Pharmacologically active antibodies for treatment of cancer and pain

In collaboration with Oblique Therapeutics AB (OTAB), Göteborg, the Team of Translational Pharmacology, FyFa, Karolinska Institutet, have developed a microfluidics/mass spectrometry platform using kinetically controlled proteolysis for identifying druggable epitopes on difficult-to-drug target proteins. These peptide sequences are identified by proteases in essentially native state proteins in motion capable of capturing structural dynamics of the target protein. The protease binding surface area is of similar size to the antigen binding region of antibodies; thus, they are highly suitable for production of high affinity, pharmacologically active antibodies. Using this platform, a library of candidate antibodies was developed. The targets involve the nociceptive human transient receptor potential cation channel subfamily V member 1 (hTRPV1) and the oncoprotein Kirsten rat sarcoma virus homologue (KRAS) which becomes highly oncogenic after a single missense mutation. TRPV1 is a highly complex ion-channel which is activated by several different modes of stimulation (multimodal) such as heat, low pH, and lipid compounds (vanilloids). The candidate mAb selected qualified due to an inhibitory action on hTRPV1 channels but spared the heat sensitivity of the channel which results in potential analgesic effects whilst avoiding heat-related side-effects. Antibody candidates directed to KRAS exhibited increased KRAS-dependent signalling aberrations as well as increased apoptosis and necrosis of cancer cells expressing the G13D (Gly exchanged for Asp at the 13th position) mutation. Furthermore, we discovered that monoclonal antibodies (mAbs) are readily taken up by cancer cells as collateral cargo from macropinocytosis which provides a venue for pharmacological activity targeting the recycling of KRAS protein in the cytoplasm. When investigating the structural properties of G13D KRAS with X-ray crystallography, we discovered that it is possible to open up G13D KRAS by stripping the Mg2+ ion from the protein's active site. Furthermore, the protein could also be opened in the liquid phase by depleting the buffer media of Mg2+ ions. The binding affinity (EC50-value) of one of the produced monoclonal antibodies (named the 4B8 antibody) was increased about 6-fold comparing Mg2+ -free to Mg2+-containing media. This methodology of stripping Mg2+ and opening up G13D KRAS in a biologically relevant solution environment was further confirmed using Size Exclusion Chromatography coupled to Small Angle X-ray Scattering (SEC-SAXS). Noticeably, when Mg2+ was stripped from the G13D-mutated version, it started to multimerize and aggregate. This aggregation behaviour was not observed in the WT-version of KRAS that was used as a control in these experiments. G13D KRAS is known to have a much higher rate of nucleotide exchange compared to e.g. WT KRAS which can be one reason to that G13D has a higher propensity to stay in an open state and having a higher capacity of Mg2+-release. We also confirmed that G13D-directed mAbs are taken up by cancer cells through macropinocytosis, in line with several recent scientific reports and observations for other KRAS binding monoclonal and polyclonal antibodies. This capacity of internalization of KRAS antibodies will be important to further explore for potential clinical applications.

List of scientific papers

I. Rational antibody design for undruggable targets using kinetically controlled biomolecular probes. Science Advances. Carolina L. Trkulja†, Oscar Jungholm†, Max Davidson†, Kent Jardemark, Monica M. Marcus, Jessica Hägglund, Anders Karlsson, Roger Karlsson, Joseph Bruton, Niklas Ivarsson, Sreesha P. Srinivasa, Alexandra Cavallin, Peder Svensson, Gavin D. M. Jeffries, Maria-Nefeli Christakopoulou, Anna Reymer, Anaswara Ashok, Gabriella Willman, Daniela Papadia, Emma Johnsson, Owe Orwar*. https://doi.org/10.1126/sciadv.abe6397
*Corresponding author. †These authors contributed equally to this work.

II. Novel druggable space in human KRAS G13D discovered using structural bioinformatics and a P-loop targeting monoclonal antibody. Scientific Reports. Oscar Jungholm†, Carolina L. Trkulja†, Martin Moche†, Sreesha P. Srinivasa, Maria-Nefeli Christakopoulou, Max Davidson, Anna Reymer, Kent Jardemark, Rafaela Lenza Fogaça, Anaswara Ashok, Gavin Jeffries, Henry Ampah-Korsah, Emilia Strandback, Juni Andréll, Tomas Nyman, Ghada Nouairia, and Owe Orwar. https://doi.org/10.1038/s41598-024-70217-9
†These authors contributed equally to this work.

III. Small angle x-ray scattering and structural bioinformatics studies reveal a Mg2+-free, open state human G13D KRAS with high propensity for homodimerization, and aggregation reactions in solution. Oscar Jungholm†, Martin Moche†, Carolina Trkulja, Henry Ampah- Korsah, Emilia Strandback, Tomas Nyman, Jill Trewhella, Sreesha Srinivasa, and Owe Orwar. [Manuscript]
†These authors contributed equally to this work