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1.
J Am Soc Nephrol ; 31(9): 2065-2082, 2020 09.
Artigo em Inglês | MEDLINE | ID: mdl-32764138

RESUMO

BACKGROUND: Circulating APOL1 lyses trypanosomes, protecting against human sleeping sickness. Two common African gene variants of APOL1, G1 and G2, protect against infection by species of trypanosomes that resist wild-type APOL1. At the same time, the protection predisposes humans to CKD, an elegant example of balanced polymorphism. However, the exact mechanism of APOL1-mediated podocyte damage is not clear, including APOL1's subcellular localization, topology, and whether the damage is related to trypanolysis. METHODS: APOL1 topology in serum (HDL particles) and in kidney podocytes was mapped with flow cytometry, immunoprecipitation, and trypanolysis assays that tracked 170 APOL1 domain-specific monoclonal antibodies. APOL1 knockout podocytes confirmed antibody specificity. RESULTS: APOL1 localizes to the surface of podocytes, with most of the pore-forming domain (PFD) and C terminus of the Serum Resistance Associated-interacting domain (SRA-ID), but not the membrane-addressing domain (MAD), being exposed. In contrast, differential trypanolytic blocking activity reveals that the MAD is exposed in serum APOL1, with less of the PFD accessible. Low pH did not detectably alter the gross topology of APOL1, as determined by antibody accessibility, in serum or on podocytes. CONCLUSIONS: Our antibodies highlighted different conformations of native APOL1 topology in serum (HDL particles) and at the podocyte surface. Our findings support the surface ion channel model for APOL1 risk variant-mediated podocyte injury, as well as providing domain accessibility information for designing APOL1-targeted therapeutics.


Assuntos
Apolipoproteína L1/análise , Membrana Celular/química , Podócitos/química , Animais , Anticorpos/imunologia , Especificidade de Anticorpos , Apolipoproteína L1/sangue , Apolipoproteína L1/química , Apolipoproteína L1/imunologia , Células CHO , Cricetulus , Humanos , Concentração de Íons de Hidrogênio , Podócitos/ultraestrutura , Domínios Proteicos
2.
Mol Pharm ; 16(1): 86-95, 2019 01 07.
Artigo em Inglês | MEDLINE | ID: mdl-30444371

RESUMO

The collection of aqueous humor (phase 1 b/2 Mahalo study) from patients dosed intravitreally with anti-factor D (AFD; FCFD4514S, lampalizumab), a humanized antibody fragment previously under investigation to treat geographic atrophy (GA) secondary to age-related macular degeneration, presented a unique opportunity to examine AFD properties in clinical samples. We investigated AFD stability and target-binding characteristics to set up strategies for engineering and evaluating optimized molecules that enable less frequent dosing. Two variants, AFD.v8 and AFD.v14, were evaluated as alternatives to AFD for longer-acting treatments. Mass spectrometry, surface plasmon resonance, and immunoassay were used to assess AFD stability and binding activity in aqueous humor samples from Mahalo patients. In vitro stability and binding activity of AFD, AFD.v8, and AFD.v14 were assessed in human vitreous humor versus buffer at 37 °C over 16 weeks and in vivo in rabbits over 28 days along with pharmacokinetic determinations. In human aqueous humor, AFD specific binding was >85% through 30 days, and deamidation was <3% through 60 days, consistent with the AFD stability and binding activity in vitreous humor from humans in vitro and rabbits in vivo. Target binding, stability, and rabbit pharmacokinetic parameters of AFD.v8 and AFD.v14 were similar to those of AFD. Physiological stability and activity of AFD translated across in vitro and in vivo studies in humans and rabbits. The two variants AFD.v8 and AFD.v14 demonstrated comparable potency and pharmacokinetics. These findings, along with previously demonstrated improved solubility of AFD.v8 and AFD.v14, provide proof-of-concept for developing other similar long-acting therapeutic variants.


Assuntos
Humor Aquoso/metabolismo , Fator D do Complemento/antagonistas & inibidores , Fragmentos Fab das Imunoglobulinas/metabolismo , Animais , Atrofia Geográfica/metabolismo , Humanos , Imunoensaio , Fragmentos Fab das Imunoglobulinas/uso terapêutico , Degeneração Macular/metabolismo , Masculino , Espectrometria de Massas , Coelhos , Ressonância de Plasmônio de Superfície , Corpo Vítreo/metabolismo
3.
Mol Pharm ; 13(9): 2996-3003, 2016 09 06.
Artigo em Inglês | MEDLINE | ID: mdl-27244474

RESUMO

We have developed a tool Fab fragment of a rabbit monoclonal antibody that is useful for early evaluation in rabbit models of technologies for long acting delivery (LAD) of proteins to the eye. Using this Fab we show that vitreal clearance can be slowed through increased hydrodynamic size. Fab (G10rabFab) and Fab' (G10rabFab') fragments of a rabbit monoclonal antibody (G10rabIgG) were expressed in Chinese hamster ovary (CHO) cells and purified using antigen-based affinity chromatography. G10rabFab retains antigen-binding upon thermal stress (37 °C) for 8 weeks in phosphate-buffered saline (PBS) and can be detected in rabbit tissues using an antigen-based ELISA. Hydrodynamic radius, measured using quasi-elastic light scattering (QELS), was increased through site-specific modification of the G10rabFab' free cysteine with linear methoxy-polyethylene glycol(PEG)-maleimide of 20000 or 40000 molecular weight. Pharmacokinetic studies upon intravitreal dosing in New Zealand white rabbits were conducted on the G10rabFab and PEGylated G10rabFab'. Results of single and multidose pharmacokinetic experiments yield reproducible results and a vitreal half-life for G10rabFab of 3.2 days. Clearance from the eye is slowed through increased hydrodynamic size, with vitreal half-life showing a linear dependence on hydrodynamic radius (RH). A linear dependence of vitreal half-life on RH suggests that molecule diffusivity makes an important contribution to vitreal clearance. A method for prediction of vitreal half-life from RH measurements is proposed.


Assuntos
Anticorpos Monoclonais/farmacocinética , Fragmentos Fab das Imunoglobulinas/administração & dosagem , Fragmentos Fab das Imunoglobulinas/metabolismo , Animais , Anticorpos Monoclonais/administração & dosagem , Células CHO , Cricetulus , Ensaio de Imunoadsorção Enzimática , Hidrodinâmica , Injeções Intravítreas , Cinética , Polietilenoglicóis/química , Coelhos
4.
J Immunol ; 193(2): 860-70, 2014 Jul 15.
Artigo em Inglês | MEDLINE | ID: mdl-24935926

RESUMO

Paired Ig-like type 2 receptor (PILR)α inhibitory receptor and its counterpart PILRß activating receptor are coexpressed on myeloid cells. In this article, we report that PILRα, but not PILRß, is elevated in human rheumatoid arthritis synovial tissue and correlates with inflammatory cell infiltration. Pilrα(-/-) mice produce more pathogenic cytokines during inflammation and are prone to enhanced autoimmune arthritis. Correspondingly, engaging PILRα with anti-PILRα mAb ameliorates inflammation in mouse arthritis models and suppresses the production of proinflammatory cytokines. Our studies suggest that PILRα mediates an important inhibitory pathway that can dampen inflammatory responses.


Assuntos
Artrite Experimental/imunologia , Citocinas/imunologia , Inflamação/imunologia , Receptores Imunológicos/imunologia , Animais , Anticorpos Monoclonais/imunologia , Anticorpos Monoclonais/farmacologia , Artrite Experimental/metabolismo , Artrite Experimental/prevenção & controle , Artrite Reumatoide/tratamento farmacológico , Artrite Reumatoide/genética , Artrite Reumatoide/imunologia , Células Cultivadas , Citocinas/metabolismo , Feminino , Citometria de Fluxo , Células HEK293 , Membro Posterior/efeitos dos fármacos , Membro Posterior/imunologia , Membro Posterior/patologia , Humanos , Imuno-Histoquímica , Inflamação/metabolismo , Inflamação/prevenção & controle , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Camundongos Knockout , Análise de Sequência com Séries de Oligonucleotídeos , Osteoartrite/tratamento farmacológico , Osteoartrite/genética , Osteoartrite/imunologia , Receptores Imunológicos/genética , Receptores Imunológicos/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Transcriptoma/genética , Transcriptoma/imunologia
5.
J Neurosci ; 34(19): 6438-47, 2014 May 07.
Artigo em Inglês | MEDLINE | ID: mdl-24806670

RESUMO

In the developing brain, initial neuronal projections are formed through extensive growth and branching of developing axons, but many branches are later pruned to sculpt the mature pattern of connections. Despite its widespread occurrence, the mechanisms controlling pruning remain incompletely characterized. Based on pharmacological and biochemical analysis in vitro and initial genetic analysis in vivo, prior studies implicated a pathway involving binding of the Amyloid Precursor Protein (APP) to Death Receptor 6 (DR6) and activation of a downstream caspase cascade in axonal pruning. Here, we further test their involvement in pruning in vivo and their mechanism of action through extensive genetic and biochemical analysis. Genetic deletion of DR6 was previously shown to impair pruning of retinal axons in vivo. We show that genetic deletion of APP similarly impairs pruning of retinal axons in vivo and provide evidence that APP and DR6 act cell autonomously and in the same pathway to control pruning. Prior analysis had suggested that ß-secretase cleavage of APP and binding of an N-terminal fragment of APP to DR6 is required for their actions, but further genetic and biochemical analysis reveals that ß-secretase activity is not required and that high-affinity binding to DR6 requires a more C-terminal portion of the APP ectodomain. These results provide direct support for the model that APP and DR6 function cell autonomously and in the same pathway to control pruning in vivo and raise the possibility of alternate mechanisms for how APP and DR6 control pruning.


Assuntos
Secretases da Proteína Precursora do Amiloide/fisiologia , Precursor de Proteína beta-Amiloide/genética , Axônios/fisiologia , Receptores do Fator de Necrose Tumoral/genética , Transdução de Sinais/fisiologia , Animais , Animais Geneticamente Modificados , Contagem de Células , Células Cultivadas , Gânglios Espinais/citologia , Gânglios Espinais/fisiologia , Imuno-Histoquímica , Imunoprecipitação , Camundongos , Degeneração Neural/genética , Degeneração Neural/patologia , Ligação Proteica , RNA Interferente Pequeno/genética , Células Ganglionares da Retina/fisiologia , Células Receptoras Sensoriais/fisiologia
6.
Mol Pharm ; 12(6): 1717-29, 2015 Jun 01.
Artigo em Inglês | MEDLINE | ID: mdl-25853436

RESUMO

B7-H4 has been implicated in cancers of the female reproductive system and investigated for its possible use as a biomarker for cancer, but there are no preclinical studies to demonstrate that B7-H4 is a molecular target for therapeutic intervention of cancer. We provide evidence that the prevalence and expression levels of B7-H4 are high in different subtypes of breast cancer and that only a few normal tissues express B7-H4 on the cell membrane. These profiles of low normal expression and upregulation in cancer provide an opportunity for the use of antibody-drug conjugates (ADCs), cytotoxic drugs chemically linked to antibodies, for the treatment of B7-H4 positive cancers. We have developed an ADC specific to B7-H4 that uses a linker drug consisting of a potent antimitotic, monomethyl auristatin E (MMAE), linked to engineered cysteines (THIOMAB) via a protease labile linker. We will refer to ADCs that use the THIOMAB format as TDCs to help distinguish the format from standard MC-vc-MMAE ADCs that are conjugated to the interchain disulfide bonds. Anti-B7-H4 (h1D11)-MC-vc-PAB-MMAE (h1D11 TDC) produced durable tumor regression in cell line and patient-derived xenograft models of triple-negative breast cancer. It also binds rat B7-H4 with similar affinity to human and allowed us to test for target dependent toxicity in rats. We found that our anti-B7-H4 TDC has toxicity findings similar to untargeted TDC. Our results validate B7-H4 as an ADC target for breast cancer and support the possible use of this TDC in the treatment of B7-H4(+) breast cancer.


Assuntos
Antineoplásicos/uso terapêutico , Imunoconjugados/uso terapêutico , Oligopeptídeos/uso terapêutico , Animais , Antineoplásicos/química , Western Blotting , Linhagem Celular Tumoral , Feminino , Citometria de Fluxo , Humanos , Imunoconjugados/química , Imuno-Histoquímica , Camundongos , Camundongos SCID , Oligopeptídeos/química , Ratos , Ratos Sprague-Dawley , Neoplasias de Mama Triplo Negativas/tratamento farmacológico , Ensaios Antitumorais Modelo de Xenoenxerto
7.
J Pharmacol Exp Ther ; 351(3): 527-37, 2014 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-25232192

RESUMO

Anti-factor D (AFD; FCFD4514S, lampalizumab) is a humanized IgG Fab fragment directed against factor D (fD), a rate-limiting serine protease in the alternative complement pathway (AP). Evaluation of AFD as a potential intravitreal (IVT) therapeutic for dry age-related macular degeneration patients with geographic atrophy (GA) is ongoing. However, it is unclear whether IVT administration of AFD can affect systemic AP activation and potentially compromise host-immune responses. We characterized the pharmacologic properties of AFD and assessed the effects of AFD administered IVT (2 or 20 mg) or intravenous (0.2, 2, or 20 mg) on systemic complement activity in cynomolgus monkeys. For the IVT groups, serum AP activity was reduced for the 20 mg dose group between 2 and 6 hours postinjection. For the intravenous groups, AFD inhibited systemic AP activity for periods of time ranging from 5 minutes (0.2 mg group) to 3 hours (20 mg group). Interestingly, the concentrations of total serum fD increased up to 10-fold relative to predose levels following administration of AFD. Furthermore, AFD was found to inhibit systemic AP activity only when the molar concentration of AFD exceeded that of fD. This occurred in cynomolgus monkeys at serum AFD levels ≥2 µg/ml, a concentration 8-fold greater than the maximum serum concentration observed following a single 10 mg IVT dose in a clinical investigation in patients with GA. Based on these findings, the low levels of serum AFD resulting from IVT administration of a clinically relevant dose are not expected to appreciably affect systemic AP activity.


Assuntos
Complemento C3a/antagonistas & inibidores , Fator D do Complemento/antagonistas & inibidores , Fragmentos Fab das Imunoglobulinas/administração & dosagem , Degeneração Macular/tratamento farmacológico , Animais , Bovinos , Complemento C3a/imunologia , Fator D do Complemento/imunologia , Relação Dose-Resposta a Droga , Feminino , Humanos , Fragmentos Fab das Imunoglobulinas/imunologia , Injeções Intravítreas , Macaca fascicularis , Degeneração Macular/sangue , Degeneração Macular/imunologia , Masculino , Camundongos , Resultado do Tratamento
8.
J Biol Chem ; 287(16): 12886-92, 2012 Apr 13.
Artigo em Inglês | MEDLINE | ID: mdl-22362762

RESUMO

By virtue of its amplifying property, the alternative complement pathway has been implicated in a number of inflammatory diseases and constitutes an attractive therapeutic target. An anti-factor D Fab fragment (AFD) was generated to inhibit the alternative complement pathway in advanced dry age-related macular degeneration. AFD potently prevented factor D (FD)-mediated proteolytic activation of its macromolecular substrate C3bB, but not proteolysis of a small synthetic substrate, indicating that AFD did not block access of the substrate to the catalytic site. The crystal structures of AFD in complex with human and cynomolgus FD (at 2.4 and 2.3 Å, respectively) revealed the molecular details of the inhibitory mechanism. The structures show that the AFD-binding site includes surface loops of FD that form part of the FD exosite. Thus, AFD inhibits FD proteolytic function by interfering with macromolecular substrate access rather than by inhibiting FD catalysis, providing the molecular basis of AFD-mediated inhibition of a rate-limiting step in the alternative complement pathway.


Assuntos
Anticorpos/imunologia , Fator D do Complemento/química , Fator D do Complemento/imunologia , Via Alternativa do Complemento/imunologia , Animais , Anticorpos/genética , Anticorpos/metabolismo , Especificidade de Anticorpos , Convertases de Complemento C3-C5/metabolismo , Complemento C3b/metabolismo , Fator D do Complemento/genética , Cristalografia , Ésteres/metabolismo , Humanos , Hibridomas , Fragmentos Fab das Imunoglobulinas/imunologia , Fragmentos Fab das Imunoglobulinas/metabolismo , Macaca fascicularis , Camundongos , Ligação Proteica/imunologia , Estrutura Terciária de Proteína , Proteínas Recombinantes/genética , Proteínas Recombinantes/imunologia , Proteínas Recombinantes/metabolismo
9.
J Biol Chem ; 287(19): 15837-50, 2012 May 04.
Artigo em Inglês | MEDLINE | ID: mdl-22396535

RESUMO

Paired immunoglobulin-like receptor (PILR) α is an inhibitory receptor that recognizes several ligands, including mouse CD99, PILR-associating neural protein, and Herpes simplex virus-1 glycoprotein B. The physiological function(s) of interactions between PILRα and its cellular ligands are not well understood, as are the molecular determinants of PILRα/ligand interactions. To address these uncertainties, we sought to identify additional PILRα ligands and further define the molecular basis for PILRα/ligand interactions. Here, we identify two novel PILRα binding partners, neuronal differentiation and proliferation factor-1 (NPDC1), and collectin-12 (COLEC12). We find that sialylated O-glycans on these novel PILRα ligands, and on known PILRα ligands, are compulsory for PILRα binding. Sialylation-dependent ligand recognition is also a property of SIGLEC1, a member of the sialic acid-binding Ig-like lectins. SIGLEC1 Ig domain shares ∼22% sequence identity with PILRα, an identity that includes a conserved arginine localized to position 97 in mouse and human SIGLEC1, position 133 in mouse PILRα and position 126 in human PILRα. We observe that PILRα/ligand interactions require conserved PILRα Arg-133 (mouse) and Arg-126 (human), in correspondence with a previously reported requirement for SIGLEC1 Arg-197 in SIGLEC1/ligand interactions. Homology modeling identifies striking similarities between PILRα and SIGLEC1 ligand binding pockets as well as at least one set of distinctive interactions in the galactoxyl-binding site. Binding studies suggest that PILRα recognizes a complex ligand domain involving both sialic acid and protein motif(s). Thus, PILRα is evolved to engage multiple ligands with common molecular determinants to modulate myeloid cell functions in anatomical settings where PILRα ligands are expressed.


Assuntos
Evolução Molecular , Glicoproteínas de Membrana/metabolismo , Ácido N-Acetilneuramínico/metabolismo , Receptores Imunológicos/metabolismo , Antígeno 12E7 , Sequência de Aminoácidos , Animais , Antígenos CD/química , Antígenos CD/genética , Antígenos CD/metabolismo , Arginina/química , Arginina/genética , Arginina/metabolismo , Sítios de Ligação/genética , Moléculas de Adesão Celular/química , Moléculas de Adesão Celular/genética , Moléculas de Adesão Celular/metabolismo , Células Cultivadas , Chlorocebus aethiops , Colectinas/química , Colectinas/genética , Colectinas/metabolismo , Sequência Conservada/genética , Células HEK293 , Humanos , Ligantes , Glicoproteínas de Membrana/química , Glicoproteínas de Membrana/genética , Camundongos , Camundongos Endogâmicos C57BL , Modelos Moleculares , Dados de Sequência Molecular , Ácido N-Acetilneuramínico/química , Proteínas do Tecido Nervoso/química , Proteínas do Tecido Nervoso/genética , Proteínas do Tecido Nervoso/metabolismo , Ligação Proteica , Estrutura Terciária de Proteína , Receptores Imunológicos/química , Receptores Imunológicos/genética , Receptores Depuradores/química , Receptores Depuradores/genética , Receptores Depuradores/metabolismo , Homologia de Sequência de Aminoácidos , Lectina 1 Semelhante a Ig de Ligação ao Ácido Siálico , Células Vero
10.
J Exp Med ; 204(6): 1319-25, 2007 Jun 11.
Artigo em Inglês | MEDLINE | ID: mdl-17548523

RESUMO

Complement is an important component of the innate and adaptive immune response, yet complement split products generated through activation of each of the three complement pathways (classical, alternative, and lectin) can cause inflammation and tissue destruction. Previous studies have shown that complement activation through the alternative, but not classical, pathway is required to initiate antibody-induced arthritis in mice, but it is unclear if the alternative pathway (AP) plays a role in established disease. Previously, we have shown that human complement receptor of the immunoglobulin superfamily (CRIg) is a selective inhibitor of the AP of complement. Here, we present the crystal structure of murine CRIg and, using mutants, provide evidence that the structural requirements for inhibition of the AP are conserved in human and mouse. A soluble form of CRIg reversed inflammation and bone loss in two experimental models of arthritis by inhibiting the AP of complement in the joint. Our data indicate that the AP of complement is not only required for disease induction, but also disease progression. The extracellular domain of CRIg thus provides a novel tool to study the effects of inhibiting the AP of complement in established disease and constitutes a promising therapeutic with selectivity for a single complement pathway.


Assuntos
Artrite Experimental/tratamento farmacológico , Reabsorção Óssea/tratamento farmacológico , Modelos Moleculares , Receptores de Complemento/genética , Animais , Artrite Experimental/complicações , Reabsorção Óssea/etiologia , Inativadores do Complemento , Cristalização , Ensaio de Imunoadsorção Enzimática , Humanos , Imuno-Histoquímica , Camundongos , Receptores de Complemento/química
11.
Nature ; 444(7116): 217-20, 2006 Nov 09.
Artigo em Inglês | MEDLINE | ID: mdl-17051150

RESUMO

The complement system is a key part of the innate immune system, and is required for clearance of pathogens from the bloodstream. After exposure to pathogens, the third component of the complement system, C3, is cleaved to C3b which, after recruitment of factor B, initiates formation of the alternative pathway convertases. CRIg, a complement receptor expressed on macrophages, binds to C3b and iC3b mediating phagocytosis of the particles, but it is unknown how CRIg selectively recognizes proteolytic C3-fragments and whether binding of CRIg to C3b inhibits convertase activation. Here we present the crystal structure of C3b in complex with CRIg and, using CRIg mutants, provide evidence that CRIg acts as an inhibitor of the alternative pathway of complement. The structure shows that activation of C3 induces major structural rearrangements, including a dramatic movement (>80 A) of the thioester-bond-containing domain through which C3b attaches to pathogen surfaces. We show that CRIg is not only a phagocytic receptor, but also a potent inhibitor of the alternative pathway convertases. The structure provides insights into the complex macromolecular structural rearrangements that occur during complement activation and inhibition. Moreover, our structure-function studies relating the structural basis of complement activation and the means by which CRIg inhibits the convertases provide important clues to the development of therapeutics that target complement.


Assuntos
Ativação do Complemento , Complemento C3b/química , Complemento C3b/metabolismo , Receptores de Complemento/química , Receptores de Complemento/metabolismo , Convertases de Complemento C3-C5/antagonistas & inibidores , Convertases de Complemento C3-C5/metabolismo , Complemento C3c/química , Complemento C3c/metabolismo , Complemento C5/antagonistas & inibidores , Complemento C5/metabolismo , Cristalografia por Raios X , Humanos , Modelos Moleculares , Mutação/genética , Ligação Proteica , Conformação Proteica , Receptores de Complemento/genética , Receptores de Complemento 3b , Relação Estrutura-Atividade
12.
J Immunol ; 181(11): 7902-8, 2008 Dec 01.
Artigo em Inglês | MEDLINE | ID: mdl-19017980

RESUMO

An important function of the complement cascade is to coat self and foreign particles with C3-proteins that serve as ligands for phagocytic receptors. Although tissue resident macrophages play an important role in complement-mediated clearance, the receptors coordinating this process have not been well characterized. In the present study, we identified a subpopulation of resident peritoneal macrophages characterized by high expression of complement receptor of the Ig superfamily (CRIg), a recently discovered complement C3 receptor. Macrophages expressing CRIg showed significantly increased binding and subsequent internalization of complement-opsonized particles compared with CRIg negative macrophages. CRIg internalized monovalent ligands and was able to bind complement-opsonized targets in the absence of Ca(2+) and Mg(2+), which differs from the beta(2)-integrin CR3 that requires divalent cations and polyvalent ligands for activation of the receptor. Although CRIg dominated in immediate binding of complement-coated particles, CRIg and CR3 contributed independently to subsequent particle phagocytosis. CRIg thus identifies a subset of tissue resident macrophages capable of increased phagocytosis of complement C3-coated particles, a function critical for immune clearance.


Assuntos
Complemento C3/imunologia , Macrófagos/imunologia , Fagocitose/imunologia , Receptores de Complemento/imunologia , Animais , Antígenos CD18/imunologia , Cálcio/imunologia , Regulação da Expressão Gênica/imunologia , Ligantes , Magnésio/imunologia , Camundongos , Camundongos Endogâmicos AKR , Camundongos Knockout , Receptores de Complemento/agonistas
13.
PLoS One ; 14(6): e0218613, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31251757

RESUMO

Innovative protein engineering and chemical conjugation technologies have yielded an impressive number of drug candidates in clinical development including >80 antibody drug conjugates, >60 bispecific antibodies, >35 Fc-fusion proteins and >10 immuno-cytokines. Despite these innovations, technological advances are needed to address unmet medical needs with new pharmacological mechanisms. Age-related eye diseases are among the most common causes of blindness and poor vision in the world. Many such diseases affect the back of the eye, where the inaccessibility of the site of action necessitates therapeutic delivery via intravitreal (IVT) injection. Treatments administered via this route typically have vitreal half-lives <10 days in humans, requiring frequent administration. Since IVT injection is burdensome to patients, there exists a strong need to develop therapeutics with prolonged residence time in the eye. We report here a strategy to increase retention of a therapeutic fragment antibody (Fab) in the eye, using an anti-complement factor D Fab previously optimized for ocular delivery. Polyethylene glycol structures, varying in length, geometry and degree of branching, were coupled to the Fab via maleimide-activated termini. A screening strategy was developed to allow for key determinants of ocular half-life to be measured in vitro. After compound selection, a scalable process was established to enable tolerability and pharmacokinetic studies in cynomolgus monkeys, demonstrating an increase in vitreal half-life with no associated adverse events. Further, we show that the technique for compound selection, analytical characterization, and scalable production is general for a range of antibody fragments. The application of the technology has broad impact in across many therapeutic areas with the first major advancement in the treatment of an important ocular disease.


Assuntos
Portadores de Fármacos/química , Sistemas de Liberação de Medicamentos , Olho , Imunoconjugados/química , Polietilenoglicóis/química , Proteínas/química , Animais , Anticorpos Monoclonais/química , Anticorpos Monoclonais/farmacologia , Avaliação Pré-Clínica de Medicamentos , Olho/efeitos dos fármacos , Feminino , Haplorrinos , Humanos , Imunoconjugados/isolamento & purificação , Imunoconjugados/farmacologia , Fragmentos Fab das Imunoglobulinas/química , Engenharia de Proteínas , Proteínas/isolamento & purificação , Proteínas/farmacologia
14.
MAbs ; 10(4): 624-635, 2018.
Artigo em Inglês | MEDLINE | ID: mdl-29494273

RESUMO

To rapidly find "best-in-class" antibody therapeutics, it has become essential to develop high throughput (HTP) processes that allow rapid assessment of antibodies for functional and molecular properties. Consequently, it is critical to have access to sufficient amounts of high quality antibody, to carry out accurate and quantitative characterization. We have developed automated workflows using liquid handling systems to conduct affinity-based purification either in batch or tip column mode. Here, we demonstrate the capability to purify >2000 antibodies per day from microscale (1 mL) cultures. Our optimized, automated process for human IgG1 purification using MabSelect SuRe resin achieves ∼70% recovery over a wide range of antibody loads, up to 500 µg. This HTP process works well for hybridoma-derived antibodies that can be purified by MabSelect SuRe resin. For rat IgG2a, which is often encountered in hybridoma cultures and is challenging to purify via an HTP process, we established automated purification with GammaBind Plus resin. Using these HTP purification processes, we can efficiently recover sufficient amounts of antibodies from mammalian transient or hybridoma cultures with quality comparable to conventional column purification.


Assuntos
Anticorpos Monoclonais/análise , Cromatografia de Afinidade/métodos , Ensaios de Triagem em Larga Escala/métodos , Imunoglobulina G/análise , Animais , Humanos , Ratos
15.
MAbs ; 9(2): 319-332, 2017.
Artigo em Inglês | MEDLINE | ID: mdl-28001487

RESUMO

Binding interactions with the neonatal Fc receptor (FcRn) are one determinant of pharmacokinetic properties of recombinant human monoclonal antibody (rhumAb) therapeutics, and a conserved binding motif in the crystallizable fragment (Fc) region of IgG molecules interacts with FcRn. Surface plasmon resonance (SPR) biosensor assays are often used to characterize interactions between FcRn and rhumAb therapeutics. In such assays, generally either the rhumAb (format 1) or the FcRn protein (format 2) is immobilized on a biosensor chip. However, because evidence suggests that, in some cases, the variable domains of a rhumAb may also affect FcRn binding, we evaluated the effect of SPR assay configuration on binding data. We sought to assess FcRn binding properties of 2 rhumAbs (rhumAb1 and rhumAb2) to FcRn proteins using these 2 biosensor assay formats. The two rhumAbs have greater than 99% sequence identity in the Fc domain but differ in their Fab regions. rhumAb2 contains a positively charged patch in the variable domain that is absent in rhumAb1. Our results showed that binding of rhumAb1 to FcRn was independent of biosensor assay configuration, while binding of rhumAb2 to FcRn was highly SPR assay configuration dependent. Further investigations revealed that the format dependency of rhumAb2-FcRn binding is linked to the basic residues that form a positively charged patch in the variable domain of rhumAb2. Our work highlights the importance of analyzing rhumAb-FcRn binding interactions using 2 alternate SPR biosensor assay configurations. This approach may also provide a simple way to identify the potential for non-Fc-driven FcRn binding interactions in otherwise typical IgGs.


Assuntos
Anticorpos Monoclonais , Técnicas Biossensoriais/métodos , Antígenos de Histocompatibilidade Classe I , Receptores Fc , Ressonância de Plasmônio de Superfície/métodos , Animais , Anticorpos Monoclonais/química , Anticorpos Monoclonais/imunologia , Afinidade de Anticorpos/imunologia , Antígenos de Histocompatibilidade Classe I/química , Antígenos de Histocompatibilidade Classe I/imunologia , Humanos , Receptores Fc/química , Receptores Fc/imunologia , Proteínas Recombinantes/química , Proteínas Recombinantes/imunologia
17.
MAbs ; 9(8): 1297-1305, 2017.
Artigo em Inglês | MEDLINE | ID: mdl-28854082

RESUMO

To date, ocular antibody therapies for the treatment of retinal diseases rely on injection of the drug into the vitreous chamber of the eye. Given the burden for patients undergoing this procedure, less frequent dosing through the use of long-acting delivery (LAD) technologies is highly desirable. These technologies usually require a highly concentrated formulation and the antibody must be stable against extended exposure to physiological conditions. Here we have increased the potential of a therapeutic antibody antigen-binding fragment (Fab) for LAD by using protein engineering to enhance the chemical and physical stability of the molecule. Structure-guided amino acid substitutions in a negatively charged complementarity determining region (CDR-L1) of an anti-factor D (AFD) Fab resulted in increased chemical stability and solubility. A variant of AFD (AFD.v8), which combines light chain substitutions (VL-D28S:D30E:D31S) with a substitution (VH-D61E) to stabilize a heavy chain isomerization site, retained complement factor D binding and inhibition potency and has properties suitable for LAD. This variant was amenable to high protein concentration (>250 mg/mL), low ionic strength formulation suitable for intravitreal injection. AFD.v8 had acceptable pharmacokinetic (PK) properties upon intravitreal injection in rabbits, and improved stability under both formulation and physiological conditions. Simulations of expected human PK behavior indicated greater exposure with a 25-mg dose enabled by the increased solubility of AFD.v8.


Assuntos
Anticorpos Monoclonais/imunologia , Fragmentos Fab das Imunoglobulinas/imunologia , Engenharia de Proteínas/métodos , Doenças Retinianas/imunologia , Sequência de Aminoácidos , Substituição de Aminoácidos , Animais , Anticorpos Monoclonais/administração & dosagem , Anticorpos Monoclonais/farmacocinética , Afinidade de Anticorpos/imunologia , Fator D do Complemento/imunologia , Regiões Determinantes de Complementaridade/genética , Regiões Determinantes de Complementaridade/imunologia , Sistemas de Liberação de Medicamentos , Estabilidade de Medicamentos , Humanos , Fragmentos Fab das Imunoglobulinas/química , Fragmentos Fab das Imunoglobulinas/genética , Modelos Moleculares , Conformação Proteica , Coelhos , Doenças Retinianas/tratamento farmacológico , Doenças Retinianas/metabolismo
18.
Nat Commun ; 7: 11473, 2016 05 05.
Artigo em Inglês | MEDLINE | ID: mdl-27145901

RESUMO

Viruses encode secreted and cell-surface expressed proteins essential to modulate host immune defenses and establish productive infections. However, to date there has been no systematic study of the extracellular interactome of any human virus. Here we utilize the E3 proteins, diverse and rapidly evolving transmembrane-containing proteins encoded by human adenoviruses, as a model system to survey the extracellular immunomodulatory landscape. From a large-scale protein interaction screen against a microarray of more than 1,500 human proteins, we find and validate 51 previously unidentified virus-host interactions. Our results uncover conserved strategies as well as substantial diversity and multifunctionality in host targeting within and between viral species. Prominent modulation of the leukocyte immunoglobulin-like and signalling lymphocyte activation molecule families and a number of inhibitory receptors were identified as hubs for viral perturbation, suggesting unrecognized immunoregulatory strategies. We describe a virus-host extracellular interaction map of unprecedented scale that provides new insights into viral immunomodulation.


Assuntos
Adenovírus Humanos/imunologia , Imunomodulação/imunologia , Mapas de Interação de Proteínas/imunologia , Proteínas Virais/imunologia , Células A549 , Adenovírus Humanos/metabolismo , Adenovírus Humanos/fisiologia , Animais , Células CHO , Linhagem Celular Tumoral , Células Cultivadas , Cricetulus , Espaço Extracelular/imunologia , Espaço Extracelular/metabolismo , Células HEK293 , Células HeLa , Interações Hospedeiro-Patógeno/imunologia , Humanos , Células Jurkat , Células K562 , Ligação Proteica , Proteoma/imunologia , Proteoma/metabolismo , Proteínas Virais/metabolismo
19.
Clin Cancer Res ; 22(6): 1469-79, 2016 Mar 15.
Artigo em Inglês | MEDLINE | ID: mdl-26589434

RESUMO

PURPOSE: Although agents targeting Delta-like ligand 4 (DLL4) have shown great promise for angiogenesis-based cancer therapy, findings in recent studies have raised serious safety concerns. To further evaluate the potential for therapeutic targeting of the DLL4 pathway, we pursued a novel strategy to reduce toxicities related to DLL4 inhibition by modulating the pharmacokinetic (PK) properties of an anti-DLL4 antibody. EXPERIMENTAL DESIGN: The F(ab')2 fragment of anti-DLL4 antibody (anti-DLL4 F(ab')2) was generated and assessed in efficacy and toxicity studies. RESULTS: Anti-DLL4 F(ab')2 enables greater control over the extent and duration of DLL4 inhibition, such that intermittent dosing of anti-DLL4 F(ab')2 can maintain significant antitumor activity while markedly mitigating known toxicities associated with continuous pathway inhibition. CONCLUSIONS: PK modulation has potentially broad implications for development of antibody-based therapeutics. Our safety studies with anti-DLL4 F(ab')2 also provide new evidence reinforcing the notion that the DLL4 pathway is extremely sensitive to pharmacologic perturbation, further underscoring the importance of exercising caution to safely harness this potent pathway in humans.


Assuntos
Inibidores da Angiogênese/farmacologia , Anticorpos Monoclonais/farmacologia , Antineoplásicos/farmacologia , Peptídeos e Proteínas de Sinalização Intracelular/antagonistas & inibidores , Proteínas de Membrana/antagonistas & inibidores , Inibidores da Angiogênese/efeitos adversos , Inibidores da Angiogênese/farmacocinética , Animais , Anticorpos Monoclonais/efeitos adversos , Anticorpos Monoclonais/farmacocinética , Antineoplásicos/efeitos adversos , Antineoplásicos/farmacocinética , Linhagem Celular Tumoral , Modelos Animais de Doenças , Relação Dose-Resposta a Droga , Feminino , Humanos , Fragmentos Fab das Imunoglobulinas , Fígado/efeitos dos fármacos , Fígado/metabolismo , Fígado/patologia , Macaca fascicularis , Camundongos , Ratos , Carga Tumoral/efeitos dos fármacos , Ensaios Antitumorais Modelo de Xenoenxerto
20.
Invest Ophthalmol Vis Sci ; 56(11): 6991-9, 2015 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-26513505

RESUMO

PURPOSE: In this work, we assessed the ability of fluorophotometry to measure the vitreal pharmacokinetics (PK) of fluorescently-labeled ranibizumab in the rabbit after intravitreal injection. We compared these values to those obtained using enzyme-linked immunosorbent assays (ELISA). Data obtained in this study were also compared to historical ranibizumab ocular PK data, either measured in-house or previously published. METHODS: Three individual in vivo studies were performed in New Zealand White rabbits to assess the feasibility of using fluorophotometry to measure rabbit ocular PK of ranibizumab; explore the dynamic range of dosing fluorescently-labeled ranibizumab; and directly compare ranibizumab concentrations and calculated PK parameters measured by vitreal fluorophotometry to those measured using ELISA. RESULTS: In direct comparisons between fluorophotometry and ELISA, the calculated clearance (CL) values were 0.26 and 0.21 mL/day, the volumes of distribution at steady state (Vss) were 0.80 and 0.94 mL, the half-lives (t1/2) were 3.1 and 2.9 days and the dose normalized areas under the curve (AUC/D) were 4.7 and 3.9 µg·day/mL/µg, respectively. These values fell within the ranges of 0.13 to 0.44 mL/day for CL, 0.5 to 1.8 mL for Vss, 2.8 to 3.5 days for t1/2, and 2.3 to 7.9 µg·day/mL/µg for AUC/D that have been either measured previously in-house or published elsewhere. CONCLUSIONS: Although not suitable for measuring retinal concentrations, fluorophotometry is a valuable, noninvasive method to measure vitreous concentrations of protein therapeutics after intravitreal injection.


Assuntos
Fluorofotometria , Fatores Imunológicos/farmacocinética , Ranibizumab/farmacocinética , Corpo Vítreo/metabolismo , Animais , Ensaio de Imunoadsorção Enzimática , Feminino , Imunofluorescência , Meia-Vida , Fatores Imunológicos/administração & dosagem , Fatores Imunológicos/análise , Injeções Intravítreas , Masculino , Coelhos , Ranibizumab/administração & dosagem , Ranibizumab/análise , Corpo Vítreo/química
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