Cart (0 Items)
Your cart is currently empty.
View ProductsCase report
Engineering Seven Bispecific Antibody Formats Yielding Nanomolar Leads Against Prostate Cancer
Download the complete case report Contact our Experts
Client
Swedish Biotech
Sector
Therapeutics
Research domain
Oncology
Target disease
Prostate cancer
Key processes
- Bispecific format choice and design
- Expression and purification
- Characterization by ELISA and Biocore
7
Bispecific formats designed
3.20x10⁻¹⁰ M
Affinity of the best bispecific candidate
Context
A Swedish biotech company trusted ProteoGenix with the design and production of various bispecific antibody formats. The customer supplied the sequences of two monoclonal antibodies, designated Ab1 and Ab2, which served as the foundation for bispecific antibody engineering.
To optimize the therapeutic potential of these parental antibodies, seven distinct bispecific formats were selected for design and development by our experts:
- Ab1-Ab2-KIH
- Ab1-Ab2-DEKK
- Ab1-Ab2-CrossMAb-KIH
- Ab1-Ab2-CrossMAb-DEKK
- Ab1-Ab2-DuetMab-KIH
- Ab1-Ab2-DuetMab-DEKK
- Ab1-Ab2-Duobody
This multi-format approach designed by our experts enabled comprehensive evaluation of different molecular architectures to identify the most suitable candidate(s) for further development.
Challenges
1
Expressing the seven bispecific antibodies with the right assembly
2
Keeping the affinity of the generated bispecific antibodies in the same range as the parental antibodies
ProteoGenix Approach
Genes coding for target proteins
- cDNAs chemically synthesized with codon optimization for expression in mammalian cells
Subcloning in expression vector
- cDNA sequences subcloned in ProteoGenix’s proprietary mammalian cells expression vector pTXs1. A total of 14 expression plasmids were used for this project.
Recombinant bispecific antibody expression & purification tests
- Endotoxin-free DNA purification was prepared for each pTXs1 expression construction.
- Transiently co-transfected in proprietary XtenCHOTM cells
- Purified on a protein G resin using a standard method
- Determination of the yields after purification and buffer exchange was done by UV280 method
Post-production reaction for IgG-like format using Duobodies
- Ab1-Duobody and Ab2-Duobody were recombined by controlled Fab-arm exchange driven by the matched mutations under tailored laboratory conditions
- General quality assessment of the generated bispecific antibodies was then performed by SDS-PAGE
Characterization of the different bispecific formats
- ELISA titration binding capacity of the bispecific antibodies to Ag1 and Ag2. Ab1 and Ab2 parental antibodies were also tested for comparison.
- KD determination was performed via Biacore T200 to evaluate the binding capacity of bispecific formats to both Ag1 and Ag2 compared to the parental antibodies The three best bispecific formats determined at previous step were selected for KD determination: Ab1-Ab2-Duobody, Ab1-Ab2-KIH and Ab1-Ab2-CrossMAb-KIH.
Results
SDS-PAGE analyses of purification profiles and final samples QCs showed that Ab1-Ab2-DEKK and Ab1-Ab2-DuetMab-DEKK formats are expressed but not assembled in full bispecific antibody structures likely due to lack of heavy chain heterodimerization. The DEKK mutation thus does seem to be an optimal bispecific format for this specific Ab1/Ab2 pair. All other bispecific formats and single Duobodies could be expressed with good yield and purity, and correctly assembled in full antibody structures.
Each bispecific antibody tested could bind to the antigens, although the two DEKK bispecific formats Ab1-Ab2-DEKK and Ab1-Ab2-DuetMab-DEKK showed very low binding consistent with the fact that they did not assemble properly. Compared to Ab1 and Ab2 parental antibodies, the best bispecific antibodies in terms of binding are Ab1-Ab2-Duobody, Ab1-Ab2-KIH and Ab1-Ab2-CrossMAb-KIH, suggesting that these bispecific constructs conserved their binding capacity.
KD determination results are coherent with ELISA titrations performed above. The binding of all three bispecific antibodies tested is confirmed against each antigen with high affinity in nanomolar range. A difference in affinity can be noted for all bispecific antibodies compared to the corresponding parental antibodies, which can be explained by reduced avidity as parental antibody has two binding sites to the antigen compared to bispecific antibodies which are monovalent for each antigen.
Key Takeway
The seven chosen bispecific antibody formats were successfully designed and expressed against two prostate cancer targets, with five formats achieving proper assembly and dual-antigen binding.
Three top candidates — Duobody, KIH, and CrossMAb-KIH — were confirmed by Biacore to retain nanomolar affinity for both antigens, with the best candidate reaching 3.20×10⁻¹⁰ M.
This demonstrates that a multi-format screening approach is an effective strategy for identifying high-affinity bispecific antibody leads while avoiding assembly pitfalls such as those observed with the DEKK-based formats.
Want to know more about this project?
Download the full case report to read the protocols, detailed results and more.
Related Services
