David Satijn

IL-8 as antibody therapeutic target in inflammatory diseases: Reduction of clinical activity in palmoplantar pustulosis

IL-8 is a chemokine that has been implicated in a number of inflammatory diseases involving neutrophil activation. HuMab 10F8 is a novel fully human mAb against IL-8, which binds a discontinuous epitope on IL-8 overlapping the receptor binding site, and which effectively neutralizes IL-8-dependent human neutrophil activation and migration. We investigated whether interference in the cytokine network by HuMab 10F8 might benefit patients suffering from palmoplantar pustulosis, a chronic inflammatory skin disease. Treatment of patients with HuMab 10F8 was well tolerated and significantly reduced clinical disease activity at all five endpoints, which included a ≥50% reduction in the formation of fresh pustules. IL-8 neutralization was monitored at the site of inflammation by assessing exudates of palmoplantar pustulosis lesions. HuMab 10F8 sequestered IL-8 in situ, as observed by rapid dose-dependent decreases of IL-8 concentrations immediately following Ab infusion. These data demonstrate a critical role for IL-8 in the pathophysiology of palmoplantar pustulosis. HuMab 10F8 is capable of interrupting IL-8 activity in vivo and represents a candidate for treatment of inflammatory diseases and other pathological conditions associated with IL-8 overproduction.

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.

High Turnover of Tissue Factor Enables Efficient Intracellular Delivery of Antibody–Drug Conjugates

Antibody–drug conjugates (ADC) are emerging as powerful cancer treatments that combine antibody-mediated tumor targeting with the potent cytotoxic activity of toxins. We recently reported the development of a novel ADC that delivers the cytotoxic payload monomethyl auristatin E (MMAE) to tumor cells expressing tissue factor (TF). By carefully selecting a TF-specific antibody that interferes with TF:FVIIa-dependent intracellular signaling, but not with the procoagulant activity of TF, an ADC was developed (TF-011-MMAE/HuMax-TF-ADC) that efficiently kills tumor cells, with an acceptable toxicology profile. To gain more insight in the efficacy of TF-directed ADC treatment, we compared the internalization characteristics and intracellular routing of TF with the EGFR and HER2. Both in absence and presence of antibody, TF demonstrated more efficient internalization, lysosomal targeting, and degradation than EGFR and HER2. By conjugating TF, EGFR, and HER2-specific antibodies with duostatin-3, a toxin that induces potent cytotoxicity upon antibody-mediated internalization but lacks the ability to induce bystander killing, we were able to compare cytotoxicity of ADCs with different tumor specificities. TF-ADC demonstrated effective killing against tumor cell lines with variable levels of target expression. In xenograft models, TF-ADC was relatively potent in reducing tumor growth compared with EGFR- and HER2-ADCs. We hypothesize that the constant turnover of TF on tumor cells makes this protein specifically suitable for an ADC approach. Mol Cancer Ther; 14(5); 1130–40. ©2015 AACR.

Efficient payload delivery by a bispecific antibody-drug conjugate targeting HER2 and CD63

Antibody–drug conjugates (ADC) are designed to be stable in circulation and to release potent cytotoxic drugs intracellularly following antigen-specific binding, uptake, and degradation in tumor cells. Efficient internalization and routing to lysosomes where proteolysis can take place is therefore essential. For many cell surface proteins and carbohydrate structures on tumor cells, however, the magnitude of these processes is insufficient to allow for an effective ADC approach. We hypothesized that we could overcome this limitation by enhancing lysosomal ADC delivery via a bispecific antibody (bsAb) approach, in which one binding domain would provide tumor specificity, whereas the other binding domain would facilitate targeting to the lysosomal compartment. We therefore designed a bsAb in which one binding arm specifically targeted CD63, a protein that is described to shuttle between the plasma membrane and intracellular compartments, and combined it in a bsAb with a HER2 binding arm, which was selected as model antigen for tumor-specific binding. The resulting bsHER2xCD63his demonstrated strong binding, internalization and lysosomal accumulation in HER2-positive tumor cells, and minimal internalization into HER2-negative cells. By conjugating bsHER2xCD63his to the microtubule-disrupting agent duostatin-3, we were able to demonstrate potent cytotoxicity of bsHER2xCD63his-ADC against HER2-positive tumors, which was not observed with monovalent HER2- and CD63-specific ADCs. Our data demonstrate, for the first time, that intracellular trafficking of ADCs can be improved using a bsAb approach that targets the lysosomal membrane protein CD63 and provide a rationale for the development of novel bsADCs that combine tumor-specific targeting with targeting of rapidly internalizing antigens. Mol Cancer Ther; 15(11); 2688–97. ©2016 AACR.

Cooperative targeting of melanoma heterogeneity with an AXL antibody-drug conjugate and BRAF/MEK inhibitors

Intratumor heterogeneity is a key factor contributing to therapeutic failure and, hence, cancer lethality. Heterogeneous tumors show partial therapy responses, allowing for the emergence of drug-resistant clones that often express high levels of the receptor tyrosine kinase AXL. In melanoma, AXL-high cells are resistant to MAPK pathway inhibitors, whereas AXL-low cells are sensitive to these inhibitors, rationalizing a differential therapeutic approach. We developed an antibody-drug conjugate, AXL-107-MMAE, comprising a human AXL antibody linked to the microtubule-disrupting agent monomethyl auristatin E. We found that AXL-107-MMAE, as a single agent, displayed potent in vivo anti-tumor activity in patient-derived xenografts, including melanoma, lung, pancreas and cervical cancer. By eliminating distinct populations in heterogeneous melanoma cell pools, AXL-107-MMAE and MAPK pathway inhibitors cooperatively inhibited tumor growth. Furthermore, by inducing AXL transcription, BRAF/MEK inhibitors potentiated the efficacy of AXL-107-MMAE. These findings provide proof of concept for the premise that rationalized combinatorial targeting of distinct populations in heterogeneous tumors may improve therapeutic effect, and merit clinical validation of AXL-107-MMAE in both treatment-naive and drug-resistant cancers in mono- or combination therapy.

Abstract 4591: Specific elimination of invasive and multidrug-resistant cancer cells by an antibody-drug conjugate targeting AXL

Upon therapeutic pressure, cancers commonly select for drug-resistant, invasive subpopulations with elevated expression of the receptor tyrosine kinase AXL. Besides the correlation between high AXL expression and induction of epithelial-to-mesenchymal transition, a process known to support metastasis, ample evidence also links AXL to resistance against a variety of targeted therapies, including inhibitors of the MAPK pathway in malignant melanoma and the EGFR pathway in lung cancer. AXL-107-MMAE (HuMax-AXL-ADC) is a therapeutic antibody-drug conjugate specific for AXL, containing the microtubule disrupting agent monomethyl auristatin E as the cytotoxic payload. AXL-107-MMAE was previously shown to induce potent cytotoxicity in vitro and in vivo, which was dependent expression of AXL on the cell surface. We evaluated the capacity of AXL-107-MMAE to target AXL-positive MAPK pathway inhibitor resistant tumor cells, using malignant melanoma as a clinically relevant example. First, AXL-107-MMAE was shown to induce cytotoxicity in BRAF-mutant tumor cell lines that showed AXL expression upon acquired resistance to BRAF-inhibitors. In contrast, no cytotoxicity was observed in the BRAF-inhibitor-sensitive, AXL-negative parental cell lines. In heterogeneous tumor cell cultures, treatment with a BRAF-inhibitor selected for AXL-high, MAPK pathway inhibitor-insensitive melanoma cells, which was prevented by combined BRAF-inhibitor and AXL-107-MMAE treatment. Interestingly, we observed marked AXL upregulation in biopsies obtained from patients after they developed resistance to MAPK pathway inhibitors compared to paired pre-treatment biopsies. The in vivo potential of AXL-107-MMAE in malignant melanoma was demonstrated using BRAFV600E-mutated xenograft model derived from a patient who developed resistance to the BRAF inhibitor vemurafenib in the clinic. This indicates that AXL expression levels in treatment-resistant malignant melanoma are sufficient to induce tumor regression with AXL-107-MMAE, at least in this model system. These findings merit clinical validation of the targeting of both treatment-naive and drug-resistant cancers with AXL-107-MMAE, either alone or in combination with other targeted therapies. Citation Format: Julia Boshuizen, Louise A. Koopman, Esther C. Breij, David Satijn, Daniel Peeper, Paul W. Parren. Specific elimination of invasive and multidrug-resistant cancer cells by an antibody-drug conjugate targeting AXL [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 4591. doi:10.1158/1538-7445.AM2017-4591

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