Synthetic Nanobodies Research Articles

Nanobody engineering: computational modelling and design for biomedical and therapeutic applications.

Nanobodies, the smallest functional antibody fragment derived from camelid heavy-chain-only antibodies, have emerged as powerful tools for diverse biomedical applications. In this comprehensive review, we discuss the structural characteristics, functional properties, and computational approaches driving the design and optimisation of synthetic nanobodies. We explore their unique antigen-binding domains, highlighting the critical role of complementarity-determining regions in target recognition and specificity. This review further underscores the advantages of nanobodies over conventional antibodies from a biosynthesis perspective, including their small size, stability, and solubility, which make them ideal candidates for economical antigen capture in diagnostics, therapeutics, and biosensing. We discuss the recent advancements in computational methods for nanobody modelling, epitope prediction, and affinity maturation, shedding light on their intricate antigen-binding mechanisms and conformational dynamics. Finally, we examine a direct example of how computational design strategies were implemented for improving a nanobody-based immunosensor, known as a Quenchbody. Through combining experimental findings and computational insights, this review elucidates the transformative impact of nanobodies in biotechnology and biomedical research, offering a roadmap for future advancements and applications in healthcare and diagnostics.

Quantitative Assessment of Energetic Contributions of Residues in a SARS-CoV-2 Viral Enzyme/Nanobody Interface.

The highly conserved protease enzyme from SARS-CoV-2 (MPro) is crucial for viral replication and is an attractive target for the design of novel inhibitory compounds. MPro is known to be conformationally flexible and has been stabilized in an extended conformation in a complex with a novel nanobody (NB2B4), which inhibits the dimerization of the enzyme via binding to an allosteric site. However, the energetic contributions of the nanobody residues stabilizing the MPro/nanobody interface remain unresolved. We probed these residues using all-atom MD simulations in combination with alchemical free energy calculations by studying the physical residue-residue interactions and discovered the role of hydrophobic and electrostatic interactions in stabilizing the complex. Specifically, we found via mutational analysis that three interfacial nanobody residues (Y59, R106, and L109) contributed significantly, two residues (L107 and P110) contributed moderately, and two residues (H112 and T113) contributed minimally to the overall binding affinity of the nanobody. We also discovered that the nanobody affinity could be enhanced via a charge-reversal mutation (D62R) that alters the local interfacial electrostatic environment of this residue in the complex. These findings are potentially useful in designing novel synthetic nanobodies as allosteric inhibitors of MPro.

Mechanism of glycoform specificity and in vivo protection by an anti-afucosylated IgG nanobody

Immunoglobulin G (IgG) antibodies contain a complex N-glycan embedded in the hydrophobic pocket between its heavy chain protomers. This glycan contributes to the structural organization of the Fc domain and determines its specificity for Fcγ receptors, thereby dictating distinct cellular responses. The variable construction of this glycan structure leads to highly-related, but non-equivalent glycoproteins known as glycoforms. We previously reported synthetic nanobodies that distinguish IgG glycoforms. Here, we present the structure of one such nanobody, X0, in complex with the Fc fragment of afucosylated IgG1. Upon binding, the elongated CDR3 loop of X0 undergoes a conformational shift to access the buried N-glycan and acts as a ‘glycan sensor’, forming hydrogen bonds with the afucosylated IgG N-glycan that would otherwise be sterically hindered by the presence of a core fucose residue. Based on this structure, we designed X0 fusion constructs that disrupt pathogenic afucosylated IgG1-FcγRIIIa interactions and rescue mice in a model of dengue virus infection.

In vitro and ex vivo proteomics of Mycobacterium marinum biofilms and the development of biofilm-binding synthetic nanobodies.

The antibiotic-tolerant biofilms present in tuberculous granulomas add an additional layer of complexity when treating mycobacterial infections, including tuberculosis (TB). For a more efficient treatment of TB, the biofilm forms of mycobacteria warrant specific attention. Here, we used Mycobacterium marinum (Mmr) as a biofilm-forming model to identify the abundant proteins covering the biofilm surface. We used biotinylation/streptavidin-based proteomics on the proteins exposed at the Mmr biofilm matrices in vitro to identify 448 proteins and ex vivo proteomics to detect 91 Mmr proteins from the mycobacterial granulomas isolated from adult zebrafish. In vitro and ex vivo proteomics data are available via ProteomeXchange with identifiers PXD033425 and PXD039416, respectively. Data comparisons pinpointed the molecular chaperone GroEL2 as the most abundant Mmr protein within the in vitro and ex vivo proteomes, while its paralog, GroEL1, with a known role in biofilm formation, was detected with slightly lower intensity values. To validate the surface exposure of these targets, we created in-house synthetic nanobodies (sybodies) against the two chaperones and identified sybodies that bind the mycobacterial biofilms in vitro and those present in ex vivo granulomas. Taken together, the present study reports a proof-of-concept showing that surface proteomics in vitro and ex vivo proteomics combined is a valuable strategy to identify surface-exposed proteins on the mycobacterial biofilm. Biofilm surface-binding nanobodies could be eventually used as homing agents to deliver biofilm-targeting treatments to the sites of persistent biofilm infection. IMPORTANCE With the currently available antibiotics, the treatment of TB takes months. The slow response to treatment is caused by antibiotic tolerance, which is especially common among bacteria that form biofilms. Such biofilms are composed of bacterial cells surrounded by the extracellular matrix. Both the matrix and the dormant lifestyle of the bacterial cells are thought to hinder the efficacy of antibiotics. To be able to develop faster-acting treatments against TB, the biofilm forms of mycobacteria deserve specific attention. In this work, we characterize the protein composition of Mmr biofilms in bacterial cultures and in mycobacteria extracted from infected adult zebrafish. We identify abundant surface-exposed targets and develop the first sybodies that bind to mycobacterial biofilms. As nanobodies can be linked to other therapeutic compounds, in the future, they can provide means to target therapies to biofilms.

Design and Characterization of Bivalent Molecules Against SARS-CoV2

Abstract The pandemic of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has compelled extraordinary attention to the development of therapeutic reagents. The favorable physical properties of nanobodies make them attractive candidates for investigations of their ability to block viral entry. By X-ray crystallography we determined the structural basis of the interaction of a panel of synthetic nanobodies (Sb14, Sb16, Sb45 and Sb68) with the SARS-CoV-2 receptor binding domain (RBD): binary complexes of Sb16–RBD and Sb45–RBD; a ternary complex of Sb14–RBD–Sb68, and Sb45–RBD–Sb68. Sb16 and Sb45 bind the RBD with identical footprints with ACE2, but their complementarity determining regions (CDR)2 and CDR3 are oriented diametrically oppositely. The ternary structure of the Sb45-RBD-Sb68 complex indicates that Sb45 binds squarely at the ACE2 interface, whereas Sb68 attaches toward the ACE2 interface's perimeter. The ternary configuration suggests that several sybodies may capture a sizable portion of the RBD's surface. These sybody structures provided structural insights that were used to create novel, highly potent biparatopic nanobodies and bivalent molecules with ACE2 and ACE2 binding scaffold mimicking miniproteins that bind the RBD with high affinity and neutralize SARS-CoV-2 and variants. Supported by the Intramural research program of the NIAID/NIH

Structures of ferroportin in complex with its specific inhibitor vamifeport.

A central regulatory mechanism of iron homeostasis in humans involves ferroportin (FPN), the sole cellular iron exporter, and the peptide hormone hepcidin, which inhibits Fe2+ transport and induces internalization and degradation of FPN. Dysregulation of the FPN/hepcidin axis leads to diverse pathological conditions, and consequently, pharmacological compounds that inhibit FPN-mediated iron transport are of high clinical interest. Here, we describe the cryo-electron microscopy structures of human FPN in complex with synthetic nanobodies and vamifeport (VIT-2763), the first clinical-stage oral FPN inhibitor. Vamifeport competes with hepcidin for FPN binding and is currently in clinical development for β-thalassemia and sickle cell disease. The structures display two distinct conformations of FPN, representing outward-facing and occluded states of the transporter. The vamifeport site is located in the center of the protein, where the overlap with hepcidin interactions underlies the competitive relationship between the two molecules. The introduction of point mutations in the binding pocket of vamifeport reduces its affinity to FPN, emphasizing the relevance of the structural data. Together, our study reveals conformational rearrangements of FPN that are of potential relevance for transport, and it provides initial insight into the pharmacological targeting of this unique iron efflux transporter.

Application Progress of the Single Domain Antibody in Medicine.

The camelid-derived single chain antibody (sdAb), also termed VHH or nanobody, is a unique, functional heavy (H)-chain antibody (HCAb). In contrast to conventional antibodies, sdAb is a unique antibody fragment consisting of a heavy-chain variable domain. It lacks light chains and a first constant domain (CH1). With a small molecular weight of only 12~15 kDa, sdAb has a similar antigen-binding affinity to conventional Abs but a higher solubility, which exerts unique advantages for the recognition and binding of functional, versatile, target-specific antigen fragments. In recent decades, with their unique structural and functional features, nanobodies have been considered promising agents and alternatives to traditional monoclonal antibodies. As a new generation of nano-biological tools, natural and synthetic nanobodies have been used in many fields of biomedicine, including biomolecular materials, biological research, medical diagnosis and immune therapies. This article briefly overviews the biomolecular structure, biochemical properties, immune acquisition and phage library construction of nanobodies and comprehensively reviews their applications in medical research. It is expected that this review will provide a reference for the further exploration and unveiling of nanobody properties and function, as well as a bright future for the development of drugs and therapeutic methods based on nanobodies.

Heteromeric LRRC8 channel assemblies and their impact on channel activity.

Volume regulated anion channels (VRAC) are activated in response to the osmotic swelling of vertebrate cells to catalyze the efflux of anions and small osmolytes. VRACs are multimeric channels that are composed of leucine-rich repeat-containing 8 (LRRC8) proteins, a protein family encompassing five paralogues in mammals (LRRC8A-E). Endogenous VRACs are known to form heteromeric assemblies of LRRC8A with at least one further LRRC8 isoform. Until recently, only structures of homomeric LRRC8 channels were described, while the structures of heteromeric channels remained elusive. Combining results from targeted quantification mass spectrometry and cryoEM with proteins obtained from heterologous overexpression, we investigated the subunit stoichiometry and structure of heteromeric LRRC8A/C channels. For these proteins, we observed a ratio of LRRC8A:C subunits of 2:1, which was confirmed in cryo-EM structures where LRRC8A and LRRC8C subunits cluster as pairs in a hexameric protein assembly. This subunit arrangement increases the pore size in LRRC8A/C channels compared to homomeric LRRC8A. Moreover, we found a tight interaction between LRR domains of LRRC8A subunit pairs of heteromers, reflecting a similar arrangement observed in homomeric LRRC8A. Conversely, the same parts of LRRC8C subunits were not resolved, indicating increased flexibility of their cytosolic regions. The structure thus suggests that the LRRC8C subunits perturb the tight packing of the LRRC8A subunits in heteromeric channels. Similar destabilizing effects were previously observed in LRRC8A homomers binding synthetic nanobodies with activating phenotype. There the binders induced a destabilization of the LRR domain leading to an increase of the open probability of homomeric channels. Collectively, our results suggest that the incorporation of LRRC8C in heteromeric LRRC8A/C channels destabilizes the tight packing of LRRC8A subunits to improve the activation properties of the channels.

Binding of synthetic nanobodies to the SARS-CoV-2 receptor-binding domain: the importance of salt bridges.

In this study, five different SARS-CoV-2 receptor-binding domain (RBD) models were created based on the crystal structures of RBD complexes with two synthetic nanobodies (Sb16 and Sb45). Microsecond all-atom MD simulations revealed that Sb16 and Sb45 substantially stabilized the flexible RBD loop (residues GLU471-SER494) due to the salt bridges and hydrogen bonding interactions between RBD and the synthetic nanobodies. However, the calculation of binding free energy displayed that Sb45 had a higher binding affinity to RBD than Sb16, in agreement with the experimental result. This is because Sb45 has stronger electrostatic attraction to RBD as compared to Sb16. In particular, the salt bridge GLU484-ARG33 in Sb45-RBD is stronger than the GLU484-LYS32 in Sb16-RBD. Furthermore, by comparing the binding affinity of Sb16 for two RBD mutants (E484K and K417N), we found that E484K mutation substantially reduced the binding affinity to Sb16, and K417N mutation had no significant effect, qualitatively in agreement with experimental studies. According to the binding free energy calculation, the strong electrostatic repulsion between LYS32 and LYS484 caused by E484K mutation destroys the salt bridge between LYS32 and GLU484 in the RBD wild type (WT). In contrast, the binding of the K417N mutant to Sb16 effectively maintains the salt bridge between LYS32 and GLU484. Therefore, our research suggests that the salt bridges between RBD and synthetic nanobodies are crucial for binding synthetic nanobodies to RBD, and a SARS-CoV-2 variant can escape neutralization from nanobodies by creating electrostatic repulsion between them.

A Cell-Permeant Nanobody-Based Degrader That Induces Fetal Hemoglobin.

Proximity-based strategies to degrade proteins have enormous therapeutic potential in medicine, but the technologies are limited to proteins for which small molecule ligands exist. The identification of such ligands for therapeutically relevant but "undruggable" proteins remains challenging. Herein, we employed yeast surface display of synthetic nanobodies to identify a protein ligand selective for BCL11A, a critical repressor of fetal globin gene transcription. Fusion of the nanobody to a cell-permeant miniature protein and an E3 adaptor creates a degrader that depletes cellular BCL11A in differentiated primary erythroid precursor cells, thereby inducing the expression of fetal hemoglobin, a modifier of clinical severity of sickle cell disease and β-thalassemia. Our strategy provides a means of fetal hemoglobin induction through reversible, temporal modulation of BCL11A. Additionally, it establishes a new paradigm for the targeted degradation of previously intractable proteins.

Synthetic nanobodies as tools to distinguish IgG Fc glycoforms

Protein glycosylation is a crucial mediator of biological functions and is tightly regulated in health and disease. However, interrogating complex protein glycoforms is challenging, as current lectin tools are limited by cross-reactivity while mass spectrometry typically requires biochemical purification and isolation of the target protein. Here, we describe a method to identify and characterize a class of nanobodies that can distinguish glycoforms without reactivity to off-target glycoproteins or glycans. We apply this technology to immunoglobulin G (IgG) Fc glycoforms and define nanobodies that specifically recognize either IgG lacking its core-fucose or IgG bearing terminal sialic acid residues. By adapting these tools to standard biochemical methods, we can clinically stratify dengue virus and SARS-CoV-2 infected individuals based on their IgG glycan profile, selectively disrupt IgG-Fcγ receptor binding both in vitro and in vivo, and interrogate the B cell receptor (BCR) glycan structure on living cells. Ultimately, we provide a strategy for the development of reagents to identify and manipulate IgG Fc glycoforms.

Studying the characteristics of nanobody CDR regions based on sequence analysis in combination with 3D structures

BackgroundSingle-domain antibodies or nanobodies have recently attracted much attention in research and applications because of their great potential and advantage over conventional antibodies. However, isolation of candidate nanobodies in the lab has been costly and time-consuming. Screening of leading nanobody candidates through synthetic libraries is a promising alternative, but it requires prior knowledge to control the diversity of the complementarity-determining regions (CDRs) while still maintaining functionality. In this work, we identified sequence characteristics that could contribute to nanobody functionality by analyzing three datasets, CDR1, CDR2, and CDR3. ResultsBy classification of amino acids based on physicochemical properties, we found that two different amino acid groups were sufficient for CDRs. The nonpolar group accounted for half of the total amino acid composition in these sequences. Observation of the highest occurrence of each amino acid revealed that the usage of some important amino acids such as tyrosine and serine was highly correlated with the length of the CDR3. Amino acid repeat motifs were also under-represented and highly restricted as 3-mers. Inspecting the crystallographic data also demonstrated conservation in structural coordinates of dominant amino acids such as methionine, isoleucine, valine, threonine, and tyrosine and certain positions in the CDR1, CDR2, and CDR3 sequences. ConclusionsWe identified sequence characteristics that contributed to functional nanobodies including amino acid groups, the occurrence of each kind of amino acids, and repeat patterns. These results provide a simple set of rules to make it easier to generate desired candidates by computational means; also, they can be used as a reference to evaluate synthetic nanobodies.

α-SAS: an integrative approach for structural modeling of biological macromolecules in solution.

Modern small-angle scattering (SAS) experiments with neutrons (SANS) or X-rays (SAXS) combined with contrast variation provide comprehensive information about the structure of large multicomponent macromolecules in solution and allow the size, shape and relative arrangement of each component to be mapped out. To obtain such information, it is essential to perform well designed experiments, in which all necessary steps, from assessing sample suitability to structure modeling, are properly executed. This paper describes α-SAS, an integrative approach that is useful for effectively planning a biological contrast-variation SAS experiment. The accurate generation of expected experimental intensities using α-SAS allows the substantial acceleratation of research into the structure and function of biomacromolecules by minimizing the time and costs associated with performing a SAS experiment. The method is validated using a few basic structures with known analytical expressions for scattering intensity and using experimental SAXS data from Arabidopsis β-amylase 1 protein and SANS data from the histidine kinase-Sda complex and from human dystrophin without and with a membrane-mimicking nanodisk. Simulation of a SANS contrast-variation experiment is performed for synthetic nanobodies that effectively neutralize SARS-CoV-2.

Combining of synthetic VHH and immune scFv libraries for pregnancy-associated glycoproteins ELISA development.

Nanobodies (VHH) from camelid antibody libraries hold great promise as therapeutic agents and components of immunoassay systems. Synthetic antibody libraries that could be designed and generated once and for various applications could yield binders to virtually any targets, even for non-immunogenic or toxic ones, in a short term. One of the most difficult tasks is to obtain antibodies with a high affinity and specificity to polyglycosylated proteins. It requires antibody libraries with extremely high functional diversity and the use of sophisticated selection techniques. Here we report a development of a novel sandwich immunoassay involving a combination of the synthetic library-derived VHH-Fc fusion protein as a capture antibody and the immune single-chain fragment variable (scFv) as a tracer for the detection of pregnancy-associated glycoprotein (PAG) of cattle (Bos taurus). We succeeded in the generation of a number of specific scFv antibodies against PAG from the mouse immune library. Subsequent selection using the immobilized scFv-Fc capture antibody allowed to isolate 1.9 nM VHH binder from the diverse synthetic library without any overlapping with the capture antibody binding site. The prototype sandwich ELISA based on the synthetic VHH and the immune scFv was established. This is the first successful example of the combination of synthetic and immune antibody libraries in a single sandwich immunoassay. Thus, our approach could be used for the express isolation of antibody pairs and the development of sandwich immunoassays for challenging antigens. KEY POINTS: • Heavily glycosylated PAG Bos Taurus were used for immune library construction and specific scFv isolation by phage display. • Nanomolar affinity VHH for PAG was selected from the original synthetic nanobodies library. • A novel VHH/scFv-based immunoassay for Bos Taurus pregnancy determination was developed.

Mechanistic basis of choline import involved in teichoic acids and lipopolysaccharide modification.

Phosphocholine molecules decorating bacterial cell wall teichoic acids and outer-membrane lipopolysaccharide have fundamental roles in adhesion to host cells, immune evasion, and persistence. Bacteria carrying the operon that performs phosphocholine decoration synthesize phosphocholine after uptake of the choline precursor by LicB, a conserved transporter among divergent species. Streptococcus pneumoniae is a prominent pathogen where phosphocholine decoration plays a fundamental role in virulence. Here, we present cryo–electron microscopy and crystal structures of S. pneumoniae LicB, revealing distinct conformational states and describing architectural and mechanistic elements essential to choline import. Together with in vitro and in vivo functional characterization, we found that LicB displays proton-coupled import activity and promiscuous selectivity involved in adaptation to choline deprivation conditions, and describe LicB inhibition by synthetic nanobodies (sybodies). Our results provide previously unknown insights into the molecular mechanism of a key transporter involved in bacterial pathogenesis and establish a basis for inhibition of the phosphocholine modification pathway across bacterial phyla.

A Novel Potent Carrier for Unconventional Protein Export in Ustilago maydis.

Recombinant proteins are ubiquitously applied in fields like research, pharma, diagnostics or the chemical industry. To provide the full range of useful proteins, novel expression hosts need to be established for proteins that are not sufficiently produced by the standard platform organisms. Unconventional secretion in the fungal model Ustilago maydis is an attractive novel option for export of heterologous proteins without N-glycosylation using chitinase Cts1 as a carrier. Recently, a novel factor essential for unconventional Cts1 secretion termed Jps1 was identified. Here, we show that Jps1 is unconventionally secreted using a fusion to bacterial β-glucuronidase as an established reporter. Interestingly, the experiment also demonstrates that the protein functions as an alternative carrier for heterologous proteins, showing about 2-fold higher reporter activity than the Cts1 fusion in the supernatant. In addition, Jps1-mediated secretion even allowed for efficient export of functional firefly luciferase as a novel secretion target which could not be achieved with Cts1. As an application for a relevant pharmaceutical target, export of functional bi-specific synthetic nanobodies directed against the SARS-CoV2 spike protein was demonstrated. The establishment of an alternative efficient carrier thus constitutes an excellent expansion of the existing secretion platform.

CD72 Nanobody-Based CAR-T Cells Have Potent Anti-Tumor Efficacy in B Cell Malignancies

Background: Approximately 50% of pediatric B-ALL patients treated with clinically approved CD19-targeting CAR-T cells do not remain in remission one year after therapy. CD22-targeting CAR-T cells appear to be curative in only a small fraction of CD19-refractory patients and this therapeutic strategy is primarily used as a bridge to stem cell transplant. Additional immunotherapeutic targets thus remain urgently needed. Our laboratory recently used cell surface proteomics to identify CD72 as a B-cell specific marker especially upregulated on poor prognosis, KMT2A/MLL-rearranged B-ALL (Nix et al., Cancer Discovery (2021)). In this published work, we used a best-in-class nanobody library displayed on yeast to develop binders to CD72. We demonstrated for the first time that fully synthetic nanobodies can generate CAR-T cells that are highly potent in vitro and in vivo. While we previously focused on these “nanoCARs” in KMT2A/MLLr B-ALL, in this follow-up study we aimed to 1) further expand our nanoCAR indications to other CD72-expressing B-cell malignancies; 2) biophysically characterize our synthetic nanobodies; 3) evaluate the potential for further humanization of the nanobody binder amino acid sequence while retaining anti-tumor efficacy; and 4) characterize the potency and T-cell immunophenotypes in the context of our lead nanobody binder (“NbD4”) placed on different CAR backbones.Methods: Flow cytometry of primary patient samples for CD72 was performed in a CLIA-certified laboratory. NbD4 nanobody was recombinantly expressed in E. coli and biolayer interferometry was used to determine the binding affinity to recombinantly-expressed CD72 extracellular domain. CAR-T cells were generated from peripheral blood donor CD4+ and CD8+ cells (1:1) ratio via lentiviral transduction. In vitro cytotoxicity assays were performed at a range of effector:tumor ratios. In vivo studies were performed in human cell line orthotopic xenografts in NSG mice. 1e6 luciferase-labeled Jeko cells were implanted at Day 0 followed by administration of 4e6 CAR-T cells at Day 6. Tumor burden was assessed by bioluminescence.Results: Flow cytometry on primary non-Hodgkin B-cell lymphoma obtained from fine needle aspiration biopsy (n = 5) confirmed CD72 surface expression (not shown), consistent with RNA-seq across larger cohorts. Biolayer interferometry demonstrated that NbD4 bound with surprisingly low affinity to recombinant CD72 (K D ~800 nM) (Fig. 1A), with both slow on rate (k on 8.38e4 M -1s -1) and rapid off rate (k off 6.82e-2 s -1). This affinity stands in contrast to that reported for FMC63 single chain variable fragment (scFv) used in clinically approved CD19-targeting CAR-T cells (K D 0.3-5 nM), despite similar in vitro and in vivo efficacy of both products. Our NbD4 framework region shows ~82% homology to a human IgG variable heavy domain, significantly higher than FMC63 (~59% homology). We made additional substitutions in the framework domain to increase human homology up to 89%. In vitro cytotoxicity assays with SEM B-ALL cells showed several humanized variants with similar efficacy to NbD4 (Fig. 1B). We further evaluated the impact of placing NbD4 on different CAR backbones, including combinations of CD28 or 4-1BB costimulatory domains and CD8 or IgG4-based transmembrane and hinge regions (Fig. 1C). In vivo, CD72 nanoCARs with Backbone 3 showed significantly increased potency (Fig. 1D). Indeed, tumors treated with Backbone 3 CAR-Ts showed complete tumor clearance and did not develop new tumors even after re-challenge with 1e6 Jeko cells at Day 52 (Fig. 1D). Preliminary characterization of effector and memory CAR-T cell phenotypes before exposure to tumor suggested that Backbone 3 had an increased number of naïve T cells compared to empty CAR and CD19 CAR-T cells (data not shown).Conclusions: Our results demonstrate that our fully synthetic CD72 nanoCARs can effectively eliminate CD72-expressing B-cell malignancy models despite low nanobody binding affinity. Humanized NbD4 variants may serve as clinical candidates with even further reduction in possible immunogenicity of the llama amino acid framework. Alterations to the CAR backbone have a major impact on anti-tumor efficacy and phenotypes of our synthetic nanobodies. CD72-targeting therapies may be effective therapeutics not only KMT2A/MLLr B-ALL but also across a broader spectrum of refractory B-cell malignancies. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

A cell-free nanobody engineering platform rapidly generates SARS-CoV-2 neutralizing nanobodies

Antibody engineering technologies face increasing demands for speed, reliability and scale. We develop CeVICA, a cell-free nanobody engineering platform that uses ribosome display for in vitro selection of nanobodies from a library of 1011 randomized sequences. We apply CeVICA to engineer nanobodies against the Receptor Binding Domain (RBD) of SARS-CoV-2 spike protein and identify >800 binder families using a computational pipeline based on CDR-directed clustering. Among 38 experimentally-tested families, 30 are true RBD binders and 11 inhibit SARS-CoV-2 pseudotyped virus infection. Affinity maturation and multivalency engineering increase nanobody binding affinity and yield a virus neutralizer with picomolar IC50. Furthermore, the capability of CeVICA for comprehensive binder prediction allows us to validate the fitness of our nanobody library. CeVICA offers an integrated solution for rapid generation of divergent synthetic nanobodies with tunable affinities in vitro and may serve as the basis for automated and highly parallel nanobody engineering.

Structures of synthetic nanobody–SARS-CoV-2 receptor-binding domain complexes reveal distinct sites of interaction

Combating the worldwide spread of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and the emergence of new variants demands understanding of the structural basis of the interaction of antibodies with the SARS-CoV-2 receptor-binding domain (RBD). Here, we report five X-ray crystal structures of sybodies (synthetic nanobodies) including those of binary and ternary complexes of Sb16–RBD, Sb45–RBD, Sb14–RBD–Sb68, and Sb45–RBD–Sb68, as well as unliganded Sb16. These structures reveal that Sb14, Sb16, and Sb45 bind the RBD at the angiotensin-converting enzyme 2 interface and that the Sb16 interaction is accompanied by a large conformational adjustment of complementarity-determining region 2. In contrast, Sb68 interacts at the periphery of the SARS-CoV-2 RBD–angiotensin-converting enzyme 2 interface. We also determined cryo-EM structures of Sb45 bound to the SARS-CoV-2 spike protein. Superposition of the X-ray structures of sybodies onto the trimeric spike protein cryo-EM map indicates that some sybodies may bind in both “up” and “down” configurations, but others may not. Differences in sybody recognition of several recently identified RBD variants are explained by these structures.

Allosteric modulation of LRRC8 channels by targeting their cytoplasmic domains

Members of the LRRC8 family form heteromeric assemblies, which function as volume-regulated anion channels. These modular proteins consist of a transmembrane pore and cytoplasmic leucine-rich repeat (LRR) domains. Despite their known molecular architecture, the mechanism of activation and the role of the LRR domains in this process has remained elusive. Here we address this question by generating synthetic nanobodies, termed sybodies, which target the LRR domain of the obligatory subunit LRRC8A. We use these binders to investigate their interaction with homomeric LRRC8A channels by cryo-electron microscopy and the consequent effect on channel activation by electrophysiology. The five identified sybodies either inhibit or enhance activity by binding to distinct epitopes of the LRR domain, thereby altering channel conformations. In combination, our work provides a set of specific modulators of LRRC8 proteins and reveals the role of their cytoplasmic domains as regulators of channel activity by allosteric mechanisms.