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Pegylated Trastuzumab Fragments Acquire an Increased in Vivo Stability but Show a Largely Reduced Affinity for the Target Antigen.

ABSTRACT: PEGylation of biomolecules is a major approach to increase blood stream half-life, stability and solubility of biotherapeutics and to reduce their immunogenicity, aggregation potential and unspecific interactions with other proteins and tissues. Antibodies have generally long half-lives due to high molecular mass and stability toward proteases, however their size lowers to some extent their potential because of a reduced ability to penetrate tissues, especially those of tumor origin. Fab or otherwise engineered smaller fragments are an alternative but are less stable and are much less well retained in circulation. We have here investigated the effects of various PEGylations on the binding properties and in vivo half-life of Fab fragments derived from the enzymatic splitting of Trastuzumab. We find that PEGylation increases the half-life of the molecules but also strongly affects the ability to recognize the target antigen in a way that is dependent on the extent and position of the chemical modification. Data thus support the concept that polyethylene glycol (PEG) conjugation on Trastuzumab Fabs increases half-life but reduces their affinity and this is a fine balance, which must be carefully considered for the design of strategies based on the use of antibody fragments.

SUBMITTER: Selis F

PROVIDER: S-EPMC4848947 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:BackgroundMajor histocompatibility complex class I (MHC-I) plays an important role in cell immune response, and stable interaction between polypeptides and MHC-I ensures efficient presentation of polypeptide-MHC-I (pMHC-I) molecular complexes to T cells. The aim of this study was to explore ways to improve the affinity and stability of the p-Human Leukocyte Antigen (HLA)-A*2402 complex.MethodsThe peptide sequences of the restricted antigen peptides for HLA-A*2402 and the results of the in vitro competitive binding test were retrieved from the literature. The affinity values were predicted using NetMHCpan v4.1 server, and the stability values were predicted using the NetMHCstab v1.0 server. Auto Vina was used to dock peptides to HLA-A*2402 protein in a flexible docking manner, while Flexpepdock was employed to optimize the docking morphology. Maestro was used to analyze the intermolecular forces and the binding affinity of the complex, while MM-GBSA was used to calculate the binding free energy values.ResultsThe intermolecular interactions that maintained the affinity and stability of peptide-HLA-A*2402 complex relied mainly on HB, followed by pi stack. The binding affinity values of molecular docking were associated with the predicted values of affinity and stability, the binding affinity and the binding free energy, as well as the intermolecular force pi-stack. The pi stack had a significant negative correlation with binding affinity and binding free energy. The replacement of the residues of the polypeptides that did not form pi-stack interactions with HLA-A*2402 improved the affinity and/or stability compared to before replacement.ConclusionsThe generation and increase in the number of pi-stacks between peptides and HLA-A*2402 molecules may help improve the affinity and stability of p-HLA-A*2402 complexes. The prediction of intermolecular forces and binding affinity of peptide-HLA by means of molecular docking is a supplement to the current commonly used prediction databases.

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Pegylated Trastuzumab Fragments Acquire an Increased in Vivo Stability but Show a Largely Reduced Affinity for the Target Antigen.

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