Functional insights of Tyr37 in framework region 2 directly contributing to the binding affinities and dissociation kinetics in single-domain VHH antibodies.

Single-domain VHH antibody is regarded as one of the promising antibody classes for therapeutic and diagnostic applications. VHH antibodies have amino acids in framework region 2 that are distinct from those in conventional antibodies, such as the Val37Phe/Tyr (V37F/Y) substitution. Correlations between the residue type at position 37 and the conformation of the CDR3 in VHH antigen recognition have been previously reported. However, few studies focused on the meaning of harboring two residue types in position 37 of VHH antibodies, and the concrete roles of Y37 have been little to be elucidated. Here, we investigated the functional states of position 37 in co-crystal structures and performed analyses of three model antibodies with either F or Y at position 37. Our analysis indicates that Y at position 37 enhances the dissociation rate, which is highly correlated with drug efficacy. Our findings help to explain the molecular mechanisms that distinguish VHH antibodies from conventional antibodies.

Combined Neutrophil-to-Lymphocyte Ratio Score Is Associated With Chemotherapeutic Response and Predicts Prognosis in Patients With Advanced Pancreatic Cancer.

BACKGROUND/AIM: Neutrophil-to-lymphocyte ratio (NLR) is a prognostic indicator for several malignancies, including pancreatic cancer. We developed a novel combined NLR score (cNLRS) based on baseline NLR and change in NLR after chemotherapy (ΔNLR), and examined its prognostic value and role in chemotherapeutic response in patients with advanced pancreatic cancer. PATIENTS AND METHODS: This study retrospectively assessed 210 advanced pancreatic cancer patients receiving chemotherapy between 2010 and 2021. The cNLRS was developed and its association with chemotherapeutic response and prognosis was investigated. RESULTS: The cNLRS consisted of baseline NLR ≥2.5 and ΔNLR ≥0, both of which were remained as independent poor predictors of prognosis adjusting for other traditional clinicopathological features. A high cNLRS served as an independent prognostic factor of reduced overall survival. Of note, the cNLRS was significantly associated with disease control rate and treatment duration not only in 1st line treatment but also in 2nd line treatment. CONCLUSION: The cNLRS established as a useful prognostic biomarker might be associated with chemotherapeutic response and could predict survival in advanced patients with pancreatic ductal adenocarcinoma treated with chemotherapy.

Structural basis for the recognition of human hemoglobin by the heme-acquisition protein Shr from Streptococcus pyogenes.

In Gram-positive bacteria, sophisticated machineries to acquire the heme group of hemoglobin (Hb) have evolved to extract the precious iron atom contained in it. In the human pathogen Streptococcus pyogenes, the Shr protein is a key component of this machinery. Herein we present the crystal structure of hemoglobin-interacting domain 2 (HID2) of Shr bound to Hb. HID2 interacts with both, the protein and heme portions of Hb, explaining the specificity of HID2 for the heme-bound form of Hb, but not its heme-depleted form. Further mutational analysis shows little tolerance of HID2 to interfacial mutations, suggesting that its interaction surface with Hb could be a suitable candidate to develop efficient inhibitors abrogating the binding of Shr to Hb.

Generation of antibodies to an extracellular region of the transporters Glut1/Glut4 by immunization with a designed antigen.

Monoclonal antibodies are one of the fastest growing class of drugs. Nevertheless, relatively few biologics target multispanning membrane proteins because of technical challenges. To target relatively small extracellular regions of multiple membrane-spanning proteins, synthetic peptides, which are composed of amino acids corresponding to an extracellular region of a membrane protein, are often utilized in antibody discovery. However, antibodies to these peptides often do not recognize parental membrane proteins. In this study, we designed fusion proteins in which an extracellular helix of the membrane protein glucose transporter 1 (Glut1) was grafted onto the scaffold protein Adhiron. In the initial design, the grafted fragment did not form a helical conformation. Molecular dynamics simulations of full-length Glut1 suggested the importance of intramolecular interactions formed by surrounding residues in the formation of the helical conformation. A fusion protein designed to maintain such intramolecular interactions did form the desired helical conformation in the grafted region. We then immunized an alpaca with the designed fusion protein and obtained VHH (variable region of heavy-chain antibodies) using the phage display method. The binding of these VHH antibodies to the recombinant Glut1 protein was evaluated by surface plasmon resonance, and their binding to Glut1 on the cell membrane was further validated by flow cytometry. Furthermore, we also succeeded in the generation of a VHH against another integral membrane protein, glucose transporter 4 (Glut4) with the same strategy. These illustrates that our combined biochemical and computational approach can be applied to designing other novel fusion proteins for generating site-specific antibodies.

Conformational features and interaction mechanisms of VH H antibodies with ß-hairpin CDR3: A case of Nb8-HigB2 interaction.

The ß-hairpin conformation is regarded as an important basic motif to form and regulate protein-protein interactions. Single-domain VH H antibodies are potential therapeutic and diagnostic tools, and the third complementarity-determining regions of the heavy chains (CDR3s) of these antibodies are critical for antigen recognition. Although the sequences and conformations of the CDR3s are diverse, CDR3s sometimes adopt ß-hairpin conformations. However, characteristic features and interaction mechanisms of ß-hairpin CDR3s remain to be fully elucidated. In this study, we investigated the molecular recognition of the anti-HigB2 VH H antibody Nb8, which has a CDR3 that forms a ß-hairpin conformation. The interaction was analyzed by evaluation of alanine-scanning mutants, molecular dynamics simulations, and hydrogen/deuterium exchange mass spectrometry. These experiments demonstrated that positions 93 and 94 (Chothia numbering) in framework region 3, which is right outside CDR3 by definition, play pivotal roles in maintaining structural stability and binding properties of Nb8. These findings will facilitate the design and optimization of single-domain antibodies.

Biophysical insight into protein-protein interactions in the Interleukin-11/Interleukin-11Rα/glycoprotein 130 signaling complex.

Interleukin-11 (IL-11) is a member of the interleukin-6 (IL-6) family of cytokines. IL-11 is a regulator of multiple events in hematopoiesis, and IL-11-mediated signaling is implicated in inflammatory disease, cancer, and fibrosis. All IL-6 family cytokines signal through the signal-transducing receptor, glycoprotein 130 (gp130), but these cytokines have distinct as well as overlapping biological functions. To understand IL-11 signaling at the molecular level, we performed a comprehensive interaction analysis of the IL-11 signaling complex, comparing it with the IL-6 complex, one of the best-characterized cytokine complexes. Our thermodynamic analysis revealed a clear difference between IL-11 and IL-6. Surface plasmon resonance analysis showed that the interaction between IL-11 and IL-11 receptor α (IL-11Rα) is entropy driven, whereas that between IL-6 and IL-6 receptor α (IL-6Rα) is enthalpy driven. Our analysis using isothermal titration calorimetry revealed that the binding of gp130 to the IL-11/IL-11Rα complex results in entropy loss, but that the interaction of gp130 with the IL-6/IL-6Rα complex results in entropy gain. Our hydrogen-deuterium exchange mass spectrometry experiments suggested that the D2 domain of gp130 was not involved in IL-6-like interactions in the IL-11/IL-11Rα complex. It has been reported that IL-6 interaction with gp130 in the signaling complex was characterized through the hydrophobic interface located in its D2 domain of gp130. Our findings suggest that unique interactions of the IL-11 signaling complex with gp130 are responsible for the distinct biological activities of IL-11 compared to IL-6.

Modulation of a conformational ensemble by a small molecule that inhibits key protein-protein interactions involved in cell adhesion.

Small molecules that regulate protein-protein interactions can be valuable drugs; however, the development of such small molecules is challenging as the molecule must interfere with an interaction that often involves a large surface area. Herein, we propose that modulating the conformational ensemble of the proteins participating in a given interaction, rather than blocking the interaction by directly binding to the interface, is a relevant strategy for interfering with a protein-protein interaction. In this study, we applied this concept to P-cadherin, a cell surface protein forming homodimers that are essential for cell-cell adhesion in various biological contexts. We first determined the crystal structure of P-cadherin with a small molecule inhibitor whose inhibitory mechanism was unknown. Molecular dynamics simulations suggest that the inhibition of cell adhesion by this small molecule results from modulation of the conformational ensemble of P-cadherin. Our study demonstrates the potential of small molecules altering the conformation ensemble of a protein as inhibitors of biological relevant protein-protein interactions.

Structural basis for cross-group recognition of an influenza virus hemagglutinin antibody that targets postfusion stabilized epitope.

Plasticity of influenza virus hemagglutinin (HA) conformation increases an opportunity to generate conserved non-native epitopes with unknown functionality. Here, we have performed an in-depth analysis of human monoclonal antibodies against a stem-helix region that is occluded in native prefusion yet exposed in postfusion HA. A stem-helix antibody, LAH31, provided IgG Fc-dependent cross-group protection by targeting a stem-helix kinked loop epitope, with a unique structure emerging in the postfusion state. The structural analysis and molecular modeling revealed key contact sites responsible for the epitope specificity and cross-group breadth that relies on somatically mutated light chain. LAH31 was inaccessible to the native prefusion HA expressed on cell surface; however, it bound to the HA structure present on infected cells with functional linkage to the Fc-mediated clearance. Our study uncovers a novel non-native epitope that emerges in the postfusion HA state, highlighting the utility of this epitope for a broadly protective antigen design.

Arginine cluster introduction on framework region in anti-lysozyme antibody improved association rate constant by changing conformational diversity of CDR loops.

Antibodies are used for many therapeutic and biotechnological purposes. Because the affinity of an antibody to the antigen is critical for clinical efficacy of pharmaceuticals, many affinity maturation strategies have been developed. Although we previously reported an affinity maturation strategy in which the association rate of the antibody toward its antigen is improved by introducing a cluster of arginine residues into the framework region of the antibody, the detailed molecular mechanism responsible for this improvement has been unknown. In this study, we introduced five arginine residues into an anti-hen egg white lysozyme antibody (HyHEL10) Fab fragment to create the R5-mutant and comprehensively characterized the interaction between antibody and antigen using thermodynamic analysis, X-ray crystallography, and molecular dynamics (MD) simulations. Our results indicate that introduction of charged residues strongly enhanced the association rate, as previously reported, and the antibody-antigen complex structure was almost the same for the R5-mutant and wild-type Fabs. The MD simulations indicate that the mutation increased conformational diversity in complementarity-determining region loops and thereby enhanced the association rate. These observations provide the molecular basis of affinity maturation by R5 mutation.

Structural delineation and computational design of SARS-CoV-2-neutralizing antibodies against Omicron subvariants.

SARS-CoV-2 Omicron subvariants have evolved to evade receptor-binding site (RBS) antibodies that exist in diverse individuals as public antibody clones. We rationally selected RBS antibodies resilient to mutations in emerging Omicron subvariants. Y489 was identified as a site of virus vulnerability and a common footprint of broadly neutralizing antibodies against the subvariants. Multiple Y489-binding antibodies were encoded by public clonotypes and additionally recognized F486, potentially accounting for the emergence of Omicron subvariants harboring the F486V mutation. However, a subclass of antibodies broadly neutralized BA.4/BA.5 variants via hydrophobic binding sites of rare clonotypes along with high mutation-resilience under escape mutation screening. A computationally designed antibody based on one of the Y489-binding antibodies, NIV-10/FD03, was able to bind XBB with any 486 mutation and neutralized XBB.1.5. The structural basis for the mutation-resilience of this Y489-binding antibody group may provide important insights into the design of therapeutics resistant to viral escape.

Targeting hemoglobin receptors IsdH and IsdB of Staphylococcus aureus with a single VHH antibody inhibits bacterial growth.

Methicillin-resistant Staphylococcus aureus, or MRSA, is one of the major causative agents of hospital-acquired infections worldwide. Novel antimicrobial strategies efficient against antibiotic-resistant strains are necessary and not only against S. aureus. Among those, strategies that aim at blocking or dismantling proteins involved in the acquisition of essential nutrients, helping the bacteria to colonize the host, are intensively studied. A major route for S. aureus to acquire iron from the host organism is the Isd (iron surface determinant) system. In particular, the hemoglobin receptors IsdH and IsdB located on the surface of the bacterium are necessary to acquire the heme moiety containing iron, making them a plausible antibacterial target. Herein, we obtained an antibody of camelid origin that blocked heme acquisition. We determined that the antibody recognized the heme-binding pocket of both IsdH and IsdB with nanomolar order affinity through its second and third complementary-determining regions. The mechanism explaining the inhibition of acquisition of heme in vitro could be described as a competitive process in which the complementary-determining region 3 from the antibody blocked the acquisition of heme by the bacterial receptor. Moreover, this antibody markedly reduced the growth of three different pathogenic strains of MRSA. Collectively, our results highlight a mechanism for inhibiting nutrient uptake as an antibacterial strategy against MRSA.

An Engineered Synthetic Receptor-Aptamer Pair for an Artificial Signal Transduction System.

Cell membrane receptors regulate cellular responses through sensing extracellular environmental signals and subsequently transducing them. Receptor engineering provides a means of directing cells to react to a designated external cue and exert programmed functions. However, rational design and precise modulation of receptor signaling activity remain challenging. Here, we report an aptamer-based signal transduction system and its applications in controlling and customizing the functions of engineered receptors. A previously reported membrane receptor-aptamer pair was used to design a synthetic receptor system that transduces cell signaling depending on exogenous aptamer input. To eliminate the cross-reactivity of the receptor with its native ligand, the extracellular domain of the receptor was engineered to ensure that the receptor was solely activated by the DNA aptamer. The present system features tunability in the signaling output level using aptamer ligands with different receptor dimerization propensities. In addition, the functional programmability of DNA aptamers enables the modular sensing of extracellular molecules without the need for genetic engineering of the receptor.

Molecular mechanism underlying the increased risk of colorectal cancer metastasis caused by single nucleotide polymorphisms in LI-cadherin gene.

LI-cadherin is a member of the cadherin superfamily. LI-cadherin mediates Ca2+-dependent cell-cell adhesion through homodimerization. A previous study reported two single nucleotide polymorphisms (SNPs) in the LI-cadherin-coding gene (CDH17). These SNPs correspond to the amino acid changes of Lys115 to Glu and Glu739 to Ala. Patients with colorectal cancer carrying these SNPs are reported to have a higher risk of lymph node metastasis than patients without the SNPs. Although proteins associated with metastasis have been identified, the molecular mechanisms underlying the functions of these proteins remain unclear, making it difficult to develop effective strategies to prevent metastasis. In this study, we employed biochemical assays and molecular dynamics (MD) simulations to elucidate the molecular mechanisms by which the amino acid changes caused by the SNPs in the LI-cadherin-coding gene increase the risk of metastasis. Cell aggregation assays showed that the amino acid changes weakened the LI-cadherin-dependent cell-cell adhesion. In vitro assays demonstrated a decrease in homodimerization tendency and MD simulations suggested an alteration in the intramolecular hydrogen bond network by the mutation of Lys115. Taken together, our results indicate that the increased risk of lymph node metastasis is due to weakened cell-cell adhesion caused by the decrease in homodimerization tendency.

A case of accessory hepatic duct entering cystic duct successfully treated by laparoscopic cholecystectomy for cholecystolithiasis.

Although laparoscopic cholecystectomy is a well-established surgical procedure, an accessory hepatic duct (AcHD) entering the cystic duct is poorly understood. A 77-year-old woman with symptomatic cholecystlithiasis was referred to our hospital. Abdominal ultrasonography indicated several small stones in the gall bladder. Magnetic resonance cholangiopancreatography (MRCP) did not reveal an anomalous cystic duct. Dissecting the gall bladder bed at operation, AcHD entering the cystic duct was suspected. Intraoperative cholangiography revealed that B5 branch entered the cystic duct. We ligated the AcHD, and divided it. Laparoscopic cholecystectomy was completed, and the patient was discharged without any complication. A week after the operation, MRCP showed that ventral branch of B5 was dilated. The patient showed no symptom for more than a year. The present case exhibited extremely rare AcHD entering the cystic duct, which was hardly recognized before surgery. It is possible to recognize such anomalous variants with standard laparoscopic approach based on 2018 Tokyo Guidelines and with attention to the possibilities of AcHD entering the cystic duct.

Structural Classification of CDR-H3 in Single-Domain VHH Antibodies.

The immune systems protect vertebrates from foreign molecules or antigens, and antibodies are important mediators of this system. The sequences and structural features of antibodies vary depending on species. Many of antibodies from vertebrates, including camelids, have both heavy and light chain variable domains, but camelids also have antibodies that lack the light chains. In antibodies that lack light chains, the C-terminal variable region is called the VHH domain. Antibodies recognize antigens through six complementarity-determining regions (CDRs). The third CDR of the heavy chain (CDR-H3) is at the center of the antigen-binding site and is diverse in terms of sequence and structure. Due to the importance of antibodies in basic science as well as in medical applications, there have been many studies of CDR-H3s of antibodies that possess both light and heavy chains. However, nature of CDR-H3s of single-domain VHH antibodies is less well studied. In this chapter, we describe current knowledge of sequence-structure-function correlations of single-domain VHH antibodies with emphasis on CDR-H3. Based on the 370 crystal structures in the Protein Data Bank, we also attempt structural classification of CDR-H3 in single-domain VHH antibodies and discuss lessons learned from the ever-increasing number of the structures.

Unveiling the affinity-stability relationship in anti-measles virus antibodies: a computational approach for hotspots prediction.

Recent years have seen an uptick in the use of computational applications in antibody engineering. These tools have enhanced our ability to predict interactions with antigens and immunogenicity, facilitate humanization, and serve other critical functions. However, several studies highlight the concern of potential trade-offs between antibody affinity and stability in antibody engineering. In this study, we analyzed anti-measles virus antibodies as a case study, to examine the relationship between binding affinity and stability, upon identifying the binding hotspots. We leverage in silico tools like Rosetta and FoldX, along with molecular dynamics (MD) simulations, offering a cost-effective alternative to traditional in vitro mutagenesis. We introduced a pattern in identifying key residues in pairs, shedding light on hotspots identification. Experimental physicochemical analysis validated the predicted key residues by confirming significant decrease in binding affinity for the high-affinity antibodies to measles virus hemagglutinin. Through the nature of the identified pairs, which represented the relative hydropathy of amino acid side chain, a connection was proposed between affinity and stability. The findings of the study enhance our understanding of the interactions between antibody and measles virus hemagglutinin. Moreover, the implications of the observed correlation between binding affinity and stability extend beyond the field of anti-measles virus antibodies, thereby opening doors for advancements in antibody research.

ACE2 N-glycosylation modulates interactions with SARS-CoV-2 spike protein in a site-specific manner.

SARS-CoV-2 has evolved continuously and accumulated spike mutations with each variant having a different binding for the cellular ACE2 receptor. It is not known whether the interactions between such mutated spikes and ACE2 glycans are conserved among different variant lineages. Here, we focused on three ACE2 glycosylation sites (53, 90 and 322) that are geometrically close to spike binding sites and investigated the effect of their glycosylation pattern on spike affinity. These glycosylation deletions caused distinct site-specific changes in interactions with the spike and acted cooperatively. Of note, the particular interaction profiles were conserved between the SARS-CoV-2 parental virus and the variants of concern (VOCs) Delta and Omicron. Our study provides insights for a better understanding of the importance of ACE2 glycosylation on ACE2/SARS-CoV-2 spike interaction and guidance for further optimization of soluble ACE2 for therapeutic use.

Preoperative serum CA19-9 predicts postoperative pancreatic fistula in PDAC patients: retrospective analysis at a single institution.

BACKGROUND: Postoperative pancreatic fistula (POPF) is a critical complication of pancreatectomy in patients with pancreatic ductal adenocarcinoma (PDAC). Recent papers reported that serum carbohydrate antigen (CA)19-9 levels predicted long-term prognosis. We investigated whether preoperative serum CA19-9 levels were associated with POPF in PDAC patients. METHODS: This cohort study was conducted at a single institution retrospectively. Clinicopathologic features were determined using medical records. RESULTS: Among of 196 consecutive patients who underwent pancreatectomy against PDAC, 180 patients whose CA19-9 levels were above the measurement sensitivity, were registered in this study. The patients consisted of 122 patients who underwent pancreaticoduodenectomy and 58 patients who underwent distal pancreatectomy. Several clinicopathological factors, including CA 19-9 level, as well as surgical factors were determined retrospectively based on the medical records. Patients with high CA19-9 levels had a significantly higher incidence of POPF than those with low levels (43.9 vs. 13.0%, P < 0.0001). The receiver operating characteristic curves calculated that the cutoff CA19-9 value to predict POPF was 428 U/mL. CA19-9, BMI, curability, and histology were statistically significant risk factors for POPF by univariate analysis. Multivariate analysis showed that CA19-9 and BMI levels were statistically significant independent risk factors for POPF. CA19-9 levels were correlated with both histology and curability. Disease free survival and overall survival of patients with higher levels of CA19-9 were significantly shorter than that of patients with lower levels of preoperative serum CA19-9. CONCLUSIONS: In patients undergoing pancreatectomy for PDAC, higher preoperative CA19-9 levels are a significant predictor for POPF.

Molecular basis for thermal stability and affinity in a VHH: Contribution of the framework region and its influence in the conformation of the CDR3.

The camelid single domain antibody, referred to VHH or Nanobody, is considered a versatile tool for various biotechnological and clinical applications because of its favorable biophysical properties. To take advantage of these characteristics and for its application in biotechnology and therapy, research on VHH engineering is currently vigorously conducted. To humanize a camelid VHH, we performed complementarity determining region (CDR) grafting using a humanized VHH currently in clinical trials, and investigated the effects of these changes on the biophysical properties of the resulting VHH. The chimeric VHH exhibited a significant decrease in affinity and thermal stability and a large conformational change in the CDR3. To elucidate the molecular basis for these changes, we performed mutational analyses on the framework regions revealing the contribution of individual residues within the framework region. It is demonstrated that the mutations resulted in the loss of affinity and lower thermal stability, revealing the significance of bulky residues in the vicinity of the CDR3, and the importance of intramolecular interactions between the CDR3 and the framework-2 region. Subsequently, we performed back-mutational analyses on the chimeric VHH. Back-mutations resulted in an increase of the thermal stability and affinity. These data suggested that back-mutations restored the intramolecular interactions, and proper positioning and/or dynamics of the CDR3, resulting in the gain of thermal stability and affinity. These observations revealed the molecular contribution of the framework region on VHHs and further designability of the framework region of VHHs without modifying the CDRs.