Tumor-infiltrating lymphocyte expansion protocols for adoptive cell therapy in cancer

Abstract

Adoptive cell therapy (ACT) with tumor infiltrating lymphocytes (TILs) has emerged as an innovative strategy in cancer immunotherapy, especially in the treatment of solid tumors. This review provides a comprehensive analysis of current TILs expansion protocols, with a focus on strategies aimed at optimizing their performance, feasibility and functionality. The classical TILs expansion protocol is based on a two-step process: a rapid pre-expansion phase (Pre-REP), followed by a rapid expansion phase (REP), in which high concentrations of interleukin-2 (IL-2) are used. In recent years, various modifications have been incorporated into the protocol with the aim of improving its efficiency, including the use of alternative combinations of cytokines such as IL-7, IL-15, and IL-21, as well as the replacement of irradiated feeder cells or anti-CD3 antibodies with other strategies. Furthermore, advances such as the targeting of TILs specific to neoantigens, the development of CAR-TILs, the application of artificial intelligence, and the genetic modification of TILs have contributed to expanding both the applicability and efficacy of this therapy. The clinical success of Lifileucel with the recent FDA approval of underscore the translational potential of TILs-based therapies. However, significant challenges remain, including time and production costs. This review examines ongoing clinical trials and combination therapeutic strategies, such as the use of immune checkpoint inhibitors. As research progresses, optimization of TILs expansion protocols will be critical to improving clinical outcomes and broadening the applicability of this personalized immunotherapy in oncology.

Keywords: Adoptive cell therapy (ACT); Immunotherapy; Neoantigen-reactive t cells; TILs expansion protocols; Tumor-infiltrating lymphocytes (TILs).

Figure 1

General process of the protocol for the production of cell therapy based on TILs. The process begins with surgical resection of the tumor and its fragmentation for the removal of tumor cells. The TILs are isolated from the tumor tissue and undergo an initial expansion phase with high doses of IL-2. Subsequently, the main expansion phase is performed using IL-2, OKT3 and feeder cells. At the end of the expansion protocol, the functionality of the TILs is assessed by specific assays. Finally, the activated TILs are infused into the patient together with IL-2 to enhance the antitumor immune response. Created in https://BioRender.com

Figure 2

Interaction between K562 cells and TILs. The diagram depicts the interaction between a K562 cell and a TILs cell. This configuration is used in the REP of TILs REP. The K562 cell is modified to express key co-stimulatory ligands, such as anti-CD64, CD80/CD86 and 4-1BBL, which interact with their respective receptors on T cells: CD3, CD28 and 4-1BB. This contributes to the efficient activation and expansion of TILs. Created in https://BioRender.com

Figure 3

Schematic representation of TILs and its interaction with presenting cells (APCs) and tumor cells. The most important markers in immunophenotyping are shown including activation markers (CD28, CD69, OX-40, 4-1BB), CD4⁺ TILs subpopulations (CCR7, CXCR3, CD45RA), cytokines and chemokines involved in their activation, as well as depletion markers (PD-1, TIM-3, CD39). Activation of CD8⁺ TILs is illustrated by interaction with the MHC-I complex of tumor cells, whereas activation of CD4⁺ TILs occur through MHC-II-mediated antigenic presentation on antigen-presenting cells. Created in https://BioRender.com

Figure 4

Historical evolution and key advances in the development of tumor TILs therapy. From the first cell expansion protocols and clinical trials in melanoma in the 1980s to current strategies in genetic engineering, gene editing and culture optimization, the figure summarizes the main scientific and clinical milestones in the evolution of this therapy. Noteworthy events include the introduction of ‘young’ TILs, the use of G-Rex platforms for expansion REP, the development of neoantigen-specific TILs, and new strategies such as IL-12-modified TILs, CAR-TILs, low-dose IL-2 protocols, and selection based on biomarkers such as CD39 and CD69. The timeline also includes emerging technologies such as CRISPR-Cas9 and combinations with metabolic factors such as adiponectin. This overview illustrates the dynamism and continuous progress in the field of TIL-based adoptive immunotherapy [122]