Bart De Goeij

Efficient generation of stable bispecific IgG1 by controlled Fab-arm exchange

The promise of bispecific antibodies (bsAbs) to yield more effective therapeutics is well recognized; however, the generation of bsAbs in a practical and cost-effective manner has been a formidable challenge. Here we present a technology for the efficient generation of bsAbs with normal IgG structures that is amenable to both antibody drug discovery and development. The process involves separate expression of two parental antibodies, each containing single matched point mutations in the CH3 domains. The parental antibodies are mixed and subjected to controlled reducing conditions in vitro that separate the antibodies into HL half-molecules and allow reassembly and reoxidation to form highly pure bsAbs. The technology is compatible with standard large-scale antibody manufacturing and ensures bsAbs with Fc-mediated effector functions and in vivo stability typical of IgG1 antibodies. Proof-of-concept studies with HER2×CD3 (T-cell recruitment) and HER2×HER2 (dual epitope targeting) bsAbs demonstrate superior in vivo activity compared with parental antibody pairs.

New developments for antibody-drug conjugate-based therapeutic approaches.

The clinical success of Adcetris(®) (brentuximab vedotin) and Kadcyla(®) (ado-trastuzumab emtansine) has sparked clinical development of novel ADCs. These powerful anti-cancer agents are designed to allow specific targeting of highly potent cytotoxic agents to tumor cells while sparing healthy tissues. Despite the use of tumor-specific antibodies, the emerging clinical data with ADCs indicates that adverse effects frequently occur before ADCs have reached their optimal therapeutic dose, resulting in a relatively narrow therapeutic window. This review summarizes the therapeutic window of ADCs currently in clinical development, along with some strategies that may help to widen the window.

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.

HER2 monoclonal antibodies that do not interfere with receptor heterodimerization-mediated signaling induce effective internalization and represent valuable components for rational antibody-drug conjugate design

The human epidermal growth factor receptor (HER)2 provides an excellent target for selective delivery of cytotoxic drugs to tumor cells by antibody-drug conjugates (ADC) as has been clinically validated by ado-trastuzumab emtansine (KadcylaTM). While selecting a suitable antibody for an ADC approach often takes specificity and efficient antibody-target complex internalization into account, the characteristics of the optimal antibody candidate remain poorly understood. We studied a large panel of human HER2 antibodies to identify the characteristics that make them most suitable for an ADC approach. As a model toxin, amenable to in vitro high-throughput screening, we employed Pseudomonas exotoxin A (ETA’) fused to an anti-kappa light chain domain antibody. Cytotoxicity induced by HER2 antibodies, which were thus non-covalently linked to ETA’, was assessed for high and low HER2 expressing tumor cell lines and correlated with internalization and downmodulation of HER2 antibody-target complexes. Our results demonstrate that HER2 antibodies that do not inhibit heterodimerization of HER2 with related ErbB receptors internalize more efficiently and show greater ETA’-mediated cytotoxicity than antibodies that do inhibit such heterodimerization. Moreover, stimulation with ErbB ligand significantly enhanced ADC-mediated tumor kill by antibodies that do not inhibit HER2 heterodimerization. This suggests that the formation of HER2/ErbB-heterodimers enhances ADC internalization and subsequent killing of tumor cells. Our study indicates that selecting HER2 ADCs that allow piggybacking of HER2 onto other ErbB receptors provides an attractive strategy for increasing ADC delivery and tumor cell killing capacity to both high and low HER2 expressing tumor cells.

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