonal antibody (mAb) against the epidermal growth factor receptor (EGFR) approved in treatment of HNSCC by the US Food and Drug Administration (FDA) in 2006; 2) progressive preclinical insights into specific, targetable immune escape mechanisms important to the survival of HNSCC tumors; and 3) previously unimagined clinical responses in non–small cell lung cancer (NSCLC), a nonimmunogenic solid tumor similar to HNSCC, to phase 1 therapy with an immune checkpoint mAb.[21, 22] In the interest of prioritizing rational clinical investigations, this review will examine the immunotherapeutic mAbs currently in human trials for cancer patients, in the specific context of immune escape mechanisms in HNSCC. Immunotherapeutic mAbs will be conceptually divided into those which target tumor antigens (TAs), immunosuppressive cytokines, tumor necrosis factor receptor (TNFR) costimulatory molecules, or immune checkpoint receptors (Table 1).
Although cytotoxic T lymphocytes (CTL) specific for p53, EGFR, or the HPV E7 oncoprotein have been detected in patients with HNSCC,[23-25] the nascent adaptive immune response is ineffective. Because of selective loss of human leukocyte antigen (HLA) I and functional deficiency in antigen processing machinery, HNSCC tumor cells avoid recognition and destruction by extant TA-specific CTLs.[11, 26] Recent evidence confirms that cetuximab, a chimeric, immunoglobulin G1 (IgG1)-isotype mAb which blocks the extracellular domain of EGFR, potentiates both innate and adaptive immune responses against endogenous TAs, indicating that TA-targeted mAbs have broader immunogenic potential than is currently being exploited.
In HNSCC, cetuximab development was compelled by the frequent finding of overexpression of EGFR correlating with advanced stage, radiation resistance, and poor survival.[27, 28] Indeed, cetuximab increased response rate and overall survival combined with radiation in locally advanced HNSCC, or with platinum-based chemotherapy in recurrent HNSCC, ultimately gaining FDA approval for both indications.[29, 30] Cetuximab is frequently described as the first molecularly targeted agent in HNSCC, which is a deserved appellation; yet, certain conundrums prompted the search for additional, immunologic mechanisms of action. First, despite overexpression of EGFR in more than 90% of HNSCC, cetuximab monotherapy demonstrates a response rate of only 10% to 15% in recurrent disease. Moreover, overexpression or amplification of EGFR does not predict clinical benefit from cetuximab, and activating EGFR mutations are not found in HNSCC, underscoring the absence of a predictive molecular marker.[33, 34] Second, nonimmunogenic small-molecule inhibitors of the intracellular tyrosine kinase domain of EGFR have not demonstrated clinical benefit in randomized trials in HNSCC.[35, 36] Third, despite rapid abrogation of EGFR phosphorylation and tumor proliferation upon mAb exposure in preclinical models, in vitro tumor cell apoptosis or lysis requires the presence of lymphocytes.
Dissection of the immunologic actions of cetuximab provides guidance for further development of TA-targeted mAbs in HNSCC. Specifically, cetuximab-coated HNSCC tumor cells activate the antibody-binding receptor (FcγR IIIa) on natural killer (NK) cells, the major effector cell of innate immunity. Binding between the constant Fc component of the IgG mAb and FcγR IIIa instigates antibody-dependent cellular cytotoxicity (ADCC).[20, 38, 39] Moreover, activation of FcγR IIIa upregulates NK cell secretion of interferon gamma (IFN-γ), an immunostimulatory cytokine which promotes maturation of dendritic cells (DC). NK-DC cross-priming enhances antigen processing and presentation by DCs, ultimately promoting activation of TA-specific CTLs. Of interest, the repertoire of TA-specific CTLs induced by cetuximab is not restricted to EGFR. Preclinical modeling also demonstrates cross-priming of CTLs specific for MAGE-3, a second TA. These findings suggest 3 broad and interacting strategies to augment the therapeutic potential of cetuximab and other TA-targeted mAbs: 1) potentiating ADCC; 2) promoting DC maturation; and 3) releasing suppression of CTLs.
Fundamentally, ADCC requires binding between FcγR IIIa on NK cells and the IgG Fc region on the mAb-coated tumor cell, an interaction which may be influenced by an intrinsic patient factor, FcγR polymorphisms, or a drug factor, the IgG isotype subclass. Differential patient response to mAbs, including rituximab in lymphoma (anti-CD20), trastuzumab in breast cancer (anti-HER2), and cetuximab in colorectal cancer, has been correlated with FcγR IIIa polymorphisms, thought to reflect Fc binding affinities.[40-42] Although these findings raise the possibility of patient selection for mAb therapy by ADCC capacity, a similar preclinical correlation in HNSCC was not validated in patients. The VV FcγR IIIa genotype favorably influenced NK cytolysis of HNSCC tumor cells in ADCC assays, but was not associated with cetuximab outcome in patients with HNSCC where it was found in only 5% of cases. Thus, enhancing mAb activity by patient selection for favorable polymorphisms is not currently suitable for the clinic.
The FcγR binding partner—the IgG Fc subclass of the mAb itself—appears increasingly important to the efficacy of TA-targeted mAbs. In HNSCC, the case in point is panitumumab, which is a fully humanized IgG2 mAb specific for the same EGFR epitope as cetuximab. In contrast to IgG1, the IgG2 isotype has a low binding affinity for FcγR IIIa and is ineffective at mediating ADCC by NK cells.[19, 20, 44] IgG2 does bind FcγR IIa on myeloid-lineage lytic cells, triggering ADCC; however, the clinical importance of this mechanism is unclear. Notably, panitumumab does not induce the NK-DC cross-priming underlying the adaptive immune response. Unlike cetuximab, panitumumab did not improve survival when combined with platinum-based chemotherapy in recurrent HNSCC, although a secondary analysis noted improvement in patients with tumors negative for p-16, a surrogate marker for HPV within the oropharynx. In a phase 2 trial in the locally advanced setting, panitumumab combined with radiotherapy was inferior to cisplatin-radiotherapy combination for the primary endpoint of 2-year locoregional control. The failure of panitumumab to make therapeutic inroads in HNSCC, despite identical antisignaling properties to cetuximab, has further elevated the hypothesis that immune mechanisms are critical to cetuximab's activity. This insight should inform development of subsequent TA-targeted mAbs in HNSCC, with prioritization of IgG1 subclass drugs.
Innate or acquired resistance to cetuximab is frequently associated with compensatory signaling by alternate growth factor receptors, eg, HER2, HER3, cMet, and insulin growth factor receptor (IGFR). In this light, TA-specific mAbs targeting parallel growth factor receptors are rational candidates for investigation in HNSCC. Representative IgG1-isotype mAbs are in clinical development. Trastuzumab and pertuzumab target HER2-overexpressing breast cancer; cotargeting HER2 with both drugs in a HER2-expressing xenograft model augmented ADCC. Thus, combinatorial mAb strategies are desirable, and investigation of immune correlates will be critical to guiding development. For example, AV-203, an anti-HER3 mAb, is under phase 1 evaluation in combination with cetuximab (Clinicaltrials.gov identifier NCT01603979). This trial includes a cetuximab-resistant HNSCC cohort, a setting where both signaling and immune resistance mechanisms may occur. Although cixutumumab, an anti-IGFR mAb, failed to improve progression-free survival alone or in combination with cetuximab as compared to cetuximab monotherapy, it is now under investigation in a window-of-opportunity trial in patients with operable HNSCC (Clinicaltrials.gov identifier NCT00957853). In this model, both signaling and immune mechanisms could be investigated in paired tumor specimens.
Although FcγR-Fc binding is required for ADCC, the NK cell's capacity for cytolysis is significantly amplified by the immunostimulatory cytokines interleukin-12 (IL-12) and TNFα, secreted by activated DCs following TA uptake and presentation. In the development of IgG1-isotype mAbs in HNSCC, judicious coinvestigation of 2 ADCC adjuncts would appear rational: exogenous cytokines, such as IL-12, or toll-like receptor (TLR) agonists. Intravenous IL-12 has been studied in phase 1 combination with paclitaxel and trastuzumab in HER2-expressing cancers, exploiting the ADCC mechanism. In operable HNSCC, neoadjuvant tumoral injections of IL-12 resulted in migration of NK cells to the primary tumor and draining lymph nodes, and increased IFN-γ secretion; these promising immunomodulatory findings underpin an ongoing phase 2 study of cetuximab and subcutaneous IL-12 in recurrent HNSCC (NCT01468896). VTX-2337, a TLR8 agonist, activates innate immunity, amplifying phagocytosis, immunostimulatory cytokine secretion, and antigen presentation by DCs.[52-56] Trials evaluating cetuximab with/without VTX-2337 (NCT01334177) or platinum-based chemotherapy and cetuximab, with/without VTX-2337 (NCT01836029), are recruiting.
The immunosuppressive cytokine profile of the HNSCC microenvironment is driven by aberrant regulation of the signal transducer and activator of transcription (STAT) family. HNSCC tumors and immune cells demonstrate deficient immunostimulatory STAT1 signaling, and excess immunosuppressive STAT3 signaling.[3, 57, 58] The STAT1/STAT3 activation imbalance results in dominant production of TGF-β1, IL-6, IL-10, and vascular endothelial growth factor (VEGF) by HNSCC tumors and tumor-associated macrophages. These cytokines inhibit NK cell cytolysis, DC maturation, and CTL activation while inducing regulatory T cells (Tregs).[7, 57, 60-62] Tumor-associated fibroblasts reinforce immunosuppression through secretion of hepatocyte growth factor (HGF), a paracrine cytokine that promotes HNSCC proliferation and metastasis through cMet signaling while inhibiting DC maturation.[63, 64] Because immunosuppressive cytokines in sera of patients with HNSCC correlate longitudinally with poor response and relapse, cytokine-neutralizing mAbs may be particularly relevant therapeutic adjuncts. Specifically, mAbs targeting VEGF and HGF are in clinical development in HNSCC, whereas mAbs neutralizing TGF-β1 and IL-6 are under study in other malignancies.
Bevacizumab is an anti-VEGF IgG1 subclass mAb which increased patient survival when combined with carboplatin-paclitaxel chemotherapy in advanced NSCLC, and VEGF and microvascular density are negative prognostic indicators in HNSCC.[66, 67] Although the phase 2 combination of bevacizumab-cetuximab had disappointing efficacy in recurrent HNSCC, a randomized phase 3 Eastern Cooperative Oncology Group trial testing frontline platinum-based chemotherapy with/without bevacizumab is ongoing (NCT00588770).
Ficlatuzumab (IgG1) and rilotumumab (IgG2) are humanized mAbs that neutralize HGF, preventing ligation of the oncogenic cMet receptor. Because HGF/cMet signaling is implicated in resistance to standard HNSCC therapies,[69-71] trials combining these agents with cetuximab, cisplatin, and/or radiation are in development. Bearing in mind that HGF inhibits DC maturation, companion immune biomarkers should be incorporated into early-phase design.
Fresolimumab is an IgG4 subclass mAb that neutralizes all TGF-β isoforms, and is under phase 1 study in myeloproliferative disorders, kidney cancer, and melanoma (NCT01291784; NCT00356460). In an HNSCC xenograft model, adding a preclinical TGF-β mAb to cetuximab prevented resistance to NK cell cytolysis mediated by TGF-β1. Siltuximab is an IgG1 chimeric mAb against IL-6, and is under phase 2 evaluation in kidney cancer, prostate cancer, and multiple myeloma (NCT00385827; NCT01484275). Because TGF-β1 and IL-6 are key immunosuppressive STAT3 cytokines, and no direct STAT3 inhibitors are available, both agents are of significant interest in HNSCC.
TNFR Family Targeted mAbs
Ultimately, effective immune eradication of tumor requires priming of the TA-specific, HLA-I restricted, CD8(+) CTLs by antigen-presenting cells (APCs). Binding of the T cell receptor (TCR) by its cognate TA-HLA I complex is insufficient for differentiation of the naive CTLs. TA recognition can be followed by anergy versus activation, depending on the balance of coinhibitory versus costimulatory intercellular signaling across the lymphocyte-APC “immune synapse” (Fig. 1). Priming, cytolytic capacity, and memory cell differentiation require predominance of costimulatory signaling by the TNFR superfamily of accessory surface receptors. Agonist mAbs to TNFR costimulatory members, including CD40, OX40, and CD137, are in early clinical development. In most cases, these mAbs demonstrate synergy with other immunomodulatory therapies, including the cytolytic modalities of chemotherapy and radiation, TA-targeted mAbs, or T cell checkpoint inhibitors.
Figure 1. Schematic shows the immune synapse. Binding of the T cell receptor (TCR; blue) to the tumor antigen–major histocompatibility complex (MHC) I complex is necessary but not sufficient for TCR activation. Costimulatory signaling by the major TCR coreceptor, CD28, is also required for full T cell activation. Moreover, activation of the T cell is modulated by the balance of positive, costimulatory signaling by the tumor necrosis factor receptor superfamily (green; including CD40, OX40, and CD137) versus negative, coinhibitory signaling by cytotoxic T-lymphocyte–associated antigen 4 (CTLA-4) and programmed death-1 (PD-1; red). Abbreviations: HNSCC, head and neck squamous cell carcinoma; mAb, monoclonal antibody.
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Signaling by CD40, a TNFR expressed by APCs, dramatically magnifies APC priming capacity. Binding of CD40 on DCs by its ligand CD40L, present solely on activated CD4(+) T helper cells, triggers immunostimulatory cytokine secretion, upregulation of antigen-processing machinery, and CTL priming. In HNSCC, CD40/CD40L expression on APCs/lymphocytes decreases with advancing stage. Moreover, monocytic CD40 expression increases postoperatively, suggesting this pathway is actively suppressed during HNSCC immune evasion. Agonist CD40 mAbs have been developed to substitute for the critical role of the T helper cell in licensing DC. A point of theoretical controversy is that HNSCC cell lines and human tumors express CD40, where ligation promotes proliferation and angiogenesis in preclinical models.[77, 78] Nonetheless, in the first clinical trial evaluating CD40 agonism with recombinant human CD40L, a patient with refractory HNSCC sustained a long-term complete response. CP-870,893, a humanized IgG2 mAb with CD40 agonist activity, demonstrated efficacy in melanoma during phase 1 evaluation. CP-870,893 may be more active when added to treatments that release TA, such as cytotoxic chemotherapy or radiation. Phase 1 combinations with carboplatin-paclitaxel or gemcitabine showed immunologic and clinical responses.[81, 82]
Tumor-infiltrating lymphocytes (TILs) in HNSCC display an anergic phenotype associated with low expression of the costimulatory TNFRs, OX40 and CD137. OX40 signaling supports survival, clonal expansion, inflammatory cytokine production, and memory function in T helper cells; conversely, suppression by Tregs is relieved.[83, 84] Although not explicitly evaluated in HNSCC preclinical models, OX40 agonism has arrested both immunogenic and nonimmunogenic solid tumors; however as with CD40, more robust responses were seen upon combination with cytolytic treatments. An OX40 agonist mAb is under phase 1 study in combination with radiation in breast and prostate cancers (NCT01642290; NCT01303705). CD137 signaling enhances proliferation, inflammatory cytokine production, cytolytic capacity, and survival of CTLs; similar to OX40, ligation of CD137 on Tregs can paradoxically release immunosuppression. CD137 on NK cells also costimulates ADCC. A significant proportion of HNSCC patients receiving cetuximab demonstrate upregulation of CD137 on NK cells, which correlating with the VV and VF FcγR IIIa polymorphisms. In HNSCC preclinical models, cetuximab induces CD137 upregulation on NK cells; sequential treatment with a CD137 mAb enhances cytolytic activity. Urelumab is a humanized IgG4 agonist mAb against CD137 which has been evaluated in dose-finding trials in melanoma, NSCLC, and lymphoma. Because serious hepatotoxicity limited dose escalation, lower-dose phase 1 studies have resumed, including a combination trial with rituximab (NCT01471210; NCT01775631). Similarly, PF-05082566 is an IgG2 CD137 agonist mAb under evaluation in combination with rituximab (NCT01307267). In HNSCC, combinatorial trials with cetuximab, chemotherapy, or radiation are warranted.
Immune Checkpoint-Targeted mAbs
The therapeutic complement to agonism of costimulatory TNFRs is blockade of coinhibitory receptors. HNSCC TILs are characterized by high expression of the coinhibitory receptors cytotoxic T lymphocyte–associated antigen 4 (CTLA-4) and programmed death 1 (PD-1), which are so-called “immune checkpoints.”[10, 90] CTLA-4 is a surface glycoprotein progressively expressed by CTLs following TCR ligation. CTLA-4 down-modulates immune response to chronic antigen stimulation, preventing autoimmunity; however, in the case of cancer, CTLA-4 promotes inappropriate tolerance and immune escape. Tregs in the HNSCC microenvironment constitutively express CTLA-4, which competes with the major costimulatory receptor CD28 for the B7 ligands, CD80, and CD86 (Fig. 1). Thus, mAb blockade of CTLA-4 frees CD28 to bind B7 and propagate the necessary TCR costimulatory signal. Inhibition of Treg CTLA-4 signaling also releases CTL suppression, potentiating TA-targeted cytolysis. This novel therapeutic paradigm culminated in the first FDA approval of an immune checkpoint inhibitor for melanoma, an IgG4 anti–CTLA-4 mAb, ipilimumab. Ipilimumab has intriguing potential to reverse immunosuppression in HNSCC, alone or in combination with other immunogenic therapies. Of particular interest to trial design, CTLA-4 inhibition potentiates the abscopal effect when combined with fractionated radiotherapy: in breast and colon cancer radiation experiments, a preclinical CTLA-4 mAb inhibited out-of-field secondary tumor growth. A phase 1 trial of cetuximab, ipilimumab, and intensity-modulated radiotherapy in the management of locally advanced HNSCC is now accruing (NCT01860430).
Similar to CTLA-4, PD-1 is an inhibitory member of the B7-CD28 family of coreceptors. Following TCR activation, PD-1 is expressed by multiple immune cells including CTLs, NK cells, B lymphocytes, monocytes, and DCs. The PD-1 ligand, PD-L1, is expressed broadly on nonhematopoietic tissue in response to IFN-γ; thus, PD-1 ligation is thought to broadly protect against autoimmunity and immune destruction of normal tissue during infection. PD-L1 is also expressed in the majority of HNSCC, and is explicitly linked to the immune-privileged, invasive front of HPV-transformed HNSCC.[96-98] Infiltration by PD-1–expressing T cells is associated with favorable prognosis in HPV-associated disease; this paradox highlights the importance of prior immune response and the therapeutic potential of restoring it by PD-1 blockade.
Therapeutic targeting of PD-1 or PD-L1 block the coinhibitory signal at the immune synapse. Nivolumab, a humanized IgG4 anti–PD-1 mAb, was investigated in advanced solid tumors. Objective response rates in this heavily treated population were notable in renal cell carcinoma (27%), melanoma (28%), and NSCLC (18%). An important preliminary observation correlated objective response with PD-L1–positive tumors. BMS-936559, a humanized IgG4 mAb which inhibits binding of PD-L1 to both PD-1 and CD80, also resulted in objective responses in a phase 1 study for patients with pretreated, advanced solid tumors, including NSCLC (10%). Anti–PD-1 and anti–PD-L1 mAbs are of pressing interest for therapeutic development in HPV-positive and HPV-negative HNSCC.
Successful development of novel immunotherapeutic mAbs in the treatment of HNSCC can be guided by recent insights into the immune mechanisms of cetuximab, the first FDA-approved immunotherapy in HNSCC, as well as dissection of immune evasion by HNSCC. In addition to blocking EGFR signaling, cetuximab induces ADCC, DC maturation, and cross-priming of EGFR-specific CTLs, all of which are immune mechanisms that could be augmented by cotreatment with other TA-targeted mAbs, exogenous IL-12 or TLR stimulation, mAbs to neutralize immunosuppressive cytokines, mAbs to enhance antigen presentation, or mAbs favorably influencing costimulatory versus coinhibitory receptor signaling. However, the strategic vision for novel therapeutic mAbs should not be limited to coadministration with cetuximab. HNSCC exploits numerous redundant and mutually reinforcing immune escape mechanisms, including an imbalanced STAT1/STAT3 cytokine profile, downregulation of antigen processing and presentation, underexpression of costimulatory receptors, and overexpression of coinhibitory receptors in TILs. Targeting these nodes of immunosuppression may be of independent therapeutic value, and holds the promise of synergy with standard cytotoxic modalities exposing TAs, including chemotherapy and radiation. Clinical trials evaluating monotherapeutic and combinatorial mAb strategies, including combinations with cytolytic therapy, are of urgent priority.