RESUMO
The bispecific antibody gefurulimab (also known as ALXN1720) was developed to provide patients with a subcutaneous treatment option for chronic disorders involving activation of the terminal complement pathway. Gefurulimab blocks the enzymatic cleavage of complement component 5 (C5) into the biologically active C5a and C5b fragments, which triggers activation of the terminal complement cascade. Heavy-chain variable region antigen-binding fragment (VHH) antibodies targeting C5 and human serum albumin (HSA) were isolated from llama immune-based libraries and humanized. Gefurulimab comprises an N-terminal albumin-binding VHH connected to a C-terminal C5-binding VHH via a flexible linker. The purified bispecific VHH antibody has the expected exact size by mass spectrometry and can be formulated at greater than 100 mg/mL. Gefurulimab binds tightly to human C5 and HSA with dissociation rate constants at pH 7.4 of 54 pM and 0.9 nM, respectively, and cross-reacts with C5 and serum albumin from cynomolgus monkeys. Gefurulimab can associate with C5 and albumin simultaneously, and potently inhibits the terminal complement activity from human serum initiated by any of the three complement pathways in Wieslab assays. Electron microscopy and X-ray crystallography revealed that the isolated C5-binding VHH recognizes the macroglobulin (MG) 4 and MG5 domains of the antigen and thereby is suggested to sterically prevent C5 binding to its activating convertase. Gefurulimab also inhibits complement activity supported by the rare C5 allelic variant featuring an R885H substitution in the MG7 domain. Taken together, these data suggest that gefurulimab may be a promising candidate for the potential treatment of complement-mediated disorders.
Assuntos
Complemento C5 , Anticorpos de Domínio Único , Humanos , Proteínas do Sistema Complemento/metabolismo , Ativação do Complemento , AlbuminasRESUMO
This study sought to understand the nature of the immune complexes that could be formed when a patient is exposed simultaneously to two different anti-complement component 5 (C5) antibodies, such as in patients converting from one bivalent, noncompetitive, C5-binding monoclonal antibody to another. Size exclusion chromatography (SEC) in combination with multiangle light scattering was used to assess the potential formation of multivalent complexes among eculizumab, C5, and each of two other anti-C5 bivalent antibodies, TPP-2799 or TP-3544, respectively having the same sequence as either crovalimab or pozelimab currently undergoing clinical trials. Each of these two antibodies bound C5 noncompetitively with eculizumab. In phosphate-buffered saline (PBS), C5-eculizumab in the absence of other antibodies measured <500 kDa; however, inclusion of other antibodies at levels ranging from equimolar and up to a fivefold excess over eculizumab and C5 yielded a series of complexes with some >1500 kDa in size, consistent with incorporation of multiple antibodies and C5 molecules. A similar pattern of complexes was also observed when fluorescently labeled eculizumab and either of the other two antibodies were spiked into human plasma, based on SEC monitored by fluorescence detection. A detailed characterization of the pharmacodynamic and pharmacokinetic properties of such complexes is warranted, as is the incorporation of mitigation processes to avoid their formation in patients converting from one bivalent, noncompetitive, C5-binding monoclonal antibody to another.
Assuntos
Complemento C5 , Hemoglobinúria Paroxística , Humanos , Ativação do Complemento , Hemoglobinúria Paroxística/tratamento farmacológico , Anticorpos Monoclonais/uso terapêutico , Complexo Antígeno-AnticorpoRESUMO
Eculizumab, a monoclonal antibody (mAb) directed against complement protein C5, is considered to be the current standard of care for patients with paroxysmal nocturnal hemoglobinuria (PNH) and atypical hemolytic uremic syndrome. This study describes the generation and preclinical attributes of ALXN1210, a new long-acting anti-C5 mAb, obtained through select modifications to eculizumab to both largely abolish target-mediated drug disposition (TMDD) and increase recycling efficiency via the neonatal Fc receptor (FcRn). To attenuate the effect of TMDD on plasma terminal half-life (t1/2), histidine substitutions were engineered into the complementarity-determining regions of eculizumab to enhance the dissociation rate of the mAb:C5 complex in the acidic early endosome relative to the slightly basic pH of blood. Antibody variants with optimal pH-dependent binding to C5 exhibited little to no TMDD in mice in the presence of human C5. To further enhance the efficiency of FcRn-mediated recycling of the antibody, two additional substitutions were introduced to increase affinity for human FcRn. These substitutions yielded an additional doubling of the t½ of surrogate anti-mouse C5 antibodies with reduced TMDD in transgenic mice expressing the human FcRn. In conclusion, ALXN1210 is a promising new therapeutic candidate currently in clinical development for treatment of patients with PNH and atypical hemolytic uremic syndrome.
Assuntos
Anticorpos Monoclonais/química , Anticorpos Monoclonais/farmacologia , Complemento C5/antagonistas & inibidores , Desenho de Fármacos , Animais , Anticorpos Monoclonais/farmacocinética , Afinidade de Anticorpos , Avaliação Pré-Clínica de Medicamentos , Hemólise/imunologia , Antígenos de Histocompatibilidade Classe I/genética , Humanos , Cinética , Camundongos , Camundongos Transgênicos , Ligação Proteica , Receptores Fc/genéticaRESUMO
BACKGROUND: In the rat brain, a single intracerebroventricular injection of neuraminidase from Clostridium perfringens induces ependymal detachment and death. This injury occurs before the infiltration of inflammatory blood cells; some reports implicate the complement system as a cause of these injuries. Here, we set out to test the role of complement. METHODS: The assembly of the complement membrane attack complex on the ependymal epithelium of rats injected with neuraminidase was analyzed by immunohistochemistry. Complement activation, triggered by neuraminidase, and the participation of different activation pathways were analyzed by Western blot. In vitro studies used primary cultures of ependymal cells and explants of the septal ventricular wall. In these models, ependymal cells were exposed to neuraminidase in the presence or absence of complement, and their viability was assessed by observing beating of cilia or by trypan blue staining. The role of complement in ependymal damage induced by neuraminidase was analyzed in vivo in two rat models of complement blockade: systemic inhibition of C5 by using a function blocking antibody and testing in C6-deficient rats. RESULTS: The complement membrane attack complex immunolocalized on the ependymal surface in rats injected intracerebroventricularly with neuraminidase. C3 activation fragments were found in serum and cerebrospinal fluid of rats treated with neuraminidase, suggesting that neuraminidase itself activates complement. In ventricular wall explants and isolated ependymal cells, treatment with neuraminidase alone induced ependymal cell death; however, the addition of complement caused increased cell death and disorganization of the ependymal epithelium. In rats treated with anti-C5 and in C6-deficient rats, intracerebroventricular injection of neuraminidase provoked reduced ependymal alterations compared to non-treated or control rats. Immunohistochemistry confirmed the absence of membrane attack complex on the ependymal surfaces of neuraminidase-exposed rats treated with anti-C5 or deficient in C6. CONCLUSIONS: These results demonstrate that the complement system contributes to ependymal damage and death caused by neuraminidase. However, neuraminidase alone can induce moderate ependymal damage without the aid of complement.
Assuntos
Ventriculite Cerebral/induzido quimicamente , Ventriculite Cerebral/patologia , Complexo de Ataque à Membrana do Sistema Complemento/metabolismo , Epêndima/lesões , Neuraminidase/toxicidade , Animais , Anticorpos/farmacologia , Células Cultivadas , Complemento C3/metabolismo , Complemento C5/imunologia , Complemento C5/metabolismo , Complemento C6/efeitos dos fármacos , Complemento C6/genética , Modelos Animais de Doenças , Epêndima/citologia , Epêndima/patologia , Regulação da Expressão Gênica/efeitos dos fármacos , Regulação da Expressão Gênica/genética , Injeções Intraventriculares , Lectinas/metabolismo , Masculino , Ratos , Ratos Transgênicos , Ratos Wistar , Transdução de Sinais/efeitos dos fármacos , Fatores de Tempo , Vimentina/metabolismoRESUMO
BACKGROUND: Eculizumab is a humanized monoclonal antibody that targets complement protein C5 and inhibits terminal complement-mediated hemolysis associated with paroxysmal nocturnal hemoglobinuria (PNH). The molecular basis for the poor response to eculizumab in a small population of Japanese patients is unclear. METHODS: We assessed the sequences of the gene encoding C5 in patients with PNH who had either a good or poor response to eculizumab. We also evaluated the functional properties of C5 as it was encoded in these patients. RESULTS: Of 345 Japanese patients with PNH who received eculizumab, 11 patients had a poor response. All 11 had a single missense C5 heterozygous mutation, c.2654G â A, which predicts the polymorphism p.Arg885His. The prevalence of this mutation among the patients with PNH (3.2%) was similar to that among healthy Japanese persons (3.5%). This polymorphism was also identified in a Han Chinese population. A patient in Argentina of Asian ancestry who had a poor response had a very similar mutation, c.2653C â T, which predicts p.Arg885Cys. Nonmutant and mutant C5 both caused hemolysis in vitro, but only nonmutant C5 bound to and was blocked by eculizumab. In vitro hemolysis due to nonmutant and mutant C5 was completely blocked with the use of N19-8, a monoclonal antibody that binds to a different site on C5 than does eculizumab. CONCLUSIONS: The functional capacity of C5 variants with mutations at Arg885, together with their failure to undergo blockade by eculizumab, account for the poor response to this agent in patients who carry these mutations. (Funded by Alexion Pharmaceuticals and the Ministry of Health, Labor, and Welfare of Japan.).
Assuntos
Anticorpos Monoclonais Humanizados/uso terapêutico , Complemento C5/genética , Resistência a Medicamentos/genética , Hemoglobinúria Paroxística/genética , Mutação de Sentido Incorreto , Anticorpos Monoclonais Humanizados/farmacocinética , Povo Asiático , Hemoglobinúria Paroxística/tratamento farmacológico , Hemoglobinúria Paroxística/etnologia , Humanos , Japão , Análise de Sequência de DNARESUMO
C3 glomerulopathy is a recently introduced pathological entity whose original definition was glomerular pathology characterized by C3 accumulation with absent or scanty immunoglobulin deposition. In August 2012, an invited group of experts (comprising the authors of this document) in renal pathology, nephrology, complement biology, and complement therapeutics met to discuss C3 glomerulopathy in the first C3 Glomerulopathy Meeting. The objectives were to reach a consensus on: the definition of C3 glomerulopathy, appropriate complement investigations that should be performed in these patients, and how complement therapeutics should be explored in the condition. This meeting report represents the current consensus view of the group.
Assuntos
Complemento C3/análise , Glomerulonefrite/imunologia , Glomérulos Renais/imunologia , Pesquisa Biomédica , Biópsia , Comportamento Cooperativo , Glomerulonefrite/diagnóstico , Glomerulonefrite/terapia , Humanos , Cooperação Internacional , Glomérulos Renais/patologia , Valor Preditivo dos Testes , PrognósticoRESUMO
Carbonic anhydrase IX (CAIX) is a cell surface glycoprotein that is expressed in many different tumors and yet restricted in normal tissues to the gastrointestinal tract. It is upregulated by hypoxia and correlates with tumor grade and poor survival in several tumor indications. Monoclonal antibodies (mAb) with single digit nanomolar binding affinity for CAIX were derived by panning with the recombinant ectodomain of CAIX against the MorphoSys HUCAL Gold library of human Fabs. Highest affinity Fabs were converted to full-length IgGs and subjected to further characterization based upon their avidity and selectivity for CAIX, their capacity to undergo internalization in CAIX-expressing cell lines, and their selective localization to CAIX-positive human xenografted tumors when administered to mice as fluorescent conjugates. Through this selection process, the 3ee9 mAb was identified, which upon conjugation to monomethyl auristatin E through a self-immolative enzyme-cleavable linker yielded the potent and selective CAIX antibody-drug conjugate CAIX-ADC (BAY 79-4620). In preclinical human xenograft models in mice representing several tumor indications, BAY 79-4620 showed potent antitumor efficacy and in some models showed partial and complete tumor shrinkage even following a single dose. The mechanism of action was shown by histology to involve the sequelae of events typical of antitubulin agents. Efficacy in murine preclinical models correlated semiquantitatively, with CAIX expression levels as determined by immunohistochemistry and ELISA. These preclinical data collectively support the development of BAY 79-4620 for the treatment of cancer patients with CAIX overexpressing tumors.
Assuntos
Anticorpos Monoclonais/farmacologia , Antígenos de Neoplasias/metabolismo , Anidrases Carbônicas/metabolismo , Inibidores Enzimáticos/farmacologia , Neoplasias/tratamento farmacológico , Oligopeptídeos/farmacologia , Animais , Anticorpos Monoclonais/imunologia , Anticorpos Monoclonais/metabolismo , Anticorpos Monoclonais/farmacocinética , Antígenos de Neoplasias/imunologia , Western Blotting , Células CHO , Anidrase Carbônica IX , Anidrases Carbônicas/imunologia , Sobrevivência Celular/efeitos dos fármacos , Cricetinae , Cricetulus , Relação Dose-Resposta a Droga , Inibidores Enzimáticos/imunologia , Inibidores Enzimáticos/farmacocinética , Células HCT116 , Células HT29 , Células HeLa , Humanos , Fragmentos Fab das Imunoglobulinas/genética , Fragmentos Fab das Imunoglobulinas/imunologia , Fragmentos Fab das Imunoglobulinas/farmacologia , Camundongos , Camundongos Endogâmicos , Camundongos Nus , Camundongos SCID , Neoplasias/enzimologia , Neoplasias/patologia , Oligopeptídeos/metabolismo , Oligopeptídeos/farmacocinética , Biblioteca de Peptídeos , Distribuição Tecidual , Resultado do Tratamento , Carga Tumoral/efeitos dos fármacos , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Thrombopoietin (TPO) receptor agonists lacking sequence homology to TPO were designed by grafting a known peptide sequence into the hinge and/or kappa constant regions of a human anti-anthrax antibody. Some of these proteins were equipotent to TPO in stimulating cMpl-r activity in vitro and in increasing platelet levels in vivo. ALXN4100TPO (4100TPO), the best agonist in this series with a K(d) of 30 nM for cMpl-r, exhibited potent activity as a radiation countermeasure in CD2F1 mice exposed to lethal total-body radiation from a cobalt-60 γ-ray source. 4100TPO (2 mg/kg, s.c.) administered once either 24 h before or 6 h after TBI showed superior protection to five daily doses given before or after TBI. Prophylactic administration (69 to 94% survival) was superior to therapeutic schedules (60% survival). 4100TPO conferred a significant survival benefit (P < 0.01) when administered 4 days before or even 12 h after exposure and across a dose range of 0.1 to 8 mg/kg. The dose reduction factors (DRFs) with a single dose of 1 mg/kg 4100TPO 24 h before or 12 h after TBI were 1.32 and 1.11, respectively (P < 0.0001). Furthermore, 4100TPO increased bone marrow cellularity and megakaryocytic development and accelerated multi-lineage hematopoietic recovery in irradiated mice, demonstrating the potential of 4100TPO as both a protector and a mitigator in the event of a radiological incident.