RESUMEN
Antibody-based drugs, which now represent the dominant biologic therapeutic modality, are used to modulate disparate signaling pathways across diverse disease indications. One fundamental premise that has driven this therapeutic antibody revolution is the belief that each monoclonal antibody exhibits exquisitely specific binding to a single-drug target. Herein, we review emerging evidence in antibody off-target binding and relate current key findings to the risk of failure in therapeutic development. We further summarize the current state of understanding of structural mechanisms underpining the different phenomena that may drive polyreactivity and polyspecificity, and highlight current thinking on how de-risking studies may be best implemented in the screening triage. We conclude with a summary of what we believe to be key observations in the field to date, and a call for the wider antibody research community to work together to build the tools needed to maximize our understanding in this nascent area.
Asunto(s)
Anticuerpos Monoclonales , Anticuerpos Monoclonales/uso terapéutico , Especificidad de Anticuerpos , Factores de RiesgoRESUMEN
We live in an era of rapidly advancing computing capacity and algorithmic sophistication. "Big data" and "artificial intelligence"find progressively wider use in all spheres of human activity, including healthcare. A diverse array of computational technologies is being applied with increasing frequency to antibody drug research and development (R&D). Their successful applications are met with great interest due to the potential for accelerating and streamlining the antibody R&D process. While this excitement is very likely justified in the long term, it is less likely that the transition from the first use to routine practice will escape challenges that other new technologies had experienced before they began to blossom. This transition typically requires many cycles of iterative learning that rely on the deconstruction of the technology to understand its pitfalls and define vectors for optimization. The study by Vasquez et al. identifies a key obstacle to such learning: the lack of transparency regarding methodology in computational antibody design reports, which has the potential to mislead the community efforts.
Asunto(s)
Anticuerpos Monoclonales/farmacología , Diseño de Fármacos , Sitios de Unión de Anticuerpos , Simulación por Computador , Epítopos/química , Humanos , Ingeniería de ProteínasRESUMEN
Monoclonal anti-programmed cell death 1 (PD1) antibodies are successful cancer therapeutics, but it is not well understood why individual antibodies should have idiosyncratic side-effects. As the humanized antibody SHR-1210 causes capillary hemangioma in patients, a unique toxicity amongst anti-PD1 antibodies, we performed human receptor proteome screening to identify nonspecific interactions that might drive angiogenesis. This screen identified that SHR-1210 mediated aberrant, but highly selective, low affinity binding to human receptors such as vascular endothelial growth factor receptor 2 (VEGFR2), frizzled class receptor 5 and UL16 binding protein 2 (ULBP2). SHR-1210 was found to be a potent agonist of human VEGFR2, which may thereby drive hemangioma development via vascular endothelial cell activation. The v-domains of SHR-1210's progenitor murine monoclonal antibody 'Mab005' also exhibited off-target binding and agonism of VEGFR2, proving that the polyspecificity was mediated by the original mouse complementarity-determining regions (CDRs), and had survived the humanization process. Molecular remodelling of SHR-1210 by combinatorial CDR mutagenesis led to deimmunization, normalization of binding affinity to human and cynomolgus PD1, and increased potency in PD1/PD-L1 blockade. Importantly, CDR optimization also ablated all off-target binding, rendering the resulting antibodies fully PD1-specific. As the majority of changes to the paratope were found in the light chain CDRs, the germlining of this domain drove the ablation of off-target binding. The combination of receptor proteome screening and optimization of the antibody binding interface therefore succeeded in generating novel, higher-potency, specificity-enhanced therapeutic IgGs from a single, clinically sub-optimal progenitor. This study showed that highly-specific off-target binding events might be an under-appreciated phenomenon in therapeutic antibody development, but that these unwanted properties can be fully ameliorated by paratope refinement.
Asunto(s)
Anticuerpos Monoclonales Humanizados/inmunología , Especificidad de Anticuerpos/inmunología , Sitios de Unión de Anticuerpos/inmunología , Ingeniería de Proteínas/métodos , Animales , Anticuerpos Monoclonales Humanizados/genética , Especificidad de Anticuerpos/genética , Sitios de Unión de Anticuerpos/genética , Regiones Determinantes de Complementariedad/genética , Regiones Determinantes de Complementariedad/inmunología , Humanos , Macaca fascicularis , Ratones , Receptor de Muerte Celular Programada 1/antagonistas & inhibidores , Receptor 2 de Factores de Crecimiento Endotelial Vascular/agonistasRESUMEN
Antibodies are critical reagents in many fundamental biochemical methods such as affinity chromatography, enzyme-linked immunosorbent assays (ELISA), flow cytometry, western blotting, immunoprecipitation, and immunohistochemistry techniques. As our understanding of the proteome becomes more complex, demand is rising for rapidly generated antibodies of higher specificity than ever before. It is therefore surprising that few investigators have moved beyond the classical methods of antibody production in their search for new reagents. Despite their long-standing efficacy, recombinant antibody generation technologies such as phage display are still largely the tools of biotechnology companies or research groups with a direct interest in protein engineering. In this chapter, we discuss the inherent limitations of classical polyclonal and monoclonal antibody generation and highlight an attractive alternative: generating high-specificity, high-affinity recombinant antibodies from alternative immune sources such as chickens, via phage display.
Asunto(s)
Técnicas de Visualización de Superficie Celular , Animales , Anticuerpos , Afinidad de Anticuerpos , Especificidad de Anticuerpos , Pollos/inmunología , Cromatografía de Afinidad , Proteínas Recombinantes de Fusión , Anticuerpos de Cadena ÚnicaRESUMEN
High-affinity, highly specific binding proteins are a key class of molecules used in the development of new affinity chromatography methods. Traditionally, antibody-based methods have relied on the use of immunoglobulins purified from immune animal sera, from egg yolks, or from murine monoclonal hybridoma supernatants. To accelerate and refine the reagent antibody generation process, we have developed optimized methods that allow the rapid assembly of scFv libraries from chickens immunized with pools of immunogens. These methods allow the simplified generation of a single, moderately sized library of single chain Fv (scFv) and the subsequent isolation of diverse, high affinity, and high specificity monoclonals for each individual immunogen, via phage display. Using these methods, antibodies can be derived that exhibit the desired selectivity, including exquisite specificity or cross-reactivity to multiple orthologues of the same protein.
Asunto(s)
Afinidad de Anticuerpos/inmunología , Especificidad de Anticuerpos/inmunología , Técnicas de Visualización de Superficie Celular , Anticuerpos de Cadena Única/biosíntesis , Anticuerpos de Cadena Única/inmunología , Animales , Antígenos/inmunología , Pollos/inmunología , Epítopos/inmunología , Ratones , Biblioteca de Péptidos , Proteínas Recombinantes de Fusión , Anticuerpos de Cadena Única/química , Anticuerpos de Cadena Única/genéticaRESUMEN
Fully-human single-chain Fv (scFv) proteins are key potential building blocks of bispecific therapeutic antibodies, but they often suffer from manufacturability and clinical development limitations such as instability and aggregation. The causes of these scFv instability problems, in proteins that should be theoretically stable, remains poorly understood. To inform the future development of such molecules, we carried out a comprehensive structural analysis of the highly stabilized anti-CXCL13 scFv E10. E10 was derived from the parental 3B4 using complementarity-determining region (CDR)-restricted mutagenesis and tailored selection and screening strategies, and carries four mutations in VL-CDR3. High-resolution crystal structures of parental 3B4 and optimized E10 scFvs were solved in the presence and absence of human CXCL13. In parallel, a series of scFv mutants was generated to interrogate the individual contribution of each of the four mutations to stability and affinity improvements. In combination, these analyses demonstrated that the optimization of E10 was primarily mediated by removing clashes between both the VL and the VH, and between the VL and CXCL13. Importantly, a single, germline-encoded VL-CDR3 residue mediated the key difference between the stable and unstable forms of the scFv. This work demonstrates that, aside from being the critical mediators of specificity and affinity, CDRs may also be the primary drivers of biotherapeutic developability.
Asunto(s)
Productos Biológicos/química , Quimiocina CXCL13/antagonistas & inhibidores , Modelos Moleculares , Anticuerpos de Cadena Única/química , Sustitución de Aminoácidos , Afinidad de Anticuerpos , Especificidad de Anticuerpos , Complejo Antígeno-Anticuerpo/química , Complejo Antígeno-Anticuerpo/metabolismo , Sitios de Unión de Anticuerpos , Productos Biológicos/metabolismo , Quimiocina CXCL13/química , Quimiocina CXCL13/metabolismo , Regiones Determinantes de Complementariedad/química , Regiones Determinantes de Complementariedad/genética , Regiones Determinantes de Complementariedad/metabolismo , Humanos , Cinética , Mutación , Agregado de Proteínas , Conformación Proteica , Estabilidad Proteica , Proteínas Recombinantes de Fusión/química , Proteínas Recombinantes de Fusión/metabolismo , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Anticuerpos de Cadena Única/genética , Anticuerpos de Cadena Única/metabolismo , Solubilidad , Difracción de Rayos XRESUMEN
Bispecific antibodies offer a promising approach for the treatment of cancer but can be challenging to engineer and manufacture. Here we report the development of PF-06671008, an extended-half-life dual-affinity re-targeting (DART®) bispecific molecule against P-cadherin and CD3 that demonstrates antibody-like properties. Using phage display, we identified anti-P-cadherin single chain Fv (scFv) that were subsequently affinity-optimized to picomolar affinity using stringent phage selection strategies, resulting in low picomolar potency in cytotoxic T lymphocyte (CTL) killing assays in the DART format. The crystal structure of this disulfide-constrained diabody shows that it forms a novel compact structure with the two antigen binding sites separated from each other by approximately 30 Å and facing approximately 90° apart. We show here that introduction of the human Fc domain in PF-06671008 has produced a molecule with an extended half-life (-4.4 days in human FcRn knock-in mice), high stability (Tm1 > 68 °C), high expression (>1 g/L), and robust purification properties (highly pure heterodimer), all with minimal impact on potency. Finally, we demonstrate in vivo anti-tumor efficacy in a human colorectal/human peripheral blood mononuclear cell (PBMC) co-mix xenograft mouse model. These results suggest PF-06671008 is a promising new bispecific for the treatment of patients with solid tumors expressing P-cadherin.
RESUMEN
Although humanized antibodies have been highly successful in the clinic, all current humanization techniques have potential limitations, such as: reliance on rodent hosts, immunogenicity due to high non-germ-line amino acid content, v-domain destabilization, expression and formulation issues. This study presents a technology that generates stable, soluble, ultrahumanized antibodies via single-step complementarity-determining region (CDR) germ-lining. For three antibodies from three separate key immune host species, binary substitution CDR cassettes were inserted into preferred human frameworks to form libraries in which only the parental or human germ-line destination residue was encoded at each position. The CDR-H3 in each case was also augmented with 1 ± 1 random substitution per clone. Each library was then screened for clones with restored antigen binding capacity. Lead ultrahumanized clones demonstrated high stability, with affinity and specificity equivalent to, or better than, the parental IgG. Critically, this was mainly achieved on germ-line frameworks by simultaneously subtracting up to 19 redundant non-germ-line residues in the CDRs. This process significantly lowered non-germ-line sequence content, minimized immunogenicity risk in the final molecules and provided a heat map for the essential non-germ-line CDR residue content of each antibody. The ABS technology therefore fully optimizes the clinical potential of antibodies from rodents and alternative immune hosts, rendering them indistinguishable from fully human in a simple, single-pass process.
Asunto(s)
Anticuerpos Monoclonales/inmunología , Anticuerpos Monoclonales/uso terapéutico , Regiones Determinantes de Complementariedad/inmunología , Células Germinativas/inmunología , Secuencia de Aminoácidos , Animales , Anticuerpos Monoclonales/química , Especificidad de Anticuerpos/inmunología , Células Clonales , Regiones Determinantes de Complementariedad/química , Simulación por Computador , Ensayo de Inmunoadsorción Enzimática , Epítopos de Linfocito T/inmunología , Humanos , Inmunoglobulina G/química , Inmunoglobulina G/inmunología , Cadenas Pesadas de Inmunoglobulina/química , Cadenas Pesadas de Inmunoglobulina/inmunología , Cadenas Ligeras de Inmunoglobulina/química , Cadenas Ligeras de Inmunoglobulina/inmunología , Región Variable de Inmunoglobulina/química , Región Variable de Inmunoglobulina/inmunología , Modelos Moleculares , Datos de Secuencia Molecular , Mutación/genética , Biblioteca de Péptidos , Estabilidad Proteica , Estructura Terciaria de Proteína , Ratas , Alineación de Secuencia , Análisis de Secuencia de Proteína , Proteínas tau/química , Proteínas tau/inmunologíaRESUMEN
While myriad molecular formats for bispecific antibodies have been examined to date, the simplest structures are often based on the scFv. Issues with stability and manufacturability in scFv-based bispecific molecules, however, have been a significant hindrance to their development, particularly for high-concentration, stable formulations that allow subcutaneous delivery. Our aim was to generate a tetravalent bispecific molecule targeting two inflammatory mediators for synergistic immune modulation. We focused on an scFv-Fc-scFv format, with a flexible (A4T)3 linker coupling an additional scFv to the C-terminus of an scFv-Fc. While one of the lead scFvs isolated directly from a naïve library was well-behaved and sufficiently potent, the parental anti-CXCL13 scFv 3B4 required optimization for affinity, stability, and cynomolgus ortholog cross-reactivity. To achieve this, we eschewed framework-based stabilizing mutations in favor of complementarity-determining region (CDR) mutagenesis and re-selection for simultaneous improvements in both affinity and thermal stability. Phage-displayed 3B4 CDR-mutant libraries were used in an aggressive "hammer-hug" selection strategy that incorporated thermal challenge, functional, and biophysical screening. This approach identified leads with improved stability and>18-fold, and 4,100-fold higher affinity for both human and cynomolgus CXCL13, respectively. Improvements were exclusively mediated through only 4 mutations in VL-CDR3. Lead scFvs were reformatted into scFv-Fc-scFvs and their biophysical properties ranked. Our final candidate could be formulated in a standard biopharmaceutical platform buffer at 100 mg/ml with<2% high molecular weight species present after 7 weeks at 4 °C and viscosity<15 cP. This workflow has facilitated the identification of a truly manufacturable scFv-based bispecific therapeutic suitable for subcutaneous administration.
Asunto(s)
Anticuerpos Biespecíficos/genética , Regiones Determinantes de Complementariedad/genética , Ingeniería de Proteínas , Anticuerpos de Cadena Única/química , Anticuerpos de Cadena Única/metabolismo , Animales , Bacteriófagos/genética , Ensayo de Inmunoadsorción Enzimática , Femenino , Humanos , Inyecciones Subcutáneas , Biblioteca de Péptidos , Estabilidad Proteica , Ratas , Anticuerpos de Cadena Única/genética , TemperaturaRESUMEN
We have generated large libraries of single-chain Fv antibody fragments (>10(10) transformants) containing unbiased amino acid diversity that is restricted to the central combining site of the stable, well-expressed DP47 and DPK22 germline V-genes. Library WySH2A was constructed to examine the potential for synthetic complementarity-determining region (CDR)-H3 diversity to act as the lone source of binding specificity. Library WySH2B was constructed to assess the necessity for diversification in both the H3 and L3. Both libraries provided diverse, specific antibodies, yielding a total of 243 unique hits against 7 different targets, but WySH2B produced fewer hits than WySH2A when selected in parallel. WySH2A also consistently produced hits of similar quality to WySH2B, demonstrating that the diversification of the CDR-L3 reduces library fitness. Despite the absence of deliberate bias in the library design, CDR length was strongly associated with the number of hits produced, leading to a functional loop length distribution profile that mimics the biases observed in the natural repertoire. A similar trend was also observed for the CDR-L3. After target selections, several key amino acids were enriched in the CDR-H3 (e.g., small and aromatic residues) while others were reduced (e.g., strongly charged residues) in a manner that was specific to position, preferentially occurred in CDR-H3 stem positions, and tended towards residues associated with loop stabilization. As proof of principle for the WySH2 libraries to produce viable lead candidate antibodies, 114 unique hits were produced against Delta-like ligand 4 (DLL4). Leads exhibited nanomolar binding affinities, highly specific staining of DLL4+ cells, and biochemical neutralization of DLL4-NOTCH1 interaction.
Asunto(s)
Especificidad de Anticuerpos , Regiones Determinantes de Complementariedad/inmunología , Regiones Determinantes de Complementariedad/uso terapéutico , Biblioteca de Péptidos , Anticuerpos de Cadena Única/biosíntesis , Anticuerpos de Cadena Única/uso terapéutico , Proteínas Adaptadoras Transductoras de Señales , Animales , Especificidad de Anticuerpos/genética , Proteínas de Unión al Calcio , Clonación Molecular , Regiones Determinantes de Complementariedad/genética , Células HEK293 , Humanos , Péptidos y Proteínas de Señalización Intercelular/genética , Péptidos y Proteínas de Señalización Intercelular/inmunología , Ratones , Modelos Moleculares , Mutación , Receptor Notch1/antagonistas & inhibidores , Receptor Notch1/genética , Receptor Notch1/inmunología , Anticuerpos de Cadena Única/genéticaRESUMEN
Antibodies are the fastest-growing segment of the biologics market. The success of antibody-based drugs resides in their exquisite specificity, high potency, stability, solubility, safety, and relatively inexpensive manufacturing process in comparison with other biologics. We outline here the structural studies and fundamental principles that define how antibodies interact with diverse targets. We also describe the antibody repertoires and affinity maturation mechanisms of humans, mice, and chickens, plus the use of novel single-domain antibodies in camelids and sharks. These species all utilize diverse evolutionary solutions to generate specific and high affinity antibodies and illustrate the plasticity of natural antibody repertoires. In addition, we discuss the multiple variations of man-made antibody repertoires designed and validated in the last two decades, which have served as tools to explore how the size, diversity, and composition of a repertoire impact the antibody discovery process.
RESUMEN
Highly specific antibodies to phosphoepitopes are valuable tools to study phosphorylation in disease states, but their discovery is largely empirical, and the molecular mechanisms mediating phosphospecific binding are poorly understood. Here, we report the generation and characterization of extremely specific recombinant chicken antibodies to three phosphoepitopes on the Alzheimer disease-associated protein tau. Each antibody shows full specificity for a single phosphopeptide. The chimeric IgG pT231/pS235_1 exhibits a K(D) of 0.35 nm in 1:1 binding to its cognate phosphopeptide. This IgG is murine ortholog-cross-reactive, specifically recognizing the pathological form of tau in brain samples from Alzheimer patients and a mouse model of tauopathy. To better understand the underlying binding mechanisms allowing such remarkable specificity, we determined the structure of pT231/pS235_1 Fab in complex with its cognate phosphopeptide at 1.9 Å resolution. The Fab fragment exhibits novel complementarity determining region (CDR) structures with a "bowl-like" conformation in CDR-H2 that tightly and specifically interacts with the phospho-Thr-231 phosphate group, as well as a long, disulfide-constrained CDR-H3 that mediates peptide recognition. This binding mechanism differs distinctly from either peptide- or hapten-specific antibodies described to date. Surface plasmon resonance analyses showed that pT231/pS235_1 binds a truly compound epitope, as neither phosphorylated Ser-235 nor free peptide shows any measurable binding affinity.
Asunto(s)
Enfermedad de Alzheimer/metabolismo , Anticuerpos/inmunología , Epítopos/inmunología , Proteínas tau/inmunología , Enfermedad de Alzheimer/genética , Secuencia de Aminoácidos , Animales , Anticuerpos/química , Anticuerpos/genética , Encéfalo/metabolismo , Pollos , Epítopos/química , Epítopos/genética , Humanos , Inmunoglobulina G/química , Inmunoglobulina G/genética , Inmunoglobulina G/inmunología , Ratones , Ratones Transgénicos , Datos de Secuencia Molecular , Fosforilación , Proteínas tau/química , Proteínas tau/genética , Proteínas tau/metabolismoRESUMEN
Examination of 1269 unique naive chicken V(H) sequences showed that the majority of positions in the framework (FW) regions were maintained as germline, with high mutation rates observed in the CDRs. Many FW mutations could be clearly related to the modulation of CDR structure or the V(H)-V(L) interface. CDRs 1 and 2 of the V(H) exhibited frequent mutation in solvent-exposed positions, but conservation of common structural residues also found in human CDRs at the same positions. In comparison with humans and mice, the chicken CDR3 repertoire was skewed toward longer sequences, was dominated by small amino acids (G/S/A/C/T), and had higher cysteine (chicken, 9.4%; human, 1.6%; and mouse, 0.25%) but lower tyrosine content (chicken, 9.2%; human, 16.8%; and mouse 26.4%). A strong correlation (R(2) = 0.97) was observed between increasing CDR3 length and higher cysteine content. This suggests that noncanonical disulfides are strongly favored in chickens, potentially increasing CDR stability and complexity in the topology of the combining site. The probable formation of disulfide bonds between CDR3 and CDR1, FW2, or CDR2 was also observed, as described in camelids. All features of the naive repertoire were fully replicated in the target-selected, phage-displayed repertoire. The isolation of a chicken Fab with four noncanonical cysteines in the V(H) that exhibits 64 nM (K(D)) binding affinity for its target proved these constituents to be part of the humoral response, not artifacts. This study supports the hypothesis that disulfide bond-constrained CDR3s are a structural diversification strategy in the restricted germline v-gene repertoire of chickens.
Asunto(s)
Sustitución de Aminoácidos , Pollos/genética , Regiones Determinantes de Complementariedad/genética , Cadenas Pesadas de Inmunoglobulina/genética , Mutación , Animales , Afinidad de Anticuerpos/genética , Camelus/genética , Camelus/inmunología , Pollos/inmunología , Regiones Determinantes de Complementariedad/inmunología , Disulfuros/inmunología , Humanos , Cadenas Pesadas de Inmunoglobulina/inmunología , Ratones , Estabilidad Proteica , Especificidad de la EspecieRESUMEN
We present a method for synthetic antibody library generation that combines the use of high-throughput immune repertoire analysis and a novel synthetic technology. The library design recapitulates positional amino acid frequencies observed in natural antibody repertoires. V-segment diversity in four heavy (V(H)) and two kappa (V(κ)) germlines was introduced based on the analysis of somatically hypermutated donor-derived repertoires. Complementarity-determining region 3 length and amino acid designs were based on aggregate frequencies of all V(H) and V(κ) sequences in the data set. The designed libraries were constructed through an adaptation of a novel gene synthesis technology that enables precise positional control of amino acid composition and incorporation frequencies. High-throughput pyrosequencing was used to monitor the fidelity of construction and characterize genetic diversity in the final 3.6×10(10) transformants. The library exhibited Fab expression superior to currently reported synthetic approaches of equivalent diversity, with greater than 93% of clones observed to successfully display both a correctly folded heavy chain and a correctly folded light chain. Genetic diversity in the library was high, with 95% of 7.0×10(5) clones sequenced observed only once. The obtained library diversity explores a comparable sequence space as the donor-derived natural repertoire and, at the same time, is able to access novel recombined diversity due to lack of segmental linkage. The successful isolation of low- and subnanomolar-affinity antibodies against a diverse panel of receptors, growth factors, enzymes, antigens from infectious reagents, and peptides confirms the functional viability of the design strategy.
Asunto(s)
Anticuerpos/química , Biblioteca de Péptidos , Técnicas Biosensibles , Ensayo de Inmunoadsorción Enzimática , Variación Genética , Humanos , Modelos TeóricosRESUMEN
This study aims at generating immune chicken phage display libraries and single-chain antibodies (scFvs) specifically directed against cell surface markers of cultured peripheral blood mononuclear cells (PBMCs) that contain endothelial progenitor cells (EPCs). In contrast to previous approaches that use well-defined recombinant antigens attached to plastic surfaces that may alter the structure of the proteins, the authors describe a method that maintains the cell surface markers on live cells while providing the opportunity to rapidly screen entire libraries for antibodies that bind to unknown cell surface markers of progenitor/stem cells. Chickens immunized with live EPCs, consisting of a heterogeneous population of lymphocytes and monocytes, demonstrated a robust immune response. After three rounds of biopanning, the authors purified and characterized three unique scFvs called UG1-3. Codon-optimized recombinant UG1 (gUG-1) shows binding by flow cytometry to circulating CD14-positive cells in peripheral blood consistent with predominant expression of a target protein on monocyte subsets. The authors describe the successful use of immunization of chickens for the generation of scFvs against a heterogenous population of EPCs displaying unknown cell surface markers and demonstrate the strong potential of phage display technology in the development of reagents for the isolation and characterization of stem/progenitor cells.
Asunto(s)
Células Endoteliales/inmunología , Biblioteca de Péptidos , Anticuerpos de Cadena Única/inmunología , Células Madre/inmunología , Secuencia de Aminoácidos , Animales , Antígenos de Superficie/inmunología , Antígenos de Superficie/metabolismo , Pollos/inmunología , Células Endoteliales/metabolismo , Epítopos/inmunología , Citometría de Flujo , Humanos , Inmunización , Leucocitos Mononucleares/inmunología , Leucocitos Mononucleares/metabolismo , Datos de Secuencia Molecular , Unión Proteica/inmunología , Alineación de Secuencia , Anticuerpos de Cadena Única/aislamiento & purificación , Células Madre/metabolismoRESUMEN
Over the past 10 years, a growing field of research supporting the value of myeloperoxidase (MPO) as a prognostic indicator in acute cardiac pathophysiologies has emerged. The availability of a rapid and disposable MPO detection platform would enable research clinicians to more readily assess MPO indications for guiding therapy and also facilitate clinicians at the patient interface to readily adopt MPO testing and potentially drive more informed prognoses. Here we describe the isolation of a high-affinity avian MPO-specific recombinant antibody panel using phage display. Rapid isolation of a suitable single-chain variable fragment (scFv) antibody was facilitated using a surface plasmon resonance (SPR)-based "off-rate ranking" screening process. The selected scFv was then successfully incorporated into a rapid, simple, and sensitive one-step lateral flow immunoassay (LFIA) for the detection of MPO. This "one-step" feature of the developed assay was made possible by the scFv's strong affinity for MPO, obviating the need for sandwich signal enhancement steps. The assay's rapid performance was also further enhanced by exploiting the intrinsic enzymatic properties of MPO in its final detection. Use of the optimized LFIA facilitated the sensitive detection of MPO in MPO-depleted serum within clinically relevant reference ranges.
Asunto(s)
Afinidad de Anticuerpos , Inmunoensayo/métodos , Peroxidasa/análisis , Proteínas Recombinantes/inmunología , Anticuerpos de Cadena Única/inmunología , Animales , Cromatografía de Afinidad , Enzimas Inmovilizadas/análisis , Enzimas Inmovilizadas/sangre , Enzimas Inmovilizadas/inmunología , Humanos , Biblioteca de Péptidos , Peroxidasa/sangre , Peroxidasa/inmunología , Proteínas Recombinantes/aislamiento & purificación , Anticuerpos de Cadena Única/aislamiento & purificación , Factores de TiempoRESUMEN
Antibodies are critical reagents in many fundamental biochemical methods such as affinity chromatography. As our understanding of the proteome becomes more complex, demand is rising for rapidly generated antibodies of higher specificity than ever before. It is therefore surprising that few investigators have moved beyond the classical methods of antibody production in their search for new reagents. Despite their long-standing efficacy, recombinant antibody generation technologies such as phage display are still largely the tools of biotechnology companies or research groups with a direct interest in protein engineering. In this chapter, we discuss the inherent limitations of classical polyclonal and monoclonal antibody generation and highlight an attractive alternative: generating high specificity, high affinity recombinant antibodies from alternative immune sources such as chickens, via phage display.
Asunto(s)
Anticuerpos/inmunología , Anticuerpos/aislamiento & purificación , Especificidad de Anticuerpos , Biblioteca de Péptidos , Animales , Anticuerpos/química , Anticuerpos/genética , Pollos/inmunología , Cromatografía de Afinidad , Humanos , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/inmunología , Proteínas Recombinantes/aislamiento & purificaciónRESUMEN
High-affinity, highly specific binding proteins are a key class of molecules used in the development of new affinity chromatography methods. Traditionally, antibody-based methods have relied on the use of whole immunoglobulins purified from immune animal sera, from egg yolks, or from murine monoclonal hybridoma supernatants. To accelerate and refine the reagent antibody generation process, we have developed optimized methods that allow the rapid assembly of scFv libraries from chickens immunized with pools of immunogens. These methods allow the simplified generation of a single moderately sized library of single-chain Fv (scFv) and the subsequent isolation of diverse, high-affinity, and high-specificity monoclonals for each individual immunogen, via phage display. Using these methods, antibodies can be derived that exhibit the desired selectivity, such as complete specificity or cross-reactivity to multiple orthologues of the same protein.
Asunto(s)
Afinidad de Anticuerpos , Especificidad de Anticuerpos , Antígenos/inmunología , Pollos/inmunología , Inmunización/métodos , Anticuerpos de Cadena Única/inmunología , Animales , Antibacterianos/farmacología , Médula Ósea/metabolismo , Clonación Molecular , Cartilla de ADN/genética , ADN Complementario/biosíntesis , ADN Complementario/genética , Humanos , Biblioteca de Péptidos , Reacción en Cadena de la Polimerasa , Anticuerpos de Cadena Única/química , Anticuerpos de Cadena Única/genética , Solubilidad , Bazo/metabolismoRESUMEN
BACKGROUND & AIMS: Late diagnosis of colorectal carcinoma results in a significant reduction of average survival times. Yet despite screening programs, about 70% of tumors are detected at advanced stages (International Union Against Cancer stages III/IV). We explored whether detection of malignant disease would be possible through identification of tumor-specific protein biomarkers in serum samples. METHODS: A discovery set of sera from patients with colorectal malignancy (n = 58) and healthy control individuals (n = 32) were screened for potential differences using surface-enhanced laser desorption/ionization time-of-flight mass spectrometry. Candidate proteins were identified and their expression levels were validated in independent sample sets using a specific immunoassay (enzyme-linked immunosorbent assay). RESULTS: By using class comparison and custom-developed algorithms we identified several m/z values that were expressed differentially between the malignant samples and the healthy controls of the discovery set. Characterization of the most prominent m/z values revealed a member of the complement system, the stable form of C3a anaphylatoxin (ie, C3a-desArg). Based on a specific enzyme-linked immunosorbent assay, serum levels of complement C3a-desArg predicted the presence of colorectal malignancy in a blinded validation set (n = 59) with a sensitivity of 96.8% and a specificity of 96.2%. Increased serum levels were also detected in 86.1% of independently collected sera from patients with colorectal adenomas (n = 36), whereas only 5.6% were classified as normal. CONCLUSIONS: Complement C3a-desArg is present at significantly higher levels in serum from patients with colorectal adenomas (P < .0001) and carcinomas (P < .0001) than in healthy individuals. This suggests that quantification of C3a-desArg levels could ameliorate existing screening tests for colorectal cancer.
Asunto(s)
Adenoma/sangre , Adenoma/diagnóstico , Anafilatoxinas/metabolismo , Neoplasias Colorrectales/sangre , Neoplasias Colorrectales/diagnóstico , Complemento C3a/metabolismo , Adulto , Anciano , Anciano de 80 o más Años , Biomarcadores de Tumor/sangre , Ensayo de Inmunoadsorción Enzimática/normas , Femenino , Humanos , Masculino , Persona de Mediana Edad , Valor Predictivo de las Pruebas , Reproducibilidad de los Resultados , Espectrometría de Masa por Láser de Matriz Asistida de Ionización DesorciónRESUMEN
Antibody-based assay systems are now accepted by regulatory authorities for detection of the toxins produced by phytoplankton that accumulate in shellfish tissues. However, the generation of suitable antibodies for sensitive assay development remains a major challenge. We have examined the potential of using the chicken immune system to generate high-affinity, high-specificity recombinant antibody fragments against phytotoxins. Following immunization of the chicken with domoic acid-bovine serum albumin, a single-chain antibody variable region (scFv) gene library was generated from single V(H) and V(L) genes isolated from the immune cells in the spleen and bone marrow. scFvs reacting with domoic acid were isolated by phage display and affinity matured by light chain shuffling, resulting in an approximate 10-fold increase in sensitivity. The isolated scFvs were effectively expressed in Escherichia coli and readily purified by affinity chromatography. They were then used to develop a convenient and sensitive indirect competitive enzyme-linked immunosorbent assay for domoic acid, with a 50% effective dose of 156 ng/ml, which could be used reliably with shellfish extracts. This study demonstrates that chickens provide a valuable model system for the simplified, rapid generation of high-affinity recombinant antibody fragments with specificity for small toxin molecules.