Background Activating mutations in the Kirsten rat sarcoma (KRAS) gene are highly prevalent oncogenic driver mutations in human cancers associated with tumorigenesis and aggressive tumor growth.¹ The global target population for mutated KRAS (mKRAS) malignancies is estimated to exceed 300,000 patients, with high prevalence found in pancreatic (81.72%), colorectal (37.97%) and non-small cell lung cancer patients (21.20%).² mKRAS mainly comprises 21 missense mutations, with G12D (29.19%), G12V (22.97%), and G12C (13.43%) being the most common.³ To date, mKRAS has been considered ‘undruggable’⁴ except for G12C mutations where a recently approved targeted therapy shows benefit, but unmet need for further efficacy improvements remains.^{5 6} T cell receptor T cell (TCR-T) therapies have demonstrated efficacy in mKRAS⁺ tumors but addressable patient populations are limited by human leukocyte antigen (HLA) restriction.^{7 8} We describe a library of optimal affinity mKRAS-specific TCRs which together recognize multiple mutations whilst covering multiple HLA allotypes. These TCRs demonstrate highly specific, sensitive and safe (3S) recognition of mKRAS⁺ solid tumor cells but do not react against wildtype KRAS⁺ cells. 3S TCRs combined with a costimulatory switch receptor (CSR) may armor and enhance TCR-T cell function in hostile tumor microenvironments.

Methods T cells of multiple healthy donors were primed using dendritic cells, providing diverse TCR sequences for comparison. TCRs identified by functional screening were co-expressed with a PD1–41BB CSR in recipient TCR-T cells for assessment of cytokine release, cytotoxicity, functional avidity, HLA-allogeneic cross-recognition and specificity of HLA restriction, as well as CD8 co-receptor (in)dependency.

Results The TCR library has specificity for different KRAS mutations in association with several HLA-A*11 subtypes, including HLA-A*11:01,11:02,11:03 and 11:04. Identification of G12V TCRs with CD8 co-receptor independency was of particular interest. Studies characterizing mKRAS-specific TCRs relevant to the three most prevalent indications are ongoing. Impact of PD1–41BB on armoring and enhancement is also investigated.

Conclusions Our multi-dimensional library of optimal affinity mKRAS-specific TCRs is designed to cover mutations prominent in several solid tumor indications. The library of TCRs recognizing multiple mKRAS mutations presented by different HLA allotypes may address the challenges of tumor heterogeneity and population diversity by expanding the patient population suitable for treatment. Dual armoring and enhancement of TCR-T cells with PD1–41BB CSR may simultaneously block PD-L1 inhibition and provide positive T cell costimulation. This would be particularly effective to improve TCR-T cell recognition of tumor cells with low antigen and high PD-L1 expression in hostile solid tumor microenvironments.

References

1. Lee JK, Sivakumar S, Schrock AB, et al. Comprehensive pan-cancer genomic landscape of KRAS altered cancers and real-world outcomes in solid tumors. npj Precis. Onc. 2022;6:91. https://doi.org/10.1038/s41698–022-00334-z

2. Alexandrov LB, Kim J, Haradhvala NJ, et al. PCAWG Mutational Signatures Working Group; Getz G, Rozen SG, Stratton MR; PCAWG Consortium. The repertoire of mutational signatures in human cancer. Nature.2020 Feb;578(7793):94–101. doi: 10.1038/s41586–020-1943–3. Epub 2020 Feb 5. Erratum in: Nature. 2023 Feb;614(7948):E41. PMID: 32025018; PMCID: PMC7054213.

3. Yang Y, Zhang H, Huang S, et al. KRAS Mutations in Solid Tumors: Characteristics, Current Therapeutic Strategy, and Potential Treatment Exploration. J Clin Med. 2023;12(2):709. Published 2023 Jan 16. doi:10.3390/jcm12020709

4. Huang L, Guo Z, Wang F, et al. KRAS mutation: from undruggable to druggable in cancer. Signal Transduct Target Ther. 2021;6(1):386. Published 2021 Nov 15. doi:10.1038/s41392–021-00780–4

5. Nakajima EC, Drezner N, Li X, et al. FDA Approval Summary: Sotorasib for KRAS G12C-Mutated Metastatic NSCLC. Clin Cancer Res. 2022;28(8):1482–1486. doi:10.1158/1078–0432.CCR-21–3074

6. Guo MZ, Marrone KA, Spira A, et al. Adagrasib: a novel inhibitor for KRASG12C-mutated non-small-cell lung cancer [published online ahead of print, 2023 May 3]. Future Oncol. 2023;10.2217/fon-2022–1106. doi:10.2217/fon-2022–1106

7. Tran E, Robbins PF, Lu YC, et al. T-Cell Transfer Therapy Targeting Mutant KRAS in Cancer. N Engl J Med. 2016;375(23):2255–2262. doi:10.1056/NEJMoa1609279

8. Sim MJW, Lu J, Spencer M, et al. High-affinity oligoclonal TCRs define effective adoptive T cell therapy targeting mutant KRAS-G12D [published correction appears in Proc Natl Acad Sci U S A. 2020 Nov 3;117(44):27743–27744]. Proc Natl Acad Sci U S A. 2020;117(23):12826–12835. doi:10.1073/pnas.1921964117