Via Research Recognition Day Program VCOM-Carolinas 2025

Biomedical Research

Lighting the way: an economical alternative to feeder cell irradiation for T-cell expansion Michael S. Benavidez Arias, MA, OMS-III, An Nguyen OMS-III, Daniel Ross, BS, NREMT, David H. Eagerton, PhD, F-ABFT and Krit Ritthipichai, DVM, MS, PhD. Edward Via College of Osteopathic Medicine, Dept. of Biomedical Affairs, Spartanburg, South Carolina. Results

Introduction

Background: T-cell therapy has emerged as an effective treatment for blood cancers with an average 80.5% objective response rate (ORR) 1 . A robust T-cell expansion process involves co-culturing T-cells with non proliferating feeder cells combined with anti-CD3 antibody and IL-2. Challenge: Although ionizing irradiation effectively inhibits feeder cell proliferation, the high operating costs limit cell therapy research to well-funded institutions. Rationale: UVC is high energy electromagnetic radiation, causing severe DNA damage. It is widely used for inactivating microorganisms and inducing cell apoptosis 2 . Given UVC generators’ cost -effectiveness, we explored their potential as an alternative to ionizing irradiators for generating non-proliferating feeder cells for T-cell expansion. Hypothesis: We hypothesize that TILs expanded using UVC-irradiated feeder cells demonstrate similar or superior viability, expansion rates, and effector functions compared to those expanded with ionizing irradiated feeder cells.

TILs expanded with UVC-Irradiated PBMCs showed comparable T-Cell expansion

UVC Effectively Suppressed Feeder Cell Proliferation by Inducing DNA Damage

>90% viability

~ 500 folds

>97% T-cells

B.

C.

A.

A.

↑ DNA damage

↑ Cell death

↓ Cell proliferation

B.

Figure 1. The impact of UVC irradiation on DNA and apoptosis. Flow cytometry analysis of phosphorylated -H2AX (DNA damage marker) in CD32hi K562 cells 15, 60, and 120 minutes after UVC irradiation (A) . Line graph demonstrating cell count in CD32hi K562 pre- and post-UVC irradiation from day 0 to 14 (B) . Early (annexin V*) and late apoptosis (Annexin V*7-AAD*) in K562 on day 14 post-UVC irradiation was determined by flow cytometry (C) . Data are presented as mean ± SEM. Two independent experiments; n=5, per group. Two-tailed Student's t-test; *P ≤ 0.05; **P ≤ 0.01; ***p ≤ 0.001 ****p ≤ 0.0001.

C.

~ 50% degranulation

Experimental Design

UVC Irradiated Cells Dampened Glucose Uptake and ATP production While Enhancing Antibody Retention on the Cell Surface ↑ Antibody retention

B.

A.

C.

1. To determine the impact of UVC on feeder cell proliferation apoptosis (Fig. 1)

↓ Glucose uptake

↓ ATP production

Figure 4. Characteristics of Tumor-infiltrating lymphocytes (TILs) expanded with irradiated feeder cells. Bar graphs depicting live cells (left), cell expansion (middle), and T-cell purity (right) (A) . Bar graphs displaying T-cell exhaustion markers in expanded TILs (B) . Bar graphs showing TIL effector function following re-stimulation with anti-human CD3/CD28 antibodies (C) . Data are presented as mean ± SEM. Two independent experiments; n=4, per group. Two-tailed Student's t-test, ns; not significant.

2. To assess antibody retention and cell metabolism of UVC-Irradiated cells (Fig. 2)

Figure 2. An alteration in antibody retention and metabolic function following UVC exposure. Luminescence assay for measuring glucose uptake at 6 and 24 hours post-UVC treatment (A) . Bar graph demonstrating ATP concentrations at 6 and 24 hours after UVC irradiation (B) . Bar graph displaying the percentage of membrane-bound antibodies on cells exposed to UVC for 6 and 24 hours at 25, 50, 75, and 100 minutes after antibody labeling (C) . Data are presented as mean ± SEM. Two independent experiments; n=5, per group. Two-tailed Student's t-test; ***p ≤ 0.001 ****p ≤ 0.0001.

Conclusion

• UVC effectively suppressed feeder cell proliferation, indicated by apoptosis in 95% of irradiated cells. • Cell viability, expansion rates, and effector functions of T cells expanded with UVC-irradiated feeder were comparable to those expanded with ionizing irradiation. • Further investigation into UVC irradiation's scalability and long-term effects on T-cell therapies could revolutionize accessibility and affordability in clinical settings, particularly in resource-limited environments.

3. To investigate the association between antibody retention and energy depletion (Fig. 3)

Glucose depletion Antibody Retention on Cellular Surfaces Was Dose-Dependently Increased Following Energy Depletion

A.

C.

B.

ATP production

Glucose uptake

4. To examine the potential of UVC irradiated feeder cells for T-cell expansion (Fig. 4)

Acknowledgements

References

1. Xiang, X., He, Q., Ou, Y., Wang, W. & Wu, Y. Efficacy and Safety of CAR-Modified T Cell Therapy in Patients with Relapsed or Refractory Multiple Myeloma: A Meta-Analysis of Prospective Clinical Trials. Front Pharmacol 11, 544754 (2020). 2. Sun, W., Jing, Z., Zhao, Z. Dose-Response Behavior of Pathogens and Surrogate Microorganisms across the Ultraviolet-C Spectrum: Inactivation Efficiencies, Action Spectra, and Mechanisms. Environ Sci Technol. 57, 10891 (2023).

• The work is supported by VCOM Seed Grant Program • The authors would like to thank Dr. Bidyut Mohanty for providing anti Gamma H2AX (phospho-Ser139) antibody.

Figure 3. Association between antibody retention and cellular energy depletion. Bar graph demonstrating the effect of cytochalasin B treatment on antibody retention post-antibody labeling (A) . Bar graph showing antibody retention when glucose is depleted (B) . Bar graph depicting antibody retention in rotenone-treated cells (C) . Data are shown as mean ± SEM. Two independent experiments; n=5, per group. Two-tailed Student's t-test; **P ≤ 0.01 ****P ≤ 0.0001. R 2 between 0.7 and 0.9 considered highly correlated.

2025 Research Recognition Day

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