(M1530-01-02) Game of Clones – Investigating the Implications of Mouse Strain and Adjuvant Selection on Sequence Diversity of Monoclonal Anti-ID Reagents

Purpose: To investigate the impact of mouse strain and adjuvant selection on the sequence diversity of the Anti-Idiotypic antibody repertoire, generated from the mouse-hybridoma based reagent generation campaign.

Methods: Anti-idiotypic (Anti-ID) antibodies were generated in 6 independent immunization campaigns. These antibodies were generated using the mouse hybridoma approach1. For each immunization campaign, three different strains of mice (A/J, Balb/C and CD-1) were used, coupled with two different adjuvants (Freund’s and TiterMaxGold). The selected mAbs were also subjected to V-gene recovery and Sanger Sequencing. For the purposes of this study, a one amino acid (AA) change in the CDR region of the antibody was considered enough for determining uniqueness2. Finally, the CDR sequences and the binding kinetics were analyzed based on the immunization strategy used on the mice from which said monoclonal antibodies were extracted. This was done to try to identify a correlation between the immunization strategy and the mAb diversity and binding strength. A visual representation of the sequence diversity based on the immunization strategy was also included using an internal Boehringer-developed tool TaDI.

Results: Preliminary analysis of immunization and sequence data on all anti-IDs from the six campaigns revealed the combination of CD1 mice with TiterMax adjuvant yielded the highest number of stable and unique anti-ID clones (n = 214). 29.32% clones exhibiting distinct sequences – with at least one distinct amino acid in the CDR or framework regions of the mAb – in each of the campaigns and lastly 63.18% clones from CD1 mice with Titermax group exhibited strong binding with < 1nM affinity.

Conclusion: The findings from our analyses suggest that both, the mouse strain as well as the adjuvant selection can have a significant impact on the diversity and binding affinity of anti-ID antibodies. Optimizing the strain and adjuvant selections can: (i) significantly enhance the efficiency of anti-ID generation workflows. (ii) improve downstream assay performance. (iii) while reducing the burden on animal use due to the need for repeat campaigns due to low affinity or non-diverse set of anti-IDs.

References: Oquendo E, Savoie J, Swenson JM, Grimaldi C. Critical reagent generation, characterization, handling and storage workflows: impact on ligand binding assays. Bioanalysis. 2021;13(10):847–60.
Oquendo E, Lin X, Ye S, Coble K, Grimaldi C. Using multiple platforms for critical reagents selection process to support pharmacokinetic ligand-binding assay development. Bioanalysis. 2021;13(10):761–9.
Alex Horspool, Kyle Martin, Nate Azevedo. TADI. 2025. BI BTD Portal. (internal Boehringer resource)

Acknowledgements: The authors would like to acknowledge and thank numerous colleagues in Biotherapeutics Discovery and Development DMPK groups, especially Alexander Horspool, Kyle Martin, Nate Azevedo, Kathryn Phoenix and Niksa Kastrapeli and Elisa Oquendo, at Boehringer Ingelheim Pharmaceuticals Inc. for excellent discussions and support.

Sequence analysis using an internal sequence alignment illustration tool - TaDI, indicated greater CDR3 diversity in CD1 mice derived clones, suggesting a strain-dependent bias in Anti-ID repertoire.


Overall, a higher number of unique anti-IDs were obtained from CD1 strain, especially with the TiterMax combination. Balb/c strain with Freund’s combination also yielded anti-IDs with distinct sequence.

Binding affinity for CD1 mice generated anti-IDs were stronger overall compared to other strains. Titermax with CD1 produced stronger binders but not with Balb/c or A/J strains.
Binding affinity tested using ELISA and confirmed using SPR.
Strong Binder - mAbs with affinities < 1nM
Weak Binder - mAbs with affinities > 1nM
ELISA setup is done using the full-length anti-ID mAb whereas the SPR setup utilizes Fab based true affinity setup.