Nanobody based dual specific CARs

Recent clinical trials have shown that adoptive chimeric antigen receptor (CAR) T cell therapy is a very potent and possibly curative option in the treatment of B cell leukemias and lymphomas. However, targeting a single antigen may not be sufficient, and relapse due to the emergence of antigen negative leukemic cells may occur. A potential strategy to counter the outgrowth of antigen escape variants is to broaden the specificity of the CAR by incorporation of multiple antigen recognition domains in tandem. As a proof of concept, we here describe a bispecific CAR in which the single chain variable fragment (scFv) is replaced by a tandem of two single-antibody domains or nanobodies (nanoCAR). High membrane nanoCAR expression levels are observed in retrovirally transduced T cells. NanoCARs specific for CD20 and HER2 induce T cell activation, cytokine production and tumor lysis upon incubation with transgenic Jurkat cells expressing either antigen or both antigens simultaneously. The use of nanobody technology allows for the production of compact CARs with dual specificity and predefined affinity

HIV-1 Vpr N-terminal tagging affects alternative splicing of the viral genome

To facilitate studies on Vpr function in replicating HIV-1, we aimed to tag the protein in an infectious virus. First we showed that N-, but not C-terminal HA/FLAG tagging of Vpr protein preserves Vpr cytopathicity. Cloning the tags into proviral DNA however ablated viral production and replication. By construction of additional viral variants we could show this defect was not protein-but RNA-dependent and sequence specific, and characterized by oversplicing of the genomic RNA. Simulation of genomic RNA folding suggested that introduction of the tag sequence induced an alternative folding structure in a region enriched in splice sites and splicing regulatory sequences. In silico predictions identified the HA/His(6)-Vpr tagging in HIV-1 to affect mRNA folding less than HA/FLAG-Vpr tagging. In vitro infectivity and mRNA splice pattern improved but did not reach wild-type values. Thus, sequence-specific insertions may interfere with mRNA splicing, possibly due to altered RNA folding. Our results point to the complexity of viral RNA genome sequence interactions. This should be taken into consideration when designing viral manipulation strategies, for both research as for biological interventions

TARP as immunotherapeutic target in AML expressed in the LSC compartment

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Human thymic CD10+ PD-1+ intraepithelial lymphocyte precursors acquire interleukin-15 responsiveness at the CD1a– CD95+ CD28– CCR7– developmental stage

Human thymic CD8αα+ CD10+ PD-1+ αβ T cells selected through early agonist selection have been proposed as the putative thymic precursors of the human CD8αα+ intestinal intraepithelial lymphocytes (IELs). However, the progeny of these thymic precursor cells in human blood or tissues has not yet been characterized. Here, we studied the phenotypical and transcriptional differentiation of the thymic IEL precursor (IELp) lineage upon in vitro exposure to cytokines prominent in the peripheral tissues such as interleukin-15 (IL-15) and the inflammatory cytokines interleukin-12 (IL-12) and interleukin-18 (IL-18). We showed that only the CD1a− fraction of the CD10+ PD-1+ IELp population was able to proliferate with IL-15, suggesting that this subset had acquired functionality. These cells downregulated PD-1 expression and completely lost CD10 expression, whereas other surface markers such as CD95 and CXCR3 remained highly expressed. RNA-seq analysis of the IL-15-cultured cells clearly showed induction of innate-like and effector genes. Induction of the cytotoxic machinery by the CD10+ PD-1+ population was acquired in the presence of IL-15 and was further augmented by inflammatory cytokines. Our data suggest that only the CD1a− CD10+ PD-1+ population exits the thymus and survives in the periphery. Furthermore, PD-1 and CD10 expression is not an intrinsic property of this lineage, but rather characterizes a transient stage in differentiation. CD95 and CXCR3 expression combined with the absence of CD28, CCR7, and CD6 expression might be more powerful markers to define this lineage in the periphery

Rapid and effective generation of nanobody based CARs using PCR and Gibson Assembly

Recent approval of chimeric antigen receptor (CAR) T cell therapy by the European Medicines Agency (EMA)/Federal and Drug Administration (FDA) and the remarkable results of CAR T clinical trials illustrate the curative potential of this therapy. While CARs against a multitude of different antigens are being developed and tested (pre)clinically, there is still a need for optimization. The use of single -chain variable fragments (scFvs) as targeting moieties hampers the quick generation of functional CARs and could potentially limit the efficacy. Instead, nanobodies may largely circumvent these difficulties. We used an available nanobody library generated after immunization of llamas against Cluster of Differentiation (CD) 20 through DNA vaccination or against the ectodomain of CD33 using soluble protein. The nanobody specific sequences were amplified by PCR and cloned by Gibson Assembly into a retroviral vector containing two different second -generation CAR constructs. After transduction in T cells, we observed high cell membrane nanoCAR expression in all cases. Following stimulation of nanoCAR-expressing T cells with antigen-positive cell lines, robust T cell activation, cytokine production and tumor cell lysis both in vitro and in vivo was observed. The use of nanobody technology in combination with PCR and Gibson Assembly allows for the rapid and effective generation of compact CARs

A novel neutralizing human monoclonal antibody broadly abrogates hepatitis C virus infection in vitro and in vivo

Infections with hepatitis C virus (HCV) represent a worldwide health burden and a prophylactic vaccine is still not available. Liver transplantation (LT) is often the only option for patients with HCV-induced end-stage liver disease. However, immediately after transplantation, the liver graft becomes infected by circulating virus, resulting in accelerated progression of liver disease. Although the effi cacy of HCV treatment using direct-acting antivirals has improved significantly, immune compromised LT-patients and patients with advanced liver disease remain difficult to treat. As an alternative approach, interfering with viral entry could prevent infection of the donor liver. We generated a human monoclonal antibody (mAb), designated 2A5, which targets the HCV envelope. The neutralizing activity of mAb 2A5 was assessed using multiple prototype and patient-derived HCV pseudoparticles (HCVpp), cell culture produced HCV (HCVcc), and a human-liver chimeric mouse model. Neutralization levels observed for mAb 2A5 were generally high and mostly superior to those obtained with AP33, a well-characterized HCV-neutralizing monoclonal antibody. Using humanized mice, complete protection was observed after genotype 1a and 4a HCV challenge, while only partial protection was achieved using gt1b and 6a isolates. Epitope mapping revealed that mAb 2A5 binding is conformation-dependent and identified the E2-region spanning amino acids 434 to 446 (epitope II) as the predominant contact domain. Conclusion : mAb 2A5 shows potent anti-HCV neutralizing activity both in vitro and in vivo and could hence represent a valuable candidate to prevent HCV recurrence in LT-patients. In addition, the detailed identification of the neutralizing epitope can be applied for the design of prophylactic HCV vaccines

The Wiskott–Aldrich syndrome protein is required for positive selection during T-cell lineage differentiation

The Wiskott–Aldrich syndrome (WAS) is an X-linked primary immune deficiency caused by a mutation in the WAS gene. This leads to altered or absent WAS protein (WASp) expression and function resulting in thrombocytopenia, eczema, recurrent infections, and autoimmunity. In T cells, WASp is required for immune synapse formation. Patients with WAS show reduced numbers of peripheral blood T lymphocytes and an altered T-cell receptor repertoire. In vitro, their peripheral T cells show decreased proliferation and cytokine production upon aCD3/aCD28 stimulation. It is unclear whether these T-cell defects are acquired during peripheral activation or are, in part, generated during thymic development. Here, we assessed the role of WASp during T-cell differentiation using artificial thymic organoid cultures and in the thymus of humanized mice. Although CRISPR/Cas9 WAS knockout hematopoietic stem and progenitor cells (HSPCs) rearranged the T-cell receptor and differentiated to T-cell receptor (TCR)+ CD4+ CD8+ double-positive (DP) cells similar to wild-type HSPCs, a partial defect in the generation of CD8 single-positive (SP) cells was observed, suggesting that WASp is involved in their positive selection. TCR repertoire analysis of the DP and CD8+ SP population, however, showed a polyclonal repertoire with no bias toward autoreactivity. To our knowledge, this is the first study of the role of WASp in human T-cell differentiation and on TCR repertoire generation

Characterization of a cAMP-stimulated cAMP phosphodiesterase in Dictyostelium discoideum

A cyclic nucleotide phosphodiesterase, PdeE, that harbors two cyclic nucleotide binding motifs and a binuclear Zn2+-binding domain was characterized in Dictyostelium. In other eukaryotes, the Dictyostelium domain shows greatest homology to the 73-kDa subunit of the pre-mRNA cleavage and polyadenylation specificity factor. The Dictyostelium PdeE gene is expressed at its highest levels during aggregation, and its disruption causes the loss of a cAMP-phosphodiesterase activity. The pdeE null mutants show a normal cAMP-induced cGMP response and a 1.5-fold increase of cAMP-induced cAMP relay. Overexpression of a PdeE-yellow fluorescent protein (YFP) fusion construct causes inhibition of aggregation and loss of the cAMP relay response, but the cells can aggregate in synergy with wild-type cells. The PdeE-YFP fusion protein was partially purified by immunoprecipitation and biochemically characterized. PdeE and its Dictyostelium ortholog, PdeD, are both maximally active at pH 7.0. Both enzymes require bivalent cations for activity. The common cofactors Zn2+ and Mg2+ activated PdeE and PdeD maximally at 10 mm, whereas Mn2+ activated the enzymes to 4-fold higher levels, with half-maximal activation between 10 and 100 gm. PdeE is an allosteric enzyme, which is similar to4-fold activated by cAMP, with half-maximal activation occurring at about 10 muM and an apparent K-m of similar to1 mm. cGMP is degraded at a 6-fold lower rate than cAMP. Neither cGMP nor 8-Br-cAMP are efficient activators of PdeE activity