RESUMEN
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.
Asunto(s)
Anticuerpos Biespecíficos/biosíntesis , Fragmentos Fab de Inmunoglobulinas/biosíntesis , Fragmentos Fc de Inmunoglobulinas/biosíntesis , Inmunoglobulina G/biosíntesis , Animales , Anticuerpos Biespecíficos/química , Anticuerpos Biespecíficos/genética , Células CHO , Cricetinae , Cricetulus , Células HEK293 , Humanos , Fragmentos Fab de Inmunoglobulinas/química , Fragmentos Fab de Inmunoglobulinas/genética , Fragmentos Fc de Inmunoglobulinas/química , Fragmentos Fc de Inmunoglobulinas/genética , Inmunoglobulina G/química , Inmunoglobulina G/genética , Células Jurkat , Proteínas Recombinantes/biosíntesis , Proteínas Recombinantes/química , Proteínas Recombinantes/genéticaRESUMEN
BACKGROUND: HexaBody®-CD38 (GEN3014) is a hexamerization-enhanced human IgG1 that binds CD38 with high affinity. The E430G mutation in its Fc domain facilitates the natural process of antibody hexamer formation upon binding to the cell surface, resulting in increased binding of C1q and potentiated complement-dependent cytotoxicity (CDC). METHODS: Co-crystallization studies were performed to identify the binding interface of HexaBody-CD38 and CD38. HexaBody-CD38-induced CDC, antibody-dependent cellular cytotoxicity (ADCC), antibody-dependent cellular phagocytosis (ADCP), trogocytosis, and apoptosis were assessed using flow cytometry assays using tumour cell lines, and MM patient samples (CDC). CD38 enzymatic activity was measured using fluorescence spectroscopy. Anti-tumour activity of HexaBody-CD38 was assessed in patient-derived xenograft mouse models in vivo. FINDINGS: HexaBody-CD38 binds a unique epitope on CD38 and induced potent CDC in multiple myeloma (MM), acute myeloid leukaemia (AML), and B-cell non-Hodgkin lymphoma (B-NHL) cells. Anti-tumour activity was confirmed in patient-derived xenograft models in vivo. Sensitivity to HexaBody-CD38 correlated with CD38 expression level and was inversely correlated with expression of complement regulatory proteins. Compared to daratumumab, HexaBody-CD38 showed enhanced CDC in cell lines with lower levels of CD38 expression, without increasing lysis of healthy leukocytes. More effective CDC was also confirmed in primary MM cells. Furthermore, HexaBody-CD38 efficiently induced ADCC, ADCP, trogocytosis, and apoptosis after Fc-crosslinking. Moreover, HexaBody-CD38 strongly inhibited CD38 cyclase activity, which is hypothesized to relieve immune suppression in the tumour microenvironment. INTERPRETATION: Based on these preclinical studies, a clinical trial was initiated to assess the clinical safety of HexaBody-CD38 in patients with MM. FUNDING: Genmab.
Asunto(s)
Antineoplásicos , Mieloma Múltiple , Humanos , Animales , Ratones , ADP-Ribosil Ciclasa 1/genética , ADP-Ribosil Ciclasa 1/metabolismo , Antineoplásicos/farmacología , Antineoplásicos/uso terapéutico , Mieloma Múltiple/tratamiento farmacológico , Mieloma Múltiple/genética , Citotoxicidad Celular Dependiente de Anticuerpos , Línea Celular Tumoral , Proteínas del Sistema Complemento/metabolismo , Microambiente TumoralRESUMEN
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.
Asunto(s)
Anticuerpos Biespecíficos/administración & dosificación , Antineoplásicos/administración & dosificación , Inmunoconjugados/administración & dosificación , Receptor ErbB-2/antagonistas & inhibidores , Tetraspanina 30/antagonistas & inhibidores , Animales , Anticuerpos Biespecíficos/inmunología , Anticuerpos Biespecíficos/farmacocinética , Afinidad de Anticuerpos/inmunología , Antineoplásicos/farmacocinética , Línea Celular Tumoral , Supervivencia Celular/efectos de los fármacos , Modelos Animales de Enfermedad , Liberación de Fármacos , Femenino , Humanos , Inmunoconjugados/farmacocinética , Fragmentos Fab de Inmunoglobulinas/inmunología , Fragmentos Fab de Inmunoglobulinas/metabolismo , Lisosomas/metabolismo , Ratones , Terapia Molecular Dirigida , Unión Proteica , Carga Tumoral/efectos de los fármacos , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
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.
Asunto(s)
Antineoplásicos/administración & dosificación , Receptores ErbB/metabolismo , Factor VIIa/metabolismo , Inmunotoxinas/administración & dosificación , Neoplasias Experimentales/tratamiento farmacológico , Animales , Anticuerpos , Antineoplásicos/farmacocinética , Apoptosis , Línea Celular Tumoral , Sistemas de Liberación de Medicamentos , Receptores ErbB/inmunología , Factor VIIa/inmunología , Humanos , Inmunotoxinas/farmacocinética , Lisosomas/metabolismo , Ratones , Neoplasias Experimentales/metabolismo , Receptor ErbB-2/inmunología , Receptor ErbB-2/metabolismo , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
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.
Asunto(s)
Aminobenzoatos/uso terapéutico , Antineoplásicos/uso terapéutico , Inmunoconjugados/uso terapéutico , Neoplasias/tratamiento farmacológico , Oligopéptidos/uso terapéutico , Tromboplastina/inmunología , Animales , Anticuerpos Monoclonales/uso terapéutico , Células Cultivadas , Células HCT116 , Células HEK293 , Humanos , Ratones , Ratones Desnudos , Terapia Molecular Dirigida , Tromboplastina/antagonistas & inhibidores , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
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 (Kadcyla(TM)). 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.