Supplementary Figures 1-9 from Tumor Necrosis Factor-α and Interleukin-1 Antagonists Alleviate Inflammatory Skin Changes Associated with Epidermal Growth Factor Receptor Antibody Therapy in Mice

Supplementary Figure 1 from Vascular Endothelial Growth Factor Receptor 3 Is Involved in Tumor Angiogenesis and Growth

Supplementary Table 1 from Vascular Endothelial Growth Factor Receptor 3 Is Involved in Tumor Angiogenesis and Growth

Target-based immunocytokine approaches have been reported to be efficacious in the control of tumor growth in preclinical and clinical studies. By targeting cytokines-activated immune effectors to local tumor sites, an antibody-based immunocytokine is able to achieve antitumor immunity in the tumor microenvironment while reducing cytokines-mediated systemic side effects. Recently, immune checkpoint antagonists to PD-1/PD-L1 have shown success in certain clinical settings across multiple cancer types. However, the full potential of the checkpoint inhibitor is limited due to impaired overall antitumor immunity. It is therefore desirable to develop immunotherapeutics with the capacity of simultaneously inhibiting immunosuppressive pathways and stimulating immune effector cells to potentiate innate and adaptive immune responses against tumor growth. To this end, we generated a bifunctional fusion protein, KD033, composed of an antibody specific for PD-L1 and IL-15 as a novel immunocytokine for achieving better immunotherapeutic efficacy against tumors. Previously, we demonstrated that KD033 has an enhanced immunological activity and stronger antitumor efficacy in some syngeneic mouse tumor models in comparison to single agents. In the present report, we show that the mechanisms of actions of the bifunctional protein in the enhancement of antitumor immune responses results from an increase in Th1 cytokine secretion, the expansion and cytotoxicity of CD8 T-cells and NK cells and a decrease in immunosuppressive cells, i.e. regulatory T cells and myeloid derived suppressive cells in a number of preclinical experimental models. In the preclinical studies, KD033 regimens, with the unique immunological properties, led to stronger anti-tumor efficacy in controlling primary tumor growth and prolonging the survival of tumor bearing mice in a number of mouse tumor models including PDX and GEMM tumor models. Importantly, the PD-L1-targeted IL-15 bifunctional protein had significantly less cytokine-related toxicity when compared to non-targeted full IgG antibody-IL-15 fusion protein in vivo. These results further elucidate the capacity of targeting IL-15-stimulated innate and adaptive immune effector cells into tumor microenvironment, thereby effectively controlling tumor progression while having minimized adverse effect in vivo. These encouraging preclinical results of the novel immunotherapeutics suggest further advancement of this innovative therapeutic candidate towards clinical development for cancer treatment. Citation Format: Yan Wu, Zhaojing Zhong, Stella Martomo, Dan Lu, Haifan Zhang, Zhanna Polonskaya, Xenia Luna, Zhikai Zhang, Zhun Wang, Leo Liu, Jeegar Patel, James Tonra, Henry Li, Larry Witte, Sam Waksal, Zhenping Zhu. Novel anti-PD-L1/IL-15 bifunctional immunotherapeutics potentiates antitumor immunity. [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 4997.

Abstract The importance of VEGF/VEGFR2 signaling in cancer growth and metastasis has been clearly highlighted by the therapeutic benefits of bevacizumab (Avastin), a humanized antibody to VEGF, and Cyramza, a fully human antibody to VEGFR2, in multiple cancer treatment modalities. Recently, immune checkpoint antagonistic antibodies to PD1 and PDL1 have shown some success in a variety of clinical settings across multiple cancer types. The overall clinical efficacy of these individual antibody therapies has been, however, rather limited and, only evident in a fraction of patients. To this end, combinations of antibodies against both VEGF/VEGFR2 and PD1/PDL1 may represent promising approaches to further enhance the antitumor efficacy of individual antibody therapies. In this study, we engineered a bispecific anti-PDL1 x anti-VEGFR2 antibody using two fully human antibodies derived from antibody phage display libraries. In this bispecific format, a high affinity single chain antibody to PDL1 was genetically fused to the C-terminus of the heavy chain of a conventional IgG antibody against VEGFR2. The bispecific antibody was efficiently expressed in mammalian cells and could be purified to homogeneity via single step affinity chromatography. The bispecific antibody retained the binding activity of its parental antibodies to PDL1 and VEGFR2, and strongly blocked both VEGF/VEGFR2 and PDL1/PD1 interaction. Further, the bispecific antibody inhibited VEGF-stimulated VEGFR2 phosphorylation and proliferation of endothelial cells, and promoted proliferation of human T cells and secretion of cytokine such as IL2 and INFγ. The bispecific antibody is currently being evaluated in vivo in relevant animal models. Citation Format: Dan Lu, Zhanna Polonskaya, Haifan Zhang, Xenia Luna, Stella Martomo, Zhikai Zhang, Zhaojing Zhong, Yan Wu, Jeegar Patel, James Tonra, Larry Witte, Sam Waksal, Zhenping Zhu. A novel anti-PDL1 x anti-VEGFR2 bispecific antibody for enhanced antitumor immunity. [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 572.

Abstract KD019 is an orally bioavailable small molecule inhibitor of molecular drivers of cancer growth and progression, including HER2/neu, Src family nonreceptor tyrosine kinases (Src) and EGFR. This profile of activity underlies the robust activity of KD019 in animal models of cancer. Although alternative agents targeting these tyrosine kinase receptors are available, only KD019 targets all of these pathways without the need for a combination strategy. A more important shortcoming of alternative agents is a lack of blood brain barrier (BBB) penetration (Brain/Plasma concentration ratio<<1), significantly limiting their ability to target oncogenic molecular pathways in intracranial tumors. The BBB permeability of KD019 was heretofore untested. In the present work, KD019 is demonstrated to achieve concentrations in the brain equivalent to that in blood utilizing quantitative whole body autoradiography (QWBA). Lister Hooded partially pigmented rats were administered a single dose of 14C labeled KD019 and tissue radioactivity was evaluated utilizing phosphor-storage imaging plates and a Fuji FLA-5100 fluorescent image analyzing system (Quotient Bioresearch, Rushden, UK). Brain/blood radioactivity ratio was approximately 1 at 6-24 hours after dosing indicating significant brain penetration of KD019. BBB penetrance was further supported by a study performed in CD1 mice utilizing LC/MS/MS to determine tissue KD019 concentrations following a single oral dose of 100 mg free base/kg unlabeled drug (Pharmaron, Beijing, China). Brain/plasma KD019 ratios were 2.3-4.4 from 1-24 hours after dosing with brain concentrations of 2137-8253 ng/g tissue, compared to 0.03-0.07 for the EGFR/HER2 inhibitor lapatinib with brain concentrations of 0-286 ng/g tissue. To demonstrate that the penetration of KD019 into the brain parenchyma would translate into anticancer effects, KD019 efficacy was tested in a GL261 orthotopic syngeneic model of glioma (Molecular Imaging, Michigan, USA). GL261-luc2 luciferase expressing cells were implanted intracranially (2 mm right lateral and 1 mm anterior from Bregma, 2-3 mm down from burr hole) into C57BL/6 albino mice. KD019 dosed orally once daily at 75 mg base/kg on Days 8-12 and 15-19 after intracranial implantation significantly reduced the intracranial tumor growth as evaluated by bioluminescence, and extended median survival time by 20%. In conclusion, KD019 is a BBB penetrant TKI with an important and novel profile of kinase activity that, based on the data presented, will be advanced further in nonclinical and clinical experiments aiming at benefiting patients with intracranial tumors, a significant unmet clinical need. KD019 is currently being tested in combination with trastuzumab in patients with HER2-positive breast cancer and brain metastases. Citation Format: James R. Tonra, Masha Poyurovsky, Kevin G. Liu, Jeegar Patel, Nishta Rao, Robert Tilton, John L. Ryan, Mark S. Berger, Larry Witte, Ji-In Kim, Samuel D. Waksal. KD019: Blood brain barrier penetrant HER2/neu, Src, and EGFR inhibitor. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 2590. doi:10.1158/1538-7445.AM2015-2590

Immune checkpoint antagonists to PD-1/PD-L1 and immunostimulating cytokines such as IL-15 have shown success to some extent in certain clinical settings across multiple cancer types. However, the full potential of the checkpoint inhibitor is limited due to impaired overall antitumor immunity, and cytokine as single agent has insufficient half-life and systemic toxicities due to the lack of target specificity. To overcome these challenging hurdles, we developed a bifunctional fusion protein, KD-033, composed of an antibody specific for PD-L1 and complex of IL-15Rα sushi domain/IL-15 as a novel immunotherapeutic agent for achieving better antitumor efficacy. Previously, we presented the generation and characteristics of a prototype of bifunctional fusion protein and its potential of in vivo antitumor activity. Here, we report a genetically modified fusion protein that has enhanced immunological activity and capability to achieve stronger antitumor efficacy in tumor models in comparison with either single agent. Our data indicate that the improved bifunctional fusion protein has favorable thermal stability and can be efficiently expressed in mammalian cells. The bifunctional fusion protein has higher affinity to PD-L1, silenced binding activity to Fc receptors and better ability to increase the secretion of Th1 cytokine, i.e. gamma IFN and the cytotoxicity of CD8 T-cells and NK cells to tumor cells as assessed in immunological assays. In preclinical study, KD033 had stronger anti-tumor efficacy in controlling primary tumor growth and prolonging the survival of tumor bearing mice in a number of mouse tumor models including those aggressive tumor models. Furthermore, the PD-L1 targeted bifunctional protein had significantly less cytokine-related toxicity when compared to non-targeted full IgG/IL-15Rα sushi domain/IL-15 fusion protein in vivo. These results demonstrate that KD033 has the capacity of targeting IL-15-stimulated innate and adaptive immune effectors into local tumor sites, thereby effectively controlling tumor progression while having minimized potential adverse effect in vivo. The preclinical studies of the novel immunotherapeutics warrant further investigation towards the clinical development of the bifunctional immunotherapeutic agent for cancer treatment. Citation Format: Yan Wu, Zhaojing Zhong, Stella Martomo, Dan Lu, Zhanna Polonskaya, Xenia Luna, Haifan Zhang, Zhikai Zhang, Zhun Wang, Leo Liu, Jeegar Patel, James Tonra, Henry Li, Larry Witte, Sam Waksal, Zhenping Zhu. Anti-PD-L1 antibody-based IL-15 immunocytokine has enhanced antitumor immunity. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2015 Nov 5-9; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2015;14(12 Suppl 2):Abstract nr C173.

Zfra is a 31-amino-acid zinc finger-like protein, which participates in tumor necrosis factor signaling. Here, we determined that synthetic full-length Zfra1−31 peptide selfpolymerized in degassed buffers without catalytic enzymes. When nude mice and BALB/c mice were pre-injected with micromolar levels of Zfra1−31 or truncated Zfra4−10 via tail veins, these mice became resistant to the growth, metastasis and stemness of prostate, breast, and lung cancer cells, melanoma cells, and many malignant cancer cells. Alteration of the Ser8 phosphorylation site to Gly8 abolished Zfra polymerization and cancer suppression in vivo. Injected Zfra was deposited mainly in the spleen. Transfer of Zfra-stimulated spleen cells to naive mice conferred resistance to cancer growth. Mechanistically, Zfra bound membrane hyaluronidase Hyal-2 and suppressed the TGF-b/Hyal-2/WWOX/Smad4 signaling, via down-regulation of Hyal-2 and activated WWOX (with Y33 phosphorylation), in the spleen for generating novel non-T/non-B memory cells, designated Hyal2+ CD3−CD19− cells. Transfer of these cells to naive mice also induced anticancer response. Similarly, agonist anti-Hyal-2 antibody mimicked the effect of Zfra in causing cancer suppression. In conclusion, Zfra self-polymerizes in the spleen to activate Hyal2+ CD3− CD19− cells for blocking cancer growth, stemness and metastasis in vivo. Supported in part by NSC and NHRI, Taiwan, and DoD, USA

The benefits of inhibiting vascular endothelial growth factor (VEGF) signaling in cancer patients are predominantly attributed to effects on tumor endothelial cells. Targeting non-endothelial stromal cells to further impact tumor cell growth and survival is being pursued through the inhibition of additional growth factor pathways important for the survival and/or proliferation of these cells. However, recent data suggest that VEGF receptor (VEGFR)-specific inhibitors may target lymphatic vessels and pericytes in addition to blood vessels. Here, in fact, we demonstrate that DC101 (40 mg/kg, thrice a week), an antibody specific to murine VEGFR2, significantly reduces all three of these stromal components in subcutaneous (SKRC-29) and orthotopic (786-O-LP) models of renal cell carcinoma (RCC) established in nu/nu athymic mice. Sunitinib (40 mg/kg, once daily), a receptor tyrosine kinase inhibitor of VEGFR2 and other growth factor receptors, also caused significant loss of tumor blood vessels in RCC models but had weaker effects than DC101 on pericytes and lymphatic vessels. In combination, sunitinib did not significantly add to the effects of DC101 on tumor blood vessels, lymphatic vessels, or pericytes. Nevertheless, sunitinib increased the effect of DC101 on tumor burden in the SKRC-29 model, perhaps related to its broader specificity. Our data have important implications for combination therapy design, supporting the conclusion that targeting VEGFR2 alone in RCC has the potential to have pleiotropic effects on tumor stroma.

BACKGROUND:Clinically relevant targets for developmental drug efficacy in animal models of cancer are critical yet understudied parameters.MATERIALS AND METHODS:Cetuximab, a chimeric antibody to epidermal growth factor receptor (EGFR), was administered to athymic mice bearing subcutaneous tumors established with 13 human colorectal cancer cell lines of varying biomarker status, defined by DNA sequencing and RT-PCR.RESULTS:If tumor growth inhibition is taken as a target, as is commonly done, then in contrast to the clinical situation where KRAS mutation strongly predicts for a lack of clinically meaningful benefit in colorectal cancer patients, cetuximab alone and in combination with irinotecan-based chemotherapy were efficacious in a similar proportion of KRAS wild-type and mutant models. It was only when tumor regression was utilized to define relevant efficacy that cetuximab monotherapy was efficacious in KRAS wild-type, but not mutant models. Adding cytotoxic therapy to cetuximab treatment increased tumor regression frequency in both genotypes to the point that once again the response was similar for KRAS wild-type and mutant models.CONCLUSION:Our data support shifting the threshold for claiming clinically relevant targeted therapy efficacy in subcutaneous xenograft models towards tumor regression, rather than tumor growth inhibition, focusing on the evaluation of tumor cells that are addicted to the pathways being targeted.

Bone marrow endothelial cells (ECs) are essential for reconstitution of hematopoiesis, but their role in self-renewal of long-term hematopoietic stem cells (LT-HSCs) is unknown. We have developed angiogenic models to demonstrate that EC-derived angiocrine growth factors support in vitro self-renewal and in vivo repopulation of authentic LT-HSCs. In serum/cytokine-free cocultures, ECs, through direct cellular contact, stimulated incremental expansion of repopulating CD34(-)F1t3(-)cKit(+)Lineage(-)Sca1(+) LT-HSCs, which retained their self-renewal ability, as determined by single-cell and serial transplantation assays. Angiocrine expression of Notch ligands by ECs promoted proliferation and prevented exhaustion of LT-HSCs derived from wild-type, but not Notch1/Notch2-deficient, mice. In transgenic notch-reporter (TNR.Gfp) mice, regenerating TNR.Gfp(+) LT-HSCs were detected in cellular contact with sinusoidal ECs. Interference with angiocrine, but not perfusion, function of SECs impaired repopulation of TNR.Gfp(+) LT-HSCs. ECs establish an instructive vascular niche for clinical-scale expansion of LT-HSCs and a cellular platform to identify stem cellactive trophogens.

Abstract Purpose: Transforming growth factor β (TGFβ) is a pleiotropic cytokine that affects tumor growth, metastasis, stroma, and immune response. We investigated the therapeutic efficacy of anti–TGFβ receptor II (TGFβ RII) antibody in controlling metastasis and tumor growth as well as enhancing antitumor immunity in preclinical tumor models. Experimental Design: We generated neutralizing antibodies to TGFβ RII and assessed the antibody effects on cancer, stroma, and immune cells in vitro. The efficacy and mechanism of action of the antibody as monotherapy and in combination with chemotherapy in suppression of primary tumor growth and metastasis were evaluated in several tumor models. Results: Anti–TGFβ RII antibody blocked TGFβ RII binding to TGFβ 1, 2, and 3, and attenuated the TGFβ-mediated activation of downstream Smad2 kinase, invasion of cancer cells, motility of endothelial and fibroblast cells, and induction of immunosuppressive cells. Treatment with the antibody significantly suppressed primary tumor growth and metastasis and enhanced natural killer and CTL activity in tumor-bearing mice. Immunohistochemistry analysis showed cancer cell apoptosis and massive necrosis, and increased tumor-infiltrating T effector cells and decreased tumor-infiltrating Gr-1+ myeloid cells in the antibody-treated tumors. Fluorescence-activated cell sorting analysis indicated the significant reduction of peripheral Gr-1+/CD11b+ myeloid cells in treated animals. Concomitant treatment with the cytotoxic agent cyclophosphamide resulted in a significantly increased antitumor efficacy against primary tumor growth and metastasis. Conclusions: These preclinical data provide a foundation to support using anti–TGFβ RII antibody as a therapeutic agent for TGFβ RII–dependent cancer with metastatic capacity. Clin Cancer Res; 16(4); 1191–205

The formation of new blood vessels (angiogenesis) represents a critical factor in the malignant growth of solid tumors and metastases. Vascular endothelial cell growth factor (VEGF) and its receptor VEGFR2 represent central molecular targets for antiangiogenic intervention, because of their integral involvement in endothelial cell proliferation and migration. In the current study, we investigated in vitro and in vivo effects of receptor blockade on various aspects of the angiogenic process using monoclonal antibodies against VEGFR2 (cp1C11, which is human specific, and DC101, which is mouse specific). Molecular blockade of VEGFR2 inhibited several critical steps involved in angiogenesis. VEGFR2 blockade in endothelial cells attenuated cellular proliferation, reduced cellular migration, and disrupted cellular differentiation and resultant formation of capillary-like networks. Further, VEGFR2 blockade significantly reduced the growth response of human squamous cell carcinoma xenografts in athymic mice. The growth-inhibitory effect of VEGFR2 blockade in tumor xenografts seems to reflect antiangiogenic influence as demonstrated by vascular growth inhibition in an in vivo angiogenesis assay incorporating tumor-bearing Matrigel plugs. Further, administration of VEGFR2-blocking antibodies in endothelial cell cultures, and in mouse xenograft models, increased their response to ionizing radiation, indicating an interactive cytotoxic effect of VEGFR2 blockade with radiation. These data suggest that molecular inhibition of VEGFR2 alone, and in combination with radiation, can enhance tumor response through molecular targeting of tumor vasculature.

Platelet-derived growth factor receptor beta (PDGFR beta) is upregulated in most of solid tumors. It is expressed by pericytes/smooth muscle cells, fibroblast, macrophage, and certain tumor cells. Several PDGF receptor-related antagonists are being developed as potential antitumor agents and have demonstrated promising antitumor activity in both preclinical and clinical settings. Here, we produced a fully human neutralizing antibody, IMC-2C5, directed against PDGFR beta from an antibody phage display library. IMC-2C5 binds to both human and mouse PDGFR beta and blocks PDGF-B from binding to the receptor. IMC-2C5 also blocks ligand-stimulated activation of PDGFR beta and downstream signaling molecules in tumor cells. In animal studies, IMC-2C5 significantly delayed the growth of OVCAR-8 and NCI-H460 human tumor xenografts in nude mice but failed to show antitumor activities in OVCAR-5 and Caki-1 xenografts. Our results indicate that the antitumor efficacy of IMC-2C5 is primarily due to its effects on tumor stroma, rather than on tumor cells directly. Combination of IMC-2C5 and DC101, an anti-mouse vascular endothelial growth factor receptor 2 antibody, resulted in significantly enhanced antitumor activity in BxPC-3, NCI-H460, and HCT-116 xenografts, compared with DC101 alone, and the trend of additive effects to DC101 treatment in several other tumor models. ELISA analysis of NCI-H460 tumor homogenates showed that IMC-2C5 attenuated protein level of vascular endothelial growth factor and basic fibroblast growth factor elevated by DC101 treatment. Finally, IMC-2C5 showed a trend of additive effects when combined with DC101/chemotherapy in MIA-PaCa-2 and NCI-H460 models. Taken together, these results lend great support to the use of PDGFR beta antagonists in combination with other antiangiogenic agents in the treatment of a broad range of human cancers.

Transforming growth factor beta (TGF \#946;) is a pleiotropic cytokine that profoundly regulates the pathogenesis of cancer and metastasis. TGF \#946; stimulates activation of TGF \#946; receptor I kinase and downstream signaling cascades that initiate broad cellular and non cellular processes i.e. survival, proliferation and differentiation, migration and motility, and deposition of the extracellular matrix and induction of cytokines contributing to tumorigenesis, metastasis, angiogenesis, and inflammation during tumor progression. Myeloid cells have been reported to be an important mediator in promoting metastasis and angiogenesis, suppressing immunity against tumor and inducing anti-VEGF resistant tumors. Therefore, targeting this multifunctional cell population will be beneficial for enhancement of antitumor therapeutic efficacy. The binding of TGF \#946; ligands to TGF \#946; receptor II (TGF \#946; RII) is one crucial step to initiate the activation of TGF \#946; signaling pathways. Here, we report that we have developed high affinity monoclonal antibodies with specific blocking activity to TGF \#946; R II binding to TGF \#946; ligands. By using the antibodies, we investigated the mechanism of actions of TGF \#946; mediated activity in promoting cancer cell invasiveness, angiogenesis and the role of Gr-1/CD11b+ myeloid cells in tumor progression and evaluated the therapeutic efficacy of anti-TGF \#946; R II antibodies in suppression of tumor growth and metastasis. Data on cell-based assays displayed the capacity of anti-TGF \#946; R II antibodies in inhibiting TGF \#946; receptor mediated signaling cascades and cellular biological functions in vitro. In vivo studies demonstrated that treatment of tumor bearing mice with the anti-TGF \#946; R II antibodies resulted in suppression of primary tumor growth and metastasis in conjunction with the blockade of cancer cell invasion and VEGF-A secretion, the inhibition of myeloid cell function and the depletion of Gr-1/CD11b/TGF \#946; R II+ myeloid cells. Most importantly, we have for first time revealed that a subset of TGF \#946; R II+ myeloid cells has a major role in promoting tumor growth and metastasis through inducing tumor cell migration and inhibiting cellular immune response. Thus, this study has provided a foundation supporting that anti-TGF \#946; R II antibody mediated abrogation of TGF \#946; R II+ myeloid cells would be an effective approach to controlling the deleterious activity of myeloid cell in cancer, and anti-TGF \#946; RII antibody-based therapeutics may represent a novel strategy for treatment of cancer, particularly for metastatic tumors. Citation Information: In: Proc Am Assoc Cancer Res; 2009 Apr 18-22; Denver, CO. Philadelphia (PA): AACR; 2009. Abstract nr 844.

Abstract Cancer patients receiving epidermal growth factor receptor (EGFR) antibody therapy often experience an acneiform rash of uncertain etiology in skin regions rich in pilosebaceous units. Currently, this condition is treated symptomatically with very limited, often anecdotal success. Here, we show that a monoclonal antibody targeting murine EGFR, ME1, caused a neutrophil-rich hair follicle inflammation in mice, similar to that reported in patients. This effect was preceded by the appearance of lipid-filled hair follicle distensions adjacent to enlarged sebaceous glands. The cytokine tumor necrosis factor-α (TNFα), localized immunohistochemically to this affected region of the pilosebaceous unit, was specifically up-regulated by ME1 in skin but not in other tissues examined. Moreover, skin inflammation was reduced by cotreatment with the TNFα signaling inhibitor, etanercept, indicating the involvement of TNFα in this inflammatory process. Interleukin-1, a cytokine that frequently acts in concert with TNFα, is also involved in this process given the efficacy of the interleukin-1 antagonist Kineret. Our results provide a mechanistic framework to develop evidence-based trials for EGFR antibody–induced skin rash in patients with cancer. [Cancer Res 2009;69(14):5643–7]