Targeting intracellular WT1 in AML with a novel RMF-peptide-MHC-specific T-cell bispecific antibody.

Antibody-based immunotherapy is a promising strategy for targeting chemoresistant leukemic cells. However, classical antibody-based approaches are restricted to targeting lineage-specific cell surface antigens. By targeting intracellular antigens, a large number of other leukemia-associated targets would become accessible. In this study, we evaluated a novel T-cell bispecific (TCB) antibody, generated by using CrossMAb and knob-into-holes technology, containing a bivalent T-cell receptor-like binding domain that recognizes the RMFPNAPYL peptide derived from the intracellular tumor antigen Wilms tumor protein (WT1) in the context of HLA-A*02. Binding to CD3ε recruits T cells irrespective of their T-cell receptor specificity. WT1-TCB elicited antibody-mediated T-cell cytotoxicity against AML cell lines in a WT1- and HLA-restricted manner. Specific lysis of primary acute myeloid leukemia (AML) cells was mediated in ex vivo long-term cocultures by using allogeneic (mean ± standard error of the mean [SEM] specific lysis, 67 ± 6% after 13-14 days; n = 18) or autologous, patient-derived T cells (mean ± SEM specific lysis, 54 ± 12% after 11-14 days; n = 8). WT1-TCB-treated T cells exhibited higher cytotoxicity against primary AML cells than an HLA-A*02 RMF-specific T-cell clone. Combining WT1-TCB with the immunomodulatory drug lenalidomide further enhanced antibody-mediated T-cell cytotoxicity against primary AML cells (mean ± SEM specific lysis on days 3-4, 45.4 ± 9.0% vs 70.8 ± 8.3%; P = .015; n = 9-10). In vivo, WT1-TCB-treated humanized mice bearing SKM-1 tumors exhibited a significant and dose-dependent reduction in tumor growth. In summary, we show that WT1-TCB facilitates potent in vitro, ex vivo, and in vivo killing of AML cell lines and primary AML cells; these results led to the initiation of a phase 1 trial in patients with relapsed/refractory AML (#NCT04580121).

High-Throughput Identification of MHC Class I Binding Peptides Using an Ultradense Peptide Array.

Rational vaccine development and evaluation requires identifying and measuring the magnitude of epitope-specific CD8 T cell responses. However, conventional CD8 T cell epitope discovery methods are labor intensive and do not scale well. In this study, we accelerate this process by using an ultradense peptide array as a high-throughput tool for screening peptides to identify putative novel epitopes. In a single experiment, we directly assess the binding of four common Indian rhesus macaque MHC class I molecules (Mamu-A1*001, -A1*002, -B*008, and -B*017) to ∼61,000 8-mer, 9-mer, and 10-mer peptides derived from the full proteomes of 82 SIV and simian HIV isolates. Many epitope-specific CD8 T cell responses restricted by these four MHC molecules have already been identified in SIVmac239, providing an ideal dataset for validating the array; up to 64% of these known epitopes are found in the top 192 SIVmac239 peptides with the most intense MHC binding signals in our experiment. To assess whether the peptide array identified putative novel CD8 T cell epitopes, we validated the method by IFN-γ ELISPOT assay and found three novel peptides that induced CD8 T cell responses in at least two Mamu-A1*001-positive animals; two of these were validated by ex vivo tetramer staining. This high-throughput identification of peptides that bind class I MHC will enable more efficient CD8 T cell response profiling for vaccine development, particularly for pathogens with complex proteomes for which few epitope-specific responses have been defined.

Discovery of a microbial transglutaminase enabling highly site-specific labeling of proteins.

Microbial transglutaminases (MTGs) catalyze the formation of Gln-Lys isopeptide bonds and are widely used for the cross-linking of proteins and peptides in food and biotechnological applications (e.g. to improve the texture of protein-rich foods or in generating antibody-drug conjugates). Currently used MTGs have low substrate specificity, impeding their biotechnological use as enzymes that do not cross-react with nontarget substrates (i.e. as bio-orthogonal labeling systems). Here, we report the discovery of an MTG from Kutzneria albida (KalbTG), which exhibited no cross-reactivity with known MTG substrates or commonly used target proteins, such as antibodies. KalbTG was produced in Escherichia coli as soluble and active enzyme in the presence of its natural inhibitor ammonium to prevent potentially toxic cross-linking activity. The crystal structure of KalbTG revealed a conserved core similar to other MTGs but very short surface loops, making it the smallest MTG characterized to date. Ultra-dense peptide array technology involving a pool of 1.4 million unique peptides identified specific recognition motifs for KalbTG in these peptides. We determined that the motifs YRYRQ and RYESK are the best Gln and Lys substrates of KalbTG, respectively. By first reacting a bifunctionalized peptide with the more specific KalbTG and in a second step with the less specific MTG from Streptomyces mobaraensis, a successful bio-orthogonal labeling system was demonstrated. Fusing the KalbTG recognition motif to an antibody allowed for site-specific and ratio-controlled labeling using low label excess. Its site specificity, favorable kinetics, ease of use, and cost-effective production render KalbTG an attractive tool for a broad range of applications, including production of therapeutic antibody-drug conjugates.

Rapid determination of RNA accessible sites by surface plasmon resonance detection of hybridization to DNA arrays.

RNA accessible sites are the regions in an RNA molecule that are available for hybridization with cDNA or RNA molecules. The identification of these accessible sites is a critical first step in identifying antisense-mediated gene suppression sites, as well as in a variety of other RNA-based analysis methods. Here, we present a rapid, hybridization-based, label-free method of identifying RNA accessible sites with surface plasmon resonance imaging (SPRi) on in situ synthesized oligonucleotide arrays prepared on carbon-on-metal substrates. The accessible sites of three pre-miRNAs, miRNA precursors of approximately 75 nt in length, were determined by hybridizing the RNA molecules to RNA-specific tiling arrays. An array composed of all possible 6mer oligonucleotide sequences was also utilized in this work, offering a universal platform capable of studying RNA molecules in a high throughput manner.

Stepwise Evolution Improves Identification of Diverse Peptides Binding to a Protein Target.

Considerable efforts have been made to develop technologies for selection of peptidic molecules that act as substrates or binders to a protein of interest. Here we demonstrate the combination of rational peptide array library design, parallel screening and stepwise evolution, to discover novel peptide hotspots. These hotspots can be systematically evolved to create high-affinity, high-specificity binding peptides to a protein target in a reproducible and digitally controlled process. The method can be applied to synthesize both linear and cyclic peptides, as well as peptides composed of natural and non-natural amino acid analogs, thereby enabling screens in a much diverse chemical space. We apply this method to stepwise evolve peptide binders to streptavidin, a protein studied for over two decades and report novel peptides that mimic key interactions of biotin to streptavidin.

Genotyping single nucleotide polymorphisms directly from genomic DNA by invasive cleavage reaction on microspheres.

Here we report proof-of-principle for a microsphere-based genotyping assay that detects single nucleotide polymorphisms (SNPs) directly from human genomic DNA samples. This assay is based on a structure-specific cleavage reaction that achieves single base discrimination with a 5'-nuclease which recognizes a tripartite substrate formed upon hybridization of target DNA with probe and upstream oligonucleotides. The assay is simple with two easy steps: a cleavage reaction, which generates fluorescent signal on microsphere surfaces, followed by flow cytometry analysis of the microspheres. Genomic DNA samples were genotyped for the SNP in the Apolipoprotein E gene at amino acid position 158. The assay successfully scored wild type, heterozygous and homozygous mutants. To our knowledge, this is the first report of a solid-support assay for detection of SNPs directly from genomic DNA without PCR amplification of the target.

Modeling of flap endonuclease interactions with DNA substrate.

Structure-specific 5' nucleases play an important role in DNA replication and repair uniquely recognizing an overlap flap DNA substrate and processing it into a DNA nick. However, in the absence of a high-resolution structure of the enzyme/DNA complex, the mechanism underlying this recognition and substrate specificity, which is key to the enzyme's function, remains unclear. Here, we propose a three-dimensional model of the structure-specific 5' flap endonuclease from Pyrococcus furiosus in its complex with DNA. The model is based on the known X-ray structure of the enzyme and a variety of biochemical and molecular dynamics (MD) data utilized in the form of distance restraints between the enzyme and the DNA. Contacts between the 5' flap endonuclease and the sugar-phosphate backbone of the overlap flap substrate were identified using enzyme activity assays on substrates with methylphosphonate or 2'-O-methyl substitutions. The enzyme footprint extends two to four base-pairs upstream and eight to nine base-pairs downstream of the cleavage site, thus covering 10-13 base-pairs of duplex DNA. The footprint data are consistent with a model in which the substrate is bound in the DNA-binding groove such that the downstream duplex interacts with the helix-hairpin-helix motif of the enzyme. MD simulations to identify the substrate orientation in this model are consistent with the results of the enzyme activity assays on the methylphosphonate and 2'-O-methyl-modified substrates. To further refine the model, 5' flap endonuclease variants with alanine point substitutions at amino acid residues expected to contact phosphates in the substrate and one deletion mutant were tested in enzyme activity assays on the methylphosphonate-modified substrates. Changes in the enzyme footprint observed for two point mutants, R64A and R94A, and for the deletion mutant in the enzyme's beta(A)/beta(B) region, were interpreted as being the result of specific interactions in the enzyme/DNA complex and were used as distance restraints in MD simulations. The final structure suggests that the substrate's 5' flap interacts with the enzyme's helical arch and that the helix-hairpin-helix motif interacts with the template strand in the downstream duplex eight base-pairs from the cleavage site. This model suggests specific interactions between the 3' end of the upstream oligonucleotide and the enzyme. The proposed structure presents the first detailed description of substrate recognition by structure-specific 5' nucleases.

A surface invasive cleavage assay for highly parallel SNP analysis.

The structure-specific invasive cleavage of single-stranded DNA by 5' nucleases is a useful means for sensitive detection of single-nucleotide polymorphisms or SNPs. The solution-phase invasive cleavage reaction has sufficient sensitivity for direct detection of as few as 600 target molecules with no prior target amplification. One approach to the parallelization of SNP analysis is to adapt the invasive cleavage reaction to an addressed array format. Two surface invasive cleavage reaction strategies were designed and tested using the polymorphic site in codon 158 of the human ApoE gene as a model system, with a synthetic oligonucleotide as target. The upstream oligonucleotide, which is required for the invasive cleavage reaction, was either added in solution (strategy 1), or co-immobilized on the surface along with the probe oligonucleotide (strategy 2). Both strategies showed target-concentration and time-dependent amplification of signal. Parameters that govern the rate of the surface-invasive cleavage reactions are discussed.

Invader technology for DNA and RNA analysis: principles and applications.

Concomitant advances made by the Human Genome Project and in the development of nucleic acid screening technologies are driving the expansion of pharmacogenomic research and molecular diagnostics. However, most current technologies are restrictive due to their complexity and/or cost, limiting the potential of personalized medicine. The invader assay, which can be used for genotyping as well as for gene expression monitoring without the need for intervening target amplification steps, presents an immediate solution that is accurate, simple to use, scaleable and cost-effective.

Invader plus method detects herpes simplex virus in cerebrospinal fluid and simultaneously differentiates types 1 and 2.

We report here on the development and validation of a prototype Invader Plus method for the qualitative detection of herpes simplex virus types 1 and 2 in cerebrospinal fluid (CSF). The method combines PCR and Invader techniques in a single, closed-tube, continuous-reaction format that gives an analytical sensitivity of approximately 10 copies per reaction. The clinical sensitivity and specificity were 100.0% and 98.6%, respectively, when the results of the method were validated against the results obtained with a PCR colorimetric microtiter plate system by use of clinical CSF specimens.

Quantitation of microRNAs using a modified Invader assay.

The short lengths of microRNAs (miRNAs) present a significant challenge for detection and quantitation using conventional methods for RNA analysis. To address this problem, we developed a quantitative, sensitive, and rapid miRNA assay based on our previously described messenger RNA Invader assay. This assay was used successfully in the analysis of several miRNAs, using as little as 50-100 ng of total cellular RNA or as few as 1,000 lysed cells. Its specificity allowed for discrimination between miRNAs differing by a single nucleotide, and between precursor and mature miRNAs. The Invader miRNA assay, which can be performed in unfractionated detergent lysates, uses fluorescence detection in microtiter plates and requires only 2-3 h incubation time, allowing for parallel analysis of multiple samples in high-throughput screening analyses.

Structure-specific DNA cleavage on surfaces.

The structure-specific invasive cleavage reaction is a useful means for sensitive and specific detection of single nucleotide polymorphisms, or SNPs, directly from genomic DNA without a need for prior target amplification. A new approach integrating this invasive cleavage assay and surface DNA array technology has been developed for potentially large-scale SNP scoring in a parallel format. Two surface invasive cleavage reaction strategies were designed and implemented for a model SNP system in codon 158 of the human ApoE gene. The upstream oligonucleotide, which is required for the invasive cleavage reaction, is either co-immobilized on the surface along with the probe oligonucleotide or alternatively added in solution. The ability of this approach to unambiguously discriminate a single base difference was demonstrated using PCR-amplified human genomic DNA. A theoretical model relating the surface fluorescence intensity to the progress of the invasive cleavage reaction was developed and agreed well with experimental results.