RESUMO
Human VH single domains represent a promising class of antibody fragments with applications as therapeutic modalities. Unfortunately, isolated human VH domains also generally display poor biophysical properties and a propensity to aggregate. This has encouraged the development of non-human antibody domains as alternative means of antigen recognition and, in particular, camelid (VHH) domains. Naturally devoid of light chain partners, these domains are characterized by favorable biophysical properties and propensity for cleft binding, a highly desirable characteristic, allowing the targeting of cryptic epitopes. In contrast, previously reported structures of human VH single domains had failed to recapitulate this property. Here we report the engineering and characterization of phage display libraries of stable human VH domains and the selection of binders against a diverse set of antigens. Unlike "camelized" human domains, the domains do not rely on potentially immunogenic framework mutations and maintain the structure of the VH/VL interface. Structure determination in complex with hen egg white lysozyme revealed an extended VH binding interface, with complementarity-determining region 3 deeply penetrating into the active site cleft, highly reminiscent of what has been observed for camelid domains. Taken together, our results demonstrate that fully human VH domains can be constructed that are not only stable and well expressed but also rival the cleft binding properties of camelid antibodies.
Assuntos
Anticorpos/química , Afinidade de Anticorpos , Especificidade de Anticorpos , Sequência de Aminoácidos , Animais , Anticorpos Monoclonais/química , Sequência de Bases , Camelus , Domínio Catalítico , Regiões Determinantes de Complementaridade/química , Ensaio de Imunoadsorção Enzimática , Epitopos/química , Temperatura Alta , Humanos , Cadeias Pesadas de Imunoglobulinas/química , Região Variável de Imunoglobulina/química , Dados de Sequência Molecular , Muramidase/química , Biblioteca de Peptídeos , Ligação Proteica , Engenharia de Proteínas/métodos , Homologia de Sequência de Aminoácidos , Ressonância de Plasmônio de SuperfícieRESUMO
Protein aggregation hinders the development of biologics and underpins the molecular basis of many human diseases. Considerable variation of aggregation propensity exists not only between different proteins, but also within a single homologous family, which complicates analyses. A classic example is observed among human antibody light chains, which aggregate in a clonally specific manner, driven by sequence diversity within their variable domains. Here, we utilise a library versus library strategy, based on phage display and a chemical library of FDA approved drugs, to overcome this limitation. Our approach allowed the identification of small molecule drugs that inhibit the aggregation of the human light chain repertoire. It also provides a general template for the small molecule targeting of diverse protein families.
Assuntos
Técnicas de Visualização da Superfície Celular/métodos , Descoberta de Drogas/métodos , Ensaios de Triagem em Larga Escala/métodos , Cadeias Leves de Imunoglobulina , Agregados Proteicos/efeitos dos fármacos , Humanos , Cadeias Leves de Imunoglobulina/química , Cadeias Leves de Imunoglobulina/efeitos dos fármacos , Cadeias Leves de Imunoglobulina/metabolismoRESUMO
Aggregation limits the recombinant production of many commercially important proteins. We have recently identified mutations that control the aggregation behavior of human antibody variable domains (Dudgeon K., Rouet R., Kokmeijer I., Schofield P., Stolp J., Langley D., Stock D. and Christ D. (2012) Proc Natl Acad Sci USA, 109, 10879-10884. This has allowed the generation of a panel of human antibody variable heavy domains with a defined range of aggregation propensities. Here we utilize this unique resource to validate a previously reported heat-denaturation method on phage (Jespers L., Schon O., Famm K. and Winter G. (2004) Nat Biotechnol, 22, 1161-1165. Our experiments revealed that the method is not only robust in respect to denaturation conditions on phage, but also highly indicative of solution behavior. In particular, it is an excellent predictor of expression and refolding yields.
Assuntos
Técnicas de Visualização da Superfície Celular/métodos , Redobramento de Proteína , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Expressão Gênica , Humanos , Multimerização Proteica , Estrutura Terciária de Proteína , Fatores de TempoRESUMO
Human antibody variable heavy (VH) domains tend to display poor biophysical properties when expressed in isolation. Consequently, the domains are often characterized by low expression levels, high levels of aggregation, and increased "stickiness." Here, we describe methods that allow the engineering of human VH domains with improved biophysical properties by phage display. The engineered domains withstand challenging conditions, such as high temperature and acidic pH. Engineered human single domains are a promising new class of antibody fragments and represent robust research tools and building blocks for the generation of antibody therapeutics.
Assuntos
Técnicas de Visualização da Superfície Celular/métodos , Anticorpos de Domínio Único/química , Expressão Gênica , Biblioteca Gênica , Humanos , Concentração de Íons de Hidrogênio , Dobramento de Proteína , Anticorpos de Domínio Único/genética , Anticorpos de Domínio Único/isolamento & purificação , Solubilidade , TemperaturaRESUMO
Human antibody single domains are a promising new class of antibody fragments. Here we describe methods for the cloning of human V(H) and V(L) genes into phage and phagemid vectors. Furthermore, we provide detailed protocols for the generation of single domain antibody libraries by Kunkel mutagenesis and the analysis of diversity by DNA sequencing and superantigen binding.
Assuntos
Biologia Molecular/métodos , Mutagênese/genética , Anticorpos de Domínio Único/genética , Técnicas de Visualização da Superfície Celular , DNA/biossíntese , Eletroforese em Gel de Ágar , Eletroporação , Ensaio de Imunoadsorção Enzimática , Escherichia coli/metabolismo , Vetores Genéticos/genética , Humanos , Cadeias Pesadas de Imunoglobulinas/genética , Cadeias Leves de Imunoglobulina/genética , Reação em Cadeia da Polimerase , Análise de Sequência de DNA , Moldes Genéticos , Transformação GenéticaRESUMO
The availability of stable human antibody reagents would be of considerable advantage for research, diagnostic, and therapeutic applications. Unfortunately, antibody variable heavy and light domains (V(H) and V(L)) that mediate the interaction with antigen have the propensity to aggregate. Increasing their aggregation resistance in a general manner has proven to be a difficult and persistent problem, due to the high level of sequence diversity observed in human variable domains and the requirement to maintain antigen binding. Here we outline such an approach. By using phage display we identified specific positions that clustered in the antigen binding site (28, 30-33, 35 in V(H) and 24, 49-53, 56 in V(L)). Introduction of aspartate or glutamate at these positions endowed superior biophysical properties (non-aggregating, well-expressed, and heat-refoldable) onto domains derived from common human germline families (V(H)3 and V(κ)1). The effects of the mutations were highly positional and independent of sequence diversity at other positions. Moreover, crystal structures of mutant V(H) and V(L) domains revealed a surprising degree of structural conservation, indicating compatibility with V(H)/V(L) pairing and antigen binding. This allowed the retrofitting of existing binders, as highlighted by the development of robust high affinity antibody fragments derived from the breast cancer therapeutic Herceptin. Our results provide a general strategy for the generation of human antibody variable domains with increased aggregation resistance.
Assuntos
Anticorpos Monoclonais/química , Neoplasias da Mama/terapia , Região Variável de Imunoglobulina/química , Engenharia de Proteínas/métodos , Anticorpos Monoclonais/genética , Anticorpos Monoclonais Humanizados/química , Antineoplásicos/química , Biofísica/métodos , Neoplasias da Mama/genética , Neoplasias da Mama/imunologia , Cristalografia , Desenho de Fármacos , Feminino , Humanos , Região Variável de Imunoglobulina/genética , Mutação/genética , Biblioteca de Peptídeos , TrastuzumabRESUMO
Here we describe protocols for the expression of human antibody fragments in Escherichia coli. Antigen-specific clones are identified by soluble fragment ELISA and concentrated by periplasmic preparation. They are then further purified by affinity chromatography. This article provides an overview of expression and purification strategies for human antibody fragments, as well as detailed protocols for the identification of high-affinity binders and for affinity maturation.
Assuntos
Afinidade de Anticorpos , Escherichia coli/genética , Engenharia Genética/métodos , Fragmentos de Imunoglobulinas/genética , Cromatografia de Afinidade , Eletroforese em Gel de Poliacrilamida , Escherichia coli/metabolismo , Humanos , Fragmentos de Imunoglobulinas/metabolismo , CinéticaRESUMO
Human antibody variable heavy (VH) domains tend to aggregate upon denaturation, for instance, by heat or acid. We have previously demonstrated that domains resisting protein aggregation can be selected from CDR-only repertoires by phage display. Here we analysed their sequences to identify determinants governing protein aggregation. We found that, while many different CDR sequences conferred aggregation-resistance, certain physico-chemical properties were strongly selected for. Thus, hydrophobicity and beta-sheet propensity were significantly lower among the selected domains, whereas net negative charge was increased. Our results provide guidelines for the design of human VH repertoires with reduced levels of protein aggregation.
Assuntos
Cadeias Pesadas de Imunoglobulinas/química , Cadeias Pesadas de Imunoglobulinas/genética , Região Variável de Imunoglobulina/química , Região Variável de Imunoglobulina/genética , Estrutura Terciária de Proteína/genética , Fenômenos Químicos , Evolução Molecular , Humanos , Concentração de Íons de Hidrogênio , Interações Hidrofóbicas e Hidrofílicas , Cadeias Pesadas de Imunoglobulinas/metabolismo , Região Variável de Imunoglobulina/metabolismo , Biblioteca de Peptídeos , Conformação Proteica , Desnaturação Proteica , Estrutura Secundária de Proteína , Alinhamento de Sequência , Análise de Sequência de DNA , TemperaturaRESUMO
A molecular mechanism to explain reduced KAI1 expression in invasive and metastatic tumour cells remains elusive. In this report, we extend an earlier study in bladder cells to confirm that a 76 bp region of the KAI1 promoter (residues -922 to -847), with binding motifs for p53, AP1 and AP2, is required for high level activity of a KAI1 reporter in prostate cancer cell lines. Gel shift and supershift experiments supported binding of p53, junB and heterodimers of AP2alpha/AP2gamma or AP2beta/AP2gamma to this sequence. Introduction of mutations into specific motifs demonstrated an essential requirement for p53 and junB to reporter activity, and that functional synergy between these two factors enhanced activity. A further elevation of reporter activity required AP2. Roles of individual p53, junB and AP2 proteins, as well as functional synergy between p53 and junB, were confirmed in transfection experiments. Western blotting analysis showed that an absence of wild-type p53, and/or a loss of junB and AP2 protein expression, correlated with downregulation of KAI1 mRNA levels in a series of prostate cancer cell lines. A loss of p53 function and/or expression of junB, combined with reduced expression of specific AP2 proteins may underly downregulated KAI1 expression in tumour cells.