KRAS G12D is a mutation in the KRAS gene that is commonly found in many types of cancer, including pancreatic, lung, and colorectal cancers. KRAS is a proto-oncogene that plays a critical role in cellular signaling pathways involved in cell growth and proliferation. When mutated, KRAS can become permanently activated, leading to uncontrolled cell growth and tumor formation.
The G12D mutation occurs when a single nucleotide change causes an amino acid substitution from glycine (G) to aspartic acid (D) at position 12 of the KRAS protein. This change disrupts the normal function of KRAS and leads to its constitutive activation, which is thought to be a key driver of tumor initiation and progression.
KRAS G12D has been identified as a particularly challenging mutation to target therapeutically due to its high prevalence and unique biochemical properties. Efforts to develop drugs specifically targeting KRAS G12D have been ongoing for many years, with limited success until recently.
One promising approach involves using small molecules that bind to the mutant KRAS protein and inhibit its activity. One such molecule, known as MRTX849, has shown promising preclinical results and is currently being evaluated in clinical trials for patients with solid tumors harboring the KRAS G12D mutation.
Another approach involves using immunotherapy to target cells expressing the mutant KRAS protein. One recent study showed that a vaccine targeting KRAS G12D was able to stimulate an immune response and slow tumor growth in mice with pancreatic cancer.
Despite these advances, targeting KRAS G12D remains a significant challenge due to its complex biology and diverse downstream effects on cellular signaling pathways. Further research is needed to fully understand the mechanisms underlying this mutation and to develop effective therapies for patients with cancers driven by KRAS G12D.
Numerous cancer forms have mutations in the oncogene KRAS, the most frequent of which are G12D, G12V, and G12C. It cannot be treated with antibody-based therapies since it is an intracellular protein. However, the fact that T cell receptors can recognize the mutant proteins disguised as peptide-HLA (pHLA) suggests that TCR-based treatments may be able to target these proteins. In order to further understand the selectivity of the TCR, Poole et al. isolated a human TCR specific to the KRASG12D decamer peptide given in the context of HLA-A*11. Then, in a study that was just published in Nature Communications, they developed a bispecific T cell-engaging ImmTAC (Immune mobilizing monoclonal TCR against cancer) molecule and evaluated the efficacy of this therapy in vitro utilizing this TCR as the targeting arm.
Human peripheral blood mononuclear cells (PBMCs) from a healthy HLA-A*11+ donor contained the KRAS G12D-specific TCR (JDI TCR). In coculture with HLA-A*11+ acute lymphoblastic leukemia SUP-B15 B cells, JDI TCR-transduced T cells were used. T cells generated IFN when cells were pulsed with the decamer peptide KRASG12D but not KRASWT. To determine selectivity, pools of self-peptides from widely expressed genes and other RAS superfamily members with high amino acid sequence similarity were bound by the soluble JDI TCR. High selectivity is indicated by the fact that no binding to these other pHLA complexes was found.
The TCR affinity can be increased to enhance targeting of antigens that are delivered at low levels. The researchers used NNK randomization of complementarity-determining regions (CDR) to improve the specific JDI TCR's affinity, and they used affinity variant phage libraries to select TCRs with improved affinity to G12D and in the G12D/WT affinity window.
The crystal structures of the JDIa41b1 TCR in complex with the HLA-peptides were solved by the researchers in order to better understand the molecular basis of TCR selectivity. These structures revealed that the TCR and pHLA exhibit nearly identical conformations in both the WT and mutant G12D complexes of the JDIa41b1 TCR. There were variations in how the peptides interacted with the HLA, with the larger aspartate residue being unable to make interactions with the HLA groove while the smaller glycine may produce a more stable epitope when attached to the TCR.
The structures of both pHLAs without bound TCR were solved and compared to the conformation of the peptide in the JDIa41b1 TCR bound form of each complex in order to determine the mechanism underlying the variations in affinity between mutant and WT complex for the JDIa41b1 TCR. The core residues (4-6) of both peptides were shifted by JDIa41b1 binding, changing their orientation from facing away from the HLA (in pHLA alone) to toward the HLA-F-pocket. As a result, more links between the KRASG12D peptide and the HLA groove could develop.
Thermodynamics and solvation states may be crucial in determining a TCR's antigen specificity and affinity. In order to evaluate biomolecular binding, the researchers then evaluated thermodynamics by performing surface plasmon resonance (SPR) at a variety of temperatures. This demonstrated that JDIa41b1 TCR interactions with both pHLAs were enthalpically driven. The contact with KRASG12D had a higher favorable value, indicating a greater increase in electrostatic interactions. On the other hand, the G12D structure was more unfavorable than the other two TCR-pHLA complexes in terms of entropy. The researchers ran molecular dynamics (MD) simulations to further evaluate the variables influencing the energy disparities. Around the mutation site and the TCR contact zone, a difference in the surface electrostatic potential of HLA-KRASG12D and -KRASWT was discovered.
The interface with pHLA may alter as the TCR affinity increases, and the added mutations must maintain or improve selectivity. The researchers used a single-chain trimer format shown on phage to pan a third-generation affinity-enhanced JDI96b35 TCR against a high-complexity HLA-A*11 pHLA library in order to evaluate this selectivity. Following three rounds of panning, 452 peptides were found, and from these, a peptide specificity profile was produced, from which 20 self-peptides that might serve as structural mimics of the KRASG12D peptide were found. After that, the JDIa96b35 TCR's affinity for peptide-HLA complexes other than KRASG12D was evaluated using this panel. 18/20 of the pHLAs did not attach, while two did so with modest affinities, indicating significant selectivity for KRASG12D.
The affinity-enhanced JDIa96b35 TCR was subsequently fused to a humanized CD3-specific scFv (IMC-KRASG12D) to create a bispecific ImmTAC molecule. In order to reroute and activate T lymphocytes to target tumor cells, ImmTAC molecules can directly engage their pHLA targets on tumor cells. The decamer KRASG12D or KRASWT-loaded SUP-B15 cells were cultured with unstimulated PBMCs in the presence of IMC-KRASG12D. IFN was generated in the cocultures containing KRASG12D, and CD25+CD69+ T cells were found to be activated.
Next, autologous T cells and IMC-KRASG12D were cocultured with healthy donor immature dendritic cells (iDCs) electroporated with mRNA expressing WT or G12D KRAS. In cultures transfected with KRASG12D, the addition of IMC-KRASG12D increased IFN production. Untransfected iDCs that had received peptide pulses had similar outcomes. In order to test the specificity of IMC-KRASG12D, the KRASG12D mutation and/or HLA-A*11 were then added to the pancreatic cancer cell line PSN-1 using CRISPR/Cas9. IFN was only created in cocultures containing PSN-1 cells that also expressed KRASG12D and HLA-A*11, demonstrating that KRASG12D is only specific when HLA-A*11 is present. Cocultures comprising IMC-KRASG12D, a number of target cell lines, including HLA-A*11+ cancer cell lines with diverse KRAS mutations, and numerous healthy donor cell lines were used in T cell tests. IFN, IL-2, and granzyme B were produced during the incubation of the KRASG12D-mutated CL40 colon cancer cell line, which also caused targeted T cell death. Other cancer cell lines that were treated with IMC-KRASG12D produced very little to no IFN and no T cell death was seen. When IMC-KRASG12D was treated with healthy cells, IFN release was also not induced.
These findings imply that an ImmTAC molecule that precisely targets the oncogenic KRAS G12D mutation can work as a T cell engager, activating T cells to preferentially attack cancer cells. This study provides proof of concept for the pHLA pathway-based targeting of carcinogenic neoantigens.
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