Broad Reactivity Research Articles

Role of SARS-CoV-2-specific memory B cells promoting immune protection after booster vaccination in solid organ transplantation.

Solid organ transplant (SOT) recipients display weak seroconversion and neutralizing antibody (NAb) responses after severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) vaccination and remain at risk of severe coronavirus disease 2019 (COVID-19). While B-cell memory is the hallmark of serological immunity, its role in driving successful vaccine responses and providing immune protection in SOT patients remains unclear. We investigated the function and interplay of SARS-CoV-2-specific memory B cells (mBc), different cytokineproducing T cells, and cross-reactive NAb in driving seroconversion and protection against COVID-19 in two cohorts. First, we studied a large cohort of 148 SOT recipients and 32 immunocompetent individuals who underwent several vaccinations. Subsequently, we assessed 25 SOT patients participating in a randomized controlled trial to compare two different immunosuppressive strategies for allowing successful seroconversion and memory-cell responses after booster vaccination. We corroborate previous findings that B- and T-cell memory responses are weaker and more delayed in SOT patients than in immunocompetent (IC) individuals; however, within the SOT cohort, we found that these responses are relatively stronger and more robust in patients not receiving mycophenolate mofetil (MMF)-based therapies. Anti- spike IgG titers strongly correlated with RBD-specific IgG-producing mBc, with both displaying broad viral cross reactivity. Prebooster SARS-CoV-2-specific mBc and IL-2- producing T cells accurately predicted Nab seroconversion (AUC, 0.828) and protection against severe COVID-19. While switching unresponsive SOT patients from calcineurin inhibitors (CNI)/MMF to a low-exposure CNI/mTOR-i regimen favored wider SARS-CoV-2-specific immune responses after a fourth booster vaccination, preformed RBD-specific mBc predicted NAb seroconversion. Our study adds new insights into the pathobiology of immune memory and highlights the pivotal role of SARS-CoV-2-specific mBc in promoting immune protection inSOT patients.

Immunohistochemical Evaluation of Cathepsin B, L, and S Expression in Breast Cancer Patients.

Cysteine cathepsins are proteases that play a role in normal cellular physiology and neoplastic transformation. Elevated expression and enzymatic activity of cathepsins in breast cancer (BCa) indicates their potential as a target for tumor imaging. In particular cathepsin B (CTSB), L (CTSL), and S (CTSS) are used as targets for near-infrared (NIR) fluorescence imaging (FI), a technique that allows real-time intraoperative tumor visualization and resection margin assessment. Therefore, this immunohistochemical study explores CTSB, CTSL, and CTSS expression levels in a large breast cancer patient cohort, to investigate in which BCa patients the use of cathepsin-targeted NIR FI may have added value. Protein expression was analyzed in tumor tissue microarrays (TMA) of BCa patients using immunohistochemistry and quantified as a total immunostaining score (TIS), ranging from 0-12. In total, the tissues of 557 BCa patients were included in the TMA. CTSB, CTSL, and CTSS were successfully scored in respectively 340, 373 and 252 tumors. All tumors showed CTSB, CTSL, and/or CTSS expression to some extent (TIS > 0). CTSB, CTSL, and CTSS expression was scored as high (TIS > 6) in respectively 28%, 80%, and 18% of tumors. In 89% of the tumors scored for all three cathepsins, the expression level of one or more of these proteases was scored as high (TIS > 6). Tumors showed significantly higher cathepsin expression levels with advancing Bloom-Richardson grade (p < 0.05). Cathepsin expression was highest in estrogen receptor (ER)-negative, human epidermal growth factor receptor 2(HER2)-positive and triple-negative (TN) tumors. There was no significant difference in cathepsin expression between tumors that were treated with neoadjuvant systemic therapy and tumors that were not. The expression of at least one of the cysteine cathepsins B, L and S in all breast tumor tissues tested suggests that cathepsin-activatable imaging agents with broad reactivity for these three proteases will likely be effective in the vast majority of breast cancer patients, regardless of molecular subtype and treatment status. Patients with high grade ER-negative, HER2-positive, or TN tumors might show higher imaging signals.

Synthesis of trisaccharide antigens featuring colitose, abequose and fucose residues and assessment of antibody binding on antigen arrays

Deoxy-hexose sugars, such as rhamnose and quinovose, and the dideoxy-hexoses colitose, abequose, and tyvelose are highly antigenic given that they are absent from animal glycoconjugates. To investigate the specificity of antibodies towards structurally similar carbohydrate epitopes found in bacteria, we synthesized trisaccharides containing colitose, abequose, and fucose motifs. Each trisaccharide was designed with a spacer ending with a primary amino group. These trisaccharide constructs were immobilized on O-succinimide coated glass slides alongside bacterial lipopolysaccharides (LPS) containing colitose, abequose, and fucose residues. We compared the recognition of the synthetic trisaccharides and natural LPS including structurally related epitopes by monoclonal and polyclonal antibodies targeting bacterial LPS. Additionally, we used arrays displaying the synthetic trisaccharides and natural LPS to assess the variability of IgA reactivity from breast milk samples towards the carbohydrate antigens. The results obtained underlined the cross-reactivity of polyclonal antibodies towards structurally related carbohydrate antigens and revealed a broad reactivity of breast milk-derived IgA towards the carbohydrate antigens tested. The significant cross-reactivity of antibodies towards structurally related LPS antigens may lead to false-positive detection of bacterial serotypes when used for diagnostic purposes.

Identification of antibody targets associated with lower HIV viral load and viremic control.

High HIV viral loads (VL) are associated with increased morbidity, mortality, and on-going transmission. HIV controllers maintain low VLs in the absence of antiretroviral therapy (ART). We previously used a massively multiplexed antibody profiling assay (VirScan) to compare antibody profiles in HIV controllers and persons living with HIV (PWH) who were virally suppressed on ART. In this report, we used VirScan to evaluate whether antibody reactivity to specific HIV targets and broad reactivity across the HIV genome was associated with VL and controller status 1-2 years after infection. Samples were obtained from participants who acquired HIV infection in a community-randomized trial in Africa that evaluated an integrated strategy for HIV prevention (HPTN 071 PopART). Controller status was determined using VL and antiretroviral (ARV) drug data obtained at the seroconversion visit and 1 year later. Viremic controllers had VLs <2,000 copies/mL at both visits; non-controllers had VLs >2,000 copies/mL at both visits. Both groups had no ARV drugs detected at either visit. VirScan testing was performed at the second HIV-positive visit (1-2 years after HIV infection). The study cohort included 13 viremic controllers and 64 non-controllers. We identified ten clusters of homologous peptides that had high levels of antibody reactivity (three in gag, three in env, two in integrase, one in protease, and one in vpu). Reactivity to 43 peptides (eight unique epitopes) in six of these clusters was associated with lower VL; reactivity to six of the eight epitopes was associated with HIV controller status. Higher aggregate antibody reactivity across the eight epitopes (more epitopes targeted, higher mean reactivity across all epitopes) and across the HIV genome was also associated with lower VL and controller status. We identified HIV antibody targets associated with lower VL and HIV controller status 1-2 years after infection. Robust aggregate responses to these targets and broad antibody reactivity across the HIV genome were also associated with lower VL and controller status. These findings provide novel insights into the relationship between humoral immunity and viral containment that could help inform the design of antibody-based approaches for reducing HIV VL.

Modular Synthesis of Azines Bearing a Guanidine Core from N-Heterocyclic Carbene (NHC)-Derived Selenoureas and Diazo Reagents.

N-Heterocyclic carbene (NHC)-derived selenoureas comprise a fundamentally important class of NHC derivatives, with key applications in coordination chemistry and the determination of NHC electronic properties. Considering the broad reactivity of chalcogen-containing compounds, it is surprising to note that the use of NHC-derived selenoureas as organic synthons remains essentially unexplored. The present contribution introduces a novel, straightforward transformation leading to azines bearing a guanidine moiety, through the reaction of a wide range of NHC-derived selenoureas with commercially available diazo compounds, in the presence of triphenylphosphine. This transformation offers a new approach to such products, having biological, materials chemistry, and organic synthesis applications. The guanidine-bearing azines are obtained in excellent yields, with all manipulations taking place in air. A reaction mechanism is proposed, based on both experimental mechanistic findings and density functional theory (DFT) calculations. A one-pot, multicomponent transesterification reaction between selenoureas, α-diazoesters, alcohols, and triphenylphosphine was also developed, providing highly functionalized azines.

New Frontiers in Nonheme Enzymatic Oxyferryl Species.

Non-heme mononuclear iron dependent (NHM-Fe) enzymes exhibit exceedingly diverse catalytic reactivities. Despite their catalytic versatilities, the mononuclear iron centers in these enzymes show a relatively simple architecture, in which an iron atom is ligated with 2-4 amino acid residues, including histidine, aspartic or glutamic acid. In the past two decades, a common high-valent reactive iron intermediate, the S=2 oxyferryl (Fe(IV)-oxo or Fe(IV)=O) species, has been repeatedly discovered in NHM-Fe enzymes containing a 2-His-Fe or 2-His-1-carboxylate-Fe center. However, for 3-His/4-His-Fe enzymes, no common reactive intermediate has been identified. Recently, we have spectroscopically characterized the first S=1 Fe(IV) intermediate in a 3-His-Fe containing enzyme, OvoA, which catalyzes a novel oxidative carbon-sulfur bond formation. In this review, we summarize the broad reactivities demonstrated by S=2 Fe(IV)-oxo intermediates, the discovery of the first S=1 Fe(IV) intermediate in OvoA and the mechanistic implication of such a discovery, and the intrinsic reactivity differences of the S=2 and the S=1 Fe(IV)-oxo species. Finally, we postulate the possible reasons to utilize an S=1 Fe(IV) species in OvoA and their implications to other 3-His/4-His-Fe enzymes.

High-Performance Workflow for Identifying Site-Specific Crosslinks Originating from a Genetically Incorporated, Photoreactive Amino Acid.

In conventional crosslinking mass spectrometry, proteins are crosslinked using a highly selective, bifunctional chemical reagent, which limits crosslinks to residues that are accessible and reactive to the reagent. Genetically incorporating a photoreactive amino acid offers two key advantages: any site can be targeted, including those that are inaccessible to conventional crosslinking reagents, and photoreactive amino acids can potentially react with a broad range of interaction partners. However, broad reactivity imposes additional challenges for crosslink identification. In this study, we incorporate benzoylphenylalanine (BPA), a photoreactive amino acid, at selected sites in an intrinsically disordered region of the human protein HSPB5. We report and characterize a workflow for identifying and visualizing residue-level interactions originating from BPA. We routinely identify 30 to 300 crosslinked peptide spectral matches with this workflow, which is up to ten times more than existing tools for residue-level BPA crosslink identification. Most identified crosslinks are assigned to a precision of one or two residues, which is supported by a high degree of overlap between replicate analyses. Based on these results, we anticipate that this workflow will support the more general use of genetically incorporated, photoreactive amino acids for characterizing the structures of proteins that have resisted high-resolution characterization.

Development of a broad-spectrum therapeutic Fc-nanobody for human noroviruses.

Human norovirus was discovered more than five decades ago and is a widespread cause of outbreaks of acute gastroenteritis. There are no approved vaccines or antivirals currently available. However, norovirus inhibitors, including capsid-specific monoclonal antibodies (Mabs) and nanobodies, have recently shown promising results. Several Mabs and nanobodies were found to inhibit norovirus replication using a human intestinal enteroid (HIE) culture system and/or could block norovirus attachment to histo-blood group antigen (HBGA) co-factors. In our pursuit to develop a single broad-spectrum norovirus therapeutic, we continued our analysis and development of a cross-reactive and HBGA interfering nanobody (NB26). To improve NB26 binding capacity and therapeutic potential, we conjugated NB26 onto a human IgG Fc domain (Fc-NB26). We confirmed that Fc-NB26 cross-reacts with genetically diverse GII genotype capsid protruding (P) domains (GII.8, GII.14, GII.17, GII.24, GII.26, and GII.NA1) using a direct enzyme-linked immunosorbent assay. Furthermore, X-ray crystallography structures of these P domains and structures of other GII genotypes reveal that the NB26 binding site is largely conserved, validating its broad reactivity. We showed that Fc-NB26 has ~100-fold higher affinity toward the norovirus P domain compared to native NB26. We also found that both NB26 and Fc-NB26 neutralize human norovirus replication in the HIE culture system. Furthermore, the mode of inhibition confirmed that like NB26, Fc-NB26 caused norovirus particle disassembly and aggregation. Overall, these new findings demonstrate that structural modifications to nanobodies can improve their therapeutic potential.IMPORTANCEDeveloping vaccines and antivirals against norovirus remains a challenge, mainly due to the constant genetic and antigenic evolution. Moreover, re-infection with genetically related and/or antigenic variants is not uncommon. We further developed our leading norovirus nanobody (NB26) that indirectly interfered with norovirus binding to HBGAs, by converting NB26 into a dimeric Fc-linked Nanobody (Fc-NB26). We found that Fc-NB26 had improved binding affinity and neutralization capacity compared with native NB26. Using X-ray crystallography, we showed this nanobody engaged highly conserved capsid residues among genetically diverse noroviruses. Development of such broadly reactive potent therapeutic nanobodies delivered as a slow-releasing prophylactic could be of exceptional value for norovirus outbreaks, especially for the prevention or treatment of severe acute gastroenteritis in high-risk groups such as the young, elderly, and immunocompromised.

Cobalt‐Driven Cross‐Dehydrocoupling of Silanes and Amines for Silylamine Synthesis

AbstractCobalt complexes featuring triazine‐based PNP ligands have proven to be exceptionally active and chemoselective pre‐catalysts in facilitating the dehydrogenative coupling between silanes and amines, leading to the synthesis of diverse aminosilanes. Notably, even challenging substrates exhibited high reactivity. The catalyst‘s unique feature of avoiding coupling with tertiary silanes enhances process chemoselectivity. It facilitates a more precise synthesis of silylamines possessing of SiH2−N and SiH−N motifs, overcoming challenges associated with broader reactivity seen in previous systems. In terms of its remarkable chemoselectivity, it is also noteworthy that the catalytic system exhibits both versatility and efficacy in converting substrates with untouched double and triple carbon‐carbon bonds. This accomplishment is particularly significant, given previous challenges brought about by the activity of commonly employed catalysts in the competitive hydrosilylation process.

Genetically Encoded Epoxide Warhead for Precise and Versatile Covalent Targeting of Proteins.

Genetically encoding a proximal reactive warhead into the protein binder/drug has emerged as an efficient strategy for covalently binding to protein targets, enabling broad applications. To expand the reactivity scope for targeting the diverse natural residues under physiological conditions, the development of a genetically encoded reactive warhead with excellent stability and broad reactivity is highly desired. Herein, we reported the genetic encoding of epoxide-containing tyrosine (EPOY) for developing covalent protein drugs. Our study demonstrates that EPOY, when incorporated into a nanobody (KN035), can cross-link with different side chains (mutations) at the same position of PD-L1 protein. Significantly, a single genetically encoded reactive warhead that is capable of covalent and site-specific targeting to 10 different nucleophilic residues was achieved for the first time. This would largely expand the scope of covalent warhead and inspire the development of covalent warheads for both small-molecule drugs and protein drugs. Furthermore, we incorporate the EPOY into a designed ankyrin repeat protein (DarpinK13) to create the covalent binders of KRAS. This covalent KRAS binder holds the potential to achieve pan-covalent targeting of KRAS based on the structural similarity among all oncogenic KRAS mutants while avoiding off-target binding to NRAS/HRAS through a covalent interaction with KRAS-specific residues (H95 and E107). We envision that covalently targeting to H95 will be a promising strategy for the development of covalent pan-KRAS inhibitors in the future.

A chimeric haemagglutinin-based universal influenza virus vaccine boosts human cellular immune responses directed towards the conserved haemagglutinin stalk domain and the viral nucleoprotein

The development of a universal influenza virus vaccine, to protect against both seasonal and pandemic influenza A viruses, is a long-standing public health goal. The conserved stalk domain of haemagglutinin (HA) is a promising vaccine target. However, the stalk is immunosubdominant. As such, innovative approaches are required to elicit robust immunity against this domain. In a previously reported observer-blind, randomised placebo-controlled phase I trial (NCT03300050), immunisation regimens using chimeric HA (cHA)-based immunogens formulated as inactivated influenza vaccines (IIV)-/+ AS03 adjuvant, or live attenuated influenza vaccines (LAIV), elicited durable HA stalk-specific antibodies with broad reactivity. In this study, we sought to determine if these vaccines could also boost T cell responses against HA stalk, and nucleoprotein (NP). We measured interferon-γ (IFN-γ) responses by Enzyme-Linked ImmunoSpot (ELISpot) assay at baseline, seven days post-prime, pre-boost and seven days post-boost following heterologous prime:boost regimens of LAIV and/or adjuvanted/unadjuvanted IIV-cHA vaccines. Our findings demonstrate that immunisation with adjuvanted cHA-based IIVs boost HA stalk-specific and NP-specific T cell responses in humans. To date, it has been unclear if HA stalk-specific T cells can be boosted in humans by HA-stalk focused universal vaccines. Therefore, our study will provide valuable insights for the design of future studies to determine the precise role of HA stalk-specific T cells in broad protection. Considering that cHA-based vaccines also elicit stalk-specific antibodies, these data support the further clinical advancement of cHA-based universal influenza vaccine candidates. This study was funded in part by the Bill and Melinda Gates Foundation (BMGF).

CARs derived from broadly neutralizing, human monoclonal antibodies identified by single B cell sorting target hepatitis B virus-positive cells.

To design new CARs targeting hepatitis B virus (HBV), we isolated human monoclonal antibodies recognizing the HBV envelope proteins from single B cells of a patient with a resolved infection. HBV-specific memory B cells were isolated by incubating peripheral blood mononuclear cells with biotinylated hepatitis B surface antigen (HBsAg), followed by single-cell flow cytometry-based sorting of live, CD19+ IgG+ HBsAg+ cells. Amplification and sequencing of immunoglobulin genes from single memory B cells identified variable heavy and light chain sequences. Corresponding immunoglobulin chains were cloned into IgG1 expression vectors and expressed in mammalian cells. Two antibodies named 4D06 and 4D08 were found to be highly specific for HBsAg, recognized a conformational and a linear epitope, respectively, and showed broad reactivity and neutralization capacity against all major HBV genotypes. 4D06 and 4D08 variable chain fragments were cloned into a 2nd generation CAR format with CD28 and CD3zeta intracellular signaling domains. The new CAR constructs displayed a high functional avidity when expressed on primary human T cells. CAR-grafted T cells proved to be polyfunctional regarding cytokine secretion and killed HBV-positive target cells. Interestingly, background activation of the 4D08-CAR recognizing a linear instead of a conformational epitope was consistently low. In a preclinical model of chronic HBV infection, murine T cells grafted with the 4D06 and the 4D08 CAR showed on target activity indicated by a transient increase in serum transaminases, and a lower number of HBV-positive hepatocytes in the mice treated. This study demonstrates an efficient and fast approach to identifying pathogen-specific monoclonal human antibodies from small donor cell numbers for the subsequent generation of new CARs.

Abstract 3611: A patient-derived organoid platform to assess the effectiveness of γδTCR cell therapy in solid tumors

Abstract Recent advances in cancer immunotherapy have positively impacted the life expectancy of patients for an extensive range of clinical indications. With new treatment strategies and druggable targets being identified at an increasing pace, the number of patients eligible for cancer immunotherapy is expected to expand steadily. However, promising therapeutic developments face hurdles in translating preclinical findings into therapy since conventional 2D cancer models hold low clinical predictive value. HUB developed an innovative alternative, building on the discovery that adult stem cells proliferate and organise into three-dimensional organotypic structures when embedded into the extracellular matrix. Patient-derived organoids are generated from healthy and malignant tissues, and they recapitulate complex characteristics of the original parental tissue, including molecular heterogeneity and morphological and functional traits. Cell therapies have achieved positive results in treating haematological cancers. Recently, the unique tumour-targeting properties of γδ T cell receptors have been leveraged to develop safer and more effective strategies. HUB and Gadeta joined efforts to explore the therapeutic potential of GDT002 - i.e. αβ T cells engineered with a Gadeta proprietary new γδTCR - to target solid cancers. A preclinical basket trial was performed using patient-derived organoids from several tumour types (colorectal, breast, head and neck, ovarian cancers) cocultured with GDT002. The results showed broad reactivity of GDT002 against solid tumor PDOs. Interestingly, GDT002 was particularly efficient at killing ovarian cancer-derived PDOs, allowing Gadeta to move GDT002 to clinical trials for this indication confidently. This study reveals that our tumor organoid co-culture with engineered T cells platform holds significant value for the preclinical development of cellular therapies. Citation Format: Andrea Bisso, Rene Overmeer, Sjoerd Baardman, Chris Coomans, Cesar Oyarce, Sylvia F. Boj. A patient-derived organoid platform to assess the effectiveness of γδTCR cell therapy in solid tumors [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 3611.

Dienes as Versatile Substrates for Transition Metal‐Catalyzed Reactions

AbstractDienes have been of great interest to synthetic chemists as valuable substrates due to their abundance and ease of synthesis. Their unique stereoelectronic properties enable broad reactivity with a wide range of transition metals to construct molecular complexity facilitating synthesis of biologically active compounds. In addition, structural diene variation can result in substrate‐controlled reactions, providing valuable mechanistic insights into reactivity and selectivity patterns. The last decade has seen a wealth of new methodologies involving diene substrates through the power of transition metal catalysis. This review summarizes recent advances and remaining opportunities for transition metal‐catalyzed transformations involving dienes.

Dienes as Versatile Substrates for Transition Metal-Catalyzed Reactions.

Dienes have been of great interest to synthetic chemists as valuable substrates due to their abundance and ease of synthesis. Their unique stereoelectronic properties enable broad reactivity with a wide range of transition metals to construct molecular complexity facilitating synthesis of biologically active compounds. In addition, structural diene variation can result in substrate-controlled reactions, providing valuable mechanistic insights into reactivity and selectivity patterns. The last decade has seen a wealth of new methodologies involving diene substrates through the power of transition metal catalysis. This review summarizes recent advances and remaining opportunities for transition metal-catalyzed transformations involving dienes.

Design and Application of New Pyridine-Derived Chiral Ligands in Asymmetric Catalysis.

ConspectusThe innovation of chiral ligands has been crucial for the asymmetric synthesis of functional molecules, as demonstrated by several types of widely applied "privileged" ligands. In this context, chiral pyridine-derived ligands, by far some of the oldest and most widely utilized ligands in catalysis, have attracted considerable research interest in the past half-century. However, the development of broadly applicable chiral pyridine units (CPUs) has been plagued by several intertwining challenges, thus delaying advancements in many asymmetric reactions.This Account aims to summarize the recent progress in new CPU-containing ligands, focusing on a rationally designed, modular, and tunable CPU developed in our laboratory. A significant problem thwarting conventional designs is the paradox between broad reactivity and stereoselectivity; that is, while enhanced stereoselectivity may be achieved by introducing chiral elements close to the N atom, the concomitant increase in local steric hindrance often limits catalytic activity and scope. Our newly developed CPU features a rigid [6-5-3] fused-ring framework and a tunable spirocyclic ketal side wall. The well-defined three-dimensional structure minimizes local (inner layer) steric hindrance and tunes the peripheral environment (outer layer) by remote substituents, thus securing reactivity and stereoselectivity. Different chelating ligands were readily assembled using this chiral structural module, with applications in mechanistically diverse transition-metal-catalyzed reactions. Thus, a series of chiral 2,2'-bipyridine ligands were successfully employed in the development of a general, efficient, and highly enantioselective nickel-catalyzed intermolecular reductive addition, Ullmann coupling of ortho-chlorinated aryl aldehydes, and carboxylation of benzylic (pseudo)halides with CO2. Notably, these chiral 2,2'-bipyridine ligands exhibited superior catalytic activity in the reactions compared to common N-based ligands. In addition, highly enantioselective iridium-catalyzed C-H borylation was developed using a CPU-containing N,B-bidentate ligand. Furthermore, mechanistically challenging, additive-free, and broad-scope transfer hydrogenative direct asymmetric reductive amination was achieved using a half-sandwich iridium catalyst supported by a chiral N,C-bidentate ligand. The new ligands demonstrated excellent performance in securing high catalytic activity and stereoselectivity, which, when combined with experimental and computational mechanistic investigations, supported the "double-layer control" design concept.Considering the broad applications of pyridine-derived ligands, the research progress described herein should inspire the creation of novel chiral catalysts and drive the development of many catalytic asymmetric reactions.

Crystal structure of human serum albumin in complex with megabody reveals unique human and murine cross-reactive binding site.

The pharmacokinetic properties of small biotherapeutics can be enhanced via conjugation to cross-reactive albumin-binding ligands in a process that improves their safety and accelerates testing through multiple pre-clinical animal models. In this context, the small and stable heavy-chain-only nanobody NbAlb1, capable of binding both human and murine albumin, has recently been successfully applied to improve the stability and prolong the in vivo plasma residence time of multiple small therapeutic candidates. Despite its clinical efficacy, the mechanism of cross-reactivity of NbAlb1 between human and murine serum albumins has not yet been investigated. To unveil the molecular basis of such an interaction, we solved the crystal structure of human serum albumin (hSA) in complex with NbAlb1. The structure was obtained by harnessing the unique features of a megabody chimeric protein, comprising NbAlb1 grafted onto a modified version of the circularly permutated and bacterial-derived protein HopQ. This structure showed that NbAlb1 contacts a yet unexplored binding site located in the peripheral region of domain II that is conserved in both human and mouse serum albumin proteins. Furthermore, we show that the binding of NbAlb1 to both serum albumin proteins is retained even at acidic pH levels, thus explaining its extended in vivo half-life. The elucidation of the molecular basis of NbAlb1 cross-reactivity to human and murine albumins might guide the design of novel nanobodies with broader reactivity toward a larger panel of serum albumins, thus facilitating the pre-clinical and clinical phases in humans.

Polymeric bis(triphenylphosphine)iminium chloride as a recyclable catalyst.

Metal-free catalysts have garnered considerable interest as an environmental and economical alternative to precious metal catalysts. Bis(triphenylphosphine)iminium chloride (PPNCl) has emerged as a prominent choice due to its air and thermal stability and broad reactivity, especially in applications where a bulky cation is needed. The high phosphorus content and synthetic effort required for catalyst synthesis increase environmental impact; the recyclability of PPNCl in catalytic processes remains largely unexplored. The potential development of a polymer-supported PPNCl catalysts therefore desirable to enable this recyclability. In this work, we synthesise polymeric PPNCl (poly(PPNCl)) for the first time. Poly(PPNCl) demonstrates a comparative catalytic reactivity to its small molecule variant when employed as a catalyst in halogen-exchange reactions and CO2/epoxide coupling. For the latter the effect of catalyst loading, CO2 pressure, reaction time and addition of co-catalyst on conversion and selectivity was investigated. Poly(PPNCl) was easily recovered from the crude product by simple precipitation and its catalytic reactivity was well-maintained over three reaction cycles, providing environmental and economic advantages for sustainable reaction development.

Inter-individual and inter-regional variability of breast milk antibody reactivity to bacterial lipopolysaccharides.

Breast milk is a vital source of nutrients, prebiotics, probiotics, and protective factors, including antibodies, immune cells and antimicrobial proteins. Using bacterial lipopolysaccharide arrays, we investigated the reactivity and specificity of breast milk antibodies towards microbial antigens, comparing samples from rural Kenya and urban Switzerland. Results showed considerable variability in antibody reactivity both within and between these locations. Kenyan breast milk demonstrated broad reactivity to bacterial lipopolysaccharides, likely due to increased microbial exposure. Antibodies primarily recognized the O-antigens of lipopolysaccharides and showed strong binding to specific carbohydrate motifs. Notably, antibodies against specific Escherichia coli O-antigens showed cross-reactivity with parasitic pathogens like Leishmania major and Plasmodium falciparum, thus showing that antibodies reacting against lipopolysaccharide O-antigens can recognize a wide range of antigens beyond bacteria. The observed diversity in antigen recognition highlights the significance of breast milk in safeguarding infants from infections, particularly those prevalent in specific geographic regions. The findings also offer insights for potential immunobiotic strategies to augment natural antibody-mediated defense against diverse pathogens.

Antibody targeting of conserved sites of vulnerability on the SARS-CoV-2 spike receptor-binding domain

Given the continuous emergence of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) variants of concern (VoCs), immunotherapeutics that target conserved epitopes on the spike (S)glycoprotein have therapeutic advantages. Here, we report the crystal structure of the SARS-CoV-2S receptor-binding domain (RBD) at 1.95Å and describe flexibility and distinct conformations of the angiotensin-converting enzyme 2 (ACE2)-binding site. We identify a set of SARS-CoV-2-reactive monoclonal antibodies (mAbs) with broad RBD cross-reactivity including SARS-CoV-2 Omicron subvariants, SARS-CoV-1, and other sarbecoviruses and determine the crystal structures of mAb-RBD complexes with Ab246 and CR3022 mAbs targeting the class IV site, WRAIR-2134, which binds the recently designated class V epitope, and WRAIR-2123, the class I ACE2-binding site. The broad reactivity of class IV and V mAbs to conserved regions of SARS-CoV-2 VoCs and other sarbecovirus provides a framework for long-term immunotherapeutic development strategies.