Mischa Houtkamp

Dual Mode of Action of a Human Anti-Epidermal Growth Factor Receptor Monoclonal Antibody for Cancer Therapy

Epidermal growth factor receptor (EGF-R) overexpression is common in a large number of solid tumors and represents a negative prognostic indicator. Overexpression of EGF-R is strongly tumor associated, and this tyrosine kinase type receptor is considered an attractive target for Ab therapy. In this study, we describe the evaluation of mAb 2F8, a high avidity human mAb (IgG1κ) directed against EGF-R, developed using human Ig transgenic mice. mAb 2F8 effectively blocked binding of EGF and TGF-α to the EGF-R. At saturating concentrations, 2F8 completely blocked EGF-R signaling and inhibited the in vitro proliferation of EGF-R-overexpressing A431 cells. At much lower concentrations, associated with low receptor occupancy, 2F8 induced efficient Ab-dependent cell-mediated cytotoxicity (ADCC) in vitro. In vivo studies showed potent antitumor effects in models with A431 tumor xenografts in athymic mice. Ex vivo analysis of the EGF-R status in tumor xenografts in 2F8-treated mice revealed that there are two therapeutic mechanisms. First, blocking of EGF-R signaling, which is most effective at complete receptor saturation and therefore requires a relatively high Ab dose. Second, at very low 2F8 receptor occupancy, we observed potent antitumor effects in mice, which are likely based on the engagement of immune effector mechanisms, in particular ADCC. Taken together, our findings indicate that ADCC represents an important effector mechanism of this Ab, which is effective at relatively low dose.

Effect of Target Dynamics on Pharmacokinetics of a Novel Therapeutic Antibody against the Epidermal Growth Factor Receptor: Implications for the Mechanisms of Action

The epidermal growth factor receptor (EGFR) is overexpressed on many solid tumors and represents an attractive target for antibody therapy. Here, we describe the effect of receptor-mediated antibody internalization on the pharmacokinetics and dose-effect relationship of a therapeutic monoclonal antibody (mAb) against EGFR (2F8). This mAb was previously found therapeutically active in mouse tumor models by two dose-dependent mechanisms of action: blockade of ligand binding and induction of antibody-dependent cell-mediated cytotoxicity. In vitro studies showed 2F8 to be rapidly internalized by EGFR-overexpressing cells. In vivo, accelerated 2F8 clearance was observed in cynomolgus monkeys at low doses but not at high doses. This enhanced clearance seemed to be receptor dependent and was included in a pharmacokinetic model designed to explain its nonlinearity. Receptor-mediated clearance was also found to affect in situ antibody concentrations in tumor tissue. Ex vivo analyses of xenograft tumors of 2F8-treated nude mice revealed that relatively high antibody plasma concentrations were required for maximum EGFR saturation in high-EGFR-expressing human A431 tumors, in contrast to lower-EGFR-expressing human xenograft tumors. In summary, receptor-mediated antibody internalization and degradation provides a saturable route of clearance that significantly affects pharmacokinetics, particularly at low antibody doses. EGFR saturation in normal tissues does not predict saturation in tumor tissue as local antibody concentrations in EGFR-overexpressing tumors may be more rapidly reduced by antibody internalization. Consequently, antibody saturation of the receptor may be affected, thereby affecting the local mechanism of action.

An Antibody–Drug Conjugate That Targets Tissue Factor Exhibits Potent Therapeutic Activity against a Broad Range of Solid Tumors

Tissue factor (TF) is aberrantly expressed in solid cancers and is thought to contribute to disease progression through its procoagulant activity and its capacity to induce intracellular signaling in complex with factor VIIa (FVIIa). To explore the possibility of using tissue factor as a target for an antibody-drug conjugate (ADC), a panel of human tissue factor-specific antibodies (TF HuMab) was generated. Three tissue factor HuMab, that induced efficient inhibition of TF:FVIIa-dependent intracellular signaling, antibody-dependent cell-mediated cytotoxicity, and rapid target internalization, but had minimal impact on tissue factor procoagulant activity in vitro, were conjugated with the cytotoxic agents monomethyl auristatin E (MMAE) or monomethyl auristatin F (MMAF). Tissue factor-specific ADCs showed potent cytotoxicity in vitro and in vivo, which was dependent on tissue factor expression. TF-011-MMAE (HuMax-TF-ADC) was the most potent ADC, and the dominant mechanism of action in vivo was auristatin-mediated tumor cell killing. Importantly, TF-011-MMAE showed excellent antitumor activity in patient-derived xenograft (PDX) models with variable levels of tissue factor expression, derived from seven different solid cancers. Complete tumor regression was observed in all PDX models, including models that showed tissue factor expression in only 25% to 50% of the tumor cells. In conclusion, TF-011-MMAE is a promising novel antitumor agent with potent activity in xenograft models that represent the heterogeneity of human tumors, including heterogeneous target expression.

Neutralization of IL-8 Prevents the Induction of Dermatologic Adverse Events Associated with the Inhibition of Epidermal Growth Factor Receptor

Epidermal growth factor receptor (EGFR) inhibitors are widely used in the treatment of cancer. EGFR-targeted treatment is known to be associated with a high incidence of dermatological adverse reactions, including papulopustular rash, which can be dose-limiting and may affect compliance to treatment. Currently, the pathways involved in EGFR inhibitor-induced rash are poorly understood and few treatment options for this adverse event are available. Here, we developed a model for induction of papulopustular rash in healthy human volunteers by subcutaneous injection of the anti-EGFR monoclonal antibody zalutumumab. The injection sites and surrounding skin were evaluated by a dermatologist for the presence or absence of papulopustular rash and skin biopsies were taken to confirm the macroscopical findings by immunohistochemistry. Locally injected zalutumumab induced a papulopustular rash, characterized by acute follicular neutrophil-rich hair follicle inflammation, and thus mimicked adverse events induced by systemic administration of EGFR inhibitors. In this model, we tested the hypothesis that neutrophils, attracted by IL-8, play a central role in the observed rash. Indeed, concomitant local repeat dose treatment with HuMab-10F8, a neutralizing human antibody against IL-8, reduced the rash. Inhibition of IL-8 can therefore ameliorate dermatological adverse events induced by treatment with EGFR inhibitors.

Three-Dimensional Histologic Validation of High-Resolution SPECT of Antibody Distributions Within Xenografts

Longitudinal imaging of intratumoral distributions of antibodies in vivo in mouse cancer models is of great importance for developing cancer therapies. In this study, multipinhole SPECT with sub–half-millimeter resolution was tested for exploring intratumoral distributions of radiolabeled antibodies directed toward the epidermal growth factor receptor (EGFr) and compared with full 3-dimensional target expression assessed by immunohistochemistry. Methods: 111In-labeled zalutumumab, a human monoclonal human EGFr-targeting antibody, was administered at a nonsaturating dose to 3 mice with xenografted A431 tumors exhibiting high EGFr expression. Total-body and focused in vivo tumor SPECT was performed at 0 and 48 h after injection and compared both visually and quantitatively with full 3-dimensional immunohistochemical staining for EGFr target expression. Results: SPECT at 48 h after injection showed that activity was predominantly concentrated in the tumor (10.5% ± 1.3% of the total-body activity; average concentration, 30.1% ± 4.6% of the injected dose per cubic centimeter). 111In-labeled EGFr-targeting antibodies were distributed heterogeneously throughout the tumor. Some hot spots were observed near the tumor rim. Immunohistochemistry indicated that the antibody distributions obtained by SPECT were morphologically similar to those obtained for ex vivo EGFr target expression. Regions showing low SPECT activity were necrotic or virtually negative for EGFr target expression. A good correlation (r = 0.86, P < 0.0001) was found between the percentage of regions showing low activity on SPECT and the percentage of necrotic tissue on immunohistochemistry. Conclusion: Multipinhole SPECT enables high-resolution visualization and quantification of the heterogeneity of 111In-zalutumumab concentrations in vivo.

Abstract 1234: An antibody-drug conjugate targeting tissue factor with broad anti-tumor efficacy in xenograft models with heterogeneous tissue factor expression.

Proceedings: AACR 104th Annual Meeting 2013; Apr 6-10, 2013; Washington, DC Tissue factor (TF) is aberrantly expressed in a wide variety of solid tumors, and expression has been associated with poor prognosis. In normal physiology, TF is the main initiator of the coagulation cascade, which starts when circulating factor VII(a) (FVII(a)) binds membrane bound TF. The TF:FVIIa complex proteolytically activates FX to generate FXa, eventually resulting in clot formation. In addition, the TF:FVIIa complex activates PAR-2 intracellular signaling, thereby stimulating the production of pro-angiogenic factors, cytokines and adhesion molecules. We developed an antibody‐drug conjugate (ADC) composed of a human TF-specific IgG1κ antibody (TF-011), a protease‐cleavable valine-citrulline (vc) linker and the microtubule disrupting agent monomethyl auristatin E (MMAE). Unconjugated TF-011 efficiently inhibited TF:FVIIa induced ERK phosphorylation and IL-8 production, but showed only minor inhibition of FXa generation or clot formation. Unconjugated TF-011 efficiently killed TF-positive tumor cells by antibody dependent cell-mediated cytotoxicity (ADCC) in vitro, and showed some anti-tumor efficacy in vivo in a prophylactic setting. Upon target binding, TF-011 was rapidly internalized and co‐localization with LAMP-1 was observed already after 1 hour. This suggests efficient lysosomal targeting, a prerequisite for intracellular release of MMAE and subsequent tumor cell killing by an ADC. Indeed, TF-011-vcMMAE efficiently induced tumor cell killing in vitro, which was dependent on and correlated with TF cell surface expression. In addition, TF-011-vcMMAE demonstrated potent anti-tumor efficacy in xenograft models for pancreatic and epidermoid cancer in vivo. Therapeutic treatment at doses as low as 0,3 mg/kg inhibited tumor growth, whereas tumor regression was observed at doses of 1 mg/kg or higher. Importantly, TF-011-vcMMAE also induced tumor cell killing in human biopsy‐derived xenograft models, which are thought to represent the genetic and histological heterogeneity of human tumors. Immunohistochemical analysis confirmed that the heterogeneity of TF expression in human tumors was reflected in human biopsy‐derived xenografts. TF-011-vcMMAE induced efficient tumor regression in xenograft models for bladder, lung, pancreatic, prostate, ovarian and cervical cancer, with the percentage of TF positive cells ranging from 25-50% to 75-100%. In two tumor models that showed TF expression in less than 25% of tumor cells, TF-011-vcMMAE showed inhibition of tumor growth. In summary, TF-011-vcMMAE is a promising new ADC that showed potent anti-tumor activity in vivo in a wide variety of models, including models that represent the heterogeneous TF expression that is observed in human tumors. The ADC potently kills tumor cells by disrupting microtubules, while preserving the effector functions of the unconjugated antibody. Citation Format: Esther C.W. Breij, David Satijn, Sandra Verploegen, Bart E. de Goeij, Danita H. Schuurhuis, Mischa Houtkamp, Wim K. Bleeker, Paul W. Parren. An antibody-drug conjugate targeting tissue factor with broad anti-tumor efficacy in xenograft models with heterogeneous tissue factor expression. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 1234. doi:10.1158/1538-7445.AM2013-1234