ABSTRACT
A new strategy of peptide half-life extension has been evaluated. We investigated libraries of a small and very stable protein scaffold called Nanofitin, capable of high affinity for protein targets. We have identified Nanofitins targeting Human and mouse Serum Albumin, which could significantly improve the pharmacokinetics of an active associated peptide, mobilizing the patient's own albumin without external source. To demonstrate the impact of this approach on half-life extension, a genetic fusion of an Exenatide peptide with an Albumin Binding Nanofitin (ABNF) was performed. Specific activity of Exenatide-ABNF was measured and unaffected by the fusion. In vivo mice results provided convincing data (t½ of 8 min for Exenatide peptide compared to 20 h for Exenatide-ABNF) with sustained pharmacological activity over 3 days. This study constitutes a proof-of-concept of in vivo half-life extension of a biologic using an ABNF. Besides, the absence of cysteine in the Nanofitin scaffold, which is therefore devoid of structuring disulfide bonds, allows manufacturing in microbial cost effective systems.
Subject(s)
Biological Products , Peptides , Albumins , Animals , Exenatide , Half-Life , Mice , Peptides/chemistryABSTRACT
Most antibodies display very low brain exposure due to the blood-brain barrier (BBB) preventing their entry into brain parenchyma. Transferrin receptor (TfR) has been used previously to ferry antibodies to the brain by using different formats of bispecific constructs. Tetravalent bispecific tandem immunoglobulin Gs (IgGs) (TBTIs) containing two paratopes for both TfR and protofibrillar forms of amyloid-beta (Aß) peptide were constructed and shown to display higher brain penetration than the parent anti-Aß antibody. Additional structure-based mutations on the TfR paratopes further increased brain exposure, with maximal enhancement up to 13-fold in wild-type mice and an additional 4-5-fold in transgenic (Tg) mice harboring amyloid plaques, the main target of our amyloid antibody. Parenchymal target engagement of extracellular amyloid plaques was demonstrated using in vivo and ex vivo fluorescence imaging as well as histological methods. The best candidates were selected for a chronic study in an amyloid precursor protein (APP) Tg mouse model showing efficacy at reducing brain amyloid load at a lower dose than the corresponding monospecific antibody. TBTIs represent a promising format for enhancing IgG brain penetration using a symmetrical construct and keeping bivalency of the payload antibody.
ABSTRACT
Given the nature of the ADCs (Antibody Drug Conjugates) as antibodies carrying cytotoxic drugs, two types of immunoassays are usually implemented to perform the analysis of preclinical and clinical study samples during the development phase. The first assay measures the conjugated antibody defined as the ADC carrying at least 1 drug molecule (i.e. drug/antibody ratio greater than or equal to 1). The other measures the total antibody, defined as the ADC irrespective of the drug load (i.e., drug/antibody ratio greater than or equal to 0). One analytical limitation of the total antibody assay is the difficulty to adequately calibrate the assay due to the lack of a representative standard reference for the different circulating entities which change in proportion with time following ADC administration. A new analytical approach that gets round the above highlighted limitation is presented with the development and the validation of a method to quantify selectively naked antibody to support the development of SAR566658 (huDS6-SPDB-DM4). Assessed on 6 separate occasions, the accuracy ranged from -4.3% to 8.9% of nominal values and the precision is 13% at most. The current assay was successfully validated for the quantitation of huDS6 in human LiHe plasma even in the presence of SAR566658 up to 2.00 µg/mL as demonstrated using in vitro spike in quality controls and in actual clinical samples. This innovative assay provides a new tool to document in vivo plasma stability of ADCs and potentially to optimize dose and regimen selection for ADC development.
