Structural basis for antibody recognition of the proximal MUC16 ectodomain.

BACKGROUND: Mucin 16 (MUC16) overexpression is linked with cancer progression, metastasis, and therapy resistance in high grade serous ovarian cancer and other malignancies. The cleavage of MUC16 forms independent bimodular fragments, the shed tandem repeat sequence which circulates as a protein bearing the ovarian cancer biomarker (CA125) and a proximal membrane-bound component which is critical in MUC16 oncogenic behavior. A humanized, high affinity antibody targeting the proximal ectodomain represents a potential therapeutic agent against MUC16 with lower antigenic potential and restricted human tissue expression. RESULTS: Here, we demonstrate the potential therapeutic versatility of the humanized antibody as a monoclonal antibody, antibody drug conjugate, and chimeric antigen receptor. We report the crystal structures of 4H11-scFv, derived from an antibody specifically targeting the MUC16 C-terminal region, alone and in complex with a 26-amino acid MUC16 segment resolved at 2.36 Å and 2.47 Å resolution, respectively. The scFv forms a robust interaction with an epitope consisting of two consecutive ß-turns and a ß-hairpin stabilized by 2 hydrogen bonds. The VH-VL interface within the 4H11-scFv is stabilized through an intricate network of 11 hydrogen bonds and a cation-π interaction. CONCLUSIONS: Together, our studies offer insight into antibody-MUC16 ectodomain interaction and advance our ability to design agents with potentially improved therapeutic properties over anti-CA125 moiety antibodies.

Attogram-level light-induced antigen-antibody binding confined in microflow.

The analysis of trace amounts of proteins based on immunoassays and other methods is essential for the early diagnosis of various diseases such as cancer, dementia, and microbial infections. Here, we propose a light-induced acceleration of antigen-antibody reaction of attogram-level proteins at the solid-liquid interface by tuning the laser irradiation area comparable to the microscale confinement geometry for enhancing the collisional probability of target molecules and probe particles with optical force and fluidic pressure. This principle was applied to achieve a 102-fold higher sensitivity and ultrafast specific detection in comparison with conventional protein detection methods (a few hours) by omitting any pretreatment procedures; 47-750 ag of target proteins were detected in 300 nL of sample after 3 minutes of laser irradiation. Our findings can promote the development of proteomics and innovative platforms for high-throughput bio-analyses under the control of a variety of biochemical reactions.

Quantifying Antibody Binding Kinetics on Fixed Cells and Tissues via Fluorescence Lifetime Imaging.

We present a method for monitoring spatially localized antigen-antibody binding events on physiologically relevant substrates (cell and tissue sections) using fluorescence lifetime imaging. Specifically, we use the difference between the fluorescence decay times of fluorescently tagged antibodies in free solution and in the bound state to track the bound fraction over time and hence deduce the binding kinetics. We make use of a microfluidic probe format to minimize the mass transport effects and localize the analysis to specific regions of interest on the biological substrates. This enables measurement of binding constants (kon) on surface-bound antigens and on cell blocks using model biomarkers. Finally, we directly measure p53 kinetics with differential biomarker expression in ovarian cancer tissue sections, observing that the degree of expression corresponds to the changes in kon, with values of 3.27-3.50 × 103 M-1 s-1 for high biomarker expression and 2.27-2.79 × 103 M-1 s-1 for low biomarker expression.

[The Basic Research of Antigen-antibody Reaction in Lung Transplantation].

Lung transplantation has become popular in Japan, showing better survival rate than other countries. However, the results are still not satisfactory compared with other solid organ transplantation. One of the reasons for this might be that knowledge on donor-specific antibodies or antibody-related rejection, which has been attracting attention these days, is less than that of kidney or liver transplantation. Our laboratory has continued basic research in this field using rodent lung transplantation model. We have previously shown that type V collagen is associated in chronic rejection as an autoimmune, and that oral administration of type V collagen induces tolerance. The murine chronic rejection model of the minor antigen mismatch was developed, and involvement of the humoral immunity and role of the complement activation were shown. We are now studying the effects of immune checkpoint molecules, which play a central role in the field of cancer therapy, on rejection after lung transplantation. We are also working to verify the effects of anti-complement drugs and molecular targeted drugs in the future treatment on rejection.

A Multifunctional Neutralizing Antibody-Conjugated Nanoparticle Inhibits and Inactivates SARS-CoV-2.

The outbreak of 2019 coronavirus disease (COVID-19), caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), has resulted in a global pandemic. Despite intensive research, the current treatment options show limited curative efficacies. Here the authors report a strategy incorporating neutralizing antibodies conjugated to the surface of a photothermal nanoparticle (NP) to capture and inactivate SARS-CoV-2. The NP is comprised of a semiconducting polymer core and a biocompatible polyethylene glycol surface decorated with high-affinity neutralizing antibodies. The multifunctional NP efficiently captures SARS-CoV-2 pseudovirions and completely blocks viral infection to host cells in vitro through the surface neutralizing antibodies. In addition to virus capture and blocking function, the NP also possesses photothermal function to generate heat following irradiation for inactivation of virus. Importantly, the NPs described herein significantly outperform neutralizing antibodies at treating authentic SARS-CoV-2 infection in vivo. This multifunctional NP provides a flexible platform that can be readily adapted to other SARS-CoV-2 antibodies and extended to novel therapeutic proteins, thus it is expected to provide a broad range of protection against original SARS-CoV-2 and its variants.

Key role of a structural water molecule for the specificity of 14F7-An antitumor antibody targeting the NeuGc GM3 ganglioside.

Tumor-associated glycolipids such as NeuGc GM3 are auspicious molecular targets in antineoplastic therapies and vaccine strategies. 14F7 is a monoclonal IgG1 with high clinical potential in cancer immunotherapy as it displays extraordinary specificity for NeuGc GM3, while it does not recognize the very similar, ubiquitous NeuAc GM3. Here we present the 2.3 Å crystal structure of the 14F7 antigen-binding domain (14F7 scFv) in complex with the NeuGc GM3 trisaccharide. Modeling analysis and previous mutagenesis data suggest that 14F7 may also bind to an alternative NeuGc GM3 conformation, not observed in the crystal structure. The most intriguing finding, however, was that a water molecule centrally placed in the complementarity-determining region directly mediates the specificity of 14F7 to NeuGc GM3. This has profound impact on the complexity of engineering in the binding site and provides an excellent example of the importance in understanding the water structure in antibody-antigen interactions.

Evidence for retained spike-binding and neutralizing activity against emerging SARS-CoV-2 variants in serum of COVID-19 mRNA vaccine recipients.

BACKGROUND: Highly efficacious vaccines against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) have been developed. However, the emergence of viral variants that are more infectious than the earlier SARS-CoV-2 strains is concerning. Several of these viral variants have the potential to partially escape neutralizing antibody responses, warranting continued immune-monitoring. METHODS: We used a panel of 30 post-mRNA vaccination sera to determine neutralization and RBD and spike binding activity against a number of emerging viral variants. The virus neutralization was determined using authentic SARS-CoV-2 clinical isolates in an assay format that mimics physiological conditions. FINDINGS: We tested seven currently circulating viral variants of concern/interest, including the three Iota sublineages, Alpha (E484K), Beta, Delta and Lambda in neutralization assays. We found only small decreases in neutralization against Iota and Delta. The reduction was stronger against a sub-variant of Lambda, followed by Beta and Alpha (E484K). Lambda is currently circulating in parts of Latin America and was detected in Germany, the US and Israel. Of note, reduction in a receptor binding domain and spike binding assay that also included Gamma, Kappa and A.23.1 was negligible. INTERPRETATION: Taken together, these findings suggest that mRNA SARS-CoV-2 vaccines may remain effective against these viral variants of concern/interest and that spike binding antibody tests likely retain specificity in the face of evolving SARS-CoV-2 diversity. FUNDING: This work is part of the PARIS/SPARTA studies funded by the NIAID Collaborative Influenza Vaccine Innovation Centers (CIVIC) contract 75N93019C00051. In addition, this work was also partially funded by the Centers of Excellence for Influenza Research and Surveillance (CEIRS, contract # HHSN272201400008C), the JPB Foundation, the Open Philanthropy Project (research grant 2020-215611 (5384), by anonymous donors and by the Serological Sciences Network (SeroNet) in part with Federal funds from the National Cancer Institute, National Institutes of Health, under Contract No. 75N91019D00024, Task Order No. 75N91020F00003.

ATP stabilised and sensitised calcium phosphate nanoparticles as effective adjuvants for a DNA vaccine against cancer.

Cancer vaccines based on DNA encoding oncogenes have shown great potential in preclinical studies. However, the efficacy of DNA vaccines is limited by their weak immunogenicity because of low cellular internalisation and insufficient activation of dendritic cells (DCs). Calcium phosphate (CP) nanoparticles (NPs) are biodegradable vehicles with low toxicity and high loading capacity of DNA but suffer from stability issues. Here we employed adenosine triphosphate (ATP) as a dual functional agent, i.e. stabiliser for CP and immunological adjuvant, and applied the ATP-modified CP (ACP) NPs to the DNA vaccine. ACP NP-enhanced cellular uptake and improved transfection efficiency of DNA vaccine, and further showed the ability to activate DCs that are critical for them to prime T cells in cancer immunotherapy. As a result, a higher level of antigen-specific antibody with stronger tumour growth inhibition was achieved in mice immunised with the ACP-DNA vaccine. Overall, this one-step synthesised ACP NPs are an efficient nano-delivery system and nano-adjuvant for cancer DNA vaccines.

Plant-produced SARS-CoV-2 receptor binding domain (RBD) variants showed differential binding efficiency with anti-spike specific monoclonal antibodies.

Severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) is responsible for the ongoing coronavirus disease (COVID-19) pandemic which is characterized by respiratory illness and severe pneumonia, and currently accounts for > 2.5 million deaths worldwide. Recently, diverse mutations in the spike protein of SARS-CoV-2 were reported in United Kingdom (Alpha) and South Africa (Beta) strains which raise concerns over the potential increase in binding affinity towards the host cell receptor and diminished host neutralization capabilities. In order to study the effect of mutation in the binding efficiency of SARS-CoV-2 receptor binding domain (RBD) with anti-SARS-CoV/CoV-2 monoclonal antibodies (mAbs), we have produced SARS-CoV-2 RBD and two variants SARS-CoV-2 RBD (Alpha RBD and Beta RBD) in Nicotiana benthamiana by transient expression. Plant-produced SARS-CoV-2 RBD-Fc, Alpha RBD-Fc and Beta RBD-Fc exhibited specific binding to human angiotensin converting enzyme 2 (ACE2) receptor determined by ELISA. Intriguingly, the binding of plant-produced SARS-CoV-2 RBD proteins to plant-produced mAbs CR3022, B38, and H4 was found to be different depending on the variant mutation. In contrary to the plant-produced SARS-CoV-2 RBD-Fc and Alpha RBD-Fc, Beta RBD-Fc variant showed weak binding affinity towards the mAbs. The result suggested that the Beta RBD variant might have acquired partial resistance to neutralizing antibodies compared to other variants. However, further studies with sera from convalescent or vaccinated individuals are required to confirm this finding.

A Single-Domain Antibody-Based Anti-PSMA Recombinant Immunotoxin Exhibits Specificity and Efficacy for Prostate Cancer Therapy.

Prostate cancer (PCa) is the second most common cancer in men, causing more than 300,000 deaths every year worldwide. Due to their superior cell-killing ability and the relative simplicity of their preparation, immunotoxin molecules have great potential in the clinical treatment of cancer, and several such molecules have been approved for clinical application. In this study, we adopted a relatively simple strategy based on a single-domain antibody (sdAb) and an improved Pseudomonas exotoxin A (PE) toxin (PE24X7) to prepare a safer immunotoxin against prostate-specific membrane antigen (PSMA) for PCa treatment. The designed anti-PSMA immunotoxin, JVM-PE24X7, was conveniently prepared in its soluble form in an Escherichia coli (E. coli) system, avoiding the complex renaturation process needed for immunotoxin preparation by the conventional strategy. The product was very stable and showed a very strong ability to bind the PSMA receptor. Cytotoxicity assays showed that this molecule at a very low concentration could kill PSMA-positive PCa cells, with an EC50 value (concentration at which the cell viability decreased by 50%) of 15.3 pM against PSMA-positive LNCaP cells. Moreover, this molecule showed very good killing selectivity between PSMA-positive and PSMA-negative cells, with a selection ratio of more than 300-fold. Animal studies showed that this molecule at a very low dosage (5 × 0.5 mg/kg once every three days) completely inhibited the growth of PCa tumors, and the maximum tolerable dose (MTD) was more than 15 mg/kg, indicating its very potent tumor-treatment ability and a wide therapeutic window. Use of the new PE toxin, PE24X7, as the effector moiety significantly reduced off-target toxicity and improved the therapeutic window of the immunotoxin. The above results demonstrate that the designed anti-PSMA immunotoxin, JVM-PE24X7, has good application value for the treatment of PCa.

Deciphering Intracellular Signaling Pathways in Tumoral Pathologies.

The heterogeneity of diseases such as cancer makes it necessary to use high-throughput screening techniques to obtain the maximum number of parameters and characteristics of tumors. These obtained biomarkers can be used for the prediction, prognosis, and treatment or search for new therapeutic targets. In this sense, microarray technology allows exhaustive analysis in a short time and from a great variety of biological samples, becoming a fundamental tool in biomedical research projects. Here, operational process of protein microarrays based on the antibody-antigen interaction is described, emphasizing their application in intracellular signaling pathways in tumoral pathologies. In addition, a final validation using nucleic acid programmable protein array (NAPPA) technology in a simple ELISA assay was included to decipher functional characterization of featured proteins from microarray screening.

Development of an Antigen-Antibody Co-Display System for Detecting Interaction of G-Protein-Coupled Receptors and Single-Chain Variable Fragments.

G-protein-coupled receptors (GPCRs), especially chemokine receptors, are ideal targets for monoclonal antibody drugs. Considering the special multi-pass transmembrane structure of GPCR, it is often a laborious job to obtain antibody information about off-targets and epitopes on antigens. To accelerate the process, a rapid and simple method needs to be developed. The split-ubiquitin-based yeast two hybrid system (YTH) was used as a blue script for a new method. By fusing with transmembrane peptides, scFv antibodies were designed to be anchored on the cytomembrane, where the GPCR was co-displayed as well. The coupled split-ubiquitin system transformed the scFv-GPCR interaction signal into the expression of reporter genes. By optimizing the topological structure of scFv fusion protein and key elements, including signal peptides, transmembrane peptides, and flexible linkers, a system named Antigen-Antibody Co-Display (AACD) was established, which rapidly detected the interactions between antibodies and their target GPCRs, CXCR4 and CXCR5, while also determining the off-target antibodies and antibody-associated epitopes. The AACD system can rapidly determine the association between GPCRs and their candidate antibodies and shorten the research period for off-target detection and epitope identification. This system should improve the process of GPCR antibody development and provide a new strategy for GPCRs antibody screening.

Implementing a method for engineering multivalency to substantially enhance binding of clinical trial anti-SARS-CoV-2 antibodies to wildtype spike and variants of concern proteins.

Infection by the severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) causes COVID-19 disease. Therapeutic antibodies are being developed that interact with the viral spike proteins to limit viral infection of epithelium. We have applied a method to dramatically improve the performance of anti-SARS-CoV-2 antibodies by enhancing avidity through multimerization using simple engineering to yield tetrameric antibodies. We have re-engineered six anti-SARS-CoV-2 antibodies using the human p53 tetramerization domain, including three clinical trials antibodies casirivimab, imdevimab and etesevimab. The method yields tetrameric antibodies, termed quads, that retain efficient binding to the SARS-CoV-2 spike protein, show up to two orders of magnitude enhancement in neutralization of pseudovirus infection and retain potent interaction with virus variant of concern spike proteins. The tetramerization method is simple, general and its application is a powerful methodological development for SARS-CoV-2 antibodies that are currently in pre-clinical and clinical investigation.

Procalcitonin measurement using antibody-conjugated fluorescent microspheres distinguishes atypical bacterial meningitis from viral encephalitis in children.

Examination of cerebrospinal fluid in atypical bacterial meningitis (ABM) is similar to that of viral encephalitis (VE), so ABM can easily be misdiagnosed as VE, which can delay diagnosis and treatment. We developed a simple, rapid hand-held lateral flow immunoassay detection system based on fluorescent microspheres (FMS) for procalcitonin (PCT) detection, which provides an indicator to differentiate between ABM and VE. With this novel method, the antigen-antibody reaction systems involve different species, making the test strips more stable than those utilizing one species. The strips exhibited a wide dynamic range (0.04-50 ng/mL) and good sensitivity (0.03 ng/mL). The function of PCT in the identification of ABM and VE in children was further studied. A significant difference in PCT levels was observed between the ABM and VE groups (P = 0.00) and between the ABM and the normal control groups (P = 0.00). PCT levels were not significantly different between the VE and normal control groups (P = 0.30). The area under the receiver operating characteristic curve of PCT for the diagnosis of ABM was 0.95. These findings collectively indicate the usefulness of the PCT detection method based on FMS for clinically differentiating between ABM and VE.

Synthetic Homogeneous Glycoforms of the SARS-CoV-2 Spike Receptor-Binding Domain Reveals Different Binding Profiles of Monoclonal Antibodies.

SARS-CoV-2 attaches to its host receptor, angiotensin-converting enzyme 2 (ACE2), via the receptor-binding domain (RBD) of the spike protein. The RBD glycoprotein is a critical target for the development of neutralizing antibodies and vaccines against SARS-CoV-2. However, the high heterogeneity of RBD glycoforms may lead to an incomplete neutralization effect and impact the immunogenic integrity of RBD-based vaccines. Investigating the role of different carbohydrate domains is of paramount importance. Unfortunately, there is no viable method for preparing RBD glycoproteins with structurally defined glycans. Herein we describe a highly efficient and scalable strategy for the preparation of six glycosylated RBDs bearing defined structure glycoforms at T323, N331, and N343. A combination of modern oligosaccharide, peptide synthesis and recombinant protein engineering provides a robust route to decipher carbohydrate structure-function relationships.

Development of a novel, fully human, anti-PCSK9 antibody with potent hypolipidemic activity by utilizing phage display-based strategy.

BACKGROUND: Proprotein convertase subtilisin/kexin type 9 (PCSK9) regulates serum LDL cholesterol (LDL-C) levels by facilitating the degradation of the LDL receptor (LDLR) and is an attractive therapeutic target for hypercholesterolemia intervention. Herein, we generated a novel fully human antibody with favourable druggability by utilizing phage display-based strategy. METHODS: A potent single-chain variable fragment (scFv) named AP2M21 was obtained by screening a fully human scFv phage display library with hPCSK9, and performing two in vitro affinity maturation processes including CDR-targeted tailored mutagenesis and cross-cloning. Thereafter, it was transformed to a full-length Fc-silenced anti-PCSK9 antibody FAP2M21 by fusing to a modified human IgG1 Fc fragment with L234A/L235A/N297G mutations and C-terminal lysine deletion, thus eliminating its immune effector functions and mitigating mAb heterogeneity. FINDINGS: Our data showed that the generated full-length anti-PCSK9 antibody FAP2M21 binds to hPCSK9 with a KD as low as 1.42 nM, and a dramatically slow dissociation rate (koff, 4.68 × 10-6 s-1), which could be attributed to its lower binding energy (-47.51 kcal/mol) than its parent counterpart FAP2 (-30.39 kcal/mol). We verified that FAP2M21 potently inhibited PCSK9-induced reduction of LDL-C uptake in HepG2 cells, with an EC50 of 43.56 nM. Further, in hPCSK9 overexpressed C57BL/6 mice, a single tail i.v. injection of FAP2M21 at 1, 3 and 10 mg/kg, dose-dependently up-regulated hepatic LDLR levels, and concomitantly reduced serum LDL-C by 3.3% (P = 0.658, unpaired Student's t-test), 30.2% (P = 0.002, Mann-Whitney U-test) and 37.2% (P = 0.002, Mann-Whitney U-test), respectively. INTERPRETATION: FAP2M21 with potent inhibitory effect on PCSK9 may serve as a promising therapeutic agent for treating hypercholesterolemia and associated cardiovascular diseases.

Substrates Modulate Charge-Reorganization Allosteric Effects in Protein-Protein Association.

Protein function may be modulated by an event occurring far away from the functional site, a phenomenon termed allostery. While classically allostery involves conformational changes, we recently observed that charge redistribution within an antibody can also lead to an allosteric effect, modulating the kinetics of binding to target antigen. In the present work, we study the association of a polyhistidine tagged enzyme (phosphoglycerate kinase, PGK) to surface-immobilized anti-His antibodies, finding a significant Charge-Reorganization Allostery (CRA) effect. We further observe that PGK's negatively charged nucleotide substrates modulate CRA substantially, even though they bind far away from the His-tag-antibody interaction interface. In particular, binding of ATP reduces CRA by more than 50%. The results indicate that CRA is affected by the binding of charged molecules to a protein and provide further insight into the significant role that charge redistribution can play in protein function.

Identification of critical chemical modifications by size exclusion chromatography of stressed antibody-target complexes with competitive binding.

Chemical modifications (attributes) in the binding regions of stressed therapeutic proteins may affect binding to target and efficacy of therapeutic proteins. The method presented here describes the criticality assessment of therapeutic antibody modifications by size-exclusion chromatography (SEC) of competitive binding between a stressed antibody and its target, human epidermal growth factor receptor-2 (HER2), followed by SEC fractionation and peptide mapping characterization of bound and unbound antibodies. When stressed antibody and its target were mixed at a stoichiometric molar ratio of 1:2, only antibody-receptor complex eluted from SEC, indicating that binding was not decreased to break the complex. When a smaller amount of the receptor was provided (1:1), the antibody species with modifications reducing binding eluted as unbound from SEC, while the antibody-receptor complex eluted as the bound fraction. Peptide mapping revealed ratios of modifications between unbound and bound fractions. Statistical analysis after triplicate measurements (n = 3) indicated that heavy chain (HC) D102 isomerization and light chain (LC) N30 deamidation were four-fold higher in unbound fraction with high statistical significance. Although HC N55 deamidation and M107 oxidation were also abundant, they were not statistically different between unbound and bound. Our findings agree with previously published potency measurements of collected CEX fractions and the crystal structure of antibody and HER2. Overall, competitive SEC of stressed antibody-receptor mixture followed by peptide mapping is a useful tool in revealing critical residues and modifications involved in the antibody-target binding, even if they elute as a complex from SEC when mixed at 1:2 stoichiometric ratio.

Changes in epigenetic profiles throughout early childhood and their relationship to the response to pneumococcal vaccination.

BACKGROUND: Pneumococcal infections are a major cause of morbidity and mortality in young children and immaturity of the immune system partly underlies poor vaccine responses seen in the young. Emerging evidence suggests a key role for epigenetics in the maturation and regulation of the immune system in health and disease. The study aimed to investigate epigenetic changes in early life and to understand the relationship between the epigenome and antigen-specific antibody responses to pneumococcal vaccination. METHODS: The epigenetic profiles from 24 healthy children were analyzed at 12 months prior to a booster dose of the 13-valent pneumococcal conjugate vaccine (PCV-13), and at 24 months of age, using the Illumina Methylation 450 K assay and assessed for differences over time and between high and low vaccine responders. RESULTS: Our analysis revealed 721 significantly differentially methylated positions between 12 and 24 months (FDR < 0.01), with significant enrichment in pathways involved in the regulation of cell-cell adhesion and T cell activation. Comparing high and low vaccine responders, we identified differentially methylated CpG sites (P value < 0.01) associated with HLA-DPB1 and IL6. CONCLUSION: These data imply that epigenetic changes that occur during early childhood may be associated with antigen-specific antibody responses to pneumococcal vaccines.