Composition
A composition and drug technology, applied in the direction of drug combination, hybrid immunoglobulin, anti-animal/human immunoglobulin, etc.
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Embodiment 1
[0466] Example 1: Potency of IL-7R binding polypeptides ID-A40U, V7R-2E9 and ID-A62U compared to prior art IL-7R binding polypeptides
[0467] Inhibitory potency and efficacy (maximal inhibition) of IL-7R binding polypeptides ID-A40U (SEQ ID NO: 24) and V7R-2E9 (SEQ ID NO: 25) produced in E. coli in L-7 / IL-7R neutralization ELISA In vitro assays were performed in and compared to the clinical anti-IL-7R antibody mAb829 (also known as "GSK2618960", an anti-IL-7Rα monoclonal antibody disclosed in Ellis et al. 2019).
[0468] A 7-point dilution series of ICVD was prepared in 1% BSA (at 2x assay concentration) starting at 300 nM and using a 3.2 dilution factor. The mAb829 contrast antibody was used as a positive control in the ELISA at concentrations ranging between 10 nM and 0.088 nM (2x assay concentration). Sufficient triplicate volumes were prepared for each ICVD dilution, while sufficient 2 triplicates (2 plates) were prepared for mAb829. 85 μL (or 170 μL) of each ICVD (or m...
Embodiment 2
[0482] Example 2: Biacore estimation of ICVD-IL-7R binding affinity
[0483] The binding kinetics of ID-A40U were compared to mAb829 clinical antibody in a Biacore study. IL-7Rα-His 6 - Fc was directly coated on the Biacore sensor plate (for mAb829 analysis) or captured by anti-human IgG Fc (for ICVD analysis) and the ICVD / Ab was flowed through the plate to detect binding. ID-A40U has 7.8x10 -11 Affinity of M (K D ), and mAb829 has 5.67x10 -10 Slightly lower affinity for M (K D ). The results indicated that ID-A40U showed strong binding to the antigen. ID-A62U contains E1D and R45L mutations relative to ID-A40U.
Embodiment 3
[0484] Example 3: Tolerance to Gastrointestinal Extracts
[0485] Ex vivo incubation in intestinal supernatant predicts the stability of ICVD in the gut of cynomolgus monkeys and humans. The activities of the major intestinal proteases trypsin and chymotrypsin are conserved among mammalian species, whereas proteases present in the large intestine are likely produced by host species-specific gut microflora. To generate test matrices reflecting these two environments, pooled mouse intestinal supernatants and pooled fecal supernatants were prepared. Both matrices are highly digestible for non-selected non-engineered ICVD.
[0486] Anti-TNF-α ICVD ID-38F has previously been shown to have high stability in these matrices (see WO2016 / 156465), and this property predicts that ID-38F has high stability during transport through the gut.
[0487] V7R-2E9 and ID-A40U were tested for survival in gastrointestinal extracts from both murine and human. ICVD was incubated with mouse small in...
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