Activated and expanded natural killer (NK) cells have substantial cytotoxicity against many tumor cells, but their efficacy to eliminate solid cancers is limited. Here, we used chimeric antigen receptors (CARs) to enhance the activity of NK cells against Ewing sarcomas (EwS) in a tumor antigen-specific manner. Expression of CARs directed against the ganglioside antigen G in activated NK cells increased their responses to G+ allogeneic EwS cells and overcame resistance of individual cell lines to NK cell lysis. Second-generation CARs with 4-1BB and 2B4 co-stimulatory signaling and third-generation CARs combining both co-stimulatory domains were all equally effective. By contrast, adoptive transfer of G-specific CAR gene-modified NK cells both by intratumoral and intraperitoneal delivery failed to eliminate G-expressing EwS xenografts. Histopathology review revealed upregulation of the immunosuppressive ligand HLA-G in tumor autopsies from mice treated with NK cells compared to untreated control mice. Supporting the relevance of this finding, co-incubation of NK cells with allogeneic EwS cells induced upregulation of the HLA-G receptor CD85j, and HLA-G1 expressed by EwS cells suppressed the activity of NK cells from three of five allogeneic donors against the tumor cells. We conclude that HLA-G is a candidate immune checkpoint in EwS where it can contribute to resistance to NK cell therapy. HLA-G deserves evaluation as a potential target for more effective immunotherapeutic combination regimens in this and other cancers.

Mammalian CYP4B1 enzymes are cytochrome P450 monooxygenases that are responsible for the bioactivation of several exogenous pro-toxins including 4-ipomeanol (4-IPO). In contrast to the orthologous rabbit enzyme, we show here that native human CYP4B1 with a serine at position 427 is unable to bio-activate 4-IPO and does not cause cytotoxicity in HepG2 cells and primary human T-cells that overexpress these enzymes. We also demonstrate that a proline residue in the meander region at position 427 in human CYB4B1 and 422 in rabbit CYP4B1 is important for protein stability and rescues the 4-IPO bioactivation of the human enzyme, but is not essential for the catalytic activity of the rabbit CYP4B1 protein. Systematic substitution of native and p.S427P human CYP4B1 with peptide regions from the highly active rabbit enzyme reveals that 18 amino acids in the wild-type rabbit CYP4B1 protein are key for conferring high 4-IPO metabolizing activity. Introduction of 12 of the 18 amino acids that are also present at corresponding positions in other human CYP4 family members into the p.S427P human CYP4B1 protein results in a mutant human enzyme (P+12) that is as stable and as active as the rabbit wild-type CYP4B1 protein. These 12 mutations cluster in the predicted B–C loop through F-helix regions and reveal new amino acid regions important to P450 enzyme stability. Finally, by minimally re-engineering the human CYP4B1 enzyme for efficient activation of 4-IPO, we have developed a novel human suicide gene system that is a candidate for adoptive cellular therapies in humans.

Engineering autologous or allogeneic T cells to express a suicide gene can control potential toxicity in adoptive T-cell therapies. We recently reported the development of a novel human suicide gene system that is based on an orphan human cytochrome P450 enzyme, CYP4B1, and the naturally occurring alkylator prodrug 4-ipomeanol. The goal of this study was to systematically develop a clinically applicable self-inactivating lentiviral vector for efficient co-expression of CYP4B1 as an ER-located protein with two distinct types of cell surface proteins, either MACS selection genes for donor lymphocyte infusions after allogeneic stem cell transplantation or chimeric antigen receptors for retargeting primary T cells. The U3 region of the myeloproliferative sarcoma virus in combination with the T2A site was found to drive high-level expression of our CYP4B1 mutant with truncated CD34 or CD271 as MACS suitable selection markers. This lentiviral vector backbone was also well suited for co-expression of CYP4B1 with a codon-optimized CD19 chimeric antigen receptor (CAR) construct. Finally, 4-ipomeanol efficiently induced apoptosis in primary T cells that co-express mutant CYP4B1 and the divergently located MACS selection and CAR genes. In conclusion, we here developed a clinically suited lentiviral vector that supports high-level co-expression of cell surface proteins with a potent novel human suicide gene.

Human CYP4B1, a cytochrome P450 monooxygenase predominantly expressed in the lung, inefficiently metabolizes classical CYP4B1 substrates, such as the naturally occurring furan pro-toxin 4-ipomeanol (4-IPO). Highly active animal forms of the enzyme convert 4-IPO to reactive alkylating metabolite(s) that bind(s) to cellular macromolecules. By substitution of 13 amino acids, we restored the enzymatic activity of human CYP4B1 toward 4-IPO and this modified cDNA is potentially valuable as a suicide gene for adoptive T-cell therapies. In order to find novel pro-toxins, we tested numerous furan analogs in in vitro cell culture cytotoxicity assays by expressing the wildtype rabbit and variants of human CYP4B1 in human liver-derived HepG2 cells. To evaluate the CYP4B1 substrate specificities and furan analog catalysis, we optimized the N-terminal sequence of the CYP4B1 variants by modification/truncation and established their heterologous expression in Escherichia coli (yielding 70 and 800 nmol·l −1 of recombinant human and rabbit enzyme, respectively). Finally, spectral binding affinities and oxidative metabolism of the furan analogs by the purified recombinant CYP4B1 variants were analyzed: the naturally occurring perilla ketone was found to be the tightest binder to CYP4B1, but also the analog that was most extensively metabolized by oxidative processes to numerous non-reactive reaction products.

Immunotherapy including chimeric antigen receptor (CAR) T cell therapy has revolutionized modern cancer therapy and has achieved remarkable remission and survival rates for several malignancies with historically dismal outcomes. The hinge of the CAR connects the antigen binding to the transmembrane domain and can be exploited to confer features to CAR T cells including additional stimulation, targeted elimination or detection and enrichment of the genetically modified cells. For establishing a novel hinge derived from human CD34, we systematically tested CD34 fragments of different lengths, all containing the binding site of the QBend-10 monoclonal antibody, in a FMC63-based CD19 CAR lentiviral construct. A final construct of 99 amino acids called C6 proved to be the best candidate for flow cytometry-based detection of CAR T cells and >95% enrichment of genetically modified T cells on MACS columns. The C6 hinge was functionally indistinguishable from the commonly used CD8α hinge in vitro as well as in in vivo experiments in NSG mice. We also showed that the C6 hinge can be used for a variety of different CARs and mediates high killing efficacy without unspecific activation by target antigen-negative cells, thus making C6 ideally suited as a universal hinge for CARs for clinical applications.