RESUMO
T cell engagers, a category of T cell-retargeting immunotherapy, are rapidly transforming clinical cancer care. However, the lack of tumor-specific targets poses a significant roadblock for broad adaptation of this therapeutic modality in many indications, often resulting in systemic on-target off-tumor toxicity. Though various tumor-derived intracellular mutations provide a massive pool of potential tumor-specific antigens, targeting them is extremely challenging, partly due to the low copy number of tumor associated antigen (TAA)-derived pMHC on tumor cell surface. Further, the interplay of binding geometry and format valency in relation to the capacity of a T cell engager to efficiently target low density cell-surface pMHC is not well understood. Using the Wilms' tumor 1 (WT1) oncoprotein as a proof-of-principle TAA, combined with an array of IgG-like T cell engager modalities that differ in their anti-TAA valency and binding geometry, we show that the ability to induce an immunological synapse formation, resulting in potent killing of WT1 positive cancer cell lines is primarily dependent on the distinct geometrical conformations between the Fab arms of anti-WT1-HLA-A*02:01 and anti-CD3. The augmented avidity conferred by the binding of two anti-WT1-HLA-A*02:01 Fab arms has only minimal influence on cell killing potency. These findings demonstrate the need for careful examination of key design parameters for the development of next-generation T cell engagers targeting low density TAA-pMHCs on tumor cells.
Assuntos
Neoplasias , Linfócitos T , Humanos , Proteínas WT1/genética , Neoplasias/genética , Neoplasias/terapia , Antígenos de Neoplasias , Imunoproteínas , Antígenos HLA-A , PeptídeosRESUMO
Bispecific antibodies represent an increasingly large fraction of biologics in therapeutic development due to their expanded scope in functional capabilities. Asymmetric monovalent bispecific IgGs (bsIgGs) have the additional advantage of maintaining a native antibody-like structure, which can provide favorable pharmacology and pharmacokinetic profiles. The production of correctly assembled asymmetric monovalent bsIgGs, however, is a complex engineering endeavor due to the propensity for non-cognate heavy and light chains to mis-pair. Previously, we introduced the DuetMab platform as a general solution for the production of bsIgGs, which utilizes an engineered interchain disulfide bond in one of the CH1-CL domains to promote orthogonal chain pairing between heavy and light chains. While highly effective in promoting cognate heavy and light chain pairing, residual chain mispairing could be detected for specific combinations of Fv pairs. Here, we present enhancements to the DuetMab design that improve chain pairing and production through the introduction of novel electrostatic steering mutations at the CH1-CL interface with lambda light chains (CH1-Cλ). These mutations work together with previously established charge-pair mutations at the CH1-CL interface with kappa light chains (CH1-Cκ) and Fab disulfide engineering to promote cognate heavy and light chain pairing and enable the reliable production of bsIgGs. Importantly, these enhanced DuetMabs do not require engineering of the variable domains and are robust when applied to a panel of bsIgGs with diverse Fv sequences. We present a comprehensive biochemical, biophysical, and functional characterization of the resulting DuetMabs to demonstrate compatibility with industrial production benchmarks. Overall, this enhanced DuetMab platform substantially streamlines process development of these disruptive biotherapeutics.
Assuntos
Anticorpos Biespecíficos , Anticorpos Biespecíficos/genética , Eletricidade Estática , Dissulfetos , Mutação , Imunoglobulina G/genéticaRESUMO
Bispecific antibodies (BsAbs) are engineered to simultaneously bind two different antigens, and offer promising clinical outcomes for various diseases. The dual binding properties of BsAbs may enable superior efficacies and/or potencies compared to standard monoclonal antibodies (mAbs) or combination mAb therapies. Characterizing BsAb binding properties is critical during biotherapeutic development, where data is leveraged to predict efficacy and potency, assess critical quality attributes and improve antibody design. Traditional single-target, single-readout approaches (e.g., ELISA) have limited usefulness for interpreting complex bispecific binding, and double the benchwork. To address these deficiencies, we developed and implemented a new dual-target/readout binding assay that accurately dissects the affinities of both BsAb binding domains directly and simultaneously. This new assay uses AlphaPlex® technology, which eliminates traditional ELISA wash steps and can be miniaturized for automated workflows. The optimized BsAb AlphaPlex assay demonstrates 99-107% accuracy within a 50-150% linear range, and detected >50% binding degradation from photo- and thermal stress conditions. To the best of our knowledge, this is the first instance of a dual-target/readout BsAb AlphaPlex assay with GMP-suitable linear range, accuracy, specificity, and stability-indicating properties. As a highly customizable and efficient assay, BsAb AlphaPlex may be applicable to numerous bispecific formats and/or co-formulations against a variety of antigens beyond the clinical therapeutic space.
Assuntos
Anticorpos Biespecíficos/imunologia , Especificidade de Anticorpos , Antígenos/imunologia , Antígeno CTLA-4/imunologia , Imunoensaio , Receptor de Morte Celular Programada 1/imunologia , Anticorpos Biespecíficos/metabolismo , Complexo Antígeno-Anticorpo , Antígenos/metabolismo , Sítios de Ligação de Anticorpos , Soluções Tampão , Antígeno CTLA-4/metabolismo , Ensaio de Imunoadsorção Enzimática , Epitopos , Humanos , Concentração de Íons de Hidrogênio , Cinética , Valor Preditivo dos Testes , Receptor de Morte Celular Programada 1/metabolismo , Ligação Proteica , Reprodutibilidade dos TestesRESUMO
The clinical benefit of PD-1 blockade can be improved by combination with CTLA4 inhibition but is commensurate with significant immune-related adverse events suboptimally limiting the doses of anti-CTLA4 mAb that can be used. MEDI5752 is a monovalent bispecific antibody designed to suppress the PD-1 pathway and provide modulated CTLA4 inhibition favoring enhanced blockade on PD-1+ activated T cells. We show that MEDI5752 preferentially saturates CTLA4 on PD-1+ T cells versus PD-1- T cells, reducing the dose required to elicit IL2 secretion. Unlike conventional PD-1/CTLA4 mAbs, MEDI5752 leads to the rapid internalization and degradation of PD-1. Moreover, we show that MEDI5752 preferentially localizes and accumulates in tumors providing enhanced activity when compared with a combination of mAbs targeting PD-1 and CTLA4 in vivo. Following treatment with MEDI5752, robust partial responses were observed in two patients with advanced solid tumors. MEDI5752 represents a novel immunotherapy engineered to preferentially inhibit CTLA4 on PD-1+ T cells. SIGNIFICANCE: The unique characteristics of MEDI5752 represent a novel immunotherapy engineered to direct CTLA4 inhibition to PD-1+ T cells with the potential for differentiated activity when compared with current conventional mAb combination strategies targeting PD-1 and CTLA4. This molecule therefore represents a step forward in the rational design of cancer immunotherapy.See related commentary by Burton and Tawbi, p. 1008.This article is highlighted in the In This Issue feature, p. 995.
Assuntos
Anticorpos Monoclonais Humanizados/uso terapêutico , Adenocarcinoma/tratamento farmacológico , Adenocarcinoma de Células Claras/tratamento farmacológico , Antígeno CTLA-4/metabolismo , Humanos , Imunoterapia , Neoplasias Renais/tratamento farmacológico , Masculino , Pessoa de Meia-Idade , Receptor de Morte Celular Programada 1/metabolismo , Neoplasias Gástricas/tratamento farmacológico , Linfócitos T/imunologiaRESUMO
Complement-dependent cytotoxicity (CDC) is a potent effector mechanism, engaging both innate and adaptive immunity. Although strategies to improve the CDC activity of antibody therapeutics have primarily focused on enhancing the interaction between the antibody crystallizable fragment (Fc) and the first subcomponent of the C1 complement complex (C1q), the relative importance of intrinsic affinity and binding valency of an antibody to the target antigen is poorly understood. Here we show that antibody binding affinity to a cell surface target antigen evidently affects the extent and efficacy of antibody-mediated complement activation. We further report the fundamental role of antibody binding valency in the capacity to recruit C1q and regulate CDC. More specifically, an array of affinity-modulated variants and functionally monovalent bispecific derivatives of high-affinity anti-epidermal growth factor receptor (EGFR) and anti-human epidermal growth factor receptor 2 (HER2) therapeutic immunoglobulin Gs (IgGs), previously reported to be deficient in mediating complement activation, were tested for their ability to bind C1q by biolayer interferometry using antigen-loaded biosensors and to exert CDC against a panel of EGFR and HER2 tumor cells of various histological origins. Significantly, affinity-reduced variants or monovalent derivatives, but not their high-affinity bivalent IgG counterparts, induced near-complete cell cytotoxicity in tumor cell lines that had formerly been shown to be resistant to complement-mediated attack. Our findings suggest that monovalent target engagement may contribute to an optimal geometrical positioning of the antibody Fc to engage C1q and deploy the complement pathway.
Assuntos
Anticorpos Biespecíficos/metabolismo , Imunoglobulina G/metabolismo , Anticorpos Biespecíficos/genética , Afinidade de Anticorpos/genética , Citotoxicidade Celular Dependente de Anticorpos , Reações Antígeno-Anticorpo , Linhagem Celular Tumoral , Ativação do Complemento , Complemento C1q/metabolismo , Receptores ErbB/imunologia , Receptores ErbB/metabolismo , Humanos , Fragmentos Fc das Imunoglobulinas/metabolismo , Imunoglobulina G/genética , Interferometria , Mutagênese Sítio-Dirigida , Ligação Proteica/genética , Receptor ErbB-2/imunologia , Receptor ErbB-2/metabolismoRESUMO
Membrane proteins play key roles in the evolution of numerous diseases and as a result have become the most dominant class of targets for therapeutic intervention. However, their poor expression and detection oftentimes prohibit drug discovery and screening efforts. Herein, we have developed an approach, named 'Tag-on-Demand' that exploits amber suppression to control the expression of 'tagged' membrane proteins for detection and selections, yet can be turned off for expression of the protein in its native form. Utilizing an engineered Chinese hamster ovary cell line capable of efficient amber suppression, we evaluated the expression of a diverse panel of model membrane proteins and demonstrated the enrichment of cells with improved expression profiles, where ~200-800% improvement in total protein expression levels were observed over pre-sorted populations after a single round of fluorescence-activated cell sorting. Furthermore, results were most striking for the typically difficult-to-express G protein-coupled receptor, CXCR2, where ~2.5-fold improvement in surface expression was observed. We anticipate that the Tag-on-Demand approach will be suitable not only for membrane protein cell line development but also for the development of intracellular and secreted protein cell lines in expression systems for which amber suppression technology exists, including bacterial, yeast, insect and cell-free expression systems.
Assuntos
Códon de Terminação/genética , Engenharia Genética/métodos , Proteínas de Membrana/genética , Animais , Células CHO , Cricetulus , Avaliação Pré-Clínica de Medicamentos , Expressão Gênica , Células HEK293 , HumanosRESUMO
Bispecific antibodies are considered attractive bio-therapeutic agents owing to their ability to target two distinct disease mediators. Cross-arm avidity targeting of antigen double-positive cancer cells over single-positive normal tissue is believed to enhance the therapeutic efficacy, restrict major escape mechanisms and increase tumor-targeting selectivity, leading to reduced systemic toxicity and improved therapeutic index. However, the interplay of factors regulating target selectivity is not well understood and often overlooked when developing clinically relevant bispecific therapeutics. We show in vivo that dual targeting alone is not sufficient to endow selective tumor-targeting, and report the pivotal roles played by the affinity of the individual arms, overall avidity and format valence. Specifically, a series of monovalent and bivalent bispecific IgGs composed of the anti-HER2 trastuzumab moiety paired with affinity-modulated VH and VL regions of the anti-EGFR GA201 mAb were tested for selective targeting and eradication of double-positive human NCI-H358 non-small cell lung cancer target tumors over single-positive, non-target NCI-H358-HER2 CRISPR knock out tumors in nude mice bearing dual-flank tumor xenografts. Affinity-reduced monovalent bispecific variants, but not their bivalent bispecific counterparts, mediated a greater degree of tumor targeting selectivity, while the overall efficacy against the targeted tumor was not substantially affected.
Assuntos
Anticorpos Biespecíficos/imunologia , Afinidade de Anticorpos , Especificidade de Anticorpos , Carcinoma Pulmonar de Células não Pequenas/imunologia , Trastuzumab/imunologia , Animais , Anticorpos Biespecíficos/administração & dosagem , Carcinoma Pulmonar de Células não Pequenas/tratamento farmacológico , Linhagem Celular Tumoral , Receptores ErbB/imunologia , Humanos , Camundongos Nus , Receptor ErbB-2/imunologia , Trastuzumab/administração & dosagem , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Antibody-mediated immune effector functions play an essential role in the anti-tumor efficacy of many therapeutic mAbs. While much of the effort to improve effector potency has focused on augmenting the interaction between the antibody-Fc and activating Fc-receptors expressed on immune cells, the role of antibody binding interactions with the target antigen remains poorly understood. We show that antibody intrinsic affinity to the target antigen clearly influences the extent and efficiency of Fc-mediated effector mechanisms, and report the pivotal role of antibody binding valence on the ability to regulate effector functions. More particularly, we used an array of affinity modulated variants of three different mAbs, anti-CD4, anti-EGFR and anti-HER2 against a panel of target cell lines expressing disparate levels of the target antigen. We found that at saturating antibody concentrations, IgG variants with moderate intrinsic affinities, similar to those generated by the natural humoral immune response, promoted superior effector functions compared to higher affinity antibodies. We hypothesize that at saturating concentrations, effector function correlates most directly with the amount of Fc bound to the cell surface. Thus, high affinity antibodies exhibiting slow off-rates are more likely to interact bivalently with the target cell, occupying two antigen sites with a single Fc. In contrast, antibodies with faster off-rates are likely to dissociate each binding arm more rapidly, resulting in a higher likelihood of monovalent binding. Monovalent binding may in turn increase target cell opsonization and lead to improved recruitment of effector cells. This unpredicted relationship between target affinity and effector function potency suggests a careful examination of antibody design and engineering for the development of next-generation immunotherapeutics.
Assuntos
Afinidade de Anticorpos/imunologia , Antígenos/imunologia , Imunoglobulina G/imunologia , Citotoxicidade Celular Dependente de Anticorpos/imunologia , Antígenos CD4/imunologia , Linhagem Celular Tumoral , Endocitose , Receptores ErbB/metabolismo , Genes Reporter , Humanos , Isoformas de Proteínas , Receptor ErbB-2/metabolismo , Receptores de IgG/metabolismoRESUMO
The three-dimensional structure of a human IgG1 Fc fragment bound to wild-type human FcγRI is reported. The structure of the corresponding complex was solved at a resolution of 2.4 Šusing molecular replacement; this is the highest resolution achieved for an unmutated FcγRI molecule. This study highlights the critical structural and functional role played by the second extracellular subdomain of FcγRI. It also explains the long-known major energetic contribution of the Fc `LLGG' motif at positions 234-237, and particularly of Leu235, via a `lock-and-key' mechanism. Finally, a previously held belief is corrected and a differing view is offered on the recently proposed direct role of Fc carbohydrates in the corresponding interaction. Structural evidence is provided that such glycan-related effects are strictly indirect.
Assuntos
Imunoglobulina G/química , Imunoglobulina G/metabolismo , Receptores de IgG/química , Receptores de IgG/metabolismo , Linhagem Celular , Cristalografia por Raios X , Humanos , Modelos Moleculares , Polissacarídeos/metabolismo , Ligação Proteica , Domínios e Motivos de Interação entre ProteínasRESUMO
High throughput screenings of single chain Fv (scFv) antibody phage display libraries are currently done as soluble scFvs produced in E.coli. Due to endotoxin contaminations from bacterial cells these preparations cannot be reliably used in mammalian cell based assays. The monovalent nature and lack of Fc in soluble scFvs prevent functional assays that are dependent on target cross linking and/or Fc functions. A convenient approach is to convert scFvs into scFv.Fc fusion proteins and express them in mammalian cell lines for screening. This approach is low throughput and is only taken after primary screening of monovalent scFvs that are expressed in bacteria. There is no platform at present that combines the benefits of both bacterial and mammalian expression system for screening phage library output. We have, therefore, developed a novel dual expression vector, called pSplice, which can be used to express scFv.Fc fusion proteins both in E.coli and mammalian cell lines. The hallmark of the vector is an engineered intron which houses the bacterial promoter and signal peptide for expression and secretion of scFv.Fc in E.coli. When the vector is transfected into a mammalian cell line, the intron is efficiently spliced out resulting in a functional operon for expression and secretion of the scFv.Fc fusion protein into the culture medium. By applying basic knowledge of mammalian introns and splisosome, we designed this vector to enable screening of phage libraries in a product like format. Like IgG, the scFv.Fc fusion protein is bi-valent for the antigen and possesses Fc effector functions. Expression in E.coli maintains the speed of the bacterial expression platform and is used to triage clones based on binding and other assays that are not sensitive to endotoxin. Triaged clones are then expressed in a mammalian cell line without the need for any additional cloning steps. Conditioned media from the mammalian cell line containing the fusion proteins are then used for different types of cell based assays. Thus this system retains the speed of the current screening system for phage libraries and adds additional functionality to it.
Assuntos
Ensaios de Triagem em Larga Escala/métodos , Biblioteca de Peptídeos , Anticorpos de Cadeia Única/análise , Animais , Bacteriófagos/genética , Células CHO , Clonagem Molecular , Cricetinae , Cricetulus , Células HEK293 , Células HL-60 , Humanos , Fragmentos Fc das Imunoglobulinas/análise , Fragmentos Fc das Imunoglobulinas/genética , Anticorpos de Cadeia Única/isolamento & purificaçãoRESUMO
Bispecific antibodies constitute a valuable class of therapeutics owing to their ability to bind 2 distinct targets. Dual targeting is thought to enhance biological efficacy, limit escape mechanisms, and increase target selectivity via a strong avidity effect mediated by concurrent binding to both antigens on the surface of the same cell. However, factors that regulate the extent of target selectivity are not well understood. We show that dual targeting alone is not sufficient to promote efficient target selectivity, and report the substantial roles played by the affinity of the individual arms, overall avidity and valence. More particularly, various monovalent bispecific IgGs composed of an anti-CD70 moiety paired with variants of the anti-CD4 mAb ibalizumab were tested for preferential binding and selective depletion of CD4(+)/CD70(+) T cells over cells expressing only one of the target antigens that resulted from antibody dependent cell-mediated cytotoxicity. Variants exhibiting reduced CD4 affinity showed a greater degree of target selectivity, while the overall efficacy of the bispecific molecule was not affected.
Assuntos
Anticorpos Monoclonais , Afinidade de Anticorpos/genética , Especificidade de Anticorpos/genética , Imunoglobulina G , Anticorpos Biespecíficos , Anticorpos Monoclonais/genética , Anticorpos Monoclonais/imunologia , Linfócitos T CD4-Positivos/imunologia , Células HEK293 , Humanos , Imunoglobulina G/genética , Imunoglobulina G/imunologia , Depleção Linfocítica/métodosRESUMO
Monovalent bispecific IgGs cater to a distinct set of mechanisms of action but are difficult to engineer and manufacture because of complexities associated with correct heavy and light chain pairing. We have created a novel design, "DuetMab," for efficient production of these molecules. The platform uses knobs-into-holes (KIH) technology for heterodimerization of 2 distinct heavy chains and increases the efficiency of cognate heavy and light chain pairing by replacing the native disulfide bond in one of the CH1-CL interfaces with an engineered disulfide bond. Using two pairs of antibodies, cetuximab (anti-EGFR) and trastuzumab (anti-HER2), and anti-CD40 and anti-CD70 antibodies, we demonstrate that DuetMab antibodies can be produced in a highly purified and active form, and show for the first time that monovalent bispecific IgGs can concurrently bind both antigens on the same cell. This last property compensates for the loss of avidity brought about by monovalency and improves selectivity toward the target cell.
Assuntos
Anticorpos Biespecíficos , Afinidade de Anticorpos/genética , Cetuximab , Imunoglobulina G , Trastuzumab , Anticorpos Biespecíficos/biossíntese , Anticorpos Biespecíficos/química , Anticorpos Biespecíficos/genética , Cetuximab/biossíntese , Cetuximab/química , Cetuximab/genética , Células HEK293 , Humanos , Imunoglobulina G/biossíntese , Imunoglobulina G/química , Imunoglobulina G/genética , Trastuzumab/biossíntese , Trastuzumab/química , Trastuzumab/genéticaRESUMO
Phage display of antibody libraries is a powerful tool for antibody discovery and evolution. Recombinant antibodies have been displayed on phage particles as scFvs or Fabs, and more recently as bivalent F(ab')(2). We recently developed a technology (E-clonal) for screening of combinatorial IgG libraries using bacterial periplasmic display and selection by fluorescence-activated cell sorting (FACS) [Mazor Y et al. (2007) Nat Biotechnol 25, 563-565]. Although, as a single-cell analysis technique, FACS is very powerful, especially for the isolation of high-affinity binders, even with state of the art instrumentation the screening of libraries with diversity > 10(8) is technically challenging. We report here a system that takes advantage of display of full-length IgGs on filamentous phage particles as a prescreening step to reduce library size and enable subsequent rounds of FACS screening in Escherichia coli. For the establishment of an IgG phage display system, we utilized phagemid-encoded IgG with the fUSE5-ZZ phage as a helper phage. These phage particles display the Fc-binding ZZ protein on all copies of the phage p3 coat protein, and are exploited as both helper phages and anchoring surfaces for the soluble IgG. We demonstrate that tandem phage selection followed by FACS allows the selection of a highly diversified profile of binders from antibody libraries without undersampling, and at the same time capitalizes on the advantages of FACS for real-time monitoring and optimization of the screening process.
Assuntos
Bacteriófagos/metabolismo , Escherichia coli/metabolismo , Citometria de Fluxo , Imunoglobulina G/isolamento & purificação , Biblioteca de Peptídeos , Afinidade de Anticorpos/imunologia , Especificidade de Anticorpos/imunologia , Antígenos de Bactérias/imunologia , Toxinas Bacterianas/imunologia , Bacteriófagos/química , Bacteriófagos/genética , Proteínas do Capsídeo/genética , Digoxina/imunologia , Ensaio de Imunoadsorção Enzimática , Escherichia coli/genética , Escherichia coli/virologia , Vetores Genéticos/genética , Imunoglobulina G/biossíntese , Imunoglobulina G/genética , Imunoglobulina G/imunologia , Cinética , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/genética , Proteínas Recombinantes/imunologia , Proteínas Recombinantes/isolamento & purificação , Esferoplastos/química , Esferoplastos/metabolismoRESUMO
We report on a simple method to rapidly generate very large libraries of genes encoding mutant proteins without the use of DNA amplification, and the application of this methodology in the construction of synthetic immunoglobulin variable heavy (V(H)) and light (V(kappa)) libraries. Four high quality, chemically synthesized polynucleotides (90-140 bases) were annealed and extended using T4 DNA polymerase. Following electroporation, >10(9) transformants could be synthesized within 1 day. Fusion to beta-lactamase and selection on ampicillin resulted in 3.7 x 10(8) V(H) and 6.9 x 10(8) V(kappa) clones highly enriched for full-length, in-frame genes. High-throughput 454 DNA sequencing of >250,000 V(H) and V(kappa) genes from the pre- and post-selection libraries revealed that, in addition to the expected reduction in reading-frame shifts and stop codons, selection for functional expression also resulted in a statistical decrease in the cysteine content. Apart from these differences, there was a good agreement between the expected and actual diversity, indicating that neither oligonucleotide synthesis nor biological constrains due to protein synthesis of V(H)/V(kappa)-beta-lactamase fusions introduce biases in the amino acid composition of the randomized regions. This methodology can be employed for the rapid construction of highly diverse libraries with the near elimination of PCR errors in invariant regions.
Assuntos
Anticorpos/genética , Anticorpos/imunologia , Técnicas de Química Combinatória/métodos , Biblioteca Gênica , Anticorpos de Cadeia Única/genética , Anticorpos de Cadeia Única/imunologia , Variação Genética , Proteínas Mutantes/genética , Proteínas Mutantes/imunologia , Polinucleotídeos/síntese química , Polinucleotídeos/genética , Seleção Genética , Transformação GenéticaRESUMO
We have developed a technology for the facile isolation of full-length IgG antibodies with desired specificity from combinatorial libraries expressed in Escherichia coli. Full-length heavy and light chains are expressed from a bicistronic operon and are secreted into the periplasm where they assemble into aglycosylated IgGs that are fully functional for antigen binding. Expression of an inner membrane-tethered Fc-binding protein is used to capture the IgG molecules and anchor them to the cell. Following outer membrane disruption, clones expressing IgGs that specifically recognize fluorescently labeled antigen are selected by flow cytometry. This technique was used for the isolation of several IgGs with nanomolar affinities toward the protective antigen of Bacillus anthracis from immune libraries. High-throughput isolation of E. coli-derived full-length IgG can greatly expedite the discovery and production of antibodies for therapeutic and diagnostic applications.
Assuntos
Técnicas de Química Combinatória/métodos , Escherichia coli/metabolismo , Imunoglobulina G/isolamento & purificação , Biblioteca de Peptídeos , Animais , Células Clonais , Clonagem Molecular , DNA Complementar/biossíntese , Ensaio de Imunoadsorção Enzimática , Citometria de Fluxo , Humanos , Cadeias Pesadas de Imunoglobulinas/genética , Cadeias Leves de Imunoglobulina/genética , Região Variável de Imunoglobulina/genética , Camundongos , Periplasma/metabolismo , Plasmídeos/genética , Reação em Cadeia da Polimerase , RNA/isolamento & purificação , Análise de Sequência de DNA , Esferoplastos/metabolismo , Baço/metabolismoRESUMO
Here we describe a protocol for the selection of full-length IgG antibodies from repertoires displayed on Escherichia coli. In the method described here, full-length heavy and light chains are assembled in the periplasm into aglycosylated IgGs that are fully functional for antigen binding. Expression of an inner membrane-tethered Fc-binding protein is used to capture the IgG molecules and anchor them to the cell. Following outer-membrane permeabilization, fluorescently labeled ligand-binding library clones are selected by multiple rounds of fluorescence-activated cell sorting. Selection of a comprehensive set of IgG clones can typically be obtained within 3-4 weeks, a timescale that is comparable with most prevalent antibody display technologies. The isolated antibodies are well expressed in bacteria and exhibit affinities per binding site in the nanomolar range.
Assuntos
Escherichia coli/genética , Imunoglobulina G/isolamento & purificação , Engenharia de Proteínas/métodos , Proteínas Recombinantes de Fusão/isolamento & purificação , Anticorpos Monoclonais/isolamento & purificação , Ensaio de Imunoadsorção Enzimática , Escherichia coli/metabolismo , Imunoglobulina G/genética , Biblioteca de Peptídeos , Periplasma/genética , Periplasma/metabolismo , Plasmídeos/genéticaRESUMO
As therapeutics, antibodies can be used "un-armed" or as immunoconjugates to direct cytotoxic moieties to tumor cells. Immunoconjugates are made by attaching chemotherapy drugs, radioisotopes or toxins to the antibody. Small recombinant antibody fragments fused to cytotoxic moieties, termed recombinant immunotoxins are also being developed as an additional approach for a targeted cancer therapy. Key parameters in determining the therapeutic potential of such targeted therapies are target specificity, affinity, stability and size. With regard to treating solid tumors, tumor penetration (which is inversely proportional to size) is currently regarded as the prime factor for efficacy, while parameters such as binding affinity and residence time in the body are thought to contribute to a lesser extent. When comparing recombinant immunotoxins and antibody-toxin immunoconjugates that target ErbB2/HER2, here we found that a bivalent antibody-toxin immunoconjugate (200 kDa) was superior to the corresponding recombinant monovalent immunotoxin (69 kDa) in killing ErbB2-expressing tumor cells in culture and as xenografts in nude mice, suggesting that higher avidity and longer residence time may outweigh tumor penetration. Our study suggests that the re-valuation of currently neglected, large IgG-effector molecule conjugates for anti-cancer therapy may be justified.
Assuntos
Anticorpos Monoclonais/química , Imunoconjugados/química , Imunoglobulina G/química , Imunotoxinas/química , Receptor ErbB-2/metabolismo , Animais , Anticorpos Monoclonais/imunologia , Antineoplásicos/farmacologia , Neoplasias da Mama/metabolismo , Linhagem Celular Tumoral , Sobrevivência Celular , Humanos , Imunoconjugados/farmacologia , Camundongos , Camundongos Nus , Modelos Biológicos , Transplante de NeoplasiasRESUMO
We describe facile isolation of full-length IgG antibodies from combinatorial libraries expressed in E. coli. Full-length heavy and light chains are secreted into the periplasm, where they assemble into aglycosylated IgGs that are captured by an Fc-binding protein that is tethered to the inner membrane. After permeabilizing the outer membrane, spheroplast clones expressing so-called E-clonal antibodies, which specifically recognize fluorescently labeled antigen, are selected using flow cytometry. Screening of a library constructed from an immunized animal yielded several antibodies with nanomolar affinities toward the protective antigen of Bacillus anthracis.
Assuntos
Anticorpos Monoclonais/isolamento & purificação , Anticorpos Monoclonais/fisiologia , Escherichia coli/genética , Escherichia coli/metabolismo , Biblioteca de Peptídeos , Engenharia de Proteínas/métodos , HumanosRESUMO
Targeted therapy encompasses a wide variety of different strategies, which can be divided into direct or indirect approaches. Direct approaches target tumor-associated antigens by monoclonal antibodies (mAbs) binding to the relevant antigens or by small-molecule drugs that interfere with these proteins. Indirect approaches rely on tumor-associated antigens expressed on the cell surface with antibody-drug conjugates or antibody-based fusion proteins containing different kinds of effector molecules. To deliver a lethal cargo into tumor cells, the targeting antibodies should efficiently internalize into the cells. Similarly, to qualify as targets for such drugs newly-discovered cell-surface molecules should facilitate the internalization of antibodies that bind to them. Internalization can be studied be several biochemical and microscopy approaches. An undisputed proof of internalization can be provided by the ability of an antibody to specifically deliver a drug into the target cells and kill it. We present a novel IgG binding toxin fusion, ZZ-PE38, in which the Fc-binding ZZ domain, derived from Streptococcal protein A, is linked to a truncated Pseudomonas exotoxin A, the preparation of complexes between ZZ-PE38 and IgGs that bind tumor cells and the specific cytotoxicity of such immunocomplexes is reported. Our results suggest that ZZ-PE38 could prove to be an invaluable tool for the evaluation of the suitability potential of antibodies and their cognate cell-surface antigens to be targeted by immunotherapeutics based on armed antibodies that require internalization.
Assuntos
ADP Ribose Transferases/metabolismo , Antígenos de Neoplasias/metabolismo , Antineoplásicos/metabolismo , Proteínas de Bactérias/metabolismo , Toxinas Bacterianas/metabolismo , Endocitose , Exotoxinas/metabolismo , Imunotoxinas/metabolismo , Mucinas/metabolismo , Proteína Estafilocócica A/metabolismo , Fatores de Virulência/metabolismo , ADP Ribose Transferases/genética , ADP Ribose Transferases/farmacologia , Afinidade de Anticorpos , Antígenos de Neoplasias/imunologia , Antineoplásicos/imunologia , Antineoplásicos/farmacologia , Proteínas de Bactérias/genética , Proteínas de Bactérias/imunologia , Toxinas Bacterianas/genética , Toxinas Bacterianas/farmacologia , Linhagem Celular Tumoral , Membrana Celular/metabolismo , Sobrevivência Celular/efeitos dos fármacos , Clonagem Molecular , Citoplasma/metabolismo , Relação Dose-Resposta a Droga , Ensaio de Imunoadsorção Enzimática , Exotoxinas/genética , Exotoxinas/farmacologia , Humanos , Imunoglobulina G/metabolismo , Imunotoxinas/genética , Imunotoxinas/imunologia , Imunotoxinas/farmacologia , Concentração Inibidora 50 , Microscopia Confocal , Mucina-1 , Mucinas/imunologia , Proteínas Recombinantes de Fusão/metabolismo , Proteína Estafilocócica A/genética , Proteína Estafilocócica A/imunologia , Transfecção , Fatores de Virulência/genética , Fatores de Virulência/farmacologia , Exotoxina A de Pseudomonas aeruginosaRESUMO
The tumor-associated antigen MUC1 is a cell surface mucin that is expressed on the apical surface of most glandular epithelial cells, including the ducts of the breast, ovary, pancrease, lung and colon. During malignancy, epithelial tissues regularly display elevated levels of MUC1 in a non-polar fashion and in an underglycosylated form, exposing cryptic peptide and carbohydrate epitopes. As such, MUC1 is regarded a potential target for immunotherapeutical intervention. Murine monoclonal H23 antibody specifically recognizes a MUC1 epitope on the surface of human breast cancer cells. We describe the cloning of the variable domains of H23 and their expression in (Escherichia coli) E. coli as maltose-binding protein-scFv (MBP-scFv) fusions. We humanized H23 and evaluated the binding properties of the murine and the humanized recombinant forms, which were similar in affinity and specificity, but lower in apparent affinity in comparison to the original monoclonal IgG. We mapped the epitope of humanized H23 by affinity-selecting a phage-displayed random peptide library on humanized H23 scFv-displaying bacteria. Our results show that humanized H23 binds an epitope corresponding to the MUC1 tandem repeat and an additional epitope not related to MUC1. These epitopes are competitive, bound with similar affinities and are recognized by the original murine H23 monoclonal antibody as well.