467 Engineered chimeric antigen receptor-expressing TILs for liver cancer treatment using a novel MSE-TIL platform

<h3>Background</h3> Tumor Infiltrating Lymphocytes (TILs) comprise various T cell clones with specificity towards tumor or viral antigens. TIL therapy has been approved for melanoma treatment with high-dose interleukin-2 (IL-2). However, the accessibility of TILs, the small fraction of antigen-specific T cells, limited persistence<i> in vivo</i>, exhausted phenotype, and toxicity of high-dose IL-2 infusion restrict its application to the treatment of other solid tumors. We developed multiple signaling elements (MSE)-TIL platform to expand and engineer TILs derived from liver cancer biopsies (figure 1). These TILs (MSE-TIL) showed increased memory T-cell percentage, improved proliferation capacity, and enhanced anti-tumor potential towards various liver cancer cells without IL-2 administration. <h3>Methods</h3> TILs were isolated from fresh or cryopreserved tumor biopsy samples and stimulated with anti-CD3/CD28 antibodies. Thereafter, TILs were modified by lentiviral transduction and further expanded with γ-irradiated feeder cells in an optimized rapid expansion process (REP) to induce memory CD8+ T cells. The final product was cryopreserved for storing and shipping. Phenotypic analyses were performed by flow cytometry. IFN-γ ELISA, RTCA Xcelligence, and IncuCyte were used to evaluate <i>in vitro</i> activation and cytotoxicity. Anti-tumor efficacy was further evaluated in immunocompromised xenograft models. <h3>Results</h3> The successful rate for MSE-TIL production from two tumor biopsies was ~ 95%, reaching 10¹¹ TILs on day 26. Compared with traditional REP, the percentage of central memory CD8⁺ T cells was increased by 5-fold to promote persistent anti-tumor function <i>in vitro</i> (figure 2). TILs were additionally engineered with CAR recognizing tumor antigens including CD133 and CD44v6, cofactors such as membrane-bound interleukins and mitochondrial homeostasis regulators, to sustain their <i>in vivo</i> proliferation and long-term anti-tumor efficacy. The lentiviral transduction rate was stably maintained at over 30% (Day 28: CAR, 48.8 ± 9.1%; cofactor, 58.6 ± 12.6%) for up to 7 weeks<i> ex vivo</i>. MSE-TIL shows potent killing efficacy towards liver cancer cell lines. Moreover, MSE-TIL can accumulate in tumors and inhibit their growth without toxicity in an immunocompromised murine model (figure 3). <h3>Conclusions</h3> Engineered MSE-TILs demonstrated persistent therapeutic efficacy with no obvious toxicities in the mouse model, supporting its further clinical applications. <h3>Ethics Approval</h3> All animal studies were conducted by Biosyngen staff at Guangzhou Regenerative Medicine and Health, Guangdong Laboratory (GRMH-GDL) as approved by the Ruiye model animal (Guangzhou) Biotechnology Co., Ltd facility as approved by its Experimental Animal Care and Use Committee (approval No. RYEth20220516-1).