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Optimal Synthetic Glycosylation of a Therapeutic Antibody.

ABSTRACT: Glycosylation patterns in antibodies critically determine biological and physical properties but their precise control is a significant challenge in biology and biotechnology. We describe herein the optimization of an endoglycosidase-catalyzed glycosylation of the best-selling biotherapeutic Herceptin, an anti-HER2 antibody. Precise MS analysis of the intact four-chain Ab heteromultimer reveals nonspecific, non-enzymatic reactions (glycation), which are not detected under standard denaturing conditions. This competing reaction, which has hitherto been underestimated as a source of side products, can now be minimized. Optimization allowed access to the purest natural form of Herceptin to date (?90?%). Moreover, through the use of a small library of sugars containing non-natural functional groups, Ab variants containing defined numbers of selectively addressable chemical tags (reaction handles at Sia C1) in specific positions (for attachment of cargo molecules or "glycorandomization") were readily generated.

SUBMITTER: Parsons TB

PROVIDER: S-EPMC4973692 | biostudies-literature | 2016 Feb

REPOSITORIES: biostudies-literature

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Optimal Synthetic Glycosylation of a Therapeutic Antibody.

Parsons Thomas B TB Struwe Weston B WB Gault Joseph J Yamamoto Keisuke K Taylor Thomas A TA Raj Ritu R Wals Kim K Mohammed Shabaz S Robinson Carol V CV Benesch Justin L P JL Davis Benjamin G BG

Angewandte Chemie (International ed. in English) 20160112 7

Glycosylation patterns in antibodies critically determine biological and physical properties but their precise control is a significant challenge in biology and biotechnology. We describe herein the optimization of an endoglycosidase-catalyzed glycosylation of the best-selling biotherapeutic Herceptin, an anti-HER2 antibody. Precise MS analysis of the intact four-chain Ab heteromultimer reveals nonspecific, non-enzymatic reactions (glycation), which are not detected under standard denaturing con ...[more]

PMID: 26756880

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Project description:BackgroundMonoclonal antibodies (mAbs) and their derivatives are the fastest expanding category of pharmaceuticals. Efficient screening and generation of appropriate therapeutic human antibodies are important and urgent issues in the field of medicine. The successful in vitro biopanning method for antibody screening largely depends on the highly diverse, reliable and humanized CDR library. To rapidly obtain potent human antibodies, we designed and constructed a highly diverse synthetic human single-chain variable fragment (scFv) antibody library greater than a giga in size by phage display. Herein, the novel TIM-3-neutralizing antibodies with immunomodulatory functions derived from this library serve as an example to demonstrate the library's potential for biomedical applications.MethodsThe library was designed with high stability scaffolds and six complementarity determining regions (CDRs) tailored to mimic human composition. The engineered antibody sequences were optimized for codon usage and subjected to synthesis. The six CDRs with variable length CDR-H3s were individually subjected to β-lactamase selection and then recombined for library construction. Five therapeutic target antigens were used for human antibody generation via phage library biopanning. TIM-3 antibody activity was verified by immunoactivity assays.ResultsWe have designed and constructed a highly diverse synthetic human scFv library named DSyn-1 (DCB Synthetic-1) containing 2.5 × 1010 phage clones. Three selected TIM-3-recognizing antibodies DCBT3-4, DCBT3-19, and DCBT3-22 showed significant inhibition activity by TIM-3 reporter assays at nanomolar ranges and binding affinities in sub-nanomolar ranges. Furthermore, clone DCBT3-22 was exceptionally superior with good physicochemical property and a purity of more than 98% without aggregation.ConclusionThe promising results illustrate not only the potential of the DSyn-1 library for biomedical research applications, but also the therapeutic potential of the three novel fully human TIM-3-neutralizing antibodies.

Project description:Polymer conjugation is a clinically proven approach to generate long acting protein drugs with decreased immune responses. Although poly(ethylene glycol) (PEG) is one of the most commonly used conjugation partners due to its unstructured conformation, its therapeutic application is limited by its poor biodegradability, propensity to induce an anti-PEG immune response, and the resultant accelerated blood clearance (ABC) effect. Moreover, the prevailing preference of unstructured polymers for protein conjugation still lacks strong animal data support with appropriate control reagents. By using two biodegradable synthetic polypeptides with similar structural compositions (l-P(EG3Glu) and dl-P(EG3Glu)) for site-specific protein modification, in the current study, we systematically investigate the effect of the polymer conformation on the in vivo pharmacological performances of the resulting conjugates. Our results reveal that the conjugate l20K-IFN, interferon (IFN) modified with the helical polypeptide l-P(EG3Glu) shows improved binding affinity, in vitro antiproliferative activity, and in vivo efficacy compared to those modified with the unstructured polypeptide analogue dl-P(EG3Glu) or PEG. Moreover, l20K-IFN triggered significantly less antidrug and antipolymer antibodies than the other two. Importantly, the unusual findings observed in the IFN series are reproduced in a human growth hormone (GH) conjugate series. Subcutaneously infused l20K-GH, GH modified with l-P(EG3Glu), evokes considerably less anti-GH and antipolymer antibodies compared to those modified with dl-P(EG3Glu) or PEG (dl20K-GH or PEG20K-GH). As a result, repeated injections of dl20K-GH or PEG20K-GH, but not l20K-GH, result in a clear ABC effect and significantly diminished drug availability in the blood. Meanwhile, immature mouse bone marrow cells incubated with the helical l20K-GH exhibit decreased drug uptake and secretion of proinflammatory cytokines compared to those treated with one of the other two GH conjugates bearing unstructured polymers. Taken together, the current study highlights an urgent necessity to systematically reassess the pros and cons of choosing unstructured polymers for protein conjugation. Furthermore, our results also lay the foundation for the development of next-generation biohybrid drugs based on helical synthetic polypeptides.

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Optimal Synthetic Glycosylation of a Therapeutic Antibody.

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Optimal Synthetic Glycosylation of a Therapeutic Antibody.

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