Targeted shock-and-kill HIV-1 gene therapy approach combining CRISPR activation, suicide gene tBid and retargeted adenovirus delivery

Infections with the human immunodeficiency virus type 1 (HIV-1) are incurable due the long-lasting, latent viral reservoir. The shock-and-kill cure approach aims to activate latent proviruses in HIV-1 infected cells and subsequently kill these cells with strategies such as therapeutic vaccines or immune enhancement. Here, we combined the dCas9-VPR CRISPR activation (CRISPRa) system with gRNA-V, the truncated Bid (tBid)-based suicide gene strategy and CD3-retargeted adenovirus (Ad) delivery vectors, in an all-in-one targeted shock-and-kill gene therapy approach to achieve specific elimination of latently HIV-1 infected cells. Simultaneous transduction of latently HIV-1 infected J-Lat 10.6 cells with a CD3-retargeted Ad-CRISPRa-V and Ad-tBid led to a 57.7 ± 17.0% reduction of productively HIV-1 infected cells and 2.4-fold ± 0.25 increase in cell death. The effective activation of latent HIV-1 provirus by Ad-CRISPRa-V was similar to the activation control TNF-α. The strictly HIV-1 dependent and non-leaky killing by tBid could be demonstrated. Furthermore, the high transduction efficiencies of up to 70.8 ± 0.4% by the CD3-retargeting technology in HIV-1 latently infected cell lines was the basis of successful shock-and-kill. This novel targeted shock-and-kill all-in-one gene therapy approach has the potential to safely and effectively eliminate HIV-1 infected cells in a highly HIV-1 and T cell specific manner

Receptor-targeted lentiviral vectors are exceptionally sensitive toward the biophysical properties of the displayed single-chain Fv

An increasing number of applications require the expression of single-chain variable fragments (scFv) fusion proteins in mammalian cells at the cell surface membrane. Here we assessed the CD30-specific scFv HRS3, which is used in immunotherapy, for its ability to retarget lentiviral vectors (LVs) to CD30 and to mediate selective gene transfer into CD30-positive cells. Fused to the C-terminus of the type-II transmembrane protein hemagglutinin (H) of measles virus and expressed in LV packaging cells, gene transfer mediated by the released LV particles was inefficient. A series of point mutations in the scFv framework regions addressing its biophysical properties, which substantially improved production and increased the melting temperature without impairing its kinetic binding behavior to CD30, also improved the performance of LV particles. Gene transfer into CD30-positive cells increased ∼100-fold due to improved transport of the H-scFv protein to the plasma membrane. Concomitantly, LV particle aggregation and syncytia formation in packaging cells were substantially reduced. The data suggest that syncytia formation can be triggered by trans-cellular dimerization of H-scFv proteins displayed on adjacent cells. Taken together, we show that the biophysical properties of the targeting ligand have a decisive role for the gene transfer efficiency of receptor-targeted LV

Targeted adenovirus-mediated transduction of human T cells in vitro and in vivo

Clinical success in T cell therapy has stimulated widespread efforts to increase safety and potency and to extend this technology to solid tumors. Yet progress in cell therapy remains restricted by the limited payload capacity, specificity of target cell transduction, and transgenic gene expression efficiency of applied viral vectors. This renders complex reprogramming or direct in vivo applications difficult. Here, we developed a synergistic combination of trimeric adapter constructs enabling T cell-directed transduction by the human adenoviral vector serotype C5 in vitro and in vivo. Rationally chosen binding partners showed receptor-specific transduction of otherwise non-susceptible human T cells by exploiting activation stimuli. This platform remains compatible with high-capacity vectors for up to 37 kb DNA delivery, increasing payload capacity and safety because of the removal of all viral genes. Together, these findings provide a tool for targeted delivery of large payloads in T cells as a potential avenue to overcome current limitations of T cell therapy

International nonproprietary names for monoclonal antibodies: an evolving nomenclature system

Appropriate nomenclature for all pharmaceutical substances is important for clinical development, licensing, prescribing, pharmacovigilance, and identification of counterfeits. Nonproprietary names that are unique and globally recognized for all pharmaceutical substances are assigned by the International Nonproprietary Names (INN) Programme of the World Health Organization (WHO). In 1991, the INN Programme implemented the first nomenclature scheme for monoclonal antibodies. To accompany biotechnological development, this nomenclature scheme has evolved over the years; however, since the scheme was introduced, all pharmacological substances that contained an immunoglobulin variable domain were coined with the stem -mab. To date, there are 879 INN with the stem -mab. Owing to this high number of names ending in -mab, devising new and distinguishable INN has become a challenge. The WHO INN Expert Group therefore decided to revise the system to ease this situation. The revised system was approved and adopted by the WHO at the 73rd INN Consultation held in October 2021, and the radical decision was made to discontinue the use of the well-known stem -mab in naming new antibody-based drugs and going forward, to replace it with four new stems: -tug, -bart, -mig, and -ment. Keywords: International Nonproprietary Name (INN); antibodies; antibody-based drugs; antibody-drug conjugates; biological drugs; biologics; nomenclature scheme; pharmaceuticals; safety; therapeutic antibodies

Programmable DARPin-based receptors for the detection of thrombotic markers

Cellular therapies remain constrained by the limited availability of sensors for disease markers. Here we present an integrated target-to-receptor pipeline for constructing a customizable advanced modular bispecific extracellular receptor (AMBER) that combines our generalized extracellular molecule sensor (GEMS) system with a high-throughput platform for generating designed ankyrin repeat proteins (DARPins). For proof of concept, we chose human fibrin degradation products (FDPs) as markers with high clinical relevance and screened a DARPin library for FDP binders. We built AMBERs equipped with 19 different DARPins selected from 160 hits, and found 4 of them to be functional as heterodimers with a known single-chain variable fragments binder. Tandem receptors consisting of combinations of the validated DARPins are also functional. We demonstrate applications of these AMBER receptors in vitro and in vivo by constructing designer cell lines that detect pathological concentrations of FDPs and respond with the production of a reporter and a therapeutic anti-thrombotic protein

Biofunctional Nanodot Arrays in Living Cells Uncover Synergistic Co-Condensation of Wnt Signalodroplets

Qualitative and quantitative analysis of transient signaling platforms in the plasma membrane has remained a key experimental challenge. Here, biofunctional nanodot arrays (bNDAs) are developed to spatially control dimerization and clustering of cell surface receptors at the nanoscale. High-contrast bNDAs with spot diameters of ≈300 nm are obtained by capillary nanostamping of bovine serum albumin bioconjugates, which are subsequently biofunctionalized by reaction with tandem anti-green fluorescence protein (GFP) clamp fusions. Spatially controlled assembly of active Wnt signalosomes is achieved at the nanoscale in the plasma membrane of live cells by capturing the co-receptor Lrp6 into bNDAs via an extracellular GFP tag. Strikingly, co-recruitment is observed of co-receptor Frizzled-8 as well as the cytosolic scaffold proteins Axin-1 and Disheveled-2 into Lrp6 nanodots in the absence of ligand. Density variation and the high dynamics of effector proteins uncover highly cooperative liquid-liquid phase separation (LLPS)-driven assembly of Wnt "signalodroplets" at the plasma membrane, pinpointing the synergistic effects of LLPS for Wnt signaling amplification. These insights highlight the potential of bNDAs for systematically interrogating nanoscale signaling platforms and condensation at the plasma membrane of live cells

Experimental determination of the complete spin structure for anti-proton + proton -> anti-\Lambda + \Lambda at anti-proton beam momentum of 1.637 GeV/c

The reaction anti-proton + proton -> anti-\Lambda + \Lambda -> anti-proton + \pi^+ + proton + \pi^- has been measured with high statistics at anti-proton beam momentum of 1.637 GeV/c. The use of a transversely-polarized frozen-spin target combined with the self-analyzing property of \Lambda/anti-\Lambda decay allows access to unprecedented information on the spin structure of the interaction. The most general spin-scattering matrix can be written in terms of eleven real parameters for each bin of scattering angle, each of these parameters is determined with reasonable precision. From these results all conceivable spin-correlations are determined with inherent self-consistency. Good agreement is found with the few previously existing measurements of spin observables in anti-proton + proton -> anti-\Lambda + \Lambda near this energy. Existing theoretical models do not give good predictions for those spin-observables that had not been previously measured.Comment: To be published in Phys. Rev. C. Tables of results (i.e. Ref. 24) are available at http://www-meg.phys.cmu.edu/~bquinn/ps185_pub/results.tab 24 pages, 16 figure

Measurement of Spin Transfer Observables in Antiproton-Proton -> Antilambda-Lambda at 1.637 GeV/c

Spin transfer observables for the strangeness-production reaction Antiproton-Proton -> Antilambda-Lambda have been measured by the PS185 collaboration using a transversely-polarized frozen-spin target with an antiproton beam momentum of 1.637 GeV/c at the Low Energy Antiproton Ring at CERN. This measurement investigates observables for which current models of the reaction near threshold make significantly differing predictions. Those models are in good agreement with existing measurements performed with unpolarized particles in the initial state. Theoretical attention has focused on the fact that these models produce conflicting predictions for the spin-transfer observables D_{nn} and K_{nn}, which are measurable only with polarized target or beam. Results presented here for D_{nn} and K_{nn} are found to be in disagreement with predictions from existing models. These results also underscore the importance of singlet-state production at backward angles, while current models predict complete or near-complete triplet-state dominance.Comment: 5 pages, 3 figure

Induction and Exhaustion of Lymphocytic Choriomeningitis Virus–specific Cytotoxic T Lymphocytes Visualized Using Soluble Tetrameric Major Histocompatibility Complex Class I–Peptide Complexes

This study describes the construction of soluble major histocompatibility complexes consisting of the mouse class I molecule, H-2Db, chemically biotinylated β2 microglobulin and a peptide epitope derived from the glycoprotein (GP; amino acids 33–41) of lymphocytic choriomeningitis virus (LCMV). Tetrameric class I complexes, which were produced by mixing the class I complexes with phycoerythrin-labeled neutravidin, permitted direct analysis of virus-specific cytotoxic T lymphocytes (CTLs) by flow cytometry. This technique was validated by (a) staining CD8+ cells in the spleens of transgenic mice that express a T cell receptor (TCR) specific for H-2Db in association with peptide GP33–41, and (b) by staining virus-specific CTLs in the cerebrospinal fluid of C57BL/6 (B6) mice that had been infected intracranially with LCMV-DOCILE. Staining of spleen cells isolated from B6 mice revealed that up to 40% of CD8+ T cells were GP33 tetramer+ during the initial phase of LCMV infection. In contrast, GP33 tetramers did not stain CD8+ T cells isolated from the spleens of B6 mice that had been infected 2 mo previously with LCMV above the background levels found in naive mice. The fate of virus-specific CTLs was analyzed during the acute phase of infection in mice challenged both intracranially and intravenously with a high or low dose of LCMV-DOCILE. The results of the study show that the outcome of infection by LCMV is determined by antigen load alone. Furthermore, the data indicate that deletion of virus-specific CTLs in the presence of excessive antigen is preceded by TCR downregulation and is dependent upon perforin

Trapping the HIV-1 V3 loop in a helical conformation enables broad neutralization

The third variable (V3) loop on the human immunodeficiency virus 1 (HIV-1) envelope glycoprotein trimer is indispensable for virus cell entry. Conformational masking of V3 within the trimer allows efficient neutralization via V3 only by rare, broadly neutralizing glycan-dependent antibodies targeting the closed prefusion trimer but not by abundant antibodies that access the V3 crown on open trimers after CD4 attachment. Here, we report on a distinct category of V3-specific inhibitors based on designed ankyrin repeat protein (DARPin) technology that reinstitute the CD4-bound state as a key neutralization target with up to >90% breadth. Broadly neutralizing DARPins (bnDs) bound V3 solely on open envelope and recognized a four-turn amphipathic α-helix in the carboxy-terminal half of V3 (amino acids 314-324), which we termed 'αV3C'. The bnD contact surface on αV3C was as conserved as the CD4 binding site. Molecular dynamics and escape mutation analyses underscored the functional relevance of αV3C, highlighting the potential of αV3C-based inhibitors and, more generally, of postattachment inhibition of HIV-1