RESUMEN
Here we describe a facile and robust genetic selection for isolating full-length IgG antibodies from combinatorial libraries expressed in the cytoplasm of redox-engineered Escherichia coli cells. The method is based on the transport of a bifunctional substrate comprised of an antigen fused to chloramphenicol acetyltransferase, which allows positive selection of bacterial cells co-expressing cytoplasmic IgGs called cyclonals that specifically capture the chimeric antigen and sequester the antibiotic resistance marker in the cytoplasm. The utility of this approach is first demonstrated by isolating affinity-matured cyclonal variants that specifically bind their cognate antigen, the leucine zipper domain of a yeast transcriptional activator, with subnanomolar affinities, which represent a ~20-fold improvement over the parental IgG. We then use the genetic assay to discover antigen-specific cyclonals from a naïve human antibody repertoire, leading to the identification of lead IgG candidates with affinity and specificity for an influenza hemagglutinin-derived peptide antigen.
Asunto(s)
Bioensayo , Inmunoglobulina G , Humanos , Inmunoglobulina G/genética , Citoplasma , Citosol , Escherichia coli/genética , Saccharomyces cerevisiaeRESUMEN
As a common protein modification, asparagine-linked (N-linked) glycosylation has the capacity to greatly influence the biological and biophysical properties of proteins. However, the routine use of glycosylation as a strategy for engineering proteins with advantageous properties is limited by our inability to construct and screen large collections of glycoproteins for cataloguing the consequences of glycan installation. To address this challenge, we describe a combinatorial strategy termed shotgun scanning glycomutagenesis in which DNA libraries encoding all possible glycosylation site variants of a given protein are constructed and subsequently expressed in glycosylation-competent bacteria, thereby enabling rapid determination of glycosylatable sites in the protein. The resulting neoglycoproteins can be readily subjected to available high-throughput assays, making it possible to systematically investigate the structural and functional consequences of glycan conjugation along a protein backbone. The utility of this approach was demonstrated with three different acceptor proteins, namely bacterial immunity protein Im7, bovine pancreatic ribonuclease A, and human anti-HER2 single-chain Fv antibody, all of which were found to tolerate N-glycan attachment at a large number of positions and with relatively high efficiency. The stability and activity of many glycovariants was measurably altered by N-linked glycans in a manner that critically depended on the precise location of the modification. Structural models suggested that affinity was improved by creating novel interfacial contacts with a glycan at the periphery of a protein-protein interface. Importantly, we anticipate that our glycomutagenesis workflow should provide access to unexplored regions of glycoprotein structural space and to custom-made neoglycoproteins with desirable properties.
Asunto(s)
Asparagina/química , Proteínas Portadoras/metabolismo , Proteínas de Escherichia coli/metabolismo , Glicoproteínas/metabolismo , Polisacáridos/metabolismo , Procesamiento Proteico-Postraduccional , Ribonucleasa Pancreática/metabolismo , Anticuerpos de Cadena Única/metabolismo , Secuencia de Aminoácidos , Animales , Proteínas Portadoras/química , Proteínas Portadoras/genética , Bovinos , Proteínas de Escherichia coli/química , Proteínas de Escherichia coli/genética , Glicoproteínas/química , Glicoproteínas/genética , Glicosilación , Humanos , Polisacáridos/química , Polisacáridos/genética , Conformación Proteica , Ingeniería de Proteínas , Receptor ErbB-2/antagonistas & inhibidores , Receptor ErbB-2/inmunología , Ribonucleasa Pancreática/química , Ribonucleasa Pancreática/genética , Anticuerpos de Cadena Única/química , Anticuerpos de Cadena Única/genéticaRESUMEN
Single-chain variable fragment (scFv) antibodies have great potential for a range of applications including as diagnostic and therapeutic agents. However, production of scFvs is challenging because proper folding and activity depend on the formation of two intrachain disulfide bonds that do not readily form in the cytoplasm of living cells. Functional expression in bacteria therefore involves targeting to the more oxidizing periplasm, but yields in this compartment can be limiting due to secretion bottlenecks and the relatively small volume compared to the cytoplasm. In the present study, we evaluated an anti-HER2 scFv, which is specific for human epidermal growth receptor 2 (HER2) overexpressed in breast cancer, for functional expression in the cytoplasm of Escherichia coli strains BL21(DE3) and SHuffle T7 Express, the latter of which is genetically engineered for cytoplasmic disulfide bond formation. Specifically, we observed much greater solubility and binding activity with SHuffle T7 Express cells, which likely resulted from the more oxidative cytoplasm in this strain background. We also found that SHuffle T7 Express cells were capable of supporting high-level soluble production of anti-HER2 scFvs with intact disulfide bonds independent of variable domain orientation, providing further evidence that SHuffle T7 Express is a promising host for laboratory and preparative expression of functional scFv antibodies.
Asunto(s)
Citoplasma/metabolismo , Escherichia coli/metabolismo , Receptor ErbB-2/inmunología , Anticuerpos de Cadena Única/química , Neoplasias de la Mama/inmunología , Neoplasias de la Mama/patología , Escherichia coli/genética , Escherichia coli/crecimiento & desarrollo , Femenino , Humanos , Anticuerpos de Cadena Única/genética , Anticuerpos de Cadena Única/inmunologíaRESUMEN
A major objective of synthetic glycobiology is to re-engineer existing cellular glycosylation pathways from the top down or construct non-natural ones from the bottom up for new and useful purposes. Here, we have developed a set of orthogonal pathways for eukaryotic O-linked protein glycosylation in Escherichia coli that installed the cancer-associated mucin-type glycans Tn, T, sialyl-Tn and sialyl-T onto serine residues in acceptor motifs derived from different human O-glycoproteins. These same glycoengineered bacteria were used to supply crude cell extracts enriched with glycosylation machinery that permitted cell-free construction of O-glycoproteins in a one-pot reaction. In addition, O-glycosylation-competent bacteria were able to generate an antigenically authentic Tn-MUC1 glycoform that exhibited reactivity with antibody 5E5, which specifically recognizes cancer-associated glycoforms of MUC1. We anticipate that the orthogonal glycoprotein biosynthesis pathways developed here will provide facile access to structurally diverse O-glycoforms for a range of important scientific and therapeutic applications.
Asunto(s)
Escherichia coli/metabolismo , Glicoproteínas/biosíntesis , Polisacáridos/metabolismo , Ingeniería de Proteínas , Antígenos de Carbohidratos Asociados a Tumores/biosíntesis , Sistema Libre de Células , Citometría de Flujo/métodos , Glicosilación , Humanos , Polisacáridos/genéticaRESUMEN
Antibody-drug conjugates utilize the antigen specificity of antibodies and the potency of chemotherapeutic and antibiotic drugs for targeted therapy. However, as cancers and bacteria evolve to resist the action of drugs, innovative controlled release methods must be engineered to deliver multidrug cocktails. In this work, we engineer lipoate-acid ligase A (LplA) acceptor peptide (LAP) tags into the constant heavy and light chain of a humanized Her2 targeted antibody, trastuzumab. These engineered LAP tags, along with the glutamine 295 (Q295) residue in the heavy chain, were used to generate orthogonally cleavable site-specific antibody conjugates via a one-pot chemoenzymatic ligation with microbial transglutaminase (mTG) and LplA. We demonstrate orthogonal cargo release from these dual-labeled antibody bioconjugates via matrix metalloproteinase-2 and cathepsin-B-mediated bond cleavage. To the best of our knowledge, this is the first demonstration of temporal control on dual-labeled antibody conjugates, and we believe this platform will allow for sequential release and cooperative drug combinations on a single antibody bioconjugate.
Asunto(s)
Antineoplásicos Inmunológicos/química , Inmunoconjugados/química , Trastuzumab/química , Antineoplásicos Inmunológicos/farmacocinética , Línea Celular Tumoral , Liberación de Fármacos , Humanos , Inmunoconjugados/farmacocinética , Modelos Moleculares , Péptido Sintasas/química , Péptidos/química , Trastuzumab/farmacocinéticaRESUMEN
The specific targeting of differentially expressed glycans in malignant cells has emerged as an attractive anticancer strategy. One such target is the oncodevelopmental antigen polysialic acid (polySia), a polymer of α2,8-linked sialic acid residues that is largely absent during postnatal development but is re-expressed during progression of several malignant human tumors, including small-cell and non-small cell lung carcinomas, glioma, neuroblastoma, and pancreatic carcinoma. In these cancers, expression of polySia correlates with tumor progression and poor prognosis and appears to modulate cancer cell adhesion, invasiveness, and metastasis. To evaluate the potential of PolySia as a target for anticancer therapy, we developed a chimeric human polySia-specific mAb that retained low nanomolar (nmol/L) target affinity and exhibited exquisite selectivity for polySia structures. The engineered chimeric mAb recognized several polySia-positive tumor cell lines in vitro and induced rapid endocytosis of polySia antigens. To determine whether this internalization could be exploited for delivery of conjugated cytotoxic drugs, we generated an antibody-drug conjugate (ADC) by covalently linking the chimeric human mAb to the tubulin-binding maytansinoid DM1 using a bioorthogonal chemical reaction scheme. The resulting polySia-directed ADC demonstrated potent target-dependent cytotoxicity against polySia-positive tumor cells in vitro. Collectively, these results establish polySia as a valid cell-surface, cancer-specific target for glycan-directed ADC and contribute to a growing body of evidence that the tumor glycocalyx is a promising target for synthetic immunotherapies. SIGNIFICANCE: These findings describe a glycan-specific antibody-drug conjugate that establishes polySia as a viable cell surface target within the tumor glycocalyx.