Picomolar Affinity Research Articles

Sema3A Antibody BI-X Prevents Cell Permeability and Cytoskeletal Collapse in HRMECs and Increases Tip Cell Density in Mouse Oxygen-Induced Retinopathy.

PurposeSemaphorin 3A (Sema3A) is an axonal guidance molecule that inhibits angiogenesis by vasorepulsion and blocks revascularization in the ischemic retina. BI-X is an intravitreal anti-Sema3A agent under clinical investigation in patients with proliferative diabetic retinopathy (PDR) and diabetic macular ischemia (DMI).MethodsSurface plasmon resonance was used to determine binding affinity of BI-X to human and murine Sema3A. In vitro, human retinal microvascular endothelial cells (HRMECs) were used to assess effects of BI-X on cell permeability and cytoskeletal collapse induced by Sema3A. In vivo, intravitreal BI-X or an anti-trinitrophenol control antibody was administered in both eyes in mice with oxygen-induced retinopathy (OIR). Retinal flat mounts were prepared, and avascular area and tip cell density were determined using confocal laser-scanning microscopy.ResultsDissociation constants for BI-X binding to human and murine Sema3A were 29 pM and 27 pM, respectively. In vitro, BI-X prevented HRMEC permeability and cytoskeletal collapse induced by Sema3A. In vivo, BI-X increased tip cell density by 33% (P < 0.001) and reduced avascular area by 12% (not significant). A significant negative correlation was evident between avascular area and tip cell density (r2 = 0.4205, P < 0.0001).ConclusionsBI-X binds to human Sema3A with picomolar affinity and prevents cell permeability and cytoskeletal collapse in HRMECs. BI-X also enhances revascularization in mice with OIR.Translational RelevanceBI-X is a potent inhibitor of human Sema3A that improves revascularization in a murine model of OIR; BI-X is currently being investigated in patients with laser-treated PDR and DMI.

Aptamer-immobilized bone-targeting nanoparticles in situ reduce sclerostin for osteoporosis treatment

Bone-targeted drug delivery to reduce systemic complications and maintain sufficient doses in bone tissues is the main challenge for osteoporosis treatment. Here, we develop a novel, and simply synthesized bone-targeting nanomedicine (DNA-MSN, DNAM) containing a PEGylated dendritic mesoporous silica nanoparticle (MSN) core (~ 65 nm) and an anti-sclerostin aptamer (Aptscl56) layer, to treat osteoporosis. The nanoparticle core protects the immobilized Aptscl56 from rapid nuclease degradation and renal filtration, prolonging in vivo half-lives. The DNAM-immobilized Aptscl56 layer exhibits dual functions to direct bone-attachment of ovariectomized mice, due to the interaction between phosphate groups in DNA aptamer Aptscl56 and bone calcium in hydroxyapatites, and to in situ capture sclerostin with picomolar affinities. Moreover, we show DNAM significantly reverses the serum level of sclerostin and osteoporotic bone loss to a normal level, improving bone histomorphology parameters and mechanical properties in the femur, and recovering serum levels of bone turnover markers, without systemic toxicity. Notably, the therapeutic effect of DNAM is superior to the "gold standard" drug alendronate, and the systemic dose of DNAM-immobilized Aptscl56 is only 25% of free Aptscl56. The present study provides insights into the regulation of unwanted circulating biohazards and represents a promising approach to deliver nanomedicines to treat osteoporosis.

Abstract 317: Isolation of highly selective antibodies against claudin 18.2 for the treatment of solid tumors

Abstract Monoclonal antibodies (MAbs) are a well-established treatment approach in oncology and other diseases. Nevertheless, many multipass membrane proteins are largely inaccessible as antibody targets due to their poor expression, membrane-dependent structure, small extracellular regions, and high sequence conservation between humans and rodents. Integral Molecular’s MPS Antibody Discovery platform specifically addresses each of these challenges. A key enabling feature of MPS is the use of chickens as an evolutionarily divergent host species for immunization, allowing a more robust immune response for targets that are highly conserved in mammals. We will present on antibodies isolated against challenging membrane protein targets in oncology including Claudin 18.2 (CLDN18.2).CLDN18.2 is a transmembrane adhesion protein undetectable in most adult healthy tissues but highly expressed in gastric, pancreatic, esophageal, and lung cancers. Antibody discovery efforts against this validated target are challenging due to the abundant expression of the splice isoform CLDN18.1 differing by only 8 amino acids in the extracellular domain. As part of the MPS platform, we used virus-like particles (Lipoparticles) to immunize chickens with a high concentration of native CLDN18.2 protein and obtain high-titer immune responses. This enabled us to generate and isolate a large and diverse collection of MAbs (48 unique clones) and select candidates for optimization. We present a panel of three highly specific, humanized CLDN18.2 MAbs with picomolar affinities that are superior to the clinical-stage benchmark. We will show in-depth profiling data for the MAb panel that were used for lead selection and de-risking clinical development. These data include biosensor binding kinetics, amino-acid resolution epitope mapping, and specificity testing against the Membrane Proteome Array (MPA) consisting of 6,000 membrane proteins. This panel of preclinical antibodies are being developed for therapeutic use in various formats, including bispecifics, antibody-drug conjugates, and CAR-T applications. Citation Format: Brad Screnci, Trevor Barnes, Kristen Shema, Rebecca Rimkunas, Shruthi Kannan, Tim Phillips, Carmen Navia, Charles Azuelos, Tom Charpentier, Jennifer Houtmann, Lisa Miller, Lewis J. Stafford, Benjamin J. Doranz, Joseph B. Rucker, Ross Chambers. Isolation of highly selective antibodies against claudin 18.2 for the treatment of solid tumors [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 317.

Abstract 318: Atomic-level specificity of Claudin 6 monoclonal antibodies isolated for treating solid tumors

Abstract The tight junction protein Claudin 6 (CLDN6) is differentially expressed on cancer cells with almost no expression in healthy tissue, making it a valuable therapeutic target for many solid tumor cancers. Despite their potential as cancer therapeutics, very few CLDN6 monoclonal antibodies (MAbs) are in development, because MAbs with high affinity and specificity for CLDN6 are difficult to isolate. CLDN6 is structurally complex, with 4 transmembrane domains and 95% extracellular sequence conservation between human and mouse. Achieving MAb specificity for CLDN6 is especially challenging because its extracellular region strongly resembles those of 23 other human CLDN family members. In particular, the widely expressed CLDN9 differs from CLDN6 by only 3 extracellular residues. Using MAb discovery strategies specifically tailored to complex membrane proteins, including the use of virus-like particles (Lipoparticles), divergent species (chicken) immunization, and optimized phage display panning, we isolated 6 rare MAbs that recognize the native structure of CLDN6 with as low as picomolar affinity. The MAbs were screened against a Membrane Proteome Array containing ~6,000 membrane proteins and demonstrated specificity for CLDN6 with minimal cross-reactivity for CLDN9 or other CLDN family members. Epitope mapping using Shotgun Mutagenesis alanine scanning across the 220 residue CLDN6 sequence distinguished the binding sites of the MAbs from clinical-stage benchmarks. Atomic-level epitope mapping using comprehensive site-specific mutagenesis identified the γ carbon on CLDN6 residue Q156 as the critical structural mechanism enabling these MAbs to differentiate between CLDN6 and CLDN9 with high specificity. The CLDN6 MAbs identified here can be used to study CLDN6-positive cancers, including ovarian, endometrial, lung, and testicular cancer, and have the potential to be developed into highly selective therapeutics. Characterization of highly specific CLDN6 MAbs isolated for treatment of solid tumors MAb IM301 IM302 Benchmark (IMAB027, Astellas) VH CDR3 length (Kabat) 18 18 8 CLDN protein binding: Biosensor KD ± error, nM CLDN6 (target) 0.6 ± 0.03 &amp;lt; 0.001 0.5 ± 0.01 CLDN9 No binding No binding 3.6 ± .09 CLDN3 No binding No binding No binding CLDN4 No binding 146 ± 20 153 ± 6 Mouse CLDN6 binding Yes Yes Yes Cyno CLDN6 binding Yes Yes Yes Conformational epitope Yes Yes Yes Epitope topology Yes Yes Yes Critical CLDN6 epitope residues E48, E154, R158 E154, R158 F35, G37, S39 Citation Format: Brad Screnci, Lewis J. Stafford, Trevor Barnes, Kristen Shema, Samantha Gilman, Rebecca Rimkunas, Suzie Al Absi, Tim Phillips, Charles Azuelos, Katherine Slovik, Paige Muprhy, Daniel B. Harmon, Tom Charpentier, Benjamin J. Doranz, Joseph B. Rucker, Ross Chambers. Atomic-level specificity of Claudin 6 monoclonal antibodies isolated for treating solid tumors [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 318.

Abstract 3034: Purine nucleoside phosphorylase regulates metabolic and immune checkpoints

Abstract Purine nucleoside phosphorylase (PNP) functions as a central regulator of nucleotide metabolism by catalyzing the degradation of purine nucleosides guanosine and deoxyguanosine. PNP inactivation has been paradoxically associated with both immunodeficiency and autoimmunity in humans but the genetic and molecular determinants of these divergent outcomes are poorly defined and whether they reflect cell type specific mechanisms is currently unknown. Moreover, the mechanisms underlying the clinical efficacy of PNP inhibitors in a subset of patients with hematological malignancies are yet to be determined, thereby severely limiting the potential of this promising new class of metabolic targeted therapies. To address these gaps in knowledge we performed a systematic evaluation of the metabolic, immunological and cell-context phenotypic effects of targeting PNP using a clinical-stage inhibitor with picomolar affinity and excellent oral bioavailability. Here we show that across cancer types PNP inhibition is synthetically lethal with sterile alpha motif and HD domain-containing protein 1 (SAMHD1) deficiency by triggering massive dGTP pool expansion, and impairing DNA replication. These cytotoxic effects require the expression of deoxycytidine kinase (dCK) which mediates the intracellular trapping of deoxyguanosine and expansion of dGTP pools, and are opposed by environmental deoxycytidine produced by bone marrow stromal cells in a SAMHD1 and nucleoside transporter dependent manner. This resistance mechanism to PNP inhibition can be counteracted by the deoxycytidine catabolizing enzyme cytidine deaminase (CDA). We further show that in addition to, and independent of its dCK and CDA-dependent cytotoxic effects in SAMHD1 deficient cells, PNP inhibition elevates serum inflammatory cytokines and cytokine transcript levels in secondary lymphoid organs. Using scRNAseq we identify CD19+ B-cells and CSF1R+ macrophages, which express the pattern recognition receptor TLR7, as the drivers of these alterations. TLR7 activation in these cell types is promoted by guanosine accumulation which functions as a ligand for TLR7 alongside uridine-containing ssRNA. Importantly, TLR7 stimulation achieved by PNP inhibition initiates transcriptional alterations that are quantitatively and qualitatively unique from synthetic TLR7 agonists, such as the production of type I interferons. We additionally demonstrate that PNP inactivation stimulates germinal center responses in secondary lymphoid organs via the expansion of activated B-cells and T follicular helper cells. In summary, we demonstrate that by modulating multiple metabolic and immune checkpoints in a multigenic, cell type and environment dependent manner, pharmacological PNP inhibition elicits specific outcomes which can be leveraged for the development of novel therapeutic applications. Citation Format: Evan R. Abt, Khalid Rashid, Thuc M. Le, Hailey R. Lee, Amanda L. Creech, Ting-Ting Wu, Thomas Mehrling, Shanta Bantia, Caius G. Radu. Purine nucleoside phosphorylase regulates metabolic and immune checkpoints [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 3034.

Abstract 345: Development of a novel and direct peptide Helicon࣪ inhibitor of β-catenin-TCF interaction with in vivo validation of transcriptional modulation and anti-tumor activity

Abstract Wnt pathway mutations can be found in nearly all colorectal cancers and a significant number of cancers of the liver, breast, prostate, endometrium and lung, among others. β-catenin is a key signaling hub in the Wnt pathway. Activated nuclear β-catenin forms a complex with TCF/LEF (T-cell factor/lymphoid enhancer binding factor) and drives the transcription of genes essential for cancer cell proliferation, survival, and metabolism. Blocking the β-catenin-TCF/LEF interaction offers an attractive therapeutic strategy to treat a large population of patients with WNT pathway mutations. However, β-catenin is considered as an “undruggable” target because it lacks tractable hydrophobic pockets for small-molecule binding. To address this challenge, we have successfully discovered and developed Helicon࣪ peptides targeting the β-catenin-TCF/LEF interaction. Using this novel modality, linear peptides are locked in a helical structure via a proprietary tethering technology to yield macrocyclic stapled peptides. Our helicons exhibit picomolar β-catenin binding affinity and nanomolar anti-proliferative cell-based activity. Cells treated with lead helicons followed by unbiased RNAseq GSEA indicate that the WNT/β-catenin pathways represent the top downregulated transcriptional signatures after treatment. Furthermore, PRISM cell line screening of more than 900 cell lines reveals that the most sensitive lines are enriched with APC and CTNNB1 mutations. Mechanistically, helicon treatment reduces nuclear β-catenin and alters the levels of cyclin D2/D3 and p27 in the sensitive lines. In vivo, our helicons display favorable pharmacokinetic properties, broad tissue distribution, and potent anti-tumor effects. Taken together, our data demonstrate β-catenin-targeting Helicon࣪ peptides have the potential to become the next-generation class of therapeutics to treat cancers with defined genetic mutations. Citation Format: Yaguang Si, Brian White, Sarah Cappucci, Jessica Ramirez, Charles Ponthier, Erica Visness, Kevin Ling, Peicheng Du, Minjung Choi, Ty Thomson, Josue Alfaro-Lopez, Pieter Beerepoot, Sorabh Agarwal, Paula Ortet, Miroslaw Lech, Zhi Li, Voké Olokpa, Ivan Jewett, Daniel La, Lihua Yu, John McGee, Martin Tremblay, Jonathan Hurov, Greg Verdine. Development of a novel and direct peptide Helicon࣪ inhibitor of β-catenin-TCF interaction with in vivo validation of transcriptional modulation and anti-tumor activity [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 345.

Abstract 324: Discovery of novel functional TROP2 antibodies for treatment of epithelial cancers

Abstract Trophoblast cell surface antigen-2 (TROP2) is a membrane-bound protein expressed during development and at lower levels in normal adult tissues. TROP2 has pleiotropic functions including formation of cell-cell junctions by mediating interactions with extracellular and intracellular proteins. In situations of TROP2 deficiency its role in normal tissues appears to be compensated by expression of EpCAM, a highly related family member. In cancer, the levels of TROP2 are increased in many epithelial tumor types. Overexpression of TROP2 confer oncogenic behavior in several in vitro and in vivo models. Both its extracellular and intracellular domains play a role in modulating interactions with ligands and/or receptors and with intracellular signaling partners to result in an oncogenic phenotype. In many solid tumors TROP2 upregulation is associated with increased tumor aggressiveness, metastasis, and decreased survival in large groups of difficult-to-treat cancers, making it an attractive target for cancer therapy. TROP2-based ADCs have been developed and one has been approved for therapeutic use in triple negative breast cancer and advanced bladder cancer. The toxicity associated with the toxin payload limits their use including in combination with chemotherapy in the earlier lines of treatment, and by the development of resistance to the payload. We isolated several anti-TROP2 heavy chain-only antibodies (HCAb) by immunization of a proprietary transgenic HCAb mouse. In this abstract, we describe the properties and activity of several of these antibodies. These antibodies are characterized by potent in vivo antitumor activity in established tumor models in NCG mice in the absence of ADCC, and by the inhibition of tumor cell viability in vitro, directly and in the absence of any effector cells. In NCG mice, which are characterized by a short antibody half-life, anti-TROP2 HCAbs displayed a T1/2 of approximately 3.5 days. These antibodies displayed very potent anti-tumor activity in established tumor xenograft models, causing tumor regression at 2 mg/kg and 80% TGI at 0.5 mg/kg in NCG mice when administered once per week. They also caused tumor regression when dosed once every three weeks in NCG mice and in tumors of very large sizes. Therefore, these antibodies directly target one or more aspects of TROP2 tumor biology that have a profound and long-lasting effect on the cancer cells. Upon glycoengineering, the afucosylated version of the antibodies displayed a strongly enhanced ADCC activity. In vitro they are characterized by different internalization rates which define several non-overlapping epitopes on TROP2. They are characterized by picomolar binding affinity to recombinant human TROP2 as determined by SPR. These antibodies bind with greater affinity to human and cyno but bind poorly to mouse, rat and rabbit. These potent naked TROP2 antibodies represent new opportunities to address TROP2 expressing cancers. Citation Format: Israel Matos, Yahya Ashraf, Hiba Zahreddine, Liying Gong, Amanda Baumholtz, Alex Zhou, Claire Bonfils, Aniel Moya-Torres, Yun Cui, Xiaowei Wang, Richard Wargachuk, Tiffany Cheng, Elijus Undzys, Shugang Yao, Jacynthe Linda Tolouse, Dominic Hou, Gordon Ngan, Luis daCruz, David Young. Discovery of novel functional TROP2 antibodies for treatment of epithelial cancers [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 324.

Molecular determinants of inhibition of the human proton channel hHv1 by the designer peptide C6 and a bivalent derivative

The human voltage-gated proton channel (hHv1) is important for control of intracellular pH. We designed C6, a specific peptide inhibitor of hHv1, to evaluate the roles of the channel in sperm capacitation and in the inflammatory immune response of neutrophils [R. Zhao et al., Proc. Natl. Acad. Sci. U.S.A. 115, E11847–E11856 (2018)]. One C6 binds with nanomolar affinity to each of the two S3–S4 voltage-sensor loops in hHv1 in cooperative fashion so that C6-bound channels require greater depolarization to open and do so more slowly. As depolarization drives hHv1 sensors outwardly, C6 affinity decreases, and inhibition is partial. Here, we identified residues essential to C6–hHv1 binding by scanning mutagenesis, five in the hHv1 S3–S4 loops and seven on C6. A structural model of the C6–hHv1 complex was then generated by molecular dynamics simulations and validated by mutant-cycle analysis. Guided by this model, we created a bivalent C6 peptide (C62) that binds simultaneously to both hHv1 subunits and fully inhibits current with picomolar affinity. The results help delineate the structural basis for C6 state-dependent inhibition, support an anionic lipid-mediated binding mechanism, and offer molecular insight into the effectiveness of engineered C6 as a therapeutic agent or lead.

A Potent Neutralizing Nanobody Targeting the Spike Receptor-Binding Domain of SARS-CoV-2 and the Structural Basis of Its Intimate Binding.

The continuous spread of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) around the world has raised unprecedented challenges to the human society. Antibodies and nanobodies possessing neutralization activity represent promising drug candidates. In this study, we report the identification and characterization of a potent SARS-CoV-2 neutralizing nanobody that targets the viral spike receptor-binding domain (S-RBD). The nanobody, termed as Nb-007, engages SARS-CoV-2 S-RBD with the two-digit picomolar binding affinity and shows outstanding virus entry-inhibition activity. The complex structure of Nb-007 bound to SARS-CoV-2 S-RBD reveals an epitope that is partially overlapping with the binding site for the human receptor of angiotensin-converting enzyme 2 (ACE2). The nanobody therefore exerts neutralization by competing with ACE2 for S-RBD binding, which is further ascertained by our in-vitro biochemical analyses. Finally, we also show that Nb-007 reserves promising, though compromised, neutralization activity against the currently-circulating Delta variant and that fusion of the nanobody with Fc dramatically increases its entry-inhibition capacity. Taken together, these data have paved the way of developing Nb-007 as a drug-reserve for potential treatment of SARS-CoV-2 related diseases.

A Class of Shark-Derived Single-Domain Antibodies can Broadly Neutralize SARS-Related Coronaviruses and the Structural Basis of Neutralization and Omicron Escape.

The identification of a novel class of shark‐derived single domain antibodies, named vnarbodies that show picomolar affinities binding to the receptor binding domain (RBD) of Wuhan and Alpha, Beta, Kappa, Delta, Delta‐plus, and Lambda variants, is reported. Vnarbody 20G6 and 17F6 have broad neutralizing activities against all these SARS‐CoV‐2 viruses as well as other sarbecoviruses, including Pangolin coronavirus and Bat coronavirus. Intranasal administration of 20G6 effectively protects mice from the challenges of SARS‐CoV‐2 Wuhan and Beta variants. 20G6 and 17F6 contain a unique “WXGY” motif in the complementary determining region 3 that binds to a hidden epitope on RBD, which is highly conserved in sarbecoviruses through a novel β‐sheet interaction. It is found that the S375F mutation on Omicron RBD disrupts the structure of β‐strand, thus impair the binding with 20G6. The study demonstrates that shark‐derived vnarbodies offer a prophylactic and therapeutic option against most SARS‐CoV‐2 variants and provide insights into antibody evasion by the Omicron variant.

Lipid A Mimetics Based on Unnatural Disaccharide Scaffold as Potent TLR4 Agonists for Prospective Immunotherapeutics and Adjuvants.

TLR4 is a key pattern recognition receptor that can sense pathogen‐ and danger‐ associated molecular patterns to activate the downstream signaling pathways which results in the upregulation of transcription factors and expression of interferons and cytokines to mediate protective pro‐inflammatory responses involved in immune defense. Bacterial lipid A is the primary TLR4 ligand with very complex, species‐specific, and barely predictable structure‐activity relationships. Given that therapeutic targeting of TLR4 is an emerging tool for management of a variety of human diseases, the development of novel TLR4 activating biomolecules other than lipid A is of vast importance. We report on design, chemical synthesis and immunobiology of novel glycan‐based lipid A‐mimicking molecules that can activate human and murine TLR4‐mediated signaling with picomolar affinity. Exploiting crystal structure ‐ based design we have created novel disaccharide lipid A mimetics (DLAMs) where the inherently flexible β(1→6)‐linked diglucosamine backbone of lipid A is exchanged with a conformationally restrained non‐reducing βGlcN(1↔1′)βGlcN scaffold. Excellent stereoselectivity in a challenging β,β‐1,1′ glycosylation was achieved by tuning the reactivities of donor and acceptor molecules using protective group manipulation strategy. Divergent streamlined synthesis of β,β‐1,1′‐linked diglucosamine‐derived glycolipids entailing multiple long‐chain (R)‐3‐ acyloxyacyl residues and up two three phosphate groups was developed. Specific 3D‐molecular shape and conformational rigidity of unnatural β,β‐1,1′‐linked diglucosamine combined with carefully optimized phosphorylation and acylation pattern ensured efficient induction of the TLR4‐mediated signaling in a species‐independent manner.

Three SARS-CoV-2 antibodies provide broad and synergistic neutralization against variants of concern, including Omicron.

The rapidly spreading Omicron variant is highly resistant to vaccines, convalescent sera, and neutralizing antibodies (nAbs), highlighting the urgent need for potent therapeutic nAbs. Here, a panel of human nAbs from severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) convalescent patients show diverse neutralization against Omicron, of which XMA01 and XMA04 maintain nanomolar affinities and excellent neutralization (half maximal inhibitory concentration [IC50]: ∼20 ng/mL). nAb XMA09 shows weak but unattenuated neutralization against all variants of concern (VOCs) as well as SARS-CoV. Structural analysis reveals that the above three antibodies could synergistically bind to the receptor-binding domains (RBDs) of both wild-type and Omicron spikes and defines the critical determinants for nAb-mediated broad neutralizations. Three nAbs confer synergistic neutralization against Omicron, resulting from the inter-antibody interaction between XMA04 and XMA01(or XMA09). Furthermore, the XMA01/XMA04 cocktail provides synergistic protection against Beta and Omicron variant infections in hamsters. In summary, our results provide insights for the rational design of antibody cocktail therapeutics or universal vaccines against Omicron.

Nanobodies Protecting From Lethal SARS-CoV-2 Infection Target Receptor Binding Epitopes Preserved in Virus Variants Other Than Omicron.

The emergence of SARS-CoV-2 variants that escape from immune neutralization are challenging vaccines and antibodies developed to stop the COVID-19 pandemic. Thus, it is important to establish therapeutics directed toward multiple or specific SARS-CoV-2 variants. The envelope spike (S) glycoprotein of SARS-CoV-2 is the key target of neutralizing antibodies (Abs). We selected a panel of nine nanobodies (Nbs) from dromedary camels immunized with the receptor-binding domain (RBD) of the S, and engineered Nb fusions as humanized heavy chain Abs (hcAbs). Nbs and derived hcAbs bound with subnanomolar or picomolar affinities to the S and its RBD, and S-binding cross-competition clustered them in two different groups. Most of the hcAbs hindered RBD binding to its human ACE2 (hACE2) receptor, blocked cell entry of viruses pseudotyped with the S protein and neutralized SARS-CoV-2 infection in cell cultures. Four potent neutralizing hcAbs prevented the progression to lethal SARS-CoV-2 infection in hACE2-transgenic mice, demonstrating their therapeutic potential. Cryo-electron microscopy identified Nb binding epitopes in and out the receptor binding motif (RBM), and showed different ways to prevent virus binding to its cell entry receptor. The Nb binding modes were consistent with its recognition of SARS-CoV-2 RBD variants; mono and bispecific hcAbs efficiently bound all variants of concern except omicron, which emphasized the immune escape capacity of this latest variant.

Isolation, characterization, and structure-based engineering of a neutralizing nanobody against SARS-CoV-2

SARS-CoV-2 engages with human cells through the binding of its Spike receptor-binding domain (S-RBD) to the receptor ACE2. Molecular blocking of this engagement represents a proven strategy to treat COVID-19. Here, we report a single-chain antibody (nanobody, DL4) isolated from immunized alpaca with picomolar affinity to RBD. DL4 neutralizes SARS-CoV-2 pseudoviruses with an IC50 of 0.101 μg mL−1 (6.2 nM). A crystal structure of the DL4-RBD complex at 1.75-Å resolution unveils the interaction detail and reveals a direct competition mechanism for DL4's ACE2-blocking and hence neutralizing activity. The structural information allows us to rationally design a mutant with higher potency. Our work adds diversity of neutralizing nanobodies against SARS-CoV-2 and should encourage protein engineering to improve antibody affinities in general.

Discovery of Potent Orally Bioavailable WD Repeat Domain 5 (WDR5) Inhibitors Using a Pharmacophore-Based Optimization.

WD repeat domain 5 (WDR5) is a nuclear scaffolding protein that forms many biologically important multiprotein complexes. The WIN site of WDR5 represents a promising pharmacological target in a variety of human cancers. Here, we describe the optimization of our initial WDR5 WIN-site inhibitor using a structure-guided pharmacophore-based convergent strategy to improve its druglike properties and pharmacokinetic profile. The core of the previous lead remained constant while a focused SAR effort on the three pharmacophore units was combined to generate a new in vivo lead series. Importantly, this new series of compounds has picomolar binding affinity, improved cellular antiproliferative activity and selectivity, and increased kinetic aqueous solubility. They also exhibit a desirable oral pharmacokinetic profile with manageable intravenous clearance and high oral bioavailability. Thus, these new leads are useful probes toward studying the effects of WDR5 inhibition.

Radiosynthesis and In Vivo Evaluation of Four Positron Emission Tomography Tracer Candidates for Imaging of Melatonin Receptors.

Melatonin is a neurohormone that modulates several physiological functions in mammals through the activation of melatonin receptor type 1 and 2 (MT1 and MT2). The melatonergic system is an emerging therapeutic target for new pharmacological interventions in the treatment of sleep and mood disorders; thus, imaging tools to further investigate its role in the brain are highly sought-after. We aimed to develop selective radiotracers for in vivo imaging of both MT1 and MT2 by positron emission tomography (PET). We identified four previously reported MT ligands with picomolar affinities to the target based on different scaffolds which were also amenable for radiolabeling with either carbon-11 or fluorine-18. [11C]UCM765, [11C]UCM1014, [18F]3-fluoroagomelatine ([18F]3FAGM), and [18F]fluoroacetamidoagomelatine ([18F]FAAGM) have been synthesized in high radiochemical purity and evaluated in wild-type rats. All four tracers showed moderate to high brain permeability in rats with maximum standardized uptake values (SUVmax of 2.53, 1.75, 3.25, and 4.47, respectively) achieved 1-2 min after tracer administration, followed by a rapid washout from the brain. Several melatonin ligands failed to block the binding of any of the PET tracer candidates, while in some cases, homologous blocking surprisingly resulted in increased brain retention. Two 18F-labeled agomelatine derivatives were brought forward to PET scans in non-human primates and autoradiography on human brain tissues. No specific binding has been detected in blocking studies. To further investigate pharmacokinetic properties of the putative tracers, microsomal stability, plasma protein binding, log D, and membrane bidirectional permeability assays have been conducted. Based on the results, we conclude that the fast first pass metabolism by the enzymes in liver microsomes is the likely reason of the failure of our PET tracer candidates. Nevertheless, we showed that PET imaging can serve as a valuable tool to investigate the brain permeability of new therapeutic compounds targeting the melatonergic system.

Design of protein-binding proteins from the target structure alone

The design of proteins that bind to a specific site on the surface of a target protein using no information other than the three-dimensional structure of the target remains a challenge1–5. Here we describe a general solution to this problem that starts with a broad exploration of the vast space of possible binding modes to a selected region of a protein surface, and then intensifies the search in the vicinity of the most promising binding modes. We demonstrate the broad applicability of this approach through the de novo design of binding proteins to 12 diverse protein targets with different shapes and surface properties. Biophysical characterization shows that the binders, which are all smaller than 65 amino acids, are hyperstable and, following experimental optimization, bind their targets with nanomolar to picomolar affinities. We succeeded in solving crystal structures of five of the binder–target complexes, and all five closely match the corresponding computational design models. Experimental data on nearly half a million computational designs and hundreds of thousands of point mutants provide detailed feedback on the strengths and limitations of the method and of our current understanding of protein–protein interactions, and should guide improvements of both. Our approach enables the targeted design of binders to sites of interest on a wide variety of proteins for therapeutic and diagnostic applications.

Transition Metals Induce Quenching of Monomeric Near-Infrared Fluorescent Proteins.

Transition metals such as zinc and copper are essential in numerous life processes, and both deficiency and toxic overload of these metals are associated with various diseases. Fluorescent metal sensors are powerful tools for studying the roles of metal ions in the physiology and pathology of biological systems. Green fluorescent protein (GFP) and its derivatives are highly utilized for protein-based sensor design, but application to anaerobic systems is limited because these proteins require oxygen to become fluorescent. Bacteriophytochrome-based monomeric near-infrared fluorescent proteins (miRFPs) covalently bind a bilin cofactor, which can be added exogenously for anaerobic cells. miRFPs can also have emission wavelengths extending to >700 nm, which is valuable for imaging applications. Here, we evaluated the suitability of miRFP670 and miRFP709 as platforms for single fluorescent protein metal ion sensors. We found that divalent metal ions like Zn2+, Co2+, Ni2+, and Cu2+ can quench from ∼6-20% (Zn2+, Co2+, and Ni2+) and up to nearly 90% (Cu2+) of the fluorescence intensity of pure miRFPs and have similar impacts in live Escherichia coli cells expressing miRFPs. The presence of a 6× histidine tag for purification influences metal quenching, but significant Cu2+-induced quenching and a picomolar binding affinity are retained in the absence of the His6 tag in both cuvettes and live bacterial cells. By comparing the Cu2+ and Cu+-induced quenching results for miRFP670 and miRFP709 and through examining absorption spectra and previously reported crystal structures, we propose a surface metal binding site near the biliverdin IXα chromophore.

Structure-Based Design of High-Affinity Fluorescent Probes for the Neuropeptide Y Y1 Receptor.

The recent crystallization of the neuropeptide Y Y1 receptor (Y1R) in complex with the argininamide-type Y1R selective antagonist UR-MK299 (2) opened up a new approach toward structure-based design of nonpeptidic Y1R ligands. We designed novel fluorescent probes showing excellent Y1R selectivity and, in contrast to previously described fluorescent Y1R ligands, considerably higher (∼100-fold) binding affinity. This was achieved through the attachment of different fluorescent dyes to the diphenylacetyl moiety in 2 via an amine-functionalized linker. The fluorescent ligands exhibited picomolar Y1R binding affinities (pKi values of 9.36-9.95) and proved to be Y1R antagonists, as validated in a Fura-2 calcium assay. The versatile applicability of the probes as tool compounds was demonstrated by flow cytometry- and fluorescence anisotropy-based Y1R binding studies (saturation and competition binding and association and dissociation kinetics) as well as by widefield and total internal reflection fluorescence (TIRF) microscopy of live tumor cells, revealing that fluorescence was mainly localized at the plasma membrane.

Affinity maturation of TCR-like antibodies using phage display guided by structural modeling.

TCR-like antibodies represent a unique type of engineered antibodies with specificity toward pHLA, a ligand normally restricted to the sensitive recognition by T cells. Here, we report a phage display-based sequential development path of such antibodies. The strategy goes from initial lead identification through in silico informed CDR engineering in combination with framework engineering for affinity and thermostability optimization, respectively. The strategy allowed the identification of HLA-DQ2.5 gluten peptide-specific TCR-like antibodies with low picomolar affinity. Our method outlines an efficient and general method for development of this promising class of antibodies, which should facilitate their utility including translation to human therapy.