Generation by phage display and characterization of drug-target complex-specific antibodies for pharmacokinetic analysis of biotherapeutics

Abstract

Anti-idiotypic antibodies play an important role in pre-clinical and clinical development of therapeutic antibodies, where they are used for pharmacokinetic studies and for the development of immunogenicity assays. By using an antibody phage display library in combination with guided in vitro selection against various marketed drugs, we generated antibodies that recognize the drug only when bound to its target. We have named such specificities Type 3, to distinguish them from the anti-idiotypic antibodies that specifically detect free antibody drug or total drug. We describe the generation and characterization of such reagents for the development of ligand binding assays for drug quantification. We also show how these Type 3 antibodies can be used to develop very specific and sensitive assays that avoid the bridging format.Abbreviations: BAP: bacterial alkaline phosphatase; CDR: complementarity-determining regions in VH or VL; Fab: antigen-binding fragment of an antibody; HRP: horseradish peroxidase; HuCAL®: Human Combinatorial Antibody Libraries; IgG: immunoglobulin G; LBA: ligand binding assay; LOQ: limit of quantitation; NHS: normal human serum; PK: pharmacokinetics; VH: variable region of the heavy chain of an antibody; VL: variable region of the light chain of an antibody.

An HPF1/PARP1-Based Chemical Biology Strategy for Exploring ADP-Ribosylation

“…After the selection rounds the antibody genes were subcloned as a pool into a vector for expression of a bivalent Fab format Fab-A-FH (Harth et al, 2019) or bivalent Fab format Fab-A-V5H (Dunn et al, 1999) as previously described (Jarutat et al, 2006). After transformation of E. coli TG1F-(TG1 without the F-plasmid) with the ligated expression vectors, 368 individual colonies were randomly picked and grown in 384-well microtiter plates.…”

“…Antibodies fulfilling the criteria defined in Table S1 were considered positive (e.g., for PARP1-S499ADPr antibodies, clones that bind PARP1-S499ADPr peptide with a signal greater than five-fold over background and do not bind to the respective unmodified PARP1 peptide nor other Ser-ADPr peptides). Such clones were sequenced, and the resulting unique antibodies were expressed and purified as previously described (Harth et al, 2019). E. coli TG1F-cultures (250 ml) containing AbD33204, AbD33205, AbD33641, AbD33644, AbD34251 were grown at 30 C until OD 600nm reached 0.5, and the antibody expression was induced by adding IPTG to a final concentration of 1 mM.…”

Periplasmic Expression of SpyTagged Antibody Fragments Enables Rapid Modular Antibody Assembly

“…ELISA ELISAs were performed as described 47 . Briefly, antigens or capture antibodies were coated in PBS overnight onto 384 microwell plates (Maxisorp, Thermo Fisher Scientific), washed 5 times with PBST (PBS, 0.05% Tween20), and blocked for one hour with PBST/5% BSA, PBST/5% milk powder or ChemiBLOCKER (Merck KGaA).…”

“…For analysis of expression via immunoblotting or ELISA, samples in lysis buffer without further purificationwere used. FcCatchers and full-length immunoglobulins were expressed and purified as described47 .…”

Rapid identification of anti-idiotypic mAbs with high affinity and diverse epitopes by rabbit single B-cell sorting-culture and cloning technology

“…In that context, anti-idiotypic reagent antibodies (anti-IDs or Ab2) have become one of the preferable options for bioanalytical applications at pre-clinical and clinical stages. Common approaches used to generate these types of reagent antibodies against Ab1 include in vivo hybridoma technology, in vitro phage-displayed immunized or synthetic antibody libraries [6][7][8]. In our experience these technologies work reasonably well for generation of anti-IDs but require extended timelines and often yield lownumber panels of super-high affinity monoclonal antibodies (mAbs) that lack epitope-binding diversity.…”