RESUMO
Antigen-binding Fc fragments (Fcabs) are a new unique class of immunotherapeutics. They are small (50 kD) fully functional antibody alternatives that bind antigen and elicit effector functions such as antibody-dependent cytotoxicity (ADCC) and complement-dependent cytotoxicity. Since Fcabs carry the natural FcRn binding site of antibodies, they have very favorable pharmacokinetics. We showed recently that Fcab H10-03-6 is a high-affinity binder of Her-2/neu (ErbB2/neu) mediating killing of Her-2/neu-overexpressing tumor cells in the presence of immune effector cells, strongly suggesting that the mechanism of killing is due to ADCC. The present study further confirms ADCC as the mechanism by which H10-03-6 mediates tumor cell killing, since H10-03-6 was shown to interact simultaneously with Her-2/neu and the Fc receptor CD16a. The epitope recognized by H10-03-6 overlaps with that of the clinically used monoclonal antibody trastuzumab. However, unlike trastuzumab, Fcab H10-03-6 did not inhibit proliferation of human tumor cells in vitro even under conditions favoring Her-2/neu crosslinking. Treatment of mice harboring human BT-474 cell xenograft tumors with Fcab H10-03-6 led to statistically significant retardation of tumor growth. For the first time, in vivo properties of an Fcab are presented, supporting the view that Fcabs could become highly efficacious immunotherapeutics for human use.
Assuntos
Anticorpos Monoclonais/química , Antineoplásicos/química , Antineoplásicos/farmacologia , Fragmentos Fc das Imunoglobulinas/química , Fragmentos Fc das Imunoglobulinas/farmacologia , Animais , Anticorpos Monoclonais/metabolismo , Anticorpos Monoclonais/farmacologia , Anticorpos Monoclonais Humanizados/uso terapêutico , Antineoplásicos/metabolismo , Sítios de Ligação , Linhagem Celular , Proliferação de Células/efeitos dos fármacos , Células HEK293 , Humanos , Fragmentos Fc das Imunoglobulinas/metabolismo , Técnicas In Vitro , Camundongos , Transplante de Neoplasias , Receptor ErbB-2/química , Receptor ErbB-2/imunologia , TrastuzumabRESUMO
An Fcab (Fc antigen binding) is a crystallizable fragment of IgG having C-terminal structural loops of CH3 domains engineered for antigen binding. Since introduction of novel binding sites might impair the immunoglobulin fold, repairing strategies are needed for improving the biophysical properties of promising binders without decreasing affinity to the antigen. Here, a directed evolution protocol was developed and applied for stabilization of a Her2/neu-binding Fcab. Distinct loop regions of the parental binder were softly randomized by parsimonious mutagenesis, followed by heat incubation of the yeast displayed protein library and selection for retained antigen binding. Selected Fcabs were expressed solubly in Pichia pastoris and human embryonic kidney 293 cells and characterized. Fcab clones that retained their affinity to Her2/neu but exhibited a significantly increased conformational stability and resistance to aggregation could be evolved. Moreover, we demonstrate that simultaneous selection for binding to the antigen and to structurally specific ligands (FcγRI and an antibody directed against the CH2 domain) yields even more stable Fcabs. To sum up, this study presents a very potent and generally applicable method for improving the fold and stability of antibodies, antibody fragments and alternative binding scaffolds.
Assuntos
Anticorpos Monoclonais/química , Evolução Molecular Direcionada , Fragmentos Fc das Imunoglobulinas/química , Imunoglobulina G/química , Receptor ErbB-2/química , Anticorpos Monoclonais/imunologia , Anticorpos Monoclonais/metabolismo , Antígenos/química , Regulação da Expressão Gênica , Células HEK293 , Humanos , Fragmentos Fc das Imunoglobulinas/imunologia , Fragmentos Fc das Imunoglobulinas/metabolismo , Imunoglobulina G/imunologia , Imunoglobulina G/metabolismo , Pichia , Ligação Proteica , Conformação Proteica , Receptor ErbB-2/imunologia , Receptor ErbB-2/metabolismoRESUMO
Antigen binding immunoglobulin Fc fragments (Fcab) are generated by engineering loop regions in the CH3 domain of human IgG1 Fc. Variants of an Fcab specific for Her-2 were designed to display either enhanced (S239D:A330L:I332E) or diminished (L234A:L235A) binding affinities to the Fc receptor CD16a based on mutations described previously. The two mutant Fcab proteins demonstrated the expected modulation of CD16a binding. Interaction with recombinant or cell surface expressed Her-2 was unaffected in both mutants compared to the parental Fcab. Binding affinities for CD16a correlated with the ADCC-potencies of the Fcab variants. Additional studies indicated that the L234A:L235A variant Fcab had equivalent structural features as the unmodified Fcab since their DSC profiles were similar and antigen binding after re-folding upon partial heat denaturation had not changed. Introduction of the S239D:A330L:I332E mutations resulted in a significant reduction of the CH2 domain melting temperature, a moderate decrease of the thermal transition of the CH3 domain and lower antigen binding after thermal stress compared to the parental Fcab. We conclude that the known correlation between CD16a binding affinity and ADCC potency is also valid in Fcab proteins and that antigen specific Fcab molecules can be further engineered for fine tuning of immuno effector functions.
Assuntos
Citotoxicidade Celular Dependente de Anticorpos , Fragmentos Fc das Imunoglobulinas/imunologia , Imunoglobulina G/imunologia , Receptor ErbB-2/imunologia , Receptores de IgG/imunologia , Linhagem Celular , Células Cultivadas , Humanos , Fragmentos Fc das Imunoglobulinas/química , Fragmentos Fc das Imunoglobulinas/genética , Imunoglobulina G/química , Imunoglobulina G/genética , Modelos Moleculares , Mutação Puntual , Estrutura Terciária de ProteínaRESUMO
Recent studies have demonstrated that IgG-Fc fragments (Fcabs) can be engineered to form antigen-binding sites with antibody properties. Thus they may serve as an attractive alternative to conventional antibodies in therapeutic applications. The critical influence of Fc glycosylation on effector functions of IgGs is well documented; however, whether this applies to Fcabs is not known. Here we used human cells, wild type, and glycoengineered plants to generate four different glycoforms of H10-03-6, an Fcab with engineered HER2/neu-binding sites. Plant-derived H10-03-6 differed in the presence/absence of single oligosaccharide residues, i.e., core fucose and xylose, and terminal galactose. All of the glycoforms had similar binding to HER2/neu expressed on human tumor cells. By contrast, glycoforms that lacked core oligosaccharide modifications (i.e., core α1,3-fucose and ß1,2-xylose) showed significantly enhanced binding to the Fcγ receptor IIIa, irrespective of whether plant or human expression systems were used. Consistent with this finding, plant-derived H10-03-6 glycoforms lacking core N-glycan residues mediated higher antibody-dependent cellular cytotoxicity against human tumor cells. No alteration in γ-receptor binding and antibody-dependent cellular cytotoxicity activity was observed upon decoration of N-glycans by terminal galactose. The results point to a significant impact of distinct N-glycan residues on effector functions of Fcabs. Moreover, the outcomes imply that the effector functions mediated by H10-03-6 can be optimized by altering the N-glycosylation profile. Biasing vaccine-induced immune responses toward optimal Fc glycosylation patterns could result in improved vaccine efficacy.
Assuntos
Anticorpos/química , Anticorpos/metabolismo , Fragmentos Fc das Imunoglobulinas/química , Fragmentos Fc das Imunoglobulinas/metabolismo , Polissacarídeos/química , Linhagem Celular Tumoral , Células Cultivadas , Cromatografia Líquida de Alta Pressão , Citometria de Fluxo , Glicosilação , Humanos , Fragmentos Fc das Imunoglobulinas/genética , Receptores de IgG/genética , Receptores de IgG/metabolismo , Relação Estrutura-Atividade , Ressonância de Plasmônio de Superfície , Nicotiana/genéticaRESUMO
We have constructed IgG1-Fc scaffolds with increased thermal stability by directed evolution and yeast surface display. As a basis a new selection strategy that allowed the application of yeast surface display for screening of stabilizing mutations in proteins of already high intrinsic thermal stability and T(m)-values up to 85°C was developed. Besides library construction by error prone PCR, strong heat stress at 79°C for 10min and screening for well-folded proteins by FACS, sorting rounds had to include an efficient plasmid DNA isolation step for amplification and further transfection. We describe the successful application of this experimental setup for selection of 17 single, double and triple IgG1-Fc variants of increased thermal stability after four selection rounds. The recombinantly produced homodimeric proteins showed a wild-type-like elution profile in size exclusion chromatography as well as content of secondary structures. Moreover, the kinetics of binding of FcRn, CD16a and Protein A to the engineered Fc-molecules was very similar to the wild-type protein. These data clearly demonstrate the importance and efficacy of the presented strategy for selection of stabilizing mutations in proteins of high intrinsic stability within reasonable time.
Assuntos
Evolução Molecular Direcionada , Fragmentos Fc das Imunoglobulinas/genética , Imunoglobulina G/genética , Proteínas Recombinantes de Fusão/genética , Saccharomyces cerevisiae/genética , Varredura Diferencial de Calorimetria , Dicroísmo Circular , Resposta ao Choque Térmico , Humanos , Fragmentos Fc das Imunoglobulinas/biossíntese , Fragmentos Fc das Imunoglobulinas/química , Imunoglobulina G/biossíntese , Imunoglobulina G/química , Mutação de Sentido Incorreto , Biblioteca de Peptídeos , Ligação Proteica , Engenharia de Proteínas , Estabilidade Proteica , Estrutura Secundária de Proteína , Receptores de IgG/química , Proteínas Recombinantes de Fusão/biossíntese , Proteínas Recombinantes de Fusão/química , Saccharomyces cerevisiae/fisiologia , TermodinâmicaRESUMO
Recently, it has been demonstrated that loops of the crystallizable fragment of IgG1 (IgG1-Fc) can be engineered to form antigen-binding sites. In this work C-terminal structural loops in the CH3 domains of homodimeric IgG1-Fc have been functionalized to form integrin-binding sites in order to probe the effect of engineering on structural integrity and thermal stability of IgG1-Fc as well as on binding to the ligands Protein A, CD16 and FcRn, respectively. The peptide sequence GCRGDCL--a disulfide-bridged cyclic heptapeptide that confers binding to human αvß3 integrin was introduced into AB, CD and/or EF loops and single and double mutants were heterologously expressed in Pichia pastoris. Integrin binding of engineered IgG-Fc was tested using both binding to coated αvß3 integrin in ELISA or to αvß3-expressing K562 cells in FACS analysis. Additionally, blocking of αvß3-mediated cell adhesion to vitronectin was investigated. The data presented in this report demonstrate that bioactive integrin-binding peptide(s) can be grafted on the C-terminal loops of IgG-Fc without impairing binding to effector molecules. Observed differences between the investigated variants in structural stability and integrin binding are discussed with respect to the known structure of IgG-Fc and its structural loops.
Assuntos
Regiões Constantes de Imunoglobulina/metabolismo , Imunoglobulina G/metabolismo , Integrinas/metabolismo , Ligação Proteica , Engenharia de Proteínas/métodos , Linhagem Celular Tumoral , Clonagem Molecular , Ensaio de Imunoadsorção Enzimática , Escherichia coli , Citometria de Fluxo , Antígenos de Histocompatibilidade Classe I/metabolismo , Humanos , Regiões Constantes de Imunoglobulina/genética , Imunoglobulina G/genética , Pichia , Receptores Fc/metabolismo , Receptores de IgG/metabolismo , Proteína Estafilocócica A/metabolismoRESUMO
In this work, we present a comprehensive evaluation of the Agilent Bioanalyzer, a microfluidics-based electrophoretic device that was used for impurity testing of a monoclonal antibody (mAb). We compared the system to SDS-PAGE, both operated under non-reducing conditions and found a significant improvement of accuracy for the Bioanalyzer. In addition, the latter exhibited a larger assay range and lower limit of quantitation (LOQ) based on a predefined total error limit of +/-30%. However, during method qualification applying a three-factor nested design with two operators performing duplicate measurements per day, each on 4 different days, we observed unpredictable recurring quantitative outliers using the chip-based system. In-depth analysis on multiple runs with various chip lots confirmed the above finding and indicated that most likely on-chip dye labeling and/or post-column background fluorescence elimination are not compatible with the large size of the intact antibody as similar findings were observed for myosin used as upper marker for time correction. Interestingly, after reducing the intact antibody into light and heavy chain, we resolved the outlier issue. Eventually, requalification of the micro-fabricated analytical device under reducing conditions revealed only 1 out of 32 quality control samples (QCs) exceeding the +/-30% total error limits.
Assuntos
Anticorpos Monoclonais/análise , Contaminação de Medicamentos , Microfluídica/normas , Eletroforese/métodos , Eletroforese/normas , Humanos , Microfluídica/métodosRESUMO
During the analytical characterization of the humanized Lewis-Y specific monoclonal antibody IGN311 (IgG1/kappa) used for passive anti-cancer therapy in humans, isoelectric focusing (IEF) experiments revealed that IGN311 batches produced in serum-containing and serum-free medium, respectively, displayed different banding patterns. The additional bands in the IEF pattern correlated with additional peaks observed by subsequent cation exchange (CEX)-HPLC analysis. Since the IEF pattern is one of the specification criteria in the quality control of monoclonal antibodies and a non-matching pattern may be indicative for lot-to-lot inconsistency, this phenomenon was investigated in detail. First, we investigated whether a difference in antibody glycosylation was the cause for the observed charge heterogeneity. De-N-glycosylation experiments demonstrated that charge heterogeneity observed in the IEF pattern is not a consequence of glycosylation. In contrast, sample treatment by carboxypeptidase B, removing the carboxy-terminal lysine residues from the two heavy chains of the antibody, resulted in reduced charge heterogeneity eliminating the two most basic bands observed in IEF. These data were supported by reversed phase HPLC-MALDI-TOF-MS analysis of enzymatically cleaved peptides of the antibody as well as by carboxy-terminal sequencing of the heavy chains. It was demonstrated that the differences in the IEF banding pattern were due to lysine clipping occurring during the production of the antibody. The antibody batch produced under serum-free conditions was less affected by lysine clipping. Both antibody variants--clipped and unclipped--elicited the same potency in a complement dependent cytotoxicity (CDC) assay demonstrating that lysine clipping of IGN311 does not impair Fc-mediated effector functions.