RESUMO
Vascular endothelial growth factor (VEGF) and angiopoietin (ANG)-2 have complementary roles in angiogenesis and promote an immunosuppressive tumor microenvironment. It is anticipated that the combination of VEGF and ANG2 blockade could provide superior activity to the blockade of either pathway alone and that the addition of VEGF/ANG2 inhibition to an anti-programmed cell death protein-1 (PD-1) antibody could change the tumor microenvironment to support T-cell-mediated tumor cytotoxicity. Here, we describe the pharmacologic and antitumor activity of BI 836880, a humanized bispecific nanobody comprising two single-variable domains blocking VEGF and ANG2, and an additional module for half-life extension in vivo. BI 836880 demonstrated high affinity and selectivity for human VEGF-A and ANG2, resulting in inhibition of the downstream signaling of VEGF/ANG2 and a decrease in endothelial cell proliferation and survival. In vivo, BI 836880 exhibited significant antitumor activity in all patient-derived xenograft models tested, showing significantly greater tumor growth inhibition (TGI) than bevacizumab (VEGF inhibition) and AMG386 (ANG1/2 inhibition) in a range of models. In a Lewis lung carcinoma syngeneic tumor model, the combination of PD-1 inhibition with VEGF inhibition showed superior efficacy versus the blockade of either pathway alone. TGI was further increased with the addition of ANG2 inhibition to VEGF/PD-1 blockade. VEGF/ANG2 inhibition had a strong antiangiogenic effect. Our data suggest that the blockade of VEGF and ANG2 with BI 836880 may offer improved antitumor activity versus the blockade of either pathway alone and that combining VEGF/ANG2 inhibition with PD-1 blockade can further enhance antitumor effects. SIGNIFICANCE STATEMENT: Vascular endothelial growth factor (VEGF) and angiopoietin (ANG)-2 play key roles in angiogenesis and have an immunosuppressive effect in the tumor microenvironment. This study shows that BI 836880, a bispecific nanobody targeting VEGF and ANG2, demonstrates substantial antitumor activity in preclinical models. Combining VEGF/ANG2 inhibition with the blockade of the PD-1 pathway can further improve antitumor activity.
Assuntos
Neoplasias , Fator A de Crescimento do Endotélio Vascular , Humanos , Fator A de Crescimento do Endotélio Vascular/metabolismo , Angiopoietina-2/metabolismo , Receptor de Morte Celular Programada 1 , Fatores de Crescimento do Endotélio Vascular/uso terapêutico , Inibidores da Angiogênese , Neoplasias/tratamento farmacológico , Morte Celular , Angiopoietina-1 , Microambiente TumoralRESUMO
In the present study, we have developed a novel one-arm single chain Fab heterodimeric bispecific IgG (OAscFab-IgG) antibody format targeting the insulin-like growth factor receptor type I (IGF-1R) and the epidermal growth factor receptor (EGFR) with one binding site for each target antigen. The bispecific antibody XGFR is based on the "knob-into-hole" technology for heavy chain heterodimerization with one heavy chain consisting of a single chain Fab to prevent wrong pairing of light chains. XGFR was produced with high expression yields and showed simultaneous binding to IGF-1R and EGFR with high affinity. Due to monovalent binding of XGFR to IGF-1R, IGF-1R internalization was strongly reduced compared with the bivalent parental antibody, leading to enhanced Fc-mediated cellular cytotoxicity. To further increase immune effector functions triggered by XGFR, the Fc portion of the bispecific antibody was glycoengineered, which resulted in strong antibody-dependent cell-mediated cytotoxicity activity. XGFR-mediated inhibition of IGF-1R and EGFR phosphorylation as well as A549 tumor cell proliferation was highly effective and was comparable with a combined treatment with EGFR (GA201) and IGF-1R (R1507) antibodies. XGFR also demonstrated potent anti-tumor efficacy in multiple mouse xenograft tumor models with a complete growth inhibition of AsPC1 human pancreatic tumors and improved survival of SCID beige mice carrying A549 human lung tumors compared with treatment with antibodies targeting either IGF-1R or EGFR. In summary, we have applied rational antibody engineering technology to develop a heterodimeric OAscFab-IgG bispecific antibody, which combines potent signaling inhibition with antibody-dependent cell-mediated cytotoxicity induction and results in superior molecular properties over two established tetravalent bispecific formats.
Assuntos
Anticorpos Biespecíficos/imunologia , Receptores ErbB/imunologia , Imunoglobulina G/imunologia , Engenharia de Proteínas , Receptor IGF Tipo 1/imunologia , Anticorpos de Cadeia Única/imunologia , Animais , Anticorpos Biespecíficos/química , Anticorpos Biespecíficos/metabolismo , Anticorpos Biespecíficos/farmacologia , Sítios de Ligação , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Receptores ErbB/metabolismo , Feminino , Regulação da Expressão Gênica/efeitos dos fármacos , Glicosilação , Humanos , Imunoglobulina G/química , Imunoglobulina G/metabolismo , Imunoglobulina G/farmacologia , Camundongos , Neoplasias Pancreáticas/patologia , Multimerização Proteica , Estrutura Quaternária de Proteína , Transporte Proteico/efeitos dos fármacos , Receptor IGF Tipo 1/metabolismo , Transdução de Sinais/efeitos dos fármacos , Anticorpos de Cadeia Única/química , Anticorpos de Cadeia Única/metabolismo , Anticorpos de Cadeia Única/farmacologia , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Cancer therapies based on in vivo stimulation, or on adoptive T cell transfer of Vγ9Vδ2 T cells, have been tested in the past decades but have failed to provide consistent clinical efficacy. New, promising concepts such as γδ Chimeric Antigen Receptor (CAR) -T cells and γδ T-cell engagers are currently under preclinical evaluation. Since the impact of factors, such as the relatively low abundance of γδ T cells within tumor tissue is still under investigation, it remains to be shown whether these effector T cells can provide significant efficacy against solid tumors. Here, we highlight key learnings from the natural role of Vγ9Vδ2 T cells in the elimination of host cells bearing intracellular bacterial agents and we translate these into the setting of tumor therapy. We discuss the availability and relevance of preclinical models as well as currently available tools and knowledge from a drug development perspective. Finally, we compare advantages and disadvantages of existing therapeutic concepts and propose a role for Vγ9Vδ2 T cells in immune-oncology next to Cluster of Differentiation (CD) 3 activating therapies.
Assuntos
Infecções/imunologia , Neoplasias/imunologia , Neoplasias/terapia , Receptores de Antígenos de Linfócitos T gama-delta/metabolismo , Linfócitos T/imunologia , Animais , Plasticidade Celular , Humanos , Neoplasias/patologia , Receptores de Reconhecimento de Padrão/metabolismoRESUMO
Antibody-producing Chinese-hamster ovary cells (CHO-DG44) were converted into cells producing antibodies with strongly enhanced ADCC (antibody-dependent cellular cytotoxicity) by knocking down FuT8 (alpha-1,6-fucosyltransferase or fucosyltransferase 8) via constitutive expression of shRNA (short-hairpin RNA) against FuT8. After the introduction of a FuT8 shRNA expression plasmid under the control of a U6 promoter, CHO-DG44 clones with less than 5% residual FuT8 mRNA expression were isolated by selection for neomycin resistance, followed by low affinity nerve growth factor receptor enrichment and selection for LCA [Lens culinaris (culinary lentil) agglutinin] resistance. The CHO-DG44 clones identified produced highly afucosylated anti-[IGF-1R (insulin-like-growth-factor-1 receptor)] antibodies (up to 88%) that exhibited considerably enhanced ADCC compared with anti-IGF-1R wild-type antibodies produced by parental CHO cells. At the same time, antibody productivity was not significantly decreased. Analysis of stability showed that the clones obtained may be suitable for up-scaling, since low residual levels of FuT8 mRNA and production of afucosylated antibodies were maintained for at least 4 weeks.
Assuntos
Anticorpos/imunologia , Citotoxicidade Celular Dependente de Anticorpos , Fucosiltransferases/genética , Interferência de RNA , RNA Interferente Pequeno , Receptor IGF Tipo 1/imunologia , Animais , Formação de Anticorpos , Células CHO , Clonagem Molecular , Cricetinae , Cricetulus , Fucosiltransferases/metabolismo , Expressão Gênica , Receptor IGF Tipo 1/metabolismo , Proteínas Recombinantes/imunologia , Proteínas Recombinantes/metabolismoRESUMO
High specificity accompanied with the ability to recruit immune cells has made recombinant therapeutic antibodies an integral part of drug development. Here we present a generic approach to generate two novel IgG-derived antibody formats that are based on a modification of the CrossMab technology. MoAbs harbor two heavy chains (HCs) resulting in one binding entity and one fragment crystallizable region (Fc), whereas DuoMabs are composed of four HCs harboring two binding entities and two Fc regions linked at a disulfide-bridged hinge. The latter bivalent format is characterized by avidity-enhanced target cell binding while simultaneously increasing the 'Fc-load' on the surface. DuoMabs were shown to be producible in high yield and purity and bind to surface cells with affinities comparable to IgGs. The increased Fc load directed at the surface of target cells by DuoMabs modulates their antibody-dependent cell-mediated cytotoxicity competency toward target cells, making them attractive for applications that require or are modulated by FcR interactions.
Assuntos
Anticorpos Biespecíficos/imunologia , Anticorpos Monoclonais/imunologia , Citotoxicidade Celular Dependente de Anticorpos , Fragmentos Fc das Imunoglobulinas/imunologia , Imunoglobulina G/imunologia , Anticorpos Biespecíficos/química , Anticorpos Monoclonais/química , Células HEK293 , Humanos , Fragmentos Fc das Imunoglobulinas/química , Imunoglobulina G/químicaRESUMO
Bisphosphonates are potent inhibitors of osteoclast-mediated bone resorption and play an important role in the treatment of osteoporosis, metastatic bone disease, and Paget disease. However, nephrotoxicity has been reported with some bisphosphonates. Nitrogen-containing bisphosphonates directly inhibit farnesyl diphosphate (FPP) synthase activity (mevalonate pathway) and reduce protein prenylation leading to osteoclast cell death. The aim here was to elucidate if this inhibition also occurs in kidney cells and may directly account for nephrotoxicity. In an exploratory study in rats receiving zoledronate or ibandronate an approximate 2-fold increase in FPP synthase mRNA levels was observed in the kidney. The involvement of the mevalonate pathway was confirmed in subsequent in vitro studies with zoledronate, ibandronate, and pamidronate, using the non-nitrogen containing bisphosphonate clodronate as a comparator. In vitro changes in FPP synthase mRNA expression, enzyme activity, and levels of prenylated proteins were assessed. Using two cell lines (a rat normal kidney cell line, NRK-52E, and a human kidney proximal tubule cell line, HK-2), ibandronate and zoledronate were identified as most cytotoxic (EC50: 23/>1000 microM and 16/82 microM, respectively) and as the most potent inhibitors of FPP synthase (IC50; 1.6/7.4 microM and 0.5/0.7 microM, respectively). In both cell lines, inhibition of FPP synthase activity occurred prior to a decrease in levels of prenylated proteins followed by cytotoxicity. This further supports that the mechanism responsible for osteoclast inhibition (therapeutic effect) might also underlie the mechanism of nephrotoxicity.
Assuntos
Difosfonatos/toxicidade , Geraniltranstransferase/antagonistas & inibidores , Imidazóis/toxicidade , Rim/efeitos dos fármacos , Animais , Conservadores da Densidade Óssea/administração & dosagem , Conservadores da Densidade Óssea/toxicidade , Linhagem Celular , Ácido Clodrônico/toxicidade , Difosfonatos/administração & dosagem , Relação Dose-Resposta a Droga , Avaliação Pré-Clínica de Medicamentos , Geraniltranstransferase/metabolismo , Humanos , Ácido Ibandrônico , Imidazóis/administração & dosagem , Concentração Inibidora 50 , Rim/citologia , Rim/enzimologia , Masculino , Pamidronato , RNA Mensageiro/efeitos dos fármacos , RNA Mensageiro/metabolismo , Ratos , Ratos Sprague-Dawley , Ácido ZoledrônicoRESUMO
The Crk-associated tyrosine kinase substrate p130cas (CAS) is a docking protein containing an SH3 domain near its N terminus, followed by a short proline-rich segment, a large central substrate domain composed of 15 repeats of the four amino acid sequence YxxP, a serine-rich region and a carboxy-terminal domain, which possesses consensus binding sites for the SH2 and SH3 domains of Src (YDYV and RPLPSPP, respectively). The SH3 domain of CAS mediates its interaction with several proteins involved in signaling pathways such as focal adhesion kinase (FAK), tyrosine phosphatases PTP1B and PTP-PEST, and the guanine nucleotide exchange factor C3G. As a homolog of the corresponding Src docking domain, the CAS SH3 domain binds to proline-rich sequences (PxxP) of its interacting partners that can adopt a polyproline type II helix. We have determined a high-resolution X-ray structure of the recombinant human CAS SH3 domain. The domain, residues 1-69, crystallized in two related space groups, P2(1) and C222(1), that provided diffraction data to 1.1 A and 2.1 A, respectively. The crystal structure shows, in addition to the conserved SH3 domain architecture, the way in which the CAS characteristic amino acids form an atypically charged ligand-binding surface. This arrangement provides a rationale for the unusual ligand recognition motif exhibited by the CAS SH3 domain. The structure enables modelling of the docking interactions to its ligands, for example from focal adhesion kinase, and supports structure-based drug design of inhibitors of the CAS-FAK interaction.
Assuntos
Proteínas/química , Proteínas/metabolismo , Domínios de Homologia de src , Sequência de Aminoácidos , Proteína Substrato Associada a Crk , Cristalografia por Raios X , Quinase 1 de Adesão Focal , Proteína-Tirosina Quinases de Adesão Focal , Humanos , Ligação de Hidrogênio , Ligantes , Modelos Moleculares , Dados de Sequência Molecular , Ligação Proteica , Proteínas Tirosina Quinases/química , Proteínas Tirosina Quinases/metabolismo , Proteína p130 Retinoblastoma-Like , Alinhamento de Sequência , Especificidade por Substrato , Água/químicaRESUMO
Protein kinase B (PKB)-selective inhibitors were designed, synthesized, and cocrystallized using the AGC kinase family protein kinase A (PKA, often called cAMP-dependent protein kinase); PKA has been used as a surrogate for other members of this family and indeed for protein kinases in general. The high homology between PKA and PKB includes very similar ATP binding sites and hence similar binding pockets for inhibitors, with only few amino acids that differ between the two kinases. A series of these sites were mutated in PKA in order to improve the surrogate model for a design of PKB-selective inhibitors. Namely, the PKA to PKB exchanges F187L and Q84E enable the design of the selective inhibitors described herein which mimic ATP but extend further into a site not occupied by ATP. In this pocket, selectivity over PKA can be achieved by the introduction of bulkier substituents. Analysis of the cocrystal structures and binding studies were performed to rationalize the selectivity and improve the design.