Hexokinase2-engineered T cells display increased anti-tumor function

Abstract

Background: T cells face significant metabolic challenges in the tumor microenvironment (TME), where cancer cells monopolize critical nutrients like glucose and amino acids. This metabolic competition supports tumor growth while impairing T-cell anti-tumor responses, partly by reducing glycolytic function. Hexokinase 2 (HK2), a key enzyme in glycolysis, plays a pivotal role in maintaining T-cell functionality.

Methods: To enhance T-cell function, primary human T cells were genetically engineered to overexpress HK2 alongside a tumor-specific receptor. These engineered T cells were tested in vitro and in vivo to evaluate their metabolic and therapeutic efficacy.

Results: HK2-engineered T cells exhibited increased glycolytic capacity, leading to enhanced cytokine secretion, activation marker expression, and metabolic activity compared to controls. In vivo studies using a human tumor xenograft model demonstrated the superior therapeutic efficacy of HK2-engineered T cells, including delayed tumor growth and improved survival.

Conclusion: HK2 overexpression improves T-cell metabolic fitness and functionality in hostile TMEs, offering a promising foundation for the development of next-generation immunotherapies targeting T-cell metabolism.

Keywords: T-cells; TCR; cellular immunotherapy; hexokinase 2; immunometabolism.

Figure 1

Human T cells can be engineered to express high levels of HK2. (A–C) Human PBLs were transduced with a retroviral vector encoding HK2 or NGFR. 72 h after transduction, transgene expression was measured by flow cytometry using antibodies specific for HK2 or NGFR. The left panel is a representative result, and the right panel presents the mean+SEM of n=4 (C) RNA from HK2 transduced T cells was extracted and reverse-transcribed. HK2 transcript expression was measured by qPCR and normalized to control. These results are presented as mean+SEM of n=5. (D) The CD4/CD8 ratio of transduced cells was determined by flow cytometry. These results are representative of n=8 independent experiments. (E) The memory phenotype of transduced cells was determined by flow cytometry based on CD45RO and CCR7 expression. EM - Effector memory (CD45RO+/CCR7-), CM - central memory (CD45RO+/CCR7+), EMRA - terminally differentiated effector memory cells re-expressing CD45RA (CD45RO-/CCR7-) or naïve cell population (CD45RO+/CCR7+). These results are presented as the mean+SEM of n=6. ∗: p < 0.05, ∗∗: p<0.01, ∗∗∗: p < 0.001.

Figure 2

HK2 overexpression can improve glycolysis and metabolic fitness. (A) Evaluation of T cell metabolic function using the Seahorse extracellular flux analyzer. Extracellular acidification rate (indicated as ECAR) was measured. The left panel shows a representative experiment and the right panel depicts the mean+SEM of n=3 (**: p<0.01, *: p<0.05 and ns – not significant). (B) Mitochondrial mass was measured by FC, gated on NGFR⁺ population using MitoSpy green. The left panel is a representative a result and the right panel is the mean + SEM of n=4 independent experiments with 4 different donors. The difference between HK2 and NGFR (control) was found statistically significant as indicated (*p<0.05, calculated using a Student’s paired t-test). (C) ATP was measured using the CellTiter-Glo® 2.0 Cell Viability Assay reagent that was added to 1x10⁵ T cells in a 96-well plate. The ATP expression levels were normalized to NGFR. These results are representative of n=8. (D) Extracellular Oxygen Consumption Assay was measured kinetically for 2 hours by time-resolved fluorescence (TR-F). The mean+SEM of the slope is shown. (E) Glucose-6-Phosphate was measured by colorimetry at 450 nm. The mean + SEM of the optical density (OD) is shown. (A–E) These results are performed with at least 3 different donors and the difference between HK2 and NGFR (control) was found statistically significant as indicated (*p<0.05, calculated using a Student’s paired t-test). (F) RNA from HK2 or NGFR (control) transduced T cells was extracted and reverse-transcribed. PFK, Pyruvate kinase, GLUT1 and GLUT3 transcript expression was measured by qPCR and normalized to control. These results are presented as mean+SEM of n=3.

Figure 3

HK2 forced expression enhances cytokine secretion and upregulates activation markers in engineered-T cells. (A–C) Primary human T cells were transduced with either NGFR vector (control) or HK2 and were stimulated with plate-bound OKT3. IFNγ (A), TNFα (B) and IL-2 (C) were secreted in the culture supernatant and measured by ELISA. These results represent the mean+SEM and normalized to control NGFR (with an average secretion of 3.1 ng/mL, 2.6 ng/mL and 0.95 ng/mL for IFNγ, TNFα and IL-2 respectively). (D) CD69, CD137, and CD25 expression was measured by flow cytometry, gated on the CD8⁺ population. The mean of n>5 is represented by a red line. (E) T cells were labeled with CFSE and stimulated with an anti-CD3 antibody for 4 days. The PF (proliferation factor = MFI day 10/MFI day 14 normalized to NGFR (control) is shown. These results were obtained in n=3 experiments. (A–E) These results are performed with at least 3 different donors and the difference between HK2 and NGFR (control) was found statistically significant as indicated (*p<0.05, calculated using a Student’s paired t-test). ∗: p < 0.05, ∗∗: p<0.01, ∗∗∗: p < 0.001.

Figure 4

HK2 can improve the anti-tumor function of TCR T cells. (A) Human PBLs were transduced with a MART1-specific TCR. After 24 h, the cultures were split and transduced with either HK2 or NGFR. 72 h after transduction, TCR F4 expression was measured by FC using a specific antibody (Vβ12). The left panel is a representative result, and the right panel presents the mean+SEM of n=6. (B–D) Following overnight co-culture with melanoma cell lines, IFNγ (B), TNFα (C) and IL-2 (D) secreted in the culture supernatant were measured by ELISA. These results are the mean+SEM of n>3 and normalized to control (NGFR+TCR with reference concentrations ranging between 5.9-11 ng/ml for IFNγ, 6.6-17.4 ng/ml for TNFα, and 0.23-0.5 ng/ml for IL2). (E) CD69 and CD137 expression levels were measured by FC, gated on the CD8⁺ population following overnight co-culture with melanoma cell lines. These results were obtained in n>3 independent experiments. (F) Following an overnight co-culture with SKMEL23 cell line, granzyme B secreted in the culture supernatant were measured by ELISA. These results were obtained in n=4 (A–F) These results are performed with at least 3 different donors and the difference between HK2 and NGFR (control) was found statistically significant as indicated (*p<0.05, calculated using a Student’s paired t-test). ∗∗: p<0.01, ns: not significant.

Figure 5

HK2 improves T cell anti-tumor function in vitro and in vivo. (A) NGFR + TCR and HK2 + TCR engineered T-cells were co-cultured with tumor cells (888A2) at different E:T (Effector : Target) ratio and incubated in an Incucyte apparatus. mCherry⁺ live cell population was followed over time and normalized to time=0. These results are the mean+SEM of n>3 independent experiments performed with at least 3 different donors. The difference between NGFR vector (control) and HK2 was found statistically significant as indicated (*p<0.05 and **p<0.01 calculated using a Student’s paired t-test). (B) Representative experiments are shown in co-culture, with 888A2, SKMEL23 and A375 (negative control) melanoma cell lines at 1:1 E:T ratio. (C) NSG mice were inoculated with 2x10⁶ 888A2 tumor cells. Then, 5x10⁶ of NGFR + TCR or HK2 + TCR-engineered T cells were IV injected in mice, one week and two weeks after tumor establishment (indicated by arrows). Tumor growth was measured in a blinded fashion using a caliper. Curves for the tumor growth average (n=6) and spider plots of the tumor volumes are shown. The difference between NGFR vector (control) and HK2 was found statistically significant as indicated (calculated using a Student’s paired t-test). (D) The survival of the respective groups is represented by a Kaplan-Meier curve (p=0.07, calculated using a Log-Rank test).