Science Spotlight

Rewiring T cell death signaling enhances T cell function and persistence

From the Greenberg lab, Clinical Research Division

T cells are an important immune cell subset capable of seeking and destroying infected or cancerous cells. Thus, adoptive cell transfer (ACT) of T cells, a strategy in which T-cells are engineered to recognize cancer-specific epitopes, is an effective strategy for targeting cancer, particularly hematopoietic (blood) cancers. While chimeric antigen receptor (CAR) T cell therapy has been particularly effective against B cell cancers, CAR-Ts can only target surface proteins with relatively high antigen expression. Alternatively, transgenic T cell receptors (TCRs) utilize the fine-tuned capacity of natural TCRs that can target both intracellular and surface proteins, including those with low antigen density. Antigen-specific TCRs can be transferred to T cells using viral vectors to make transgenic TCR-expressing T cells (TCR-Ts) that recognize a variety of antigens. However, TCR-Ts are often suppressed by inhibitory signals in the tumor microenvironment of solid tumors and can have trouble persisting long term. Dr. Shannon Oda, a former postdoc of the Greenberg lab (Clinical Research Division) now with her own lab at Seattle Children’s, wanted to explore whether rewiring inhibitory signals could enhance TCR-T cell function and persistence. The results of her studies were recently published in the Journal of Experimental Medicine.

Piggybacking off a similar study from Dr. Oda and Dr. Greenberg, the authors used an immunomodulatory fusion protein (IFP) created by a fusion of the ectodomain of Fas and endodomain of 4-1BB. Fas is an important receptor found on various immune cells that leads to activation of apoptotic pathways, leading to immune cell death and contraction of the immune response. However, many cancer cells express high levels of Fas ligand (FasL), leading to premature immune cell death and evasion of a productive immune response. 4-1BB is a costimulatory T cell signal that enhances TCR signaling leading to enhanced T cell function and survival. The authors aimed to switch the inhibitory Fas signal into a pro-survival 4-1BB signal to enhance T cell proliferation and function by fusing the two signaling proteins. To test their hypothesis, the authors first used TCR-Ts specific for an LCMV envelope epitope and transduced these cells with their Fas-4-1BB IFP (F-4 IFP). When LCMV-specific F-4 IFP cells were co-cultured with antigen presenting cells (APCs) pulsed with LCMV peptide, they proliferated better than T cell expressing only the LCMV-specific TCR. Additionally, the LCMV-specific F-4 IFP cells did not proliferate when co-cultured with APCs without peptide, or when treated with a Fas-stimulating antibody, indicating the proliferation response required antigen-specific TCR signaling. Peptide stimulated LCMV-specific F-4 IFP cells also produced more IL-2, an important cytokine produced downstream of 4-1BB signaling.  

Next, the authors wanted to test if their T cell could function by recognizing endogenously presented antigen, rather than a synthetic antigenic peptide. They used a TCR against a different antigen derived from the Friend murine virus-induced leukemia line (FBL), which expresses native FasL. When FBL-specific F-4 IFP cells were co-cultured with FBL cells, Fas expression could be visualized by microscopy at the immunologic synapse, indicating T cell activation and accumulation of the F-4 IFP. Additionally, when stimulated with a low amount of FBL cells, FBL-specific F-4 IFP cells proliferated while T cells expression the TCR only did not, indicating a decreased threshold for activation and proliferation. FBL-specific F-4 IFP cells were serially challenged with FBL cells every 24 hrs, a serial killing assay used to determine the robustness of the cells. FBL-specific F-4 IFP cells lysed target cells effectively over multiple cell additions, while their control counterparts killed fewer target cells with each addition. F-4-IFP cells displayed increased 4-1BB associated signaling markers, such as Bcl-2 and pAkt, and enhanced metabolic capacity, both indicating increased functional and proliferative capacity. Additionally, the authors tested whether expression of a human version of their F-4 IFP functioned similarly to their murine version. They found that human F-4 IFP cells had enhanced proliferation, produced more IFNg upon peptide stimulation, lysed significantly more human target cells in a serial lysis assay, and expressed more CD107a, a marker of T cell activation and degranulation, after co-culture with target cells. These results confirmed that the human version of their IFP was functional.

T cells engineered with the Fas-4-1BB fusion have better proliferation and function
T cells engineered with the Fas-4-1BB fusion have better proliferation and function Figured provided by Dr. Shannon Oda

Finally, the authors wanted to determine if their F-4 IFP cells could control tumor growth in vivo. They used two mouse models, one with FBL cells modeling disseminated disease and another using mice that spontaneously develop pancreatic cancer (KPC mice). In mice with FBL disease, untreated mice succumbed to disease within 25 days. However, 71% of mice treated with FBL-specific F-4 IFP cells survived to at least 100 days, a significant increase in overall survival. For KPC mice, the authors used a TCR targeting mesothelin (MSLN), an antigen upregulated in many solid cancers. In a previous study, MSLN-specific T cells quickly became exhausted and did not persist well in KPC mice. MSLN-specific F-4 IFP cells accumulated in the tumor better than their TCR only control cell counterparts at 7- and 28-days post transfer into tumor-bearing KPC mice. Additionally, while PD-1 expression was the same between T cell types, F-4 IFP cells displayed lower levels of Tim3 and Lag3, additional marker of T cell exhaustion. F-4 IFP T cells enhanced median survival in KPC mice to 65 days vs. 37 days with TCR alone T cells, and all mice receiving F-4 IFP cells had detectible T cells. In contrast, the sole surviving mouse in the TCR alone group did not have detectible T cells after 28 days. These studies demonstrate that enhanced survival and function of adoptively transferred T cells is possible by rewiring the inhibitory Fas signal into a pro-survival 4-1BB signal, which leads to better tumor control.

Dr. Oda explained the significance of their findings: “The combination of replacing a death signal with pro-survival/activation signals resulted in many enhancements to T cell function, including better proliferation, resistance to becoming exhausted from repetitive killing of tumor cells, and improved metabolism to survive in the hostile tumor environment; in other words, a “supercharged” T cell.” Dr. Oda and Dr. Greenberg are optimistic that their supercharged T cells will make the difference for cancer patients and are eagerly looking towards future clinical trials. To this, Dr. Greenberg added: “We have clinical trials planned for pancreatic cancer that should be open for enrollment in 2021 and will use a mesothelin-targeted TCR as we used in these studies. We have additional trials targeting other tumors planned to begin in 2021, and this IFP strategy should be a valuable engineering component for designing T cells that can effectively treat patients’ malignancies.”

This work was supported by the National Institutes of Health National Cancer Institute, the Leukemia & Lymphoma Society, the Ovarian Cancer Research Alliance, and Juno Therapeutics.

UW/Fred Hutch Cancer Consortium members Shannon Oda and Philip Greenberg contributed to this work.

Oda S, Anderson K, Ravikumar P, Bonson P, Garcia N, Jenkins C, Zhuang S, Daman A, Chiu E, Bates B, Greenberg P. 2020. A Fas-4-1BB fusion protein converts a death to a pro-survival signal and enhances T cell therapy. J Exp Med. Dec 7;217(12):e20191166. doi: 10.1084/jem.20191166.