Over the years, immunotherapy has improved in modulating immune cells or exploiting tumor evasion mechanisms such as immune checkpoint blockade [1], [2], [3], [4], [5]. Adoptive T cell therapy is a type of immunotherapy that uses engineered T cells with modified receptors to treat cancer or viral diseases [6]. Receptors on T cells initiate an immune response by first recognizing an antigen presented by the major histocompatibility complex as a peptide (pMHC) [1], [2], [7]. Upon binding to the pMHC, the TCR recruits molecules from the TCR-CD3 complex to transduce signals for the activation of the effector function of the T cell [8]. The affinity of TCRs to pMHC is usually weak [9], [10], leading to eventual tumor immune escape as a result of the limited effector or cytotoxic function of the T cell against the tumor. The effector function of T cells does not solely rely on the binding of TCR to the presented pMHC on the tumor surface. However, it also depends on cell adhesion, accessory membrane, and co-stimulatory molecules such as CD28 [5], which independently binds to ligands on the tumor surface to strengthen the bond between the T cell and the cancer cell, ensuring higher TCR avidity [8], [11], [12]. The CD3-complex, including the essential molecules needed for T cells to assume its functional state are achieved in vitro or ex vivo by activating T cells. Thus, the stimulation and activation of T cells are crucial for their cytotoxic effector function. In vitro or ex vivo activation of T cells are done by stimulating T cells with CD3/C28 T-activator. The CD3/CD28 ensures the co-stimulation and signaling of T cells for an appropriat immune response [5], [13], [14].

Conventionally, FBS-supplemented growth media have been used to culture and expand antigen-specific T cells. The use of FBS as a supplement is met with many issues, including the binding of TCRs to specific proteins enriched in the FBS, the transfer of zoonotic diseases to patients transfused with T cells expanded in FBS-supplemented medium coupled with the possible rejection of these cells as a result of foreign metabolites that may bound to them [15], [16], [17], [18]. Given the drawbacks associated with FBS use, alternatives such as human-derived sera, sera-free medium, and xeno-free medium have been proposed as possible alternatives [18], [19]. This study investigated the use of these alternative media and how in vitro stimulation of T cells with different T cell activator products may affect the clonal expansion of antigen-specific T cells and their effector function. The move away from FBS as a supplement to other alternatives has evolved.

In 2012, Jung and colleagues successfully expanded human mesenchymal stem cells in a serum-free medium [17]. After that, Smith’s group, 2015, confirmed using serum-free media as a substitute for FBS-supplemented media for culturing T cells [20]. In 2017, Dos Santos et al. characterized human AB serum for primary stromal cell expansion [19]. Following that, Moreira’s group successfully expanded mesenchymal stromal cells using human AB serum as a supplement in 2020 [18]. Mochzuki and Nakahara also established a xeno-free medium for culturing and expanding stem cells in 2018 [16]. Fast forward to 2023, Eberhardt and colleagues investigated and demonstrated the superior function of CAR-T cells cultured in serum-free medium over those cultured and expanded in FBS-supplemented medium [21].

T cell receptor-engineered T cell (TCR-T) therapy is a type of adoptive T cell therapy where T cells are engineered to possess modified receptors [10], [22]. Mostly, these receptors are specific for particular antigens by targeting specific epitopes [23]. When the TCR is specific for a distinct epitope of an antigen, it is known as an antigen-specific TCR (asTCR). asTCRs are transduced onto T cells to manufacture TCR-Ts, the TCR-Ts are expanded to obtain larger quantities for treatment in vitro or in vivo. Clinically, a more considerable amount of TCR-T cells is a prerequisite for immunotherapy due to the low density of pMHC on the surface of target cells [10], [24], [25], [26], necessitating multiple administration of TCR-Ts; thus, the expansion of TCR-Ts in larger folds is a crucial step for TCR-T therapy. Effectively expanding TCR-T has been linked to several factors, including the growth medium, serum, T cell activator, cytokine supplement, quality of primary cells, etcetera [25], [26], [27], [28], [29]. In this study, we sought to investigate how selected growth media and sera affect TCR-T expansion and, ultimately, how they affect the effector function of TCR-T cells in vitro.

The expression of antigens on cancer provides an avenue for antigen-specific TCR-Ts to kill cancer cells or tumors. Cancer/testis antigens (CTAs) have been shown to express in various solid malignancies [30], [31], [32]. CTAs and their subtypes are known for their immunogenicity characteristics combined with a higher reexpression level in several types of cancer. Cancer testis antigen gene 1 (CTAG1) and cancer testis antigen gene 2 (CTAG2) are two different types of CTAs reported to be expressed simultaneously in many kinds of cancer. Interestingly, CTAG1 and CTAG2 share multiple epitopes because of their unique similarity of over 50 % identity match [33], [34]. Therefore, we hypothesized that targeting a single epitope on these two antigens may create an avenue for dual targeting using the same TCR-T, which is specific for an epitope found on both CTAs. CTAG1 and CTAG2 expression profiles on cancer cell lines were obtained from the Expression Atlas server (https://www.ebi.ac.uk/gxa/home). This study sought to evaluate the in vitro antitumor response efficacy of various cancers with or without CTAG1 and CTAG2 expression. The study utilized an asTCR isolated from our previous study that is specific for a common epitope (CTAG1/2(157-165)) on CTAG1 and CTAG2 antigens [10]. This asTCR is human leucocyte antigen (HLA), HLA-A*02:01-restricted. It was used to produce TCR-Ts to assess how growth conditions and activation factors may affect the generation and expansion of high-quality TCR-T cells and any residual effect that may impact their effector function in vitro. Our results revealed that the materials used to manufacture TCR-Ts may affect their expansion quality, effector, and cytotoxic function. Furthermore, our study demonstrated that multiple or dual targeting of different antigens by antigen-specific TCR-Ts using a common epitope may yield a superior cancer-killing effect than single-antigen targeted therapy.