RESUMO
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
In this study we present novel bispecific antibodies that simultaneously target the insulin-like growth factor receptor type I (IGF-1R) and epidermal growth factor receptor (EGFR). For this purpose disulfide stabilized scFv domains of the EGFR/ADCC antibody GA201 were fused via serine-glycine connectors to the C-terminus of the heavy (XGFR2) or light chain (XGFR4), or the N-termini of the light (XGFR5) or heavy chain (XGFR3) of the IGF-1R antibody R1507 as parental IgG1 antibody. The resulting bispecific IGF-1R-EGFR antibodies XGFR2, XGFR3 and XGFR4 were successfully generated with yields and stability comparable to conventional IgG1 antibodies. They effectively inhibited IGF-1R and EGFR phosphorylation and 3D proliferation of H322M and H460M2 tumor cells, induced strong down-modulation of IGF-1R as well as enhanced EGFR down-modulation compared to the parental EGFR antibody GA201 and were ADCC competent. The bispecific XGFR derivatives showed a strong format dependent influence of N- or C-terminal heavy and light chain scFv attachment on ADCC activity and an increase in receptor downregulation over the parental combination in vitro. XGFR2 and XGFR4 were selected for in vivo evaluation and showed potent anti-tumoral efficacy comparable to the combination of monospecific IGF-1R and EGFR antibodies in subcutaneous BxPC3 and H322M xenograft models. In summary, we have managed to overcome issues of stability and productivity of bispecific antibodies, discovered important antibody fusion protein design related differences on ADCC activity and receptor downmodulation and show that IGF-1R-EGFR antibodies represent an attractive therapeutic strategy to simultaneously target two key components de-regulated in multiple cancer types, with the ultimate goal to avoid the formation of resistance to therapy.
Assuntos
Anticorpos Biespecíficos/imunologia , Anticorpos Biespecíficos/uso terapêutico , Receptores ErbB/imunologia , Imunoglobulina G/imunologia , Imunoglobulina G/uso terapêutico , Neoplasias/terapia , Receptor IGF Tipo 1/imunologia , Animais , Anticorpos Biespecíficos/genética , Afinidade de Anticorpos , Linhagem Celular , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Clonagem Molecular , Receptores ErbB/metabolismo , Feminino , Humanos , Imunoglobulina G/genética , Imunoterapia , Camundongos , Camundongos SCID , Modelos Moleculares , Neoplasias/imunologia , Neoplasias/metabolismo , Fosforilação/efeitos dos fármacos , Engenharia de Proteínas , Receptor IGF Tipo 1/metabolismo , Proteínas Recombinantes/genética , Proteínas Recombinantes/imunologia , Proteínas Recombinantes/uso terapêutico , Anticorpos de Cadeia Única/genética , Anticorpos de Cadeia Única/imunologia , Anticorpos de Cadeia Única/uso terapêuticoRESUMO
Pro-inflammatory cytokines regulate the growth, differentiation, and activation of immune cells and can play a role in antitumor responses. GM-CSF and IL-2 induce tumor rejection in animal models when expressed by tumor cells, and IL-2 is used for the treatment of melanoma and renal cell cancer. However, high doses of GM-CSF and IL-2 are associated with severe side effects in cancer patients. We generated a dual cytokine fusion protein for simultaneous targeted delivery of human GM-CSF and IL-2 to human tumors. The fusion protein is based on a heterodimeric core structure formed by human CH1 and C kappa domains (heterominibody) with C-terminally fused human cytokines and N-terminally fused human single-chain Ab fragments (scFv) specific for the tumor-associated surface antigen epithelial cell adhesion molecule (Ep-CAM). The dual cytokine heterominibody (DCH) was well expressed and secreted by CHO cells, preserved the specific proliferative activities of the two cytokines, and showed Ep-CAM-specific binding to tumor cells. DCH induced potent tumor cell lysis in vitro by two distinct mechanisms. One was activating PBMCs to lyse tumor cells, which was superior to cytotoxicity induced by equimolar ratios of free recombinant human IL-2 and GM-CSF. The other mechanism was redirected lysis, as seen with isolated human T cells, which was solely dependent on the IL-2 fusion part. The therapeutic principle of dual cytokine targeting may warrant in vivo testing of murine-specific analogues in appropriate mouse models and further preclinical development of the less immunogenic, human cytokine- and human Ep-CAM-specific DCH molecule described here.
Assuntos
Antineoplásicos/administração & dosagem , Moléculas de Adesão Celular/genética , Sistemas de Liberação de Medicamentos , Fator Estimulador de Colônias de Granulócitos e Macrófagos/administração & dosagem , Interleucina-2/administração & dosagem , Animais , Anticorpos/administração & dosagem , Antígenos de Neoplasias/administração & dosagem , Antígenos de Neoplasias/genética , Antígenos de Neoplasias/imunologia , Antineoplásicos/imunologia , Células CHO , Moléculas de Adesão Celular/administração & dosagem , Moléculas de Adesão Celular/biossíntese , Moléculas de Adesão Celular/imunologia , Linhagem Celular Tumoral , Cricetinae , Citotoxicidade Imunológica , Modelos Animais de Doenças , Combinação de Medicamentos , Eletroforese em Gel de Poliacrilamida , Molécula de Adesão da Célula Epitelial , Células Epiteliais/química , Células Epiteliais/citologia , Variação Genética , Fator Estimulador de Colônias de Granulócitos e Macrófagos/genética , Humanos , Interleucina-2/análogos & derivados , Interleucina-2/genética , Proteínas Recombinantes de Fusão/administração & dosagem , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes , Linfócitos T/imunologiaRESUMO
The epidermal growth factor receptor (EGFR) and the insulin-like growth factor-1 receptor (IGF-1R) play critical roles in tumor growth, providing a strong rationale for the combined inhibition of IGF-1R and EGFR signaling in cancer therapy. We describe the design, affinity maturation, in vitro and in vivo characterization of the bispecific anti-IGF-1R/EGFR antibody XGFR*. XGFR* is based on the bispecific IgG antibody XGFR, which enabled heterodimerization of an IGF-1R binding scFab heavy chain with an EGFR-binding light and heavy chain by the "knobs-into-holes" technology. XGFR* is optimized for monovalent binding of human EGFR and IGF-1R with increased binding affinity for IGF-1R due to affinity maturation and highly improved protein stability to oxidative and thermal stress. It bears an afucosylated Fc-portion for optimal induction of antibody-dependent cell-mediated cytotoxicity (ADCC). Stable Chinese hamster ovary cell clones with production yields of 2-3 g/L were generated, allowing for large scale production of the bispecific antibody. XGFR* potently inhibits EGFR- and IGF-1R-dependent receptor phosphorylation, reduces tumor cell proliferation in cells with heterogeneous levels of IGF-1R and EGFR receptor expression and induces strong ADCC in vitro. A comparison of pancreatic and colorectal cancer lines demonstrated superior responsiveness to XGFR*-mediated signaling and tumor growth inhibition in pancreatic cancers that frequently show a high degree of IGF-1R/EGFR co-expression. XGFR* showed potent anti-tumoral efficacy in the orthotopic MiaPaCa-2 pancreatic xenograft model, resulting in nearly complete tumor growth inhibition with significant number of tumor remissions. In summary, the bispecific anti-IGF-1R/EGFR antibody XGFR* combines potent signaling and tumor growth inhibition with enhanced ADCC induction and represents a clinical development candidate for the treatment of pancreatic cancer.
Assuntos
Anticorpos Biespecíficos/farmacologia , Antineoplásicos/farmacologia , Receptores ErbB/antagonistas & inibidores , Neoplasias Pancreáticas/imunologia , Receptor IGF Tipo 1/antagonistas & inibidores , Animais , Anticorpos Biespecíficos/biossíntese , Afinidade de Anticorpos , Citotoxicidade Celular Dependente de Anticorpos , Células CHO , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Cricetinae , Cricetulus , Humanos , Camundongos , Transdução de Sinais/efeitos dos fármacos , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Cytokine targeting to tumor-associated antigens via antibody cytokine fusion proteins has demonstrated potent antitumor activity in numerous animal models and has led to the clinical development of 2 antibody-interleukin-2 (IL-2) fusion proteins. We previously reported on the construction and in vitro properties of a "dual" cytokine fusion protein for simultaneous targeted delivery of human granulocyte macrophage-colony stimulating factor (GM-CSF) and IL-2 to human tumors. The fusion protein is based on a heterodimerized core structure formed by human CH1 and Ckappa domains (heterominibody) with C-terminally fused human cytokines and N-terminally fused single-chain antibody fragments specific for the tumor-associated surface antigen epithelial cell adhesion molecule (Ep-CAM). For testing the antitumor activity in syngeneic mouse xenograft models, we developed "dual cytokine heterominibodies" with murine cytokines (mDCH). mDCH fusion proteins and, as controls, "single cytokine heterominibodies" (SCH) carrying either murine GM-CSF (mGM-CSF) or murine IL-2 (mIL-2) were constructed, of which all retained the specific activities of cytokines and binding to the Ep-CAM antigen on human Ep-CAM transfected mouse colon carcinoma CT26-KSA cells. Over a 5-day treatment course, DCH fusion proteins induced significant inhibition of established pulmonary CT26-KSA metastases in immune-competent Balb/c mice at low daily doses of 1 mug of fusion protein per mouse. However, with the tested dosing schemes, antitumor activity of mDCH was largely independent of cytokine targeting to tumors as demonstrated by a control protein with mutated Ep-CAM binding sites. Single cytokine fusion proteins mSCH-GM-CSF and mSCH-IL-2 showed similar antitumor activity as the dual cytokine fusion protein mDCH, indicating that GM-CSF and IL-2 in one molecule did not significantly synergize in tumor rejection under our experimental conditions. Our results seem to contradict the notion that IL-2 and GM-CSF can synergize in antitumor activity and that with conventional dose regimens, their specific targeting to tumors, as tested here with 2 antibodies of different affinities, enhances their antitumor activity.