KRAS mutations are among the most common oncogenic drivers in human cancer, implicated in approximately 30% of tumors. The G12D variant is particularly prevalent and has been notoriously difficult to target with precision tools. In our latest publication, we report the development of a new site-directed monoclonal antibody that selectively recognizes KRAS G12D with high affinity and exceptional specificity. Using our partner's proprietary Epivolve™ platform, we engineered an antibody that demonstrates over 1,000-fold greater binding affinity for the G12D mutant compared to wild-type KRAS.
This blog post outlines the approach, highlights key validation data, and explores how these antibodies could advance cancer research, diagnostics, and future therapeutic strategies.
Effective antibody development begins with understanding the structural landscape of the target protein. In this case, that meant taking a close look at KRAS, specifically the G12D mutation, one of the most frequent oncogenic alterations across cancer types.
Figure 1 illustrates the structural environment surrounding this critical mutation. Using high-resolution crystallographic data (PDB ID: 7T47), the team mapped the KRAS protein in complex with both a GTP analog and MRTX1133, a known non-covalent KRAS G12D inhibitor. The G12D residue, shown in red, lies within a highly conserved region of the protein and is part of the phosphate-binding loop (P-loop), highlighted in cyan. These visualizations were generated using UCSF Chimera to ensure precise modeling of epitope accessibility.
This foundational analysis ensured that the antibodies we developed would not only recognize KRAS G12D but do so with high structural specificity, setting the stage for precise mutation targeting.
To generate antibodies capable of distinguishing KRAS G12D from its wild-type and variant forms, we used AbbraTech’s proprietary Epivolve™ platform — a multi-stage immunization strategy designed for precision epitope targeting.
We began by producing seven recombinant KRAS variants (G12G, G12V, G12D, G12R, G12A, G12S, and G12C) using a truncated version of the human KRAS protein (residues 2–128), with each variant reaching over 90% purity as confirmed by SDS-PAGE (Figure 2). These recombinant proteins provided the foundation for both immunization and screening.
Each variant was used to immunize a dedicated rabbit cohort through Epivolve’s stepwise protocol. This included sequential administration of epitope-focused peptides (Mod1), native peptides (NAT), and full-length proteins, designed to direct the immune response to the G12D site within the protein’s natural context.
ELISA analysis of antisera from different time points showed a clear progression in immune targeting. In the KRAS G12D cohort (rabbit AB232), antibody titers increased from an optical density (OD) of ~1.0 after the Mod1 stage to >1.3 following the final protein boost (Figure 3). Notably, by the NAT stage (before animals had even encountered the full-length KRAS proteins) we observed strong antibody responses to the intact G12D variant. This indicates that the immune system had been successfully trained to recognize the G12 epitope within the native protein conformation.
These findings validated Epivolve’s capacity to elicit site-directed immune responses with strong specificity for mutation-bearing KRAS, a critical first step in producing high-fidelity monoclonal antibodies.
With strong site-directed antibody responses confirmed, the next step was isolating monoclonal antibodies with high affinity and specificity for KRAS G12D. Using single B cell sorting from the best-performing immunized rabbit, we recovered 35 heavy and light chain pairs with potential G12D specificity.
These antibody candidates were recombinantly expressed and screened using ELISA to assess binding to both the immunogen peptides and full-length KRAS protein variants. Several clones showed strong selective binding to G12D, with minimal to no cross-reactivity with wild-type KRAS or other G12 variants.
The lead clone, designated ATM00004, was characterized in greater detail. Surface Plasmon Resonance (SPR) was used to quantify binding kinetics, revealing an equilibrium dissociation constant (KD) in the low nanomolar range. Most notably, ATM00004 showed over 1,000-fold higher affinity for KRAS G12D compared to wild-type KRAS, confirming its ability to distinguish a single amino acid substitution in a structurally conserved protein family.
This degree of selectivity is not only uncommon but also essential for applications where off-target recognition can compromise interpretation, such as in diagnostic assays or therapeutic research.
Beyond binding kinetics, functional performance in real-world assays is essential to demonstrate the utility of a site-directed antibody. We evaluated clone ATM00004 across multiple assay formats, including Western blot and immunocytochemistry (ICC), to confirm its specificity for endogenously expressed KRAS G12D in biologically relevant systems.
In Western blot analysis, ATM00004 recognized purified KRAS G12D protein with strong signal intensity, while showing no detectable binding to wild-type KRAS or other G12 variants. This specificity held true in complex lysates from cancer cell lines known to harbor the G12D mutation. Only samples expressing the target mutation yielded clear signal, underscoring the antibody’s precision.
These data confirm that ATM00004 performs reliably in both denaturing and native assay conditions, making it a valuable tool for detecting KRAS G12D in cellular models, tissue samples, or potentially even diagnostic workflows.
KRAS G12D continues to be one of the most challenging and consequential mutations in cancer biology. The development of site-directed antibodies like ATM00004 marks a significant advancement in the ability to detect and study this mutation with confidence.
Using the Epivolve™ platform, we engineered antibodies that combine nanomolar affinity, mutation-specific precision, and validated performance across multiple assay types. These tools open new possibilities for researchers working in oncology, diagnostics, and translational medicine.
The full publication, Site-directed antibodies targeting driver mutations of the KRAS protein, is now available. To learn more or explore the KRAS G12D Mutant Recombinant Antibody [29B] (ATM00004) for your own research, we invite you to download the publication or contact our team.