RESUMO
Neutralizing antibodies to adeno-associated virus (AAV) vectors are highly prevalent in humans1,2, and block liver transduction3-5 and vector readministration6; thus, they represent a major limitation to in vivo gene therapy. Strategies aimed at overcoming anti-AAV antibodies are being studied7, which often involve immunosuppression and are not efficient in removing pre-existing antibodies. Imlifidase (IdeS) is an endopeptidase able to degrade circulating IgG that is currently being tested in transplant patients8. Here, we studied if IdeS could eliminate anti-AAV antibodies in the context of gene therapy. We showed efficient cleavage of pooled human IgG (intravenous Ig) in vitro upon endopeptidase treatment. In mice passively immunized with intravenous Ig, IdeS administration decreased anti-AAV antibodies and enabled efficient liver gene transfer. The approach was scaled up to nonhuman primates, a natural host for wild-type AAV. IdeS treatment before AAV vector infusion was safe and resulted in enhanced liver transduction, even in the setting of vector readministration. Finally, IdeS reduced anti-AAV antibody levels from human plasma samples in vitro, including plasma from prospective gene therapy trial participants. These results provide a potential solution to overcome pre-existing antibodies to AAV-based gene therapy.
Assuntos
Anticorpos Neutralizantes/imunologia , Dependovirus/genética , Terapia Genética , Vetores Genéticos/efeitos adversos , Animais , Anticorpos Anti-Idiotípicos/genética , Anticorpos Anti-Idiotípicos/imunologia , Anticorpos Neutralizantes/genética , Anticorpos Antivirais/imunologia , Capsídeo/imunologia , Dependovirus/imunologia , Endopeptidases/imunologia , Vetores Genéticos/uso terapêutico , Humanos , Imunoglobulina G/farmacologia , Fígado/imunologia , Fígado/metabolismo , CamundongosRESUMO
Murine antibody 10H10 raised against human tissue factor is unique in that it blocks the signaling pathway, and thus inhibits angiogenesis and tumor growth without interfering with coagulation. As a potential therapeutic, the antibody was humanized in a two-step procedure. Antigen-binding loops were grafted onto selected human frameworks and the resulting chimeric antibody was subjected to affinity maturation by using phage display libraries. The results of humanization were analyzed from the structural perspective through comparison of the structure of a humanized variant with the parental mouse antibody. This analysis revealed several hot spots in the framework region that appear to affect antigen binding, and therefore should be considered in human germline selection. In addition, some positions in the Vernier zone, e.g., residue 71 in the heavy chain, that are traditionally thought to be crucial appear to tolerate amino acid substitutions without any effect on binding. Several humanized variants were produced using both short and long forms of complementarity-determining region (CDR) H2 following the difference in the Kabat and Martin definitions. Comparison of such pairs indicated consistently higher thermostability of the variants with short CDR H2. Analysis of the binding data in relation to the structures singled out the ImMunoGeneTics information system® germline IGHV1-2*01 as dubious owing to two potentially destabilizing mutations as compared to the other alleles of the same germline and to other human germlines.
Assuntos
Anticorpos Monoclonais Humanizados/química , Afinidade de Anticorpos/fisiologia , Tromboplastina/imunologia , Animais , Anticorpos Monoclonais Humanizados/imunologia , Regiões Determinantes de Complementaridade/química , Humanos , Camundongos , Modelos Moleculares , Engenharia de Proteínas/métodosRESUMO
Toll-like receptor 3 (TLR3) binds and signals in response to dsRNA and poly(I:C), a synthetic double stranded RNA analog. Activation of TLR3 triggers innate responses that may play a protective or detrimental role in viral infections or in immune-mediated inflammatory diseases through amplification of inflammation. Two monoclonal antibodies, CNTO4685 (rat anti-mouse TLR3) and CNTO5429 (CDRs from CNTO4685 grafted onto a mouse IgG1 scaffold) were generated and characterized. These mAbs bind the extracellular domain of mouse TLR3, inhibit poly(I:C)-induced activation of HEK293T cells transfected with mTLR3, and reduce poly(I:C)-induced production of CCL2 and CXCL10 by primary mouse embryonic fibroblasts. CNTO5429 decreased serum IL-6 and TNFα levels post-intraperitoneal poly(I:C) administration, demonstrating in vivo activity. In summary, specific anti-mTLR3 mAbs have been generated to assess TLR3 antagonism in mouse models of inflammation.
Assuntos
Anticorpos Monoclonais/imunologia , Poli I-C/imunologia , Receptor 3 Toll-Like/imunologia , Animais , Linhagem Celular , Células Cultivadas , Humanos , Inflamação/imunologia , Espaço Intracelular/imunologia , Macrófagos/imunologia , Camundongos , Camundongos Endogâmicos C57BL , Receptor 3 Toll-Like/genéticaRESUMO
The functional role of IL-12 and IL-23 in host defense and disease following viral infection of the CNS was determined. Instillation of mouse hepatitis virus (MHV, a positive-strand RNA virus) into the CNS of mice results in acute encephalitis followed by a chronic immune-mediated demyelinating disease. Antibody-mediated blocking of either IL-23 (anti-IL-23p19) or IL-12 and IL-23 (anti-IL-12/23p40) signaling did not mute T-cell trafficking into the CNS or antiviral effector responses and mice were able to control viral replication within the brain. Therapeutic administration of either anti-IL-23p19 or anti-IL-12/23p40 to mice with viral-induced demyelination did not attenuate T-cell or macrophage infiltration into the CNS nor improve clinical disease or diminish white matter damage. In contrast, treatment of mice with anti-IL-12/23p40 or anti-IL-23p19 resulted in inhibition of the autoimmune model of demyelination, experimental autoimmune encephalomyelitis (EAE). These data indicate that (1) IL-12 and IL-23 signaling are dispensable in generating a protective T-cell response following CNS infection with MHV, and (2) IL-12 and IL-23 do not contribute to demyelination in a model independent of autoimmune T-cell-mediated pathology. Therefore, therapeutic targeting of IL-12 and/or IL-23 for the treatment of autoimmune diseases may offer unique advantages by reducing disease severity without muting protective responses following viral infection.
Assuntos
Sistema Nervoso Central/imunologia , Infecções por Coronavirus/imunologia , Interleucina-12/imunologia , Interleucina-23/imunologia , Vírus da Hepatite Murina/imunologia , Linfócitos T/imunologia , Animais , Doenças Autoimunes , Sistema Nervoso Central/patologia , Infecções por Coronavirus/patologia , Doenças Desmielinizantes/imunologia , Encefalomielite/imunologia , Feminino , CamundongosRESUMO
One of the hallmarks of idiopathic pulmonary fibrosis with a usual interstitial pneumonia histological pathology (IPF/UIP) is excess collagen deposition, due to enhanced fibroblast extracellular matrix synthetic activity. Studies using murine models of lung fibrosis have elucidated a pro-fibrotic pathway involving IL-13 driving CCL2, which in turn drives TGFbeta1 in lung fibroblasts. Therefore, we sought to determine whether this pathway exists in the human fibrotic setting by evaluating human IPF/UIP fibroblasts. IPF/UIP fibroblasts have an increased baseline fibrotic phenotype compared to non-fibrotic fibroblasts. Interestingly, non-fibrotic fibroblasts responded in a pro-fibrotic manner to TGFbeta1 but were relatively non-responsive to IL-13 or CCL2, whereas, IPF/UIP cells were hyper-responsive to TGFbeta1, IL-13 and CCL2. Interestingly, TGFbeta1, CCL2 and IL-13 all upregulated TGFbeta receptor and IL-13 receptor expression, suggesting an ability of the mediators to modulate the function of each other. Furthermore, in vivo, neutralization of both JE and MCP5, the two functional orthologs of CCL2, during bleomycin-induced pulmonary fibrosis significantly reduced collagen deposition as well as JE and CCR2 expression. Also in the bleomycin model, CTGF, which is highly induced following TGFbeta stimulation, was attenuated with anti-JE/anti-MCP5 treatment. Overall this study demonstrates an interplay between TGFbeta1, IL-13 and CCL2 in IPF/UIP, where these three mediators feedback on each other, promoting the fibrotic response.
Assuntos
Quimiocina CCL2/farmacologia , Fibroblastos/efeitos dos fármacos , Fibroblastos/patologia , Interleucina-13/farmacologia , Fibrose Pulmonar/patologia , Fator de Crescimento Transformador beta1/farmacologia , Actinas/metabolismo , Animais , Anticorpos/farmacologia , Linhagem Celular , Colágeno/biossíntese , Feminino , Regulação da Expressão Gênica/efeitos dos fármacos , Humanos , Peptídeos e Proteínas de Sinalização Intercelular/genética , Peptídeos e Proteínas de Sinalização Intercelular/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Proteínas Quimioatraentes de Monócitos/metabolismo , Testes de Neutralização , Fenótipo , Fibrose Pulmonar/genética , Receptores de Fatores de Crescimento/genética , Receptores de Fatores de Crescimento/metabolismoRESUMO
Toll-like receptors are a family of pattern-recognition receptors that contribute to the innate immune response. Toll-like receptor 3 (TLR3) signals in response to foreign, endogenous and synthetic ligands including viral dsRNA, bacterial RNA, mitochondrial RNA, endogenous necrotic cell mRNA and the synthetic dsRNA analog, poly(I:C). We have generated a monoclonal antibody (mAb CNTO2424) that recognizes the extracellular domain (ECD) of human TLR3 in a conformation-dependent manner. CNTO2424 down-regulates poly(I:C)-induced production of IL-6, IL-8, MCP-1, RANTES, and IP-10 in human lung epithelial cells. In addition, mAb CNTO2424 was able to interfere with the known TLR3-dependent signaling pathways, namely NF-kappaB, IRF-3/ISRE, and p38 MAPK. The generation of this neutralizing anti-TLR3 mAb provides a unique tool to better understand TLR3 signaling and potential cross-talk between TLR3 and other molecules.
Assuntos
Anticorpos Monoclonais , Receptor 3 Toll-Like/antagonistas & inibidores , Receptor 3 Toll-Like/imunologia , Animais , Anticorpos Bloqueadores/metabolismo , Anticorpos Monoclonais/metabolismo , Sítios de Ligação de Anticorpos , Linhagem Celular , Linhagem Celular Transformada , Feminino , Humanos , Camundongos , Camundongos Endogâmicos BALB C , Projetos Piloto , Receptor 3 Toll-Like/metabolismoRESUMO
While well established in bacterial hosts, the effect of coding sequence variation on protein expression in mammalian systems is poorly characterized outside of viral proteins or proteins from distant phylogenetic families. The potential impact is substantial given the extensive use of mammalian expression systems in research and manufacturing of protein biotherapeutics. We are studying the effect of codon engineering on expression of recombinant antibodies with an emphasis on developing manufacturing cell lines. CNTO 888, a human mAb specific for human MCP-1, was obtained by antibody phage display in collaboration with MorphoSys AG. The isolated DNA sequence of the antibody was biased towards bacterial codons, reflecting the engineering of the Fab library for phage display expression in Escherichia coli. We compared the expression of CNTO 888 containing the parental V-region sequences with two engineered coding variants. In the native codon exchanged (NCE) variant, the V-region codons were replaced with those used in naturally derived human antibody genes. In the human codon optimized (HCO) variant the V-region codons were those used at the highest frequency based on a human codon usage table. The antibody expression levels from stable transfections in mammalian host cells were measured. The HCO codon variant of CNTO 888 yielded the highest expressing cell lines and the highest average expression for the screened populations. This had a significant positive effect on the process to generate a CNTO 888 production cell line and indicates the potential to improve antibody expression in mammalian expression systems by codon engineering.