Antibody Candidates Research Articles

Evaluation of a panel of therapeutic antibody clinical candidates for efficacy against SARS-CoV-2 in Syrian hamsters

The COVID-19 pandemic spurred the rapid development of a range of therapeutic antibody treatments. As part of the US government's COVID-19 therapeutic response, a research team was assembled to support assay and animal model development to assess activity for therapeutics candidates against SARS-CoV-2. Candidate treatments included monoclonal antibodies, antibody cocktails, and products derived from blood donated by convalescent patients. Sixteen candidate antibody products were obtained directly from manufacturers and evaluated for neutralization activity against the WA-01 isolate of SARS-CoV-2. Products were further tested in the Syrian hamster model using prophylactic (−24 h) or therapeutic (+8 h) treatment approaches relative to intranasal SARS-CoV-2 exposure. In vivo assessments included daily clinical scores and body weights. Viral RNA and viable virus titers were quantified in serum and lung tissue with histopathology performed at 3d and 7d post-virus-exposure. Sham-treated, virus-exposed hamsters showed consistent clinical signs with concomitant weight loss and had detectable viral RNA and viable virus in lung tissue. Histopathologically, interstitial pneumonia with consolidation was present. Therapeutic efficacy was identified in treated hamsters by the absence or diminution of clinical scores, body weight loss, viral loads, and improved semiquantitative lung histopathology scores. This work serves as a model for the rapid, systematic in vitro and in vivo assessment of the efficacy of candidate therapeutics at various stages of clinical development. These efforts provided preclinical efficacy data for therapeutic candidates. Furthermore, these studies were invaluable for the phenotypic characterization of SARS CoV-2 disease in hamsters and of utility to the broader scientific community.

Production of neutralizing antibody fragment variants in the cytoplasm of E. coli for rapid screening: SARS-CoV-2 a case study

Global health challenges such as the coronavirus pandemic warrant the urgent need for a system that allows efficient production of diagnostic and therapeutic interventions. Antibody treatments against SARS-CoV-2 were developed with an unprecedented pace and this enormous progress was achieved mainly through recombinant protein production technologies combined with expeditious screening approaches. A heterologous protein production system that allows efficient soluble production of therapeutic antibody candidates against rapidly evolving variants of deadly pathogens is an important step in preparedness towards future pandemic challenges. Here, we report cost and time-effective soluble production of SARS-CoV-2 receptor binding domain (RBD) variants as well as an array of neutralizing antibody fragments (Fabs) based on Casirivimab and Imdevimab using the CyDisCo system in the cytoplasm of E. coli. We also report variants of the two Fabs with higher binding affinity against SARS-CoV-2 RBD and suggest this cytoplasmic production of disulfide containing antigens and antibodies can be broadly applied towards addressing future global public health threats.

Learnings about Aβ from human brain recommend the use of a live-neuron bioassay for the discovery of next generation Alzheimer’s disease immunotherapeutics

Despite ongoing debate, the amyloid β-protein (Aβ) remains the prime therapeutic target for the treatment of Alzheimer’s disease (AD). However, rational drug design has been hampered by a lack of knowledge about neuroactive Aβ. To help address this deficit, we developed live-cell imaging of iPSC-derived human neurons (iNs) to study the effects of the most disease relevant form of Aβ-oligomeric assemblies (oAβ) extracted from AD brain. Of ten brains studied, extracts from nine caused neuritotoxicity, and in eight cases this was abrogated by Aβ immunodepletion. Here we show that activity in this bioassay agrees relatively well with disruption of hippocampal long-term potentiation, a correlate of learning and memory, and that measurement of neurotoxic oAβ can be obscured by more abundant non-toxic forms of Aβ. These findings indicate that the development of novel Aβ targeting therapeutics may benefit from unbiased activity-based discovery. To test this principle, we directly compared 5 clinical antibodies (aducanumab, bapineuzumab, BAN2401, gantenerumab, and SAR228810) together with an in-house aggregate-preferring antibody (1C22) and established relative EC50s in protecting human neurons from human Aβ. The results yielded objective numerical data on the potency of each antibody in neutralizing human oAβ neuritotoxicity. Their relative efficacies in this morphological assay were paralleled by their functional ability to rescue oAβ-induced inhibition of hippocampal synaptic plasticity. This novel paradigm provides an unbiased, all-human system for selecting candidate antibodies for advancement to human immunotherapy.

Somatostatin Containing δ-Cell Number Is Reduced in Type-2 Diabetes.

Recent developments suggest that increased glucagon and decreased somatostatin secretion from the pancreas contribute to hyperglycaemia in type-2 diabetes (T2D) patients. There is a huge need to understand changes in glucagon and somatostatin secretion to develop potential anti-diabetic drugs. To further describe the role of somatostatin in the pathogenesis of T2D, reliable means to detect islet δ-cells and somatostatin secretion are necessary. In this study, we first tested currently available anti-somatostatin antibodies against a mouse model that fluorescently labels δ-cells. We found that these antibodies only label 10-15% of the fluorescently labelled δ-cells in pancreatic islets. We further tested six antibodies (newly developed) that can label both somatostatin 14 (SST14) and 28 (SST28) and found that four of them were able to detect above 70% of the fluorescent cells in the transgenic islets. This is quite efficient compared to the commercially available antibodies. Using one of these antibodies (SST10G5), we compared the cytoarchitecture of mouse and human pancreatic islets and found fewer δ-cells in the periphery of human islets. Interestingly, the δ-cell number was also reduced in islets from T2D donors compared to non-diabetic donors. Finally, with the aim to measure SST secretion from pancreatic islets, one of the candidate antibodies was used to develop a direct-ELISA-based SST assay. Using this novel assay, we could detect SST secretion under low and high glucose conditions from the pancreatic islets, both in mice and humans. Overall, using antibody-based tools provided by Mercodia AB, our study indicates reduced δ-cell numbers and SST secretion in diabetic islets.

BJTJ-1837, a novel FXI activation-blocking antibody

BackgroundFactor (F)XI contributes to thrombosis development while it plays a limited role in normal hemostasis. FXI targeting has the potential for preventing and treating thrombosis with little bleeding risk. ObjectivesThe aim of this study was to develop novel antibody therapeutics against FXI for the treatment of thrombosis-related diseases. MethodsMouse hybridoma technology was applied to screen for anti-FXI antibodies. Surface plasma resonance, enzyme inhibition, activated partial thromboplastin time, and prothrombin time assays were conducted to characterize the binding affinity and activity of antibodies. A cynomolgus monkey arterial venous shunt model was applied to validate the antithrombotic activities. ResultsA humanized antibody, BJTJ-1837, reported here bound to the protease domain of FXI and activated FXI with high affinity. BJTJ-1837 fully inhibited the activation of FXI by activated FXII and thrombin. BJTJ-1837 also demonstrated strong anticoagulant activity in human and cynomolgus monkey plasma as measured by activated partial thromboplastin time. Moreover, BJTJ-1837 showed favorable antithrombotic activity with a dose-dependent protection in an arterial venous shunt thrombosis model in cynomolgus monkeys without the bleeding adverse effect. Furthermore, BJTJ-1837 displayed favorable pharmacokinetic and pharmacodynamic properties and good developability. ConclusionAs a potential antithrombotic therapeutic agent with a safe profile, BJTJ-1837 is a very promising FXI activation-blocking antibody candidate.

Human antibodies targeting ENPP1 as candidate therapeutics for cancers.

Ectonucleotide pyrophosphatase/phosphodiesterase 1 (ENPP1) is a type II transmembrane glycoprotein expressed in many tissues. High expression levels of ENPP1 have been observed in many cancer types such as lung cancer, ovarian cancer, and breast cancer. Such overexpression has been associated with poor prognosis in these diseases. Hence, ENPP1 is a potential target for immunotherapy across multiple cancers. Here, we isolated and characterized two high-affinity and specific anti-ENPP1 Fab antibody candidates, 17 and 3G12, from large phage-displayed human Fab libraries. After conversion to IgG1, the binding of both antibodies increased significantly due to avidity effects. Based on these antibodies, we generated antibody-drug conjugates (ADCs), IgG-based bispecific T-cell engagers (IbTEs), and CAR T-cells which all exhibited potent killing of ENPP1-expressing cells. Thus, these various antibody-derived modalities are promising therapeutic candidates for cancers expressing human ENPP1.

Improving antibody optimization ability of generative adversarial network through large language model

Generative adversarial networks (GANs) have successfully generated functional protein sequences. However, traditional GANs often suffer from inherent randomness, resulting in a lower probability of obtaining desirable sequences. Due to the high cost of wet-lab experiments, the main goal of computer-aided antibody optimization is to identify high-quality candidate antibodies from a large range of possibilities, yet improving the ability of GANs to generate these desired antibodies is a challenge. In this study, we propose and evaluate a new GAN called the Language Model Guided Antibody Generative Adversarial Network (AbGAN-LMG). This GAN uses a language model as an input, harnessing such models’ powerful representational capabilities to improve the GAN’s generation of high-quality antibodies. We conducted a comprehensive evaluation of the antibody libraries and sequences generated by AbGAN-LMG for COVID-19 (SARS-CoV-2) and Middle East Respiratory Syndrome (MERS-CoV). Results indicate that AbGAN-LMG has learned the fundamental characteristics of antibodies and that it improved the diversity of the generated libraries. Additionally, when generating sequences using AZD-8895 as the target antibody for optimization, over 50% of the generated sequences exhibited better developability than AZD-8895 itself. Through molecular docking, we identified 70 antibodies that demonstrated higher affinity for the wild-type receptor-binding domain (RBD) of SARS-CoV-2 compared to AZD-8895. In conclusion, AbGAN-LMG demonstrates that language models used in conjunction with GANs can enable the generation of higher-quality libraries and candidate sequences, thereby improving the efficiency of antibody optimization. AbGAN-LMG is available at http://39.102.71.224:88/.

Top Five Considerations When Choosing an Antibody.

The choice of an antibody for a protein-based research study is one of the most crucial steps in any project. Seemingly straightforward, the process is actually quite nuanced and filled with potential pitfalls. In this chapter, we will discuss five major topics that require consideration in the antibody selection process. These include overall study objectives and resources, details of both species and clonality, suitability in applications, and available detection methods. Each section will provide background information on the topic as well as specifics of antibody use in the laboratory. This chapter may be used as a guide to help vet antibody candidates for your project so you can stain with confidence.

A human monoclonal antibody against HBsAg for the prevention and treatment of chronic HBV and HDV infection

Elimination of chronic HBV/HDV infection remains a major global health challenge. Targeting excessive hepatitis B surface antigen (HBsAg) release may provide an interesting window of opportunity to break immune tolerance and to achieve a functional cure using additional antivirals. We evaluated a HBsAg-specific human monoclonal antibody, as part of either a prophylactic or therapeutic strategy, against HBV/HDV infection in cell culture models and in human-liver chimeric mice. To assess prophylactic efficacy, mice were passively immunized prior to infection with HBV or HBV/HDV (coinfection and superinfection setting). Therapeutic efficacy was assessed in HBV and HBV/HDV-coinfected mice receiving 4 weeks of treatment. Viral parameters (HBV DNA, HDV RNA and HBsAg) were assessed in mouse plasma. The antibody could effectively prevent HBV/HDV infection in a dose-dependent manner with IC50 values of ∼3.5ng/ml. Passive immunization showed complete protection of mice from both HBV and HBV/HDV coinfection. Moreover, HDV superinfection was either completely prevented or at least attenuated in HBV-infected mice. Finally, antibody treatment in mice with established HBV/HDV infection resulted in a significant decline in viremia and a concomitant drop in on-treatment HBsAg, with a moderate viral rebound following treatment cessation. We present data on a valuable antibody candidate that could complement other antivirals in strategies aimed at achieving functional cure of chronic HBV and HDV infection. Patients chronically infected with HBV may eventually develop liver cancer and are at great risk of being superinfected with HDV, which worsens and accelerates disease progression. Unfortunately, current treatments can rarely eliminate both viruses from chronically infected patients. In this study, we present data on a novel antibody that is able to prevent chronic HBV/HDV infection in a mouse model with a humanized liver. Moreover, antibody treatment of HBV/HDV-infected mice strongly diminishes viral loads during therapy. This antibody is a valuable candidate for further clinical development.

IM-3 IDENTIFICATION OF THERAPEUTIC TARGET ANTIGENS USING PATIENT DERIVED GLIOBLASTOMA AND THEIR APPLICATION TO CAR-T THERAPY

Abstract Introduction Recently, CAR-T cell (chimertic antigen receptor T-cell) therapy has been attracting and has shown high therapeutic efficacy, especially in the hematological tumors. The T cells from patient are then genetically engineered to specifically bind to and attack the target tumor cell antigen. In the brain, EGFRvIII or HER2 have been used as antigens, but have yet to show positive results. It is necessary to find antibodies that are more specific and have a therapeutic effect. Methods A primary culture line was prepared from glioblastoma surgical specimens, and immunized to Balb/c mice with it.The B cells from the mice were fused with myeloma cells, immortalized, and cultured monoclonally to obtain a number of monoclonal antibodies reacted glioblastoma cells. The obtained antibodies were reacted with glioblastoma cells and normal brain cells, and those that specifically react to glioblastoma were selected by flowcytometry to obtain antibody candidates that could be specifically expressed on the surface of glioblastoma cells. Results Approximately 20,000 antibody-producing strains were generated. From these, about 3,200 strains were selected as the reacted with immunized glioblastoma. About 560 strains was selected that reacted with at least one other glioblastoma, and from these, we selected 30 strains that did not react with normal brain cells. Finally, the cells were frozen and thawed at least once, and 13 stably growing strains selected. Discussion We have been identified one antibody among approximately 500 strains in past our study, and have confirmed that it is B7H3, a pan-tumor antibody. We will generate more antibody candidates in the future, as well as identify antibodies in 13 strains that have already been selected and confirmed to be glioblastoma-specific antibodies. We will also create CAR-T cells targeting these antibodies and confirm their anti-tumor effects.

Targeting Myeloid Leukemia Using Novel TCR-like Chimeric Antigen Receptor-T Cells Against Cathepsin G

Cathepsin G (CG) is a serine protease that is normally contained within primary granules of neutrophils and is expressed during the promyelocyte stage of myeloid cell development. We reported CG to be highly expressed in acute myeloid leukemia (AML) blasts compared with normal myeloid progenitors and leukocytes. We also showed CG to be a promising immunotherapeutic target in AML, having eluted a CG-derived peptide, FLLPTGAEA (hereafter referred to as CG1), from the surface of primary HLA-A*0201 (HLA-A2+) AML blasts and AML cell lines. Additionally, we showed CG expression in acute lymphoblastic leukemia (ALL) through endogenous expression by some ALL subsets and by cross-presentation from the marrow microenvironment. We also showed that CG1-specific cytotoxic T-lymphocytes (CTL) killed HLA-A2+ primary AML blasts and cell lines, but not normal myeloid progenitor cells. Furthermore, we detected CG1-CTL in peripheral blood mononuclear cells (PBMC) from AML patients following allogenic stem cell transplant. Here we report on the engineering, preclinical efficacy, and safety of CG1-targeting chimeric antigen receptor (CAR) T cells, which employ a novel T cell receptor-like (TCR-L) CAR construct that binds cell surface CG1/HLA-A2. Methods Time-of-flight mass spectrometry was used to quantify CG1 expression on the cell surface. The antibody targeting CG1/HLA-A2 was developed by immunizing Harbour Mice with CG1/HLA-A2 complex, FACS-sorting of single B cells, screening B cell antibody-containing supernatant for binding to CG1/HLA-A2, and fusion cloning of VH and VL sequences from positively screened B cells. OCTET BLI assays were used to determine the binding of different alanine substituted CG1 monomers to lead antibodies. The affinity of each monomer was calculated based on BLI data. T2 binding assays were used to validate the binding of antibody monomers to cell surface CG1/HLA-A2 complexes. Immunohistochemistry (IHC) using human fresh and frozen PBMC and marrow was used to show the specificity of anti-CG1/HLA-A2 antibodies. CG1 ScFV was cloned into pORBIT-LENTI-IRES-EGFR-III vector co-expressing either CD28 (CG1-CD28-CAR) or 4-1BB (CG1-41BB-CAR). HLA-A2+ normal donor (ND) T- cells were activated with anti-CD3 and anti-CD28 antibodies before lentiviral transduction with CG1 CAR constructs. Transduced cells were expanded and then characterized in vitro to determine CAR T quality. In vitro flow cytometry cytotoxicity assays were used to confirm in vitro killing of leukemia by CG1-CAR T cells. NSG mice engrafted with AML cell lines and AML patient-derived xenografts (PDX) were used to demonstrate the in vivo efficacy/pharmacokinetics/pharmacodynamics of CG1-CAR T cells from different donors. Results We eluted CG1 peptide from the cell surface in 7 of 8 HLA-A2+ myeloid leukemia cell lines. CG1 was absent on the cell surface of HLA-A2+ non-myeloid solid tumor and lymphoid cell lines, as well as in normal HLA-A2+ neutrophils and PBMC. Screening of different antibody clones for binding to CG1 peptide with different alanine amino acid substitutions identified top 5 antibody candidates with high affinity (single to sub-digit nM) for CG1-HLA-A2. T2 binding assays demonstrated high binding specificity of these five CG1 antibodies to CG1 monomers. The lead antibodies for CAR-T development were selected based on high antibody binding affinities (0.14-5 nM), specificity for CG1/HLA-A2, and no binding to ND marrow and PBMC. In vitro cytotoxicity assays confirmed in vitro killing of HLA-A2-transduced U937 (U937-A2) cells by CG1-CAR T cells. Both CG1-CD28- and the CG1-41BB-CAR constructs demonstrated high levels of killing. Lastly, in vivo AML mouse models using ML-2 and U937-A2 cell lines, and AML PDX validated the in vivo activity of CG1-CD28- and CG1-41BB-CAR T cells (Figure). Conclusion Targeting CG1 using immunotherapy appears to be a valid approach in the setting of AML. We first showed that CG was highly expressed and presented on AML blasts. We developed TCRL-scFv that targets CG1/HLA-A2 on the cell surface and generated two different CAR constructs based on the scFv antibody sequence. We then showed potent anti-leukemia activity and specificity of CG1-CAR T cells against human leukemia in vitro and in vivo. Together, these data provide the pre-clinical proof of concept work that lays the foundation for further development of CG1-CAR T cells as a potential immunotherapy for patients with AML. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Streptavidin-Drug Conjugates Streamline Identification of Optimal Toxic Payloads for Antibody-Based Hematopoietic Stem Cell Transplantation Conditioning

INTRODUCTION: Allogeneic hematopoietic stem cell transplantation (allo-HSCT) provides the best and possibly only chance for cure for acute myeloid leukemia (AML). In preparation for allo-HSCT, patients undergo conditioning with chemotherapy and/or irradiation to deplete host hematopoietic stem cells (HSCs), suppress the host immune system, and destroy residual leukemia cells. However, conditioning associated toxicities impose a formidable barrier to patients undergoing this potentially curative therapy. Antibody-drug conjugates (ADCs) have emerged as a promising and potentially safer alternative for allo-HSCT conditioning by allowing targeted depletion of the host HSC niche. Previously, we used saporin-conjugated CD45- or cKit-ADCs as the backbone of a novel conditioning regimen which enabled engraftment between fully mismatched mice. However, saporin ADCs were nonmyeloablative and showed modest antitumor activity. We hypothesized that fully myeloablative ADCs capable of enhanced antileukemia activity could be produced using alternative toxic payloads to saporin. However, direct conjugation of multiple candidate payloads and/or antibodies is costly, time-consuming, and laborious. Here, we describe the production of a panel of streptavidin (SAv)-drug conjugates which allows efficient ADC generation from any biotinylated protein. This flexible platform enabled the rapid production of CD45-ADCs conjugated to multiple toxic payloads, facilitating in vitro screening of their ability to target HSCs and leukemia cells. METHODS: Azide-alkyne cycloaddition ("Click" chemistry) was used to covalently link SAv-azide (Protein Mods, Inc) with dibenzocyloocytne (DBCO)-coupled drug payloads (Levena Biopharma) (Fig. 1). We conjugated three toxic payloads to SAv for these studies: monomethyl auristatin E (MMAE), Duocarmycin SA (Duo), and PNU-159682 (PNU). All of these payloads contain cleavable valine-citrulline linkers for payload release following ADC internalization. For the conjugation reactions, SAv-azide was combined with DBCO-coupled payload and incubated 2 hours at 20oC, with 20% DMSO added as cosolvent. Free payload and DMSO were removed using size exclusion chromatography and dialysis. ADCs were freshly prepared before use by mixing SAv-drug conjugates in a 1:1 molar ratio for 15 minutes with biotinylated antibodies. For our initial studies, we evaluated murine anti-CD45.2 (Clone 104) and human anti-CD45 (Clones BC8 and HI30). Non-biotinylated antibodies combined with SAv-drug conjugates were used as negative controls. All ADCs were tested for cytotoxicity in vitro against 1) murine YAC-1 (mCD45.2+) and human Jurkat (mCD45.2-) cell lines using XTT viability assays, 2) primary HSCs from B6 mice using methylcellulose assays, and 3) AML1 primary murine leukemia cells (Dnmt3aR878H/+ and FLT3-ITD; provided by Dr. Tim Ley) using methylcellulose assays. IC50 values for each assay were derived with nonlinear regression of dose response data. RESULTS: Mouse CD45-Duo and CD45-PNU were highly effective at killing AML1 cells (IC50 values: 39.2pM and 61.1pM, respectively; Fig. 2), as well as B6 HSCs (IC50 values: 0.85nM and 0.13nM, respectively) in vitro. Notably, mouse CD45-MMAE was ineffective against YAC-1 cells, B6 HSCs, and AML1 cells, suggesting that MMAE is not an effective payload for ADC-based conditioning in the mouse. Importantly, nonbiotinylated antibodies mixed with SAv-drug conjugates were largely ineffective, indicating the need for the antibody to bind the SAv-drug conjugate to achieve specific toxicity. Preliminary studies using human CD45-MMAE and CD45-Duo demonstrated that while both payloads could yield ADCs able to kill Jurkat cells, ADCs made using human antibody clone BC8 were considerably more effective than ADCs made with clone HI30. CONCLUSIONS: Our novel SAv-drug conjugate successfully enabled rapid generation of ADCs with a number of potential toxic payloads. This system identified multiple effective candidate payloads for use in mouse and human ADCs for targeting HSCs and leukemia cells in vitro and in vivo. Our ongoing and future studies with this system are focused in three areas: 1) production and testing of mouse and human cKit ADCs, 2) using mouse ADCs to condition for murine HSCT and target AML1 cells in vivo, and 3) evaluating killing of human cord blood-derived HSCs and patient-derived AML cells with human ADCs. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Developability assessment at early-stage discovery to enable development of antibody-derived therapeutics.

Developability refers to the likelihood that an antibody candidate will become a manufacturable, safe and efficacious drug. Although the safety and efficacy of a drug candidate will be well considered by sponsors and regulatory agencies, developability in the narrow sense can be defined as the likelihood that an antibody candidate will go smoothly through the chemistry, manufacturing and control (CMC) process at a reasonable cost and within a reasonable timeline. Developability in this sense is the focus of this review. To lower the risk that an antibody candidate with poor developability will move to the CMC stage, the candidate's developability-related properties should be screened, assessed and optimized as early as possible. Assessment of developability at the early discovery stage should be performed in a rapid and high-throughput manner while consuming small amounts of testing materials. In addition to monoclonal antibodies, bispecific antibodies, multispecific antibodies and antibody-drug conjugates, as the derivatives of monoclonal antibodies, should also be assessed for developability. Moreover, we propose that the criterion of developability is relative: expected clinical indication, and the dosage and administration route of the antibody could affect this criterion. We also recommend a general screening process during the early discovery stage of antibody-derived therapeutics. With the advance of artificial intelligence-aided prediction of protein structures and features, computational tools can be used to predict, screen and optimize the developability of antibody candidates and greatly reduce the risk of moving a suboptimal candidate to the development stage.

Structure-Based Optimization of Antibody-Based Biotherapeutics for Improved Developability: A Practical Guide for Molecular Modelers.

A great effort to avoid known developability risks is now more often being made earlier during the lead candidate discovery and optimization phase of biotherapeutic drug development. Predictive computational strategies, used in the early stages of antibody discovery and development, to mitigate the risk of late-stage failure of antibody candidates, are highly valuable. Various structure-based methods exist for accurately predicting properties critical to developability, and, in this chapter, we discuss the history of their development and demonstrate how they can be used to filter large sets of candidates arising from target affinity screening and to optimize lead candidates for developability. Methods for modeling antibody structures from sequence and detecting post-translational modifications and chemical degradation liabilities are also discussed.

Prevention of Shiga toxin 1-caused colon injury by plant-derived recombinant IgA

Immunoglobulin A (IgA) is a candidate antibody for oral passive immunization against mucosal pathogens like Shiga toxin-producing Escherichia coli (STEC). We previously established a mouse IgG monoclonal antibody (mAb) neutralizing Shiga toxin 1 (Stx1), a bacterial toxin secreted by STEC. We designed cDNA encoding an anti-Stx1 antibody, in which variable regions were from the IgG mAb and all domains of the heavy chain constant region from a mouse IgA mAb. Considering oral administration, we expressed the cDNA in a plant expression system aiming at the production of enough IgA at low cost. The recombinant-IgA expressed in Arabidopsis thaliana formed the dimeric IgA, bound to the B subunit of Stx1, and neutralized Stx1 toxicity to Vero cells. Colon injury was examined by exposing BALB/c mice to Stx1 via the intrarectal route. Epithelial cell death, loss of crypt and goblet cells from the distal colon were observed by electron microscopy. A loss of secretory granules containing MUC2 mucin and activation of caspase-3 were observed by immunohistochemical methods. Pretreatment of Stx1 with the plant-based recombinant IgA completely suppressed caspase-3 activation and loss of secretory granules. The results indicate that a plant-based recombinant IgA prevented colon damage caused by Stx1 in vivo.

N-terminal mutant huntingtin deposition correlates with CAG repeat length and symptom onset, but not neuronal loss in Huntington's disease

Huntington's disease (HD) is caused by a CAG repeat expansion mutation in the gene encoding the huntingtin (Htt) protein, with mutant Htt protein subsequently forming aggregates within the brain. Mutant Htt is a current target for novel therapeutic strategies for HD, however, the lack of translation from preclinical research to disease-modifying treatments highlights the need to improve our understanding of the role of Htt protein in the human brain. This study aims to undertake an immunohistochemical screen of 12 candidate antibodies against various sequences along the Htt protein to characterize Htt distribution and expression in post-mortem human brain tissue microarrays (TMAs).Immunohistochemistry was performed on middle temporal gyrus TMAs comprising of up to 28 HD and 27 age-matched control cases, using 12 antibodies specific to various sequences along the Htt protein. From this study, six antibodies directed to the Htt N-terminus successfully immunolabeled human brain tissue. Htt aggregates and Htt protein expression levels for the six successful antibodies were subsequently quantified with a customized automated image analysis pipeline on the TMAs. A 2.5–12 fold increase in the number of Htt aggregates were detected in HD cases using antibodies MAB5374, MW1, and EPR5526, despite no change in overall Htt protein expression compared to control cases, suggesting a redistribution of Htt into aggregates in HD. MAB5374, MW1, and EPR5526 Htt aggregate numbers were positively correlated with CAG repeat length, and negatively correlated with the age of symptom onset in HD. However, the number of Htt aggregates did not correlate with the degree of striatal degeneration or the degree of cortical neuron loss. Together, these results suggest that longer CAG repeat lengths correlate with Htt aggregation in the HD human brain, and greater Htt cortical aggregate deposition is associated with an earlier age of symptom onset in HD. This study also reinforces that antibodies MAB5492, MW8, and 2B7 which have been utilized to characterize Htt in animal models of HD do not specifically immunolabel Htt aggregates in HD human brain tissue exclusively, thereby highlighting the need for validated means of Htt detection to support drug development for HD.

Baculovirus-Free SARS-CoV-2 Virus-like Particle Production in Insect Cells for Rapid Neutralization Assessment.

Virus-like particles (VLPs) resemble authentic virus while not containing any genomic information. Here, we present a fast and powerful method for the production of SARS-CoV-2 VLP in insect cells and the application of these VLPs to evaluate the inhibition capacity of monoclonal antibodies and sera of vaccinated donors. Our method avoids the baculovirus-based approaches commonly used in insect cells by employing direct plasmid transfection to co-express SARS-CoV-2 envelope, membrane, and spike protein that self-assemble into VLPs. After optimization of the expression plasmids and vector ratios, VLPs with an ~145 nm diameter and the typical “Corona” aura were obtained, as confirmed by nanoparticle tracking analysis (NTA) and transmission electron microscopy (TEM). Fusion of the membrane protein to GFP allowed direct quantification of binding inhibition to angiotensin II-converting enzyme 2 (ACE2) on cells by therapeutic antibody candidates or sera from vaccinated individuals. Neither VLP purification nor fluorescent labeling by secondary antibodies are required to perform these flow cytometric assays.

SARS-CoV-2 neutralizing camelid heavy-chain-only antibodies as powerful tools for diagnostic and therapeutic applications.

IntroductionThe ongoing COVID-19 pandemic situation caused by SARS-CoV-2 and variants of concern such as B.1.617.2 (Delta) and recently, B.1.1.529 (Omicron) is posing multiple challenges to humanity. The rapid evolution of the virus requires adaptation of diagnostic and therapeutic applications.ObjectivesIn this study, we describe camelid heavy-chain-only antibodies (hcAb) as useful tools for novel in vitro diagnostic assays and for therapeutic applications due to their neutralizing capacity.MethodsFive antibody candidates were selected out of a naïve camelid library by phage display and expressed as full length IgG2 antibodies. The antibodies were characterized by Western blot, enzyme-linked immunosorbent assays, surface plasmon resonance with regard to their specificity to the recombinant SARS-CoV-2 Spike protein and to SARS-CoV-2 virus-like particles. Neutralization assays were performed with authentic SARS-CoV-2 and pseudotyped viruses (wildtype and Omicron).ResultsAll antibodies efficiently detect recombinant SARS-CoV-2 Spike protein and SARS-CoV-2 virus-like particles in different ELISA setups. The best combination was shown with hcAb B10 as catcher antibody and HRP-conjugated hcAb A7.2 as the detection antibody. Further, four out of five antibodies potently neutralized authentic wildtype SARS-CoV-2 and particles pseudotyped with the SARS-CoV-2 Spike proteins of the wildtype and Omicron variant, sublineage BA.1 at concentrations between 0.1 and 0.35 ng/mL (ND50).ConclusionCollectively, we report novel camelid hcAbs suitable for diagnostics and potential therapy.

Prediction of the structural interface between fibroblast growth factor23 and Burosumab using alanine scanning and molecular docking

Burosumab, an FGF23 targeting monoclonal antibody, was approved by the FDA in 2018 for use in children and adults with X-linked hypophosphatemia (or XLH). While several clinical studies have demonstrated the long-term safety and efficacy of Burosumab, the molecular basis of FGF23-Burosumab interaction which underpins its mechanism of action remains unknown. In this study, we employed molecular docking combined with alanine scanning of epitope and paratope to predict a model of FGF23-Burosumab interaction. Then, we used the model to understand the species-species cross-reactivity of Burosumab and to reverse engineer mouse FGF23 with 'back to human' mutations to bind Burosumab. Finally, we redesigned the CDRs with two mutations to engineer an affinity enhanced variant of the antibody. Our study provides insights into the FGF23-Burosumab interaction and demonstrates that alanine-scanning coupled with molecular docking can be used to optimize antibody candidates (e.g., structure-guided affinity maturation) for therapeutic use.

Potently neutralizing and protective anti-human metapneumovirus antibodies target diverse sites on the fusion glycoprotein

Human metapneumovirus (hMPV) is a leading cause of acute lower respiratory tract infections in high-risk populations, yet there are no vaccines or anti-viral therapies approved for the prevention or treatment of hMPV-associated disease. Here, we used a high-throughput single-cell technology to interrogate memory B cell responses to the hMPV fusion (F)glycoprotein in young adult and elderly donors. Across all donors, the neutralizing antibody response was primarily directed to epitopes expressed on both pre- and post-fusion F conformations. However, we identified rare, highly potent broadly neutralizing antibodies that recognize pre-fusion-specific epitopes and structurally characterized an antibody that targets a site of vulnerability at the pre-fusion F trimer apex. Additionally, monotherapy with neutralizing antibodies targeting three distinct antigenic sites provided robust protection against lower respiratory tract infection in a small animal model. This study provides promising monoclonal antibody candidates for passive immunoprophylaxis and informs the rational design of hMPV vaccine immunogens.