A novel antibody-TCR (AbTCR) platform combines Fab-based antigen recognition with gamma/delta-TCR signaling to facilitate T-cell cytotoxicity with low cytokine release.

The clinical use of genetically modified T-cell therapies has led to unprecedented response rates in leukemia and lymphoma patients treated with anti-CD19 chimeric antigen receptor (CAR)-T. Despite this clinical success, FDA-approved T-cell therapies are currently limited to B-cell malignancies, and challenges remain with managing cytokine-related toxicities. We have designed a novel antibody-T-cell receptor (AbTCR) platform where we combined the Fab domain of an antibody with the γ and δ chains of the TCR as the effector domain. We demonstrate the ability of anti-CD19-AbTCR-T cells to trigger antigen-specific cytokine production, degranulation, and killing of CD19-positive cancer cells in vitro and in xenograft mouse models. By using the same anti-CD19 binding moiety on an AbTCR compared to a CAR platform, we demonstrate that AbTCR activates cytotoxic T-cell responses with a similar dose-response as CD28/CD3ζ CAR, yet does so with less cytokine release and results in T cells with a less exhausted phenotype. Moreover, in comparative studies with the clinically validated CD137 (4-1BB)-based CAR, CTL019, our anti-CD19-AbTCR shows less cytokine release and comparable tumor inhibition in a patient-derived xenograft leukemia model.

Targeting Alpha-Fetoprotein (AFP)-MHC Complex with CAR T-Cell Therapy for Liver Cancer.

PURPOSE: The majority of tumor-specific antigens are intracellular and/or secreted and therefore inaccessible by conventional chimeric antigen receptor (CAR) T-cell therapy. Given that all intracellular/secreted proteins are processed into peptides and presented by class I MHC on the surface of tumor cells, we used alpha-fetoprotein (AFP), a specific liver cancer marker, as an example to determine whether peptide-MHC complexes can be targets for CAR T-cell therapy against solid tumors. EXPERIMENTAL DESIGN: We generated a fully human chimeric antigen receptor, ET1402L1-CAR (AFP-CAR), with exquisite selectivity and specificity for the AFP158-166 peptide complexed with human leukocyte antigen (HLA)-A*02:01. RESULTS: We report that T cells expressing AFP-CAR selectively degranulated, released cytokines, and lysed liver cancer cells that were HLA-A*02:01+/AFP+ while sparing cells from multiple tissue types that were negative for either expressed proteins. In vivo, intratumoral injection of AFP-CAR T cells significantly regressed both Hep G2 and AFP158-expressing SK-HEP-1 tumors in SCID-Beige mice (n = 8 for each). Moreover, intravenous administration of AFP-CAR T cells in Hep G2 tumor-bearing NSG mice lead to rapid and profound tumor growth inhibition (n = 6). Finally, in an established intraperitoneal liver cancer xenograft model, AFP-CAR T cells showed robust antitumor activity (n = 6). CONCLUSIONS: This study demonstrates that CAR T-cell immunotherapy targeting intracellular/secreted solid tumor antigens can elicit a potent antitumor response. Our approach expands the spectrum of antigens available for redirected T-cell therapy against solid malignancies and offers a promising new avenue for liver cancer immunotherapy. Clin Cancer Res; 23(2); 478-88. ©2016 AACR.

The hammerhead ribozyme: structure, catalysis, and gene regulation.

The hammerhead ribozyme has long been considered a prototype for understanding RNA catalysis, but discrepancies between the earlier crystal structures of a minimal hammerhead self-cleaving motif and various biochemical investigations frustrated attempt to understand hammerhead ribozyme catalysis in terms of structure. With the discovery that a tertiary contact distal from the ribozyme's active site greatly enhances its catalytic prowess, and the emergence of new corresponding crystal structures of full-length hammerhead ribozymes, a unified understanding of catalysis in terms of the structure is now possible. A mechanism in which the invariant residue G12 functions as a general base, and the 2'-OH moiety of the invariant G8, itself forming a tertiary base pair with the invariant C3, is the general acid, appears consistent with both the crystal structure and biochemical experimental results. Originally discovered in the context of plant satellite RNA viruses, the hammerhead more recently has been found embedded in the 3'-untranslated region of mature mammalian mRNAs, suggesting additional biological roles in genetic regulation.

The sarcin-ricin loop of 23S rRNA is essential for assembly of the functional core of the 50S ribosomal subunit.

The sarcin-ricin loop (SRL) of 23S rRNA in the large ribosomal subunit is a factor-binding site that is essential for GTP-catalyzed steps in translation, but its precise functional role is thus far unknown. Here, we replaced the 15-nucleotide SRL with a GAAA tetraloop and affinity purified the mutant 50S subunits for functional and structural analysis in vitro. The SRL deletion caused defects in elongation-factor-dependent steps of translation and, unexpectedly, loss of EF-Tu-independent A-site tRNA binding. Detailed chemical probing analysis showed disruption of a network of rRNA tertiary interactions that hold together the 23S rRNA elements of the functional core of the 50S subunit, accompanied by loss of ribosomal protein L16. Our results reveal an influence of the SRL on the higher-order structure of the 50S subunit, with implications for its role in translation.

A discontinuous hammerhead ribozyme embedded in a mammalian messenger RNA.

Structured RNAs embedded in the untranslated regions (UTRs) of messenger RNAs can regulate gene expression. In bacteria, control of a metabolite gene is mediated by the self-cleaving activity of a ribozyme embedded in its 5' UTR. This discovery has raised the question of whether gene-regulating ribozymes also exist in eukaryotic mRNAs. Here we show that highly active hammerhead ribozymes are present in the 3' UTRs of rodent C-type lectin type II (Clec2) genes. Using a hammerhead RNA motif search with relaxed delimitation of the non-conserved regions, we detected ribozyme sequences in which the invariant regions, in contrast to the previously identified continuous hammerheads, occur as two fragments separated by hundreds of nucleotides. Notably, a fragment pair can assemble to form an active hammerhead ribozyme structure between the translation termination and the polyadenylation signals within the 3' UTR. We demonstrate that this hammerhead structure can self-cleave both in vitro and in vivo, and is able to reduce protein expression in mouse cells. These results indicate that an unrecognized mechanism of post-transcriptional gene regulation involving association of discontinuous ribozyme sequences within an mRNA may be modulating the expression of several CLEC2 proteins that function in bone remodelling and the immune response of several mammals.

Intersubunit movement is required for ribosomal translocation.

Translocation of tRNA and mRNA during protein synthesis is believed to be coupled to structural changes in the ribosome. The "ratchet model," based on cryo-EM reconstructions of ribosome complexes, invokes relative movement of the 30S and 50S ribosomal subunits in this process; however, evidence that directly demonstrates a requirement for intersubunit movement during translocation is lacking. To address this problem, we created an intersubunit disulfide cross-link to restrict potential movement. The cross-linked ribosomes were unable to carry out polypeptide synthesis; this inhibition was completely reversed upon reduction of the disulfide bridge. In vitro assays showed that the cross-linked ribosomes were specifically blocked in elongation factor G-dependent translocation. These findings show that intersubunit movement is required for ribosomal translocation, accounting for the universal two-subunit architecture of ribosomes.