Recurrent mutations in the CTNNB1 gene have been identified with the potential to generate promising candidate neoantigens (Bugter et al.,2021; Parrish et al.,2022). We identified two T cell receptors recognizing neoantigens from a recurrent mutation in CTNNB1. One of the TCRs is restricted by the HLA-A*02:01 allele, while the other TCR is restricted by HLA-A*24:02. Our in vitro data indicates that T cells expressing the identified CTNNB1mut TCRs efficiently kill cells that naturally express the mutation such as the Hutu80 cell line (small intestine adenocarcinoma). To test the therapeutic effect in vivo, we plan to engraft them into NXG mice after establishing solid tumors that express the target mutation. We also plan to combine the methods listed below: Anti-VEGFR2 antibody (DC101) injection: To facilitate T-cell infiltration into solid tumors, one approach is normalizing the tumor vasculature through VEGF blockade (Park et al.,2023; Sun et al.,2021). DC101, which targets mouse VEGFR2, has demonstrated success in normalizing abnormal tumor angiogenesis (Bocci et al.,2004; Lowery et al.,2019; Mashima et al.,2021; Park et al.,2023; Sun et al.,2021). The aim is to enhance T-cell infiltration and induce in vivo antitumor responses. Reprogramming of target cancer cells: Dendritic cells type 1 (cDC1) are specialized in recruiting T cells and presenting tumor antigens in tumors, thereby supporting tumor rejection. Our collaborator, Professor Filipe Pereira has previously identified a combination of transcription factors PU.1, IRF8, and BATF3 (PIB), as sufficient to reprogram tumor cells into cDC1-like cells (Rosa et al.,2018). The in vivo use of reprogramming tumor cells already got the ethical permit in Sweden (Link attached). The combinatorial approach may improve T-cell recruitment and killing in in vivo model. CRISPR-Cas9 mutation knock-in of cell lines: The CTNNB1 target mutation is frequently detected in cancers that originate from endometrial or pancreatic cancer (Tate et al.,2019). Since there are no established pancreatic or endometrial cancer cell lines that naturally harbor the TCR targeting CTNNB1 mutation, we will perform CRISPR-Cas9 knock-in to introduce the target mutation to the pancreatic cancer cell line (PANC-1) and endometrial cancer cell line (KLE) cells in vitro. These tumor models will provide valuable information about the possible use of the CTNNB1-TCR for treating “hard-to-treat” solid cancers expressing the CTNNB1 target mutation.

Earmarking/biopsi according to SOP "ID-merking og uttak av biopsi" ved KPM/OUS. Injection of tumor cells: Subcutanously in the flank (0.1 ml cell suspension in saline). Injection of DC101 and isotype control IgG antibody: Intraperitoneal injection at 20mg/kg. The weight of mice varies from 20-30g, and the injection volume of 1mg/ml stock of DC101 or isotype control IgG antibody will be 400-600&#956;l. Injection of T cell suspension: Intravenously in the tail vein (0.1 ml cell suspension in saline). Injection of Proleukin (IL-2): 2500 U in 250&#956;l by i.p. three time per week after T cell injection. Injection of Luciferin: Each mouse should receive 150 mg Luciferin/kg body weight by i.p. The weight of mice varies from 20-30g, and the injection volume of 15mg/ml stock of Luciferin will be 200-300&#956;l. Blood sampling: Blood (0.05 ml) will be drawn from the saphenous vein puncture using a restraining tube to immobilize the mouse. We will take the blood one time per week after T cell injection. The total blood volume over a period will follow the NC3Rs guidelines: Maximum <10% TBV (= 0.14 ml) on any single occasion AND <15% TBV ( = 0.21 ml) in 28 days. the maximum duration of weekly blood samples will be less than 8 weeks. The duration of the whole experiment will be up to 24 weeks. Mice will be weighed and inspected for any sign of reduced general condition, level of activity, shaggy fur, erythema of the skin, and signs of pain. Please check the attached score sheet for more details.

So far, extensive in vitro experiments have been carried out. However, the in vivo effect is unknown. In vivo experiments in humans are not possible to perform, thus, we apply to use a transplant model in immunodeficient NXG mice. These experiments will shed light onto the in vivo homing and interactions of T cells at a systems level as well as with tumor cells in an intact tissue microenvironment and this information will be relevant for future translational studies on treatment resistant solid cancer patients, in which all other treatment options have been exhausted. The model gives information regarding efficacy as well as safety.