RESUMO
Building a strong public health nursing (PHN) work-force capable of advancing population health and reducing inequities is critical. Though undergraduate nursing education is expected to provide introductory knowledge and practice of PHN in Canada, this is not always sufficient to adequately prepare nursing graduates for the complexity of PHN practice. To be practice ready for the full scope of PHN roles and interventions, new baccalaureate nurses and new registered nurses in public health are required to apply PHN competencies, theory, and knowledge of nursing and public health sciences, and to practice within the mandates of provincial and territorial public health legislation. To advance practice readiness a formal continuing education program is essential to foster these critical roles in PHN. This article describes the development of a postgraduate continuing education program for preparation to practice in PHN.
RESUMO
We describe the development of an innovative baccalaureate nursing education strategy for public health nursing. Virtual simulation pedagogy is known to be effective for acute care nursing practice while less known for public health nursing. Three Canadian nursing schools, the Canadian Association of Schools of Nursing (CASN), and the Canadian Alliance of Nurse Educators using Simulation (CAN-Sim) partnered to develop three public health nursing virtual simulation games. Learners work through unfolding population health scenarios, simulating public health nursing practice focused on entry level public health nursing competencies. Each game fosters clinical reasoning and collaborative, community decision-making to respond to population health issues during community assessment, evidence-informed health promotion planning, and evaluation processes. A companion guide was developed to support best practices in implementing virtual simulation and promote optimum student learning using the public health nursing games.
Assuntos
Bacharelado em Enfermagem , Educação em Enfermagem , Estudantes de Enfermagem , Humanos , Enfermagem em Saúde Pública/educação , Canadá , Escolaridade , Instituições Acadêmicas , Competência ClínicaRESUMO
The serum half-life and clearance of therapeutic monoclonal antibodies (mAbs) are critical factors that impact their efficacy and optimal dosing regimen. The pH-dependent binding of an mAb to the neonatal Fc receptor (FcRn) has long been recognized as an important determinant of its pharmacokinetics. However, FcRn affinity alone is not a reliable predictor of mAb half-life, suggesting that other biologic or biophysical mechanisms must be accounted for. mAb thermal stability, which reflects its unfolding and aggregation propensities, may also relate to its pharmacokinetic properties. However, no rigorous statistical regression methods have been used to identify combinations of physical parameters that best predict biologic properties. In this work, a panel of eight mAbs with published human pharmacokinetic data were selected for biophysical analyses of FcRn binding and thermal stability. Biolayer interferometry was used to characterize FcRn/mAb binding at acidic and neutral pH, while differential scanning calorimetry was used to determine thermodynamic unfolding parameters. Individual binding or stability parameters were generally weakly correlated with half-life and clearance values. Least absolute shrinkage and selection operator regression was used to identify the combination of two parameters with the best correlation to half-life and clearance as being the FcRn binding response at pH 7.0 and the change in heat capacity. Leave-one-out subsampling yielded a root mean square difference between observed and predicted half-life of just 2.7 days (16%). Thus, the incorporation of multiple biophysical parameters into a cohesive model may facilitate early-stage prediction of in vivo half-life and clearance based on simple in vitro experiments.
Assuntos
Anticorpos Monoclonais/sangue , Antígenos de Histocompatibilidade Classe I/metabolismo , Imunoglobulina G/sangue , Modelos Biológicos , Receptores Fc/metabolismo , Fenômenos Biofísicos , Meia-Vida , Humanos , Inativação Metabólica , Cinética , Aprendizado de Máquina , Valor Preditivo dos Testes , Ligação ProteicaRESUMO
PURPOSE: To evaluate transcytosis of immunoglobulin G (IgG) by the neonatal Fc receptor (FcRn) in adult primate intestine to determine whether this is a means for oral delivery of monoclonal antibodies (mAbs). METHODS: Relative regional expression of FcRn and localization in human intestinal mucosa by RT-PCR, ELISA & immunohistochemistry. Transcytosis of full-length mAbs (sandwich ELISA-based detection) across human intestinal segments mounted in Ussing-type chambers, human intestinal (caco-2) cell monolayers grown in transwells, and serum levels after regional intestinal delivery in isoflurane-anesthetized cynomolgus monkeys. RESULTS: In human intestine, there was an increasing proximal-distal gradient of mucosal FcRn mRNA and protein expression. In cynomolgus, serum mAb levels were greater after ileum-proximal colon infusion than after administration to stomach or proximal small intestine (1-5 mg/kg). Serum levels of wild-type mAb dosed into ileum/proximal colon (2 mg/kg) were 124 ± 104 ng/ml (n = 3) compared to 48 ± 48 ng/ml (n = 2) after a non-FcRn binding variant. In vitro, mAb transcytosis in polarized caco-2 cell monolayers and was not enhanced by increased apical cell surface IgG binding to FcRn. An unexpected finding in primate small intestine, was intense FcRn expression in enteroendocrine cells (chromagranin A, GLP-1 and GLP-2 containing). CONCLUSIONS: In adult primates, FcRn is expressed more highly in distal intestinal epithelial cells. However, mAb delivery to that region results in low serum levels, in part because apical surface FcRn binding does not influence mAb transcytosis. High FcRn expression in enteroendocrine cells could provide a novel means to target mAbs for metabolic diseases after systemic administration.
Assuntos
Regulação da Expressão Gênica , Antígenos de Histocompatibilidade Classe I/biossíntese , Imunoglobulina G/metabolismo , Mucosa Intestinal/metabolismo , Receptores Fc/biossíntese , Transcitose/fisiologia , Adulto , Animais , Células CACO-2 , Feminino , Humanos , Macaca fascicularis , Masculino , Pessoa de Meia-Idade , Técnicas de Cultura de Órgãos , RNA Mensageiro/biossíntese , Ratos , Adulto JovemRESUMO
TAVO101 is a humanized anti-human thymic stromal lymphopoietin (TSLP) monoclonal antibody under clinical development for the treatment of atopic dermatitis (AD) and other allergic inflammatory conditions. The crystallizable fragment region of the antibody was engineered for half-life extension and attenuated effector functions. This Phase 1, double-blinded, randomized, placebo-controlled study assessed the safety, tolerability, pharmacokinetics, and immunogenicity of TAVO101 in healthy adult subjects in seven ascending dose cohorts. Subjects received a single intravenous administration of TAVO101 or placebo with a 195-day follow-up. TAVO101 was safe and well tolerated. The incidences and severities of treatment-emergent adverse events were mostly mild and comparable between the active and placebo groups, with no trends of dose relationship. There were no severe adverse events, deaths, or treatment-related withdrawals. TAVO101 exhibited a linear pharmacokinetic profile, low clearance, and a median elimination half-life of 67 days in healthy subjects. All TAVO101-treated subjects tested negative for anti-drug antibodies. To support development in AD, TAVO101 was studied in an oxazolone-induced AD model in hTSLP transgenic mice and demonstrated efficacy. This long-acting anti-TSLP antibody has the potential for stronger and sustained allergic inflammatory disease control. The results from this study warranted further clinical development of TAVO101 in patients.
RESUMO
The successful elimination of pathogenic cells and microorganisms by the humoral immune system relies on effective interactions between host immunoglobulins and Fc gamma receptors on effector cells, in addition to the complement system. Essential Ig motifs that direct those interactions reside within the conserved IgG lower hinge/CH2 interface. We noted that a group of tumor-related and microbial proteases cleaved human IgG1s in that region, and the "nick" of just one of the heavy chains profoundly inhibited IgG1 effector functions. We focused on IgG1 monoclonal antibodies (mAbs) since IgG1 is the most abundant human subclass and demonstrates robust Fc-mediated effector functions. The loss of Fc-mediated cell killing activities was correlated with diminished binding to the Fc gamma family of receptors, but a similar decrease in affinity was not observed toward the FcRn receptor that maintains IgG in circulation. Endogenous human IgG cleavage products of comparable size to mAbs with the single cleavage were detected by Western blot analysis in synovial fluid from patients with rheumatoid arthritis and in breast carcinoma extracts. Their detection is problematic under physiological conditions, since there is no loss of structure, and antigen-binding capability is unaffected. These findings suggest that within the hostile proteolytic microenvironments associated with many diseases, key effector functions of host IgGs, or therapeutic Abs, may be compromised.
Assuntos
Imunoglobulina G/química , Imunoglobulina G/metabolismo , Peptídeo Hidrolases/metabolismo , Animais , Anticorpos Monoclonais/química , Anticorpos Monoclonais/metabolismo , Afinidade de Anticorpos , Citotoxicidade Celular Dependente de Anticorpos , Proteínas de Bactérias/metabolismo , Sítios de Ligação , Neoplasias da Mama/enzimologia , Membrana Celular/imunologia , Feminino , Humanos , Fragmentos de Imunoglobulinas/química , Fragmentos de Imunoglobulinas/metabolismo , Técnicas In Vitro , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Ratos , Receptores de IgG/metabolismo , Serina Endopeptidases/metabolismo , Staphylococcus aureus/enzimologia , Streptococcus pyogenes/enzimologiaRESUMO
A number of proteases of potential importance to human physiology possess the ability to selectively degrade and inactivate Igs. Proteolytic cleavage within and near the hinge domain of human IgG1 yielded products including Fab and F(ab')(2) possessing full Ag binding capability but absent several functions needed for immune destruction of cellular pathogens. In parallel experiments, we showed that the same proteolytically generated Fabs and F(ab')(2)s become self-Ags that were widely recognized by autoantibodies in the human population. Binding analyses using various Fab and F(ab')(2), as well as single-chain peptide analogues, indicated that the autoantibodies targeted the newly exposed sequences where proteases cleave the hinge. The point of cleavage may be less of a determinant for autoantibody binding than the exposure of an otherwise cryptic stretch of hinge sequence. It was noted that the autoantibodies possessed an unusually high proportion of the IgG3 isotype in contrast to Abs induced against foreign immunogens in the same human subjects. In light of the recognized potency of IgG3 effector mechanisms, we adopted a functional approach to determine whether human anti-hinge (HAH) autoantibodies could reconstitute the (missing) Fc region effector functions to Fab and F(ab')(2). Indeed, in in vitro cellular assays, purified HAH autoantibodies restored effector functions to F(ab')(2) in both Ab-dependent cellular cytotoxicity and complement-dependent cytotoxicity assays. The results indicate that HAH autoantibodies selectively bind to proteolytically cleaved IgGs and can thereby provide a surrogate Fc domain to reconstitute cell lytic functions.
Assuntos
Autoanticorpos/imunologia , Fragmentos Fab das Imunoglobulinas/imunologia , Imunoglobulina G/metabolismo , Peptídeo Hidrolases/metabolismo , Complexo Antígeno-Anticorpo , Autoanticorpos/metabolismo , Autoantígenos , Sítios de Ligação de Anticorpos , Humanos , Fragmentos Fab das Imunoglobulinas/metabolismoRESUMO
Although it is now clear that certain Fc glycan structures on immunoglobulin G (IgG) antibodies (Abs) can have a dramatic influence on binding to selected Fcgamma receptors (FcgammaR) and on Fc-mediated immune functions, the effects of all known Fc glycan structures still have not been exhaustively studied. We report that in vitro analyses of pairs of monoclonal human IgG Abs that differ in the amount of sialic acid in their Fc glycans revealed that, for each of the three Ab pairs we examined, higher levels of sialylation were associated with reduced activity in Ab-dependent cellular cytotoxicity (ADCC) assays. This relationship between sialylation and ADCC activity was observed regardless of whether the differences in the extent of sialylation were derived by different Ab production processes, use of a lectin column to separate monoclonal Ab preparations into differentially sialylated fractions, or use of direct in vitro glycoengineering methods to convert a lesser sialylated Ab into a highly sialylated Ab. Subsequent investigations revealed that, depending on the individual Ab and how the differences in sialylation were derived, the lower ADCC potency of the more sialylated variants was apparently due to lower-affinity binding to FcgammaRIIIa on natural killer (NK) cells and/or, more interestingly, lower-affinity binding to cell-surface antigen. Our data provide the first example of an Fc glycan structure impacting antigen binding and suggest that avoiding Fc glycan sialylation can offer another means of optimizing ADCC activity of Abs.
Assuntos
Citotoxicidade Imunológica , Fragmentos Fc das Imunoglobulinas/imunologia , Imunoglobulina G/imunologia , Ácido N-Acetilneuramínico/análise , Polissacarídeos/química , Anticorpos Monoclonais/química , Anticorpos Monoclonais/imunologia , Antígenos/imunologia , Sequência de Carboidratos , Células Cultivadas , Testes Imunológicos de Citotoxicidade , Humanos , Fragmentos Fc das Imunoglobulinas/química , Imunoglobulina G/química , Dados de Sequência Molecular , Engenharia de Proteínas , Receptores de IgG/imunologiaRESUMO
BACKGROUND: Tissue factor (TF) expression on islets can result in an instant blood-mediated inflammatory reaction (IBMIR) that contributes to early islet loss. We tested whether peritransplant protection of islets from IBMIR with a monoclonal anti-TF antibody (CNTO859) would enhance engraftment in our nonhuman primate marginal mass model. METHODS: Each of six pairs of cynomolgus monkeys (CM) with streptozotocin-induced diabetes was closely matched for metabolic control and was transplanted with 5,000 IEQ/kg allogeneic, ABO-compatible islets from the same donor under the cover of steroid-free immunosuppression. For each pair, experimental animals received islets cultured with 20 microg/mL anti-TF and were dosed with 6 mg/kg anti-TF intravenously, 10-25 min before islet infusion; control monkeys received an equal number of islets from the same preparation cultured without anti-TF and no in vivo treatment. RESULTS: Early fasting C-peptide (CP) values were different between (P<0.01), but not within, pairs and correlated with in vitro functional capacity of islets as assessed by perifusion (r=0.60; P=0.022). Compared to their matched controls, experimental animals had decreased posttransplant markers of coagulation, higher fasting CP levels (1 month posttransplant and end of study) and prolonged graft function. CONCLUSIONS: These data suggest that pretreatment of islets and the recipient with anti-TF may limit the effects of IBMIR, thereby enhancing islet engraftment and survival.
Assuntos
Sobrevivência de Enxerto/imunologia , Transplante das Ilhotas Pancreáticas/imunologia , Tromboplastina/fisiologia , Tolerância ao Transplante/imunologia , Animais , Anticorpos Monoclonais/imunologia , Anticorpos Monoclonais/farmacologia , Sobrevivência Celular/efeitos dos fármacos , Diabetes Mellitus Experimental/cirurgia , Relação Dose-Resposta a Droga , Fibrinólise/imunologia , Ilhotas Pancreáticas/efeitos dos fármacos , Ilhotas Pancreáticas/fisiologia , Macaca fascicularis , Modelos Biológicos , Estreptozocina , Tromboplastina/efeitos dos fármacosRESUMO
The purpose of this study was to evaluate the pharmacokinetics (PK) of anti-oncostatin M (OSM) IgG1 monoclonal antibodies, CNTO 1119 and its Fc variant (CNTO 8212), which incorporates the LS(Xtend) mutation to extend terminal half-life (T1/2 ), after a single intravenous (IV) or subcutaneous (SC) administration in cynomolgus monkeys, and to predict human PK. In study 1, single doses of CNTO 1119 and CNTO 8212 were administered IV or SC at 3 mg/kg to cynomolgus monkeys (n = 3 per group). In study 2, single doses of CNTO 8212 were administered IV at 1, 5 or 20 mg/kg, or SC at 5 mg/kg to cynomolgus monkeys (n = 5 per group). Serial blood samples were collected for assessment of serum concentrations of CNTO 1119 and/or CNTO 8212. A two-compartment population PK model with first-order elimination was utilized to simultaneously describe the serum concentrations of CNTO 1119 and CNTO 8212 over time after IV and SC administration in cynomolgus monkeys. The typical population PK parameter estimates for CNTO 1119 in cynomolgus monkeys were clearance (CL) = 2.81 mL/day/kg, volume of distribution of central compartment (V1 ) = 31.3 mL/kg, volume of distribution of peripheral compartment (V2 ) = 23.3 mL/kg, absolute bioavailability (F) = 0.84 and T1/2 = 13.4 days. In comparison, the typical population PK parameter estimates for CNTO 8212 in cynomolgus monkeys were CL = 1.41 mL/day/kg, V1 = 39.8 mL/kg, V2 = 32.6 mL/kg, F = 0.75 and T1/2 = 35.7 days. The mean CL of CNTO 8212 was ~50% lower compared with that for CNTO 1119 in cynomolgus monkeys. The overall volume of distribution (V1 +V2 ) for CNTO 8212 was about 32% larger compared with that for CNTO 1119, but generally similar to the vascular volume in cynomolgus monkeys. The T1/2 of CNTO 8212 was significantly (p < 0.05) longer by about 2.7-fold than that for CNTO 1119 in cynomolgus monkeys. Thus, the modification of the Fc portion of an anti-OSM IgG1 mAb for higher FcRn binding affinity resulted in lower systemic clearance and a longer terminal half-life in cynomolgus monkeys. CNTO 8212 demonstrated linear PK after a single IV dose (1-20 mg/kg) in cynomolgus monkeys. The predicted human PK parameters suggest that CNTO 8212 is likely to exhibit slow clearance and long terminal half-life in human beings and may likely allow less frequent dosing in the clinical setting.
Assuntos
Anticorpos Monoclonais/farmacologia , Antígenos de Histocompatibilidade Classe I/metabolismo , Fatores Imunológicos/farmacologia , Oncostatina M/antagonistas & inibidores , Receptores Fc/metabolismo , Administração Intravenosa , Animais , Anticorpos Monoclonais/genética , Disponibilidade Biológica , Desenho de Fármacos , Meia-Vida , Humanos , Fibrose Pulmonar Idiopática/tratamento farmacológico , Fatores Imunológicos/genética , Injeções Subcutâneas , Macaca fascicularis , Masculino , Mutação , Oncostatina M/imunologia , Ligação ProteicaRESUMO
Immunostimulatory receptors belonging to the tumor necrosis factor receptor (TNFR) superfamily are emerging as promising targets for cancer immunotherapies. To optimize the agonism of therapeutic antibodies to these receptors, Fc engineering of antibodies was applied to facilitate the clustering of cell surface TNFRs to activate downstream signaling pathways. One engineering strategy is to identify Fc mutations that facilitate antibody multimerization on the cell surface directly. From the analyses of the crystal packing of IgG1 structures, we identified a novel set of Fc mutations, T437R and K248E, that facilitated antibody multimerization upon binding to antigens on cell surface. In a NF-κB reporter assay, the engineered T437R/K248E mutations could facilitate enhanced agonism of an anti-OX40 antibody without the dependence on FcγRIIB crosslinking. Nonetheless, the presence of cells expressing FcγRIIB could facilitate a boost of the agonism of the engineered antibody with mutations on IgG1 Fc, but not on the silent IgG2σ Fc. The Fc engineered antibody also showed enhanced effector functions, including antibody-dependent cell-meditated cytotoxicity, antibody-dependent cellular phagocytosis, and complement-dependent cytotoxicity, depending on the IgG subtypes. Also, the engineered antibodies showed normal FcRn binding and pharmacokinetic profiles in mice. In summary, this study elucidated a novel Fc engineering approach to promote antibody multimerization on a cell surface, which could enhance agonism and improve effector function for anti-TNFR antibodies as well as other therapeutic antibodies.
Assuntos
Fragmentos Fc das Imunoglobulinas/imunologia , Imunoterapia/métodos , Engenharia de Proteínas/métodos , Receptores OX40/agonistas , Animais , Citotoxicidade Celular Dependente de Anticorpos , Humanos , Camundongos , MutaçãoRESUMO
Methods to rapidly generate high quality bispecific antibodies (BsAb) having normal half-lives are critical for therapeutic programs. Here, we identify 3 mutations (T307P, L309Q, and Q311R or "TLQ") in the Fc region of human IgG1 which disrupt interaction with protein A while enhancing interaction with FcRn. The mutations are shown to incrementally alter the pH at which a mAb elutes from protein A affinity resin. A BsAb comprised of a TLQ mutant and a wild-type IgG1 can be efficiently separated from contaminating parental mAbs by differential protein A elution starting from either a) purified parental mAbs, b) in-supernatant crossed parental mAbs, or c) co-transfected mAbs. We show that the Q311R mutation confers enhanced FcRn interaction in vitro, and Abs harboring either the Q311R or TLQ mutations have serum half-lives as long as wild-type human IgG1. The mutant Abs have normal thermal stability and Fcγ receptor interactions. Together, the results lead to a method for high-throughput generation of BsAbs suitable for in vivo studies.
Assuntos
Anticorpos Biespecíficos/genética , Fragmentos Fc das Imunoglobulinas/genética , Imunoglobulina G/genética , Mutação , Receptores de IgG/química , Proteína Estafilocócica A/química , Sequência de Aminoácidos , Substituição de Aminoácidos , Animais , Anticorpos Biespecíficos/biossíntese , Anticorpos Biespecíficos/química , Anticorpos Biespecíficos/isolamento & purificação , Sítios de Ligação , Cromatografia de Afinidade , Expressão Gênica , Células HEK293 , Meia-Vida , Humanos , Concentração de Íons de Hidrogênio , Fragmentos Fc das Imunoglobulinas/biossíntese , Fragmentos Fc das Imunoglobulinas/química , Fragmentos Fc das Imunoglobulinas/isolamento & purificação , Imunoglobulina G/biossíntese , Imunoglobulina G/química , Imunoglobulina G/isolamento & purificação , Cinética , Camundongos , Modelos Moleculares , Ligação Proteica , Engenharia de Proteínas/métodos , Domínios e Motivos de Interação entre Proteínas , Estabilidade Proteica , Estrutura Secundária de Proteína , Receptores de IgG/imunologia , Receptores de IgG/metabolismo , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/isolamento & purificação , Proteína Estafilocócica A/imunologia , Proteína Estafilocócica A/metabolismoRESUMO
The increased number of bispecific antibodies (BsAb) under therapeutic development has resulted in a need for mouse surrogate BsAbs. Here, we describe a one-step method for generating highly pure mouse BsAbs suitable for in vitro and in vivo studies. We identify two mutations in the mouse IgG2a and IgG2b Fc region: one that eliminates protein A binding and one that enhances protein A binding by 8-fold. We show that BsAbs harboring these mutations can be purified from the residual parental monoclonal antibodies in one step using protein A affinity chromatography. The structural basis for the effects of these mutations was analyzed by X-ray crystallography. While the mutation that disrupted protein A binding also inhibited FcRn interaction, a bispecific mutant in which one subunit retained the ability to bind protein A could still interact with FcRn. Pharmacokinetic analysis of the serum half-lives of the mutants showed that the mutant BsAb had a serum half-life comparable to a wild-type Ab. The results describe a rapid method for generating panels of mouse BsAbs that could be used in mouse studies.
Assuntos
Anticorpos Biespecíficos/imunologia , Anticorpos Monoclonais/imunologia , Antígenos de Histocompatibilidade Classe I/imunologia , Receptores Fc/imunologia , Proteína Estafilocócica A/imunologia , Animais , Anticorpos Biespecíficos/genética , Anticorpos Biespecíficos/metabolismo , Anticorpos Monoclonais/genética , Anticorpos Monoclonais/metabolismo , Cristalografia por Raios X , Antígenos de Histocompatibilidade Classe I/metabolismo , Humanos , Imunoglobulina G/imunologia , Imunoglobulina G/metabolismo , Camundongos , Modelos Moleculares , Proteínas Mutantes/química , Proteínas Mutantes/imunologia , Proteínas Mutantes/metabolismo , Mutação , Ligação Proteica/imunologia , Domínios Proteicos , Receptores Fc/metabolismo , Proteína Estafilocócica A/metabolismoRESUMO
Engineering of fragment crystallizable (Fc) domains of therapeutic immunoglobulin (IgG) antibodies to eliminate their immune effector functions while retaining other Fc characteristics has numerous applications, including blocking antigens on Fc gamma (Fcγ) receptor-expressing immune cells. We previously reported on a human IgG2 variant termed IgG2σ with barely detectable activity in antibody-dependent cellular cytotoxicity, phagocytosis, complement activity, and Fcγ receptor binding assays. Here, we extend that work to IgG1 and IgG4 antibodies, alternative subtypes which may offer advantages over IgG2 antibodies. In several in vitro and in vivo assays, the IgG1σ and IgG4σ variants showed equal or even lower Fc-related activities than the corresponding IgG2σ variant. In particular, IgG1σ and IgG4σ variants demonstrate complete lack of effector function as measured by antibody-dependent cellular cytotoxicity, complement-dependent cytotoxicity, antibody-dependent cellular phagocytosis, and in vivo T-cell activation. The IgG1σ and IgG4σ variants showed acceptable solubility and stability, and typical human IgG1 pharmacokinetic profiles in human FcRn-transgenic mice and cynomolgus monkeys. In silico T-cell epitope analyses predict a lack of immunogenicity in humans. Finally, crystal structures and simulations of the IgG1σ and IgG4σ Fc domains can explain the lack of Fc-mediated immune functions. These variants show promise for use in those therapeutic antibodies and Fc fusions for which the Fc domain should be immunologically "silent".
RESUMO
Cell microencapsulation holds promise for the treatment of many diseases by the continuous delivery of therapeutic products. The biocompatibility of the microcapsules and their biomaterials components is a critical issue for the long-term efficacy of this technology. The objective of this paper is to provide detailed information about the principal factors affecting the biocompatibility of alginates and alginate-poly-l-lysine microcapsules, which are the most frequently employed biomaterials and encapsulation devices for cell immobilization, respectively. Some of these factors include the alginate composition and purification, the selection of the polycation, the interactions between the alginates and the polycation, the microcapsule fabrication process, the uniformity of the devices and the implantation procedure. Improved knowledge will lead to the production of standardized transplantation-grade biomaterials and biocompatible microcapsules.
Assuntos
Alginatos , Materiais Biocompatíveis , Cápsulas , Terapia Baseada em Transplante de Células e Tecidos , Polilisina/análogos & derivados , Alginatos/química , Alginatos/metabolismo , Animais , Materiais Biocompatíveis/química , Materiais Biocompatíveis/metabolismo , Cápsulas/química , Cápsulas/metabolismo , Portadores de Fármacos/química , Portadores de Fármacos/metabolismo , Estrutura Molecular , Tamanho da Partícula , Polilisina/química , Polilisina/metabolismo , Propriedades de SuperfícieRESUMO
Alginate is frequently used for cell encapsulation, but its biocompatibility is neither optimal nor reproducible. Purifying the alginate is critical for achieving a suitable biocompatibility. However, published purification methods vary in efficiency and may induce changes in polymer biofunctionality. Applying X-ray photoelectron spectroscopy, we showed that commercial alginates, purified by in-house and industrial methods, contained elemental impurities that contributed 0.41-1.73% of their atomic composition. Residual contaminants were identified to be proteins (nitrogen/COOH), endotoxins (phosphorus), and fucoidans (sulphur). Studies using attenuated total reflectance Fourier transform infrared spectroscopy suggested that trace contamination did not alter the alginate molecular structure. Alginate hydrophilicity increased by 19-40% after purification, in correlation with a reduction in protein and polyphenol content. Solution viscosity of the alginate increased by 28-108% after purification, in correlation with a reduction in protein content. These results demonstrate that commercial alginates contain potentially immunogenic contaminants that are not completely eliminated by current purification methods. Moreover, these contaminants alter the functional properties of the alginate in a manner that may compromise biocompatibility: Hydrophilicity may affect protein adsorption and solution viscosity influences the morphology of alginate-based microcapsules. These findings highlight the need to improve and better control alginate purity to ensure a reproducible biofunctionality and optimal biocompatibility of alginate and microcapsules.
Assuntos
Alginatos/isolamento & purificação , Materiais Biocompatíveis/isolamento & purificação , Contaminação de Medicamentos , Teste de Materiais , Viscosidade , MolhabilidadeRESUMO
Alginate, a polysaccharide extracted from brown seaweed, is widely used for the microencapsulation of islets of Langerhans, allowing their transplantation without immunosuppression. This natural polymer is known to be largely contaminated. The implantation of islets encapsulated using unpurified alginate leads to the development of fibrotic cell overgrowth around the microcapsules and normalization of the blood glucose is restricted to a very short period if it is achieved at all. Several research groups have developed their own purification method and obtained relatively good results. No comparative evaluation of the efficiencies of these methods has been published. We conducted an evaluative study of five different alginate preparations: a pharmaceutical-grade alginate in its raw state, the same alginate after purification according to three different published methods, and a commercially available purified alginate. The results showed that all purification methods reduced the amounts of known contaminants, that is, polyphenols, endotoxins, and proteins, although with varying efficiencies. Increased viscosity of alginate solutions was observed after purification of the alginates. Despite a general efficiency in decreasing contamination levels, all of the purified alginates contained relatively high residual amounts of protein contaminants. Because proteins may be immunogenic, these residual proteins may have a role in persisting microcapsule immunogenicity.
Assuntos
Alginatos/isolamento & purificação , Contaminação de Medicamentos/prevenção & controle , Fracionamento Químico , Composição de Medicamentos/normas , Endotoxinas/isolamento & purificação , Flavonoides/isolamento & purificação , Humanos , Transplante das Ilhotas Pancreáticas , Teste de Materiais , Fenóis/isolamento & purificação , Polifenóis , Proteínas/isolamento & purificação , ViscosidadeRESUMO
Alginate-poly-L-lysine-alginate (APA) microcapsules are currently being investigated as a means to immuno-isolate transplanted cells, but their biocompatibility is limited. In this study, we verified the hypothesis that poly-L-lysine (PLL), which is immunogenic when unbound, is exposed at the APA microcapsule surface. To do so, we analysed the microcapsule membrane at the micrometric/nanometric scale using attenuated total reflectance Fourier transform infrared spectroscopy, X-ray photoelectron spectroscopy, and time-of-flight secondary ion mass spectrometry. The results indicate that PLL and alginate molecules interact within the membrane. PLL exists in considerable amounts near the surface, contributing to the majority of the carbon within the outermost 100 Angstroms of the membrane. PLL was also detected at the true surface (the outermost monolayer) of the microcapsules. The exposure of PLL does not appear to result from defects in the outer alginate coating. This physicochemical model of APA microcapsules could explain their immunogenicity and will play an important role in the optimization of the microcapsule design.
Assuntos
Alginatos/química , Materiais Biocompatíveis/química , Técnicas de Cultura de Células/métodos , Modelos Químicos , Modelos Moleculares , Nanotecnologia/métodos , Polilisina/química , Alginatos/análise , Materiais Biocompatíveis/análise , Ácido Glucurônico/análise , Ácido Glucurônico/química , Ácidos Hexurônicos/análise , Ácidos Hexurônicos/química , Teste de Materiais/métodos , Microquímica/métodos , Microesferas , Polilisina/análise , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz , Espectrometria por Raios X , Espectroscopia de Infravermelho com Transformada de Fourier , Propriedades de SuperfícieRESUMO
Various oral platelet GPIIb/IIIa receptor antagonists have undergone clinical investigations, but to date without success. Various factors have been proposed to explain their failure such as low affinity for the receptor, large peak/trough ratio, low bioavailability, partial agonist activity and pro-aggregatory effect. Efforts to discover a truly effective, safe, oral antagonist led to the discovery of UR-3216 (Fig. 1). The active form of UR-3216, UR-2922, possessed a high affinity for the human platelet receptor (K(d) <1 nM) with a slow dissociation rate (k(off)= 90 min) in vitro. UR-2922 induced no ligand-induced binding sites (LIBS) expression or prothrombotic activity in human platelets, distinctly different from orbofiban and other small molecule antagonists. To date, UR-2922 is the only high affinity GPIIb/IIIa antagonist without LIBS expression. In vivo characteristics of UR-3216 showed prolonged duration of efficacy (>24 h) with its favorable pharmacokinetic profile, superior to all the other oral GPIIb/IIIa antagonists. UR-3216 showed high bioavailability, rapid bioconversion to the active form and biliary excretion. UR-3216 is a novel, orally active GPIIb/IIIa antagonist of a new generation, which has substantially improved the crucial compounding factors and will be useful for the treatment of cardiovascular diseases.
Assuntos
Nitrocompostos/administração & dosagem , Piperidinas/administração & dosagem , Inibidores da Agregação Plaquetária/administração & dosagem , Complexo Glicoproteico GPIIb-IIIa de Plaquetas/antagonistas & inibidores , Pró-Fármacos/administração & dosagem , Administração Oral , Animais , Humanos , Nitrocompostos/farmacocinética , Nitrocompostos/farmacologia , Piperidinas/farmacocinética , Piperidinas/farmacologia , Inibidores da Agregação Plaquetária/farmacocinética , Inibidores da Agregação Plaquetária/farmacologia , Pró-Fármacos/farmacocinéticaRESUMO
Transgenic mice expressing human neonatal Fc receptor (FcRn) instead of mouse FcRn are available for IgG antibody pharmacokinetic (PK) studies. Given the interest in a rodent model that offers reliable predictions of antibody PK in monkeys and humans, we set out to test whether the PK of IgG antibodies in such mice correlated with the PK of the same antibodies in primates. We began by using a single research antibody to study the influence of: (1) different transgenic mouse lines that differ in FcRn transgene expression; (2) homozygous vs. hemizygous FcRn transgenic mice; (3) the presence vs. absence of coinjected high-dose human intravenous immunoglobulin (IVIG), and (4) the presence vs. absence of coinjected high-dose human serum albumin (HSA). Results of those studies suggested that use of hemizygous Tg32 mice (Tg32 hemi) not treated with IVIG or HSA offered potential as a predictive model for PK in humans. Mouse PK studies were then done under those conditions with a panel of test antibodies whose PK in mice and primates is not significantly affected by target binding, and for which monkey or human PK data were readily available. Results from the studies revealed significant correlations between terminal half-life or clearance values observed in the mice and the corresponding values reported in humans. A significant relationship in clearance values between mice and monkeys was also observed. These correlations suggest that the Tg32 hemi mouse model, which is both convenient and cost-effective, can offer value in predicting antibody half-life and clearance in primates.