Chimeric antigen receptors (CARs) redirect T cells to recognize a specific target. CAR components play a pivotal role in antigen specificity, structure stability, expression on cell surface, and induction of cellular activation, which together determine the success of CAR T-cell therapy. CAR products targeting B-cell lymphoma encouraged the development of new CAR applications beyond cancer. For example, our group developed a CAR to specifically target glucuronoxylomannan (GXM) in the capsule of Cryptococcus species, called GXMR-CAR or GXMR-IgG4-28ζ. Cryptococcus are fungi that cause the life-threatening disease cryptococcosis, and GXMR-IgG4-28ζ redirected T cells to target yeast and titan cell forms of Cryptococcus spp. Here, we replaced the IgG4-hinge and CD28-transmembrane domains from GXMR-CAR with a CD8α molecule as the hinge/transmembrane and used CD28 or 4-1BB molecules as co-stimulatory domains, creating GXMR-8-28ζ and GXMR-8-BBζ, respectively. Jurkat cells expressing GXMR-CAR containing CD8α as the hinge/transmembrane improved the CAR expression and induced a tonic signaling. GXMR-8-28ζ and GXMR-8-BBζ induced high levels of IL-2 and up-regulation of CD69 expression in the presence of reference strains of C. neoformans and C. gattii. Moreover, GXMR-8-28ζ and GXMR-8-BBζ showed increased strength in response to incubation with clinical isolates of Cryptococcuss spp., and 4-1BB co-stimulatory domain triggered a more pronounced cellular activation. Dasatinib, a tyrosine kinase inhibitor, attenuated the GXMR-CAR signaling cascade’s engagement in the presence or absence of its ligand. This study optimized novel second-generation GXMR-CARs containing the CD8-hinge/transmembrane domain that improved CAR expression, antigen recognition, and signal strength in T-cell activation.

Currently, the majority of commercial recombinant therapeutic proteins are produced in nonhuman expression systems. Human cells are capable of producing recombinant proteins with post‐translational modifications that are more similar to native proteins which reduce immunogenicity. This article describes the potential of the human cell lines Sk‐Hep‐1, HKB‐11, and Huh‐7, cultured in serum‐free suspension conditions, to produce a complex recombinant glycoprotein, human erythropoietin (EPO). Recombinant cell lines were generated by lentiviral transduction and sorted by flow cytometry. All the recombinant cells presented high‐specific cell growth rates and a high level of rhEPO production. Maximum rhEPO concentrations achieved by Sk‐Hep‐1, HKB‐11, and Huh‐7 cells were 112.5 μg/mL, 112.7 μg/mL, and 571 μg/mL, respectively. The levels of rhEPO production by Huh‐7 cells were higher than the levels commonly reported in the literature for serum‐free suspension cultures. The rhEPO produced demonstrated biological activity measured through in vitro differentiation of CD34+ cells. In view of this, we demonstrate that Sk‐Hep‐1, HKB‐11, and Huh‐7 cell lines have good characteristics to be used as host cells for the production of complex recombinant glycoproteins, with special emphasis on Huh‐7 due to enhanced EPO production.

Chimeric antigen receptor (CAR)-cell-based therapies have demonstrated remarkable clinical success in hematologic malignancies; however, their application in solid tumors remains limited, largely due to the scarcity of suitable surface antigens. TCR-like CARs offer a promising strategy to overcome this barrier by targeting peptide-HLA complexes derived from intracellular tumor-associated antigens. In this study, we investigated NK cells expressing a TCR-like CAR directed against the MAGE-A4 peptide presented by HLA-A*02:01 on tumor cells. The CAR construct incorporated CD3ζ and GITR intracellular signaling domains and was assessed in both NK-92 cells and primary peripheral blood NK cells. Our results demonstrated that MAGE-A4 CAR expression enhanced the metabolic fitness of NK-92 cells, as evidenced by increased glycolysis, basal respiration, and ATP production. Functionally, CAR-engineered NK cells exhibited significantly enhanced cytotoxicity against MAGE-A4⁺ tumor cell lines (A375, U2OS, and U266), but not against MAGE-A4⁻ targets (HCT116), confirming antigen specificity. Enhanced tumor cell killing was associated with increased CD107a degranulation, elevated granzyme B and perforin expression, and augmented TNF-α and IFN-γ production. In vivo, MAGE-A4 CAR NK-92 cells demonstrated potent antitumor activity in an A375 xenograft model. Consistent with these findings, primary NK cells expressing the MAGE-A4 CAR also displayed enhanced antitumor activity and cytokine secretion in vitro. Collectively, these findings demonstrate that TCR-like targeting of the intracellular tumor antigen MAGE-A4 using CAR-engineered NK cells enhances metabolic fitness, effector function, and antitumor activity, highlighting the potential of this approach as a promising immunotherapeutic strategy for MAGE-A4⁺ solid tumors.