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
Bispecific antibodies that bind cell-surface targets as well as digoxigenin (Dig) were generated for targeted payload delivery. Targeting moieties are IgGs that bind the tumor antigens Her2, IGF1R, CD22, or LeY. A Dig-binding single-chain Fv was attached in disulfide-stabilized form to C termini of CH3 domains of targeting antibodies. Bispecific molecules were expressed in mammalian cells and purified in the same manner as unmodified IgGs. They are stable without aggregation propensity and retain binding specificity/affinity to cell-surface antigens and Dig. Digoxigeninylated payloads were generated that retain full functionality and can be complexed to bispecific antibodies in a defined 21 ratio. Payloads include small compounds (Dig-Cy5, Dig-Doxorubicin) and proteins (Dig-GFP). Complexed payloads are targeted by the bispecifics to cancer cells and because these complexes are stable in serum, they can be applied for targeted delivery. Because Dig bispecifics also effectively capture digoxigeninylated compounds under physiological conditions, separate administration of uncharged Dig bispecifics followed by application of Dig payload is sufficient to achieve antibody-mediated targeting in vitro and in vivo.
Assuntos
Anticorpos Biespecíficos/uso terapêutico , Antineoplásicos/administração & dosagem , Digoxigenina/imunologia , Sistemas de Liberação de Medicamentos/métodos , Anticorpos Biespecíficos/imunologia , Antígenos de Neoplasias/imunologia , Carbocianinas/administração & dosagem , Linhagem Celular Tumoral , Doxorrubicina/administração & dosagem , Proteínas de Fluorescência Verde/administração & dosagem , Humanos , Métodos , Anticorpos de Cadeia ÚnicaRESUMO
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
Mesothelin overexpression in lung adenocarcinomas correlates with the presence of activating KRAS mutations and poor prognosis. Hence SS1P, a mesothelin-targeted immunotoxin, could offer valuable treatment options for these patients, but its use in solid tumor therapy is hampered by high immunogenicity and non-specific toxicity. To overcome both obstacles we developed RG7787, a de-immunized cytotoxic fusion protein comprising a humanized SS1 Fab fragment and a truncated, B-cell epitope silenced, 24 kD fragment of Pseudomonas exotoxin A (PE24). Reactivity of RG7787 with sera from immunotoxin-treated patients was >1000 fold reduced. In vitro RG7787 inhibited cell viability of lung cancer cell lines with picomolar potency. The pharmacokinetic properties of RG7787 in rodents were comparable to SS1P, yet it was tolerated up to 10 fold better without causing severe vascular leak syndrome or hepatotoxicity. A pharmacokinetic/pharmacodynamic model developed based on NCI-H596 xenograft studies showed that for RG7787 and SS1P, their in vitro and in vivo potencies closely correlate. At optimal doses of 2-3 mg/kg RG7787 is more efficacious than SS1P. Even large, well established tumors (600 mm(3)) underwent remission during three treatment cycles with RG7787. Also in two patient-derived lung cancer xenograft models, Lu7336 and Lu7187, RG7787 showed anti-tumor efficacy. In monotherapy two treatment cycles were moderately efficacious in the Lu7336 model but showed good anti-tumor activity in the KRAS mutant Lu7187 model (26% and 80% tumor growth inhibition, respectively). Combination of RG7787 with standard chemotherapies further enhanced efficacy in both models achieving near complete eradication of Lu7187 tumors.
Assuntos
ADP Ribose Transferases/uso terapêutico , Toxinas Bacterianas/uso terapêutico , Exotoxinas/uso terapêutico , Proteínas Ligadas por GPI/metabolismo , Neoplasias Pulmonares/tratamento farmacológico , Engenharia de Proteínas , Pseudomonas/metabolismo , Proteínas Recombinantes de Fusão/uso terapêutico , Fatores de Virulência/uso terapêutico , Animais , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Feminino , Humanos , Fígado/efeitos dos fármacos , Fígado/patologia , Neoplasias Pulmonares/patologia , Mesotelina , Camundongos SCID , Modelos Biológicos , Ratos , Proteínas Recombinantes de Fusão/farmacocinética , Proteínas Recombinantes de Fusão/farmacologia , Ensaios Antitumorais Modelo de Xenoenxerto , Exotoxina A de Pseudomonas aeruginosaRESUMO
Preserving the chemical and structural integrity of therapeutic antibodies during manufacturing and storage is a major challenge during pharmaceutical development. Oxidation of Fc methionines Met252 and Met428 is frequently observed, which leads to reduced affinity to FcRn and faster plasma clearance if present at high levels. Because oxidation occurs in both positions simultaneously, their individual contribution to the concomitant changes in pharmacokinetic properties has not been clearly established. A novel pH-gradient FcRn affinity chromatography method was applied to isolate three antibody oxidation variants from an oxidized IgG1 preparation based on their FcRn binding properties. Physico-chemical characterization revealed that the three oxidation variants differed predominantly in the number of oxMet252 per IgG (0, 1, or 2), but not significantly in the content of oxMet428. Corresponding to the increase in oxMet252 content, stepwise reduction of FcRn affinity in vitro, as well as faster clearance and shorter terminal half-life, in huFcRn-transgenic mice were observed. A single Met252 oxidation per antibody had no significant effect on pharmacokinetics (PK) compared with unmodified IgG. Importantly, only molecules with both heavy chains oxidized at Met252 exhibited significantly faster clearance. In contrast, Met428 oxidation had no apparent negative effect on PK and even led to somewhat improved FcRn binding and slower clearance. This minor effect, however, seemed to be abrogated by the dominant effect of Met252 oxidation. The novel approach of functional chromatographic separation of IgG oxidation variants followed by physico-chemical and biological characterization has yielded the first experimentally-backed explanation for the unaltered PK properties of antibody preparations containing relatively high Met252 and Met428 oxidation levels.
Assuntos
Anticorpos Monoclonais/metabolismo , Anticorpos Monoclonais/farmacocinética , Fragmentos Fc das Imunoglobulinas/metabolismo , Metionina/metabolismo , Animais , Anticorpos Monoclonais/uso terapêutico , Afinidade de Anticorpos , Cromatografia de Afinidade , Feminino , Antígenos de Histocompatibilidade Classe I/genética , Antígenos de Histocompatibilidade Classe I/metabolismo , Humanos , Concentração de Íons de Hidrogênio , Fragmentos Fc das Imunoglobulinas/sangue , Fragmentos Fc das Imunoglobulinas/uso terapêutico , Taxa de Depuração Metabólica , Camundongos Endogâmicos C57BL , Camundongos Knockout , Camundongos Transgênicos , Oxirredução/efeitos dos fármacos , Ligação Proteica , Receptores Fc/genética , Receptores Fc/metabolismo , Espectrometria de Massas por Ionização por ElectrosprayRESUMO
Although biotherapeutics have vast potential for treating brain disorders, their use has been limited due to low exposure across the blood-brain barrier (BBB). We report that by manipulating the binding mode of an antibody fragment to the transferrin receptor (TfR), we have developed a Brain Shuttle module, which can be engineered into a standard therapeutic antibody for successful BBB transcytosis. Brain Shuttle version of an anti-Aß antibody, which uses a monovalent binding mode to the TfR, increases ß-Amyloid target engagement in a mouse model of Alzheimer's disease by 55-fold compared to the parent antibody. We provide in vitro and in vivo evidence that the monovalent binding mode facilitates transcellular transport, whereas a bivalent binding mode leads to lysosome sorting. Enhanced target engagement of the Brain Shuttle module translates into a significant improvement in amyloid reduction. These findings have major implications for the development of biologics-based treatment of brain disorders.
Assuntos
Peptídeos beta-Amiloides/metabolismo , Barreira Hematoencefálica/metabolismo , Encéfalo/metabolismo , Transporte Proteico/fisiologia , Anticorpos de Cadeia Única/metabolismo , Doença de Alzheimer/genética , Doença de Alzheimer/patologia , Doença de Alzheimer/terapia , Peptídeos beta-Amiloides/imunologia , Precursor de Proteína beta-Amiloide/genética , Animais , Barreira Hematoencefálica/efeitos dos fármacos , Encéfalo/efeitos dos fármacos , Encéfalo/imunologia , Linhagem Celular Transformada , Modelos Animais de Doenças , Relação Dose-Resposta a Droga , Inibidores Enzimáticos/farmacologia , Humanos , Lisossomos/efeitos dos fármacos , Lisossomos/metabolismo , Macrolídeos/farmacologia , Camundongos , Camundongos Transgênicos , Modelos Imunológicos , Presenilina-1/genética , Ligação Proteica/efeitos dos fármacos , Ligação Proteica/imunologia , Transporte Proteico/efeitos dos fármacos , Receptores da Transferrina/imunologia , Receptores da Transferrina/metabolismo , Anticorpos de Cadeia Única/farmacologia , Anticorpos de Cadeia Única/uso terapêutico , Frações Subcelulares/efeitos dos fármacos , Frações Subcelulares/metabolismo , Fatores de Tempo , Transcitose/efeitos dos fármacos , Transcitose/genética , Transcitose/imunologiaRESUMO
We have designed bispecific antibodies that bind one target (anti-Her3) in a bivalent IgG-like manner and contain one additional binding entity (anti-cMet) composed of one V(H) and one V(L) domain connected by a disulfide bond. The molecules are assembled by fusing a V(H,Cys44) domain via flexible connector peptides to the C-terminus of one H-chain (heavy chain), and a V(L,Cys100) to another H-chain. To ensure heterodimerization during expression in mammalian cells, we introduced complementary knobs-into-holes mutations into the different H-chains. The IgG-shaped trivalent molecules carry as third binding entity one disulfide-stabilized Fv (dsFv) without a linker between V(H) and V(L). Tethering the V(H) and V(L) domains at the C-terminus of the C(H)3 domain decreases the on-rates of the dsFv to target antigens without affecting off-rates. Steric hindrance resolves upon removal of one side of the double connection by proteolysis: this improves flexibility and accessibility of the dsFv and fully restores antigen access and affinity. This technology has multiple applications: (i) in cases where single-chain linkers are not desired, dsFvs without linkers can be generated by addition of furin site(s) in the connector that are processed during expression within mammalian cells; (ii) highly active (toxic) entities which affect expression can be produced as inactive dsFvs and subsequently be activated (e.g. via PreScission cleavage) during purification; (iii) entities can be generated which are targeted by the unrestricted binding entity and can be activated by proteases in target tissues. For example, Her3-binding molecules containing linkers with recognition sequences for matrix metalloproteases or urokinase, whose inactivated cMet binding site is activated by proteolytic processing.
Assuntos
Anticorpos Biespecíficos/química , Anticorpos Biespecíficos/metabolismo , Anticorpos de Cadeia Única/química , Anticorpos de Cadeia Única/metabolismo , Anticorpos Biespecíficos/genética , Anticorpos Biespecíficos/imunologia , Sítios de Ligação de Anticorpos , Linhagem Celular , Dissulfetos/química , Expressão Gênica , Humanos , Metaloproteinase 2 da Matriz/imunologia , Metaloproteinase 9 da Matriz/imunologia , Peptídeo Hidrolases/metabolismo , Engenharia de Proteínas , Proteólise , Receptor ErbB-3/imunologia , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/imunologia , Proteínas Recombinantes/metabolismo , Anticorpos de Cadeia Única/genética , Anticorpos de Cadeia Única/imunologia , Ativador de Plasminogênio Tipo Uroquinase/imunologiaRESUMO
Bispecific antibodies (bsAbs) that bind to cell surface antigens and to digoxigenin (Dig) were used for targeted small interfering RNA (siRNA) delivery. They are derivatives of immunoglobulins G (IgGs) that bind tumor antigens, such as Her2, IGF1-R, CD22, and LeY, with stabilized Dig-binding variable domains fused to the C-terminal ends of the heavy chains. siRNA that was digoxigeninylated at its 3'end was bound in a 2:1 ratio to the bsAbs. These bsAb-siRNA complexes delivered siRNAs specifically to cells that express the corresponding antigen as demonstrated by flow cytometry and confocal microscopy. The complexes internalized into endosomes and Dig-siRNAs separated from bsAbs, but Dig-siRNA was not released into the cytoplasm; bsAb-targeting alone was thus not sufficient for effective mRNA knockdown. This limitation was overcome by formulating the Dig-siRNA into nanoparticles consisting of dynamic polyconjugates (DPCs) or into lipid-based nanoparticles (LNPs). The resulting complexes enabled bsAb-targeted siRNA-specific messenger RNA (mRNA) knockdown with IC(50) siRNA values in the low nanomolar range for a variety of bsAbs, siRNAs, and target cells. Furthermore, pilot studies in mice bearing tumor xenografts indicated mRNA knockdown in endothelial cells following systemic co-administration of bsAbs and siRNA formulated in LNPs that were targeted to the tumor vasculature.Molecular Therapy - Nucleic Acids (2012) 1, e45; doi:10.1038/mtna.2012.39; published online 18 September 2012.