Periodic Reporting for period 4 - TIL-FIT (Increasing the fitness of tumor-infiltrating T cells for cellular immunotherapy)
Berichtszeitraum: 2023-07-01 bis 2023-12-31
Adoptive T cell therapies (ACTs) are emerging as a promising strategy to treat cancer. Tumor-infiltrating lymphocytes (TILs) are expanded ex vivo, selected for recognition of neoantigens, further expanded and then infused back into patients. This procedure requires extensive culturing and expansion of TILs during which many T cell clonotypes are lost. As tumor-reactive TILs are often exhausted and tend to be overgrown by functional, non-specific T cells in culture, the chance to identify potent tumor-reactive T cells dramatically decreases. Moreover, extensive expansion of T cells diminishes their anti-tumor activity and persistence in the body after adoptive transfers. Thus, improving the fitness of T cells is crucial to increase the success rate of ACTs and make this therapy accessible to a broad spectrum of cancer patients. Our first aim is to increase the fitness of T cells by designing metabolic and pharmacological interventions based on proteomic profiles of TILs from patients with liver cancer. Second, we will use machine-learning algorithms for the extraction of signatures to predict whether TILs grow well in culture, require and respond to metabolic interventions, or cannot be revitalized and do not grow at all. To deal with non-growing T cells, we aim at establishing a microfluidics-based workflow to graft the entire T cell receptor (TCR) repertoire from thousands of non-growing TILs onto fast-growing Jurkat cells. After selecting Jurkat cells that recognize neoantigens, their TCRs will be expressed on naïve T cells obtained from the patient’s blood that are fit and suitable for ACT. This project will contribute to a better understanding of the T cell response to liver cancer and help to increase the success of personalized ACTs for solid tumors.
Our first aim was to increase the fitness of TILs by designing metabolic and pharmacological interventions based on proteomic profiles of TILs from patients with liver cancer. We profiled proteomes of CD4+ and CD8+ T cells from tumors, liver, and blood of 48 patients with hepatocellular carcinoma. We discovered that the signaling scaffold protein AFAP1L2, typically only found in activated NK cells, is also upregulated in chronically stimulated CD8+ T cells in tumors. Ablation of AFAP1L2 in CD8+ T cells increased their viability upon repeated stimulation and enhanced their anti-tumor activity synergistically with PD-L1 blockade in mouse models. This part of the project revealed AFAP1L2 as a target in T cells to improve their survival and antitumor activity. The results were published (Canale et al. Cell Genomics 2023) and presented at various seminars and scientific conferences.
Because T cells isolated from tumors often do not grow, we aimed to establish a microfluidics-based workflow to graft the entire T cell receptor (TCR) repertoire from thousands of non-growing TILs onto fast growing Jurkat cells. For this, we developed a workflow for the amplification and linkage of TCR alpha and beta chains from thousands of single T cells in picoliter droplets, facilitating the creation of highly diverse TCR libraries. Sequencing of TCR libraries generated with this workflow showed a highly diverse repertoire indicating that a wide array of TCRs could be captured, which is critical for our goal of expressing large libraries of functional TCRs in Jurkat T cells. We also demonstrated the feasibility of expressing functional TCRs in Jurkat cells. These engineered Jurkat cells can recognize peptide antigens on antigen-presenting cells but with reduced avidity as compared to primary T cells. Currently, the workflow is being optimized.
Because T cells isolated from tumors often do not grow, we aimed to establish a microfluidics-based workflow to graft the entire T cell receptor (TCR) repertoire from thousands of non-growing TILs onto fast growing Jurkat cells. For this, we developed a workflow for the amplification and linkage of TCR alpha and beta chains from thousands of single T cells in picoliter droplets, facilitating the creation of highly diverse TCR libraries. Sequencing of TCR libraries generated with this workflow showed a highly diverse repertoire indicating that a wide array of TCRs could be captured, which is critical for our goal of expressing large libraries of functional TCRs in Jurkat T cells. We also demonstrated the feasibility of expressing functional TCRs in Jurkat cells. These engineered Jurkat cells can recognize peptide antigens on antigen-presenting cells but with reduced avidity as compared to primary T cells. Currently, the workflow is being optimized.
To analyze and express TCR repertoires of TILs we developed a multi-step workflow to reverse transcribe, PCR amplify, and link TCR alpha and beta chains from single cells in picoliter droplets. We anticipate that this technology will become an important tool to preserve, sequence, and express TCR repertoires from non-growing TILs. This may facilitate the rapid identification of TCRs that recognize tumor antigens for cellular therapies.