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Generation of a Monoclonal Antibody to Detect Elastin-like Polypeptides.

ABSTRACT: The identification and use of antibodies dominate the biologic, clinical diagnostic, and therapeutic landscapes. In particular, antibodies have become essential tools in a variety of protein analytical experiments and to study the disposition of biologic therapeutics. One emerging class of peptide biologics is known as the elastin-like polypeptides (ELPs), which are repetitive protein polymers inspired by human tropoelastin. A major limitation in the clinical translation of ELP biologics has been a lack of a monoclonal antibody (mAb) to characterize their identity during expression. To facilitate these studies, we successfully generated a new mAb that is specific toward ELPs and ELP fusion proteins. A purified antibody was evaluated in an ELISA, western blotting, and immunofluorescence assay. The optimal anti-ELP mAb proved to be highly reactive and specific toward ELPs. Moreover, they were able to detect ELPs with a variety of aliphatic guest residues. ELPs phase-separate in response to heating; furthermore, when incubated at a great excess of ELPs, the anti-ELP mAb partially blocks phase separation. These findings are direct evidence that novel murine mAbs can be raised against purified ELPs. This new reagent will enable purification, experimental detection, and characterization of these biopolymers.

SUBMITTER: Kouhi A

PROVIDER: S-EPMC7219198 | biostudies-literature | 2019 Aug

REPOSITORIES: biostudies-literature

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Generation of a Monoclonal Antibody to Detect Elastin-like Polypeptides.

Kouhi Aida A Yao Zhiyuan Z Zheng Long L Li Zhe Z Hu Peisheng P Epstein Alan L AL MacKay J Andrew JA

Biomacromolecules 20190719 8

The identification and use of antibodies dominate the biologic, clinical diagnostic, and therapeutic landscapes. In particular, antibodies have become essential tools in a variety of protein analytical experiments and to study the disposition of biologic therapeutics. One emerging class of peptide biologics is known as the elastin-like polypeptides (ELPs), which are repetitive protein polymers inspired by human tropoelastin. A major limitation in the clinical translation of ELP biologics has bee ...[more]

PMID: 31276401

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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.

Dataset Information

Generation of a Monoclonal Antibody to Detect Elastin-like Polypeptides.

Publications

Generation of a Monoclonal Antibody to Detect Elastin-like Polypeptides.

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