Comprehensive Mapping of Immune Nanobody Repertoires with NanoMAP 2267451

Nanobodies Open New Avenues in Cancer Treatment: From Molecular Engineering to Therapeutic Platforms

Nanobody-based Tools for in-depth Research of Immuno-Oncology Targets 2334607

Immuno-oncology is a rapidly growing field with an urgent need for well-characterized and highly specific research reagents, particularly in immune checkpoint targeting and acute myeloid leukemia (AML). We have developed recombinant monoclonal nanobodies (VHHs) targeting these critical areas to facilitate research with minimal artifacts and to support the development of new drug candidates. This work addresses current challenges in the field by providing tools that enhance the specificity and reliability of research outcomes. Based on our extensive experience in the field of nanobodies, we have developed VHHs that bind to key immune checkpoint proteins, including TIGIT, TIM3, LAG3, PD1, PD-L1 and CTLA4. Additionally, we have generated VHHs specific to AML typic targets, such as MSLN and FLT3. Each candidate was characterized regarding its affinity, specificity and bound epitope across different applications, including flow cytometry, immunofluorescence or live-cell imaging. n/a VHHs offer several advantages over conventional antibodies: Their unique structure allows VHHs to bind epitopes different from conventional antibodies. Their monoclonal monovalent nature prevents cross-linking of target proteins, minimizing unwanted side effects such as rapid receptor internalization or activation of intracellular signaling cascades. The absence of an Fc region provides flexibility in combining VHHs with other primary and secondary antibody reagents, while also preventing unwanted Fc signaling, even during extended experiments. Directed and precise labeling with dyes allows quantification of the bound target molecules. n/a Technological Innovations in Immunology (TECH)

Efficient discovery of an agonistic anti-OX40 nanobody by epitope-directed approach to address enrichment-driven epitope bias

Background Conventional antibody discovery approaches that do not account for enrichment-driven biases, such as epitope immunogenicity, PCR amplification bias, or protein expression efficiency, may result in under-representation of rare yet functionally relevant clones, necessitating labor-intensive in vitro screening to identify agonistic antibodies among a large number of dominant clones. Thus, efficient screening methods for agonistic antibodies are urgently needed. OX40 is a promising target for cancer immunotherapy due to its role in enhancing T-cell activation and survival. However, effective anti-OX40 agonistic antibodies have not yet been developed. Methods We developed a novel screening strategy that involves the selection of nanobody clone pools enriched by biopanning against gp34-engaged and non-engaged OX40-expressing cells, next-generation sequencing, and computational clustering and subtraction analysis to identify clones recognizing the ligand-receptor interface. Representative nanobody clones underwent in vitro validation, including epitope mapping, binding affinity measurements, and functional assessments. Furthermore, we engineered the selected nanobody to enhance its in vivo efficacy. We also performed structural analysis of the nanobody-OX40 complex. Results Our epitope-directed approach efficiently identified nanobody clones recognizing functionally relevant epitopes distinct from dominant immunogenic regions. Notably, clone Nb479 demonstrated robust agonistic activity, closely mimicking the natural ligand gp34 with extensive OX40-binding interactions. Trimerization of Nb479 facilitated potent OX40 activation without the need for a cross-linking scaffold. Conjugation of the Nb479 trimer with an anti-serum albumin nanobody exhibited significantly improved pharmacokinetics in vivo and enhanced antitumor activity in a mouse model treated with CD19 chimeric antigen receptor T cells. Conclusion This study presents an innovative epitope-directed approach that greatly accelerates the discovery of functionally potent agonistic nanobodies by effectively circumventing enrichment-driven epitope bias. Our approach and engineered multivalent anti-OX40 nanobody offer a powerful platform to advance immunotherapeutic strategies for cancer treatment.

Digital Nanopore Counting Enabling Direct Identification of Trace-Level Neoantigens in Native Biological Samples

Tumor-specific neoantigens have been the most promising targets for TCR-based cancer therapies; however, their detection is greatly hindered by low abundance, sequence variability, and potential modifications. To address these, we developed a digital nanopore framework to unambiguously identify and effectively quantify candidate neoantigens directly in cell samples via event-resolved molecular identification and counting. By engineering a multirecognition-region nanopore and developing a universal water-osmosis driving force, we simultaneously enhanced the detection throughput and specificity. Combined with enriched current signatures and a gradient-boosting decision-tree classifier, the platform identifies individual ovalbumin-derived model neoantigens with single-amino-acid resolution from digitally assigned events in both exogenously incubated and endogenously expressed cell samples. We show that the limit of quantification is improved to the fmol level in native biological environments, enabling the direct identification of neoantigens from as few as 5000 cells within 30 min. We further demonstrate the identification of a native Epstein–Barr virus-associated neoantigen, highlighting the potential of this approach in ultrasensitive analysis of rare biomolecular species directly in biological samples.

Intracellular screening of nanobodies reveals an intrabody that inhibits ITCH E3 ubiquitin ligase

Nanobodies are a class of small, monomeric camelid antibody fragments that can bind target antigens with high affinity and specificity. Their small size, structural simplicity, and limited reliance on disulfide bonding makes them attractive for intracellular expression for labeling and perturbing cellular processes in live cells. However, screening campaigns carried out exclusively in vitro often yield antigen binders that fail to perform well in live cells due to low expression, misfolding, or mistargeting. We demonstrate that traditional in vitro screening of a nanobody library combined with an intracellular bioluminescence resonant energy transfer (BRET) proximity sensor approach for sequence down-selection can yield strong in vitro binders that also perform well as intrabodies, in this case capable of binding to, and inhibiting the enzymatic activity of, ITCH E3 ubiquitin ligase in human cells. This strategy allows a more direct and scalable path toward intrabody discovery.

Development and application of nbLIBRA-seq for high-throughput discovery of antigen-specific nanobodies.

Nanobodies are of high interest in many fields of medicine and biotechnology, although nanobody discovery has been limited by laborious screening techniques. Here, we demonstrate the successful adaptation of linking B cell receptor to antigen specificity through sequencing (LIBRA-seq) to immunized alpacas for the rapid identification of antigen-specific nanobodies, derived from heavy-chain antibodies. We validated LIBRA-seq for nanobody discovery (nbLIBRA-seq) in two different disease settings. First, we identified over 300 antigen-specific heavy-chain antibodies against human transferrin receptor (TfR1) from a single alpaca blood sample. Second, we tested the efficiency of nbLIBRA-seq with multiple antigens in the screening library. Using fusion glycoproteins from the related respiratory syncytial virus (RSV) and human metapneumovirus (hMPV), 1,125 antigen-specific heavy-chain-expressing B cells were recovered. Our results illustrate the potential of nbLIBRA-seq to rapidly identify antigen-specific heavy-chain antibodies for a range of diverse targets, a capability that will be critical for the efficient development of nanobody-based therapeutics.