High-affinity Antibodies Research Articles

Polarization toward Tfh2 cell involved in development of MBC and antibody responses against Plasmodium vivax infection.

Plasmodium vivax is the dominant Plasmodium spp. causing malaria throughout tropical and sub-tropical countries. Humoral immunity is induced during P. vivax infection. However, data on longevity of antibody and memory B cell (MBC) responses is lacking. Follicular helper T cells (Tfh) are drivers of high-affinity and long-lived antibody responses. Understanding of Tfh-mediated immunity against malaria is valuable for vaccine development. We enrolled 31 acutely infected P. vivax patients in low malaria transmission areas of Thailand to detect frequencies, phenotypes and kinetics of different subsets of circulating Tfh (cTfh) and MBCs, and to evaluate their association with humoral immunity following natural P. vivax infection. Expansion of cTfh2 cells, activated and atypical MBCs were shown during acute malaria. To relate increased cTfh2 cells to humoral immunity, P. vivax-specific MBCs and antibodies were assessed. High anti-PvCSP and -PvDBPII seropositivity was detected and most subjects produced MBCs specific to these antigens. The increased cTfh2 cells were positively related to atypical MBCs, plasmablasts/plasma cells, and anti-PvDBPII IgM and IgG levels. Distributions of memory cTfh cell subsets were altered from central memory (CM) to effector memory (EM) during infection. The highest ratios of cTfh-EM/cTfh-CM were represented in cTfh2 cells. Positive correlation of cTfh17-EM with activated and atypical MBCs was observed, while cTfh2-CM and cTfh17-CM cells were positively related to PvDBPII-specific MBCs and IgM levels. Present study demonstrated that P. vivax infection induced cTfh polarization into cTfh2 subset, and alteration of memory cTfh2 phenotype from CM to EM phase. These P. vivax-induced cTfh responses significantly associated with generation of MBCs and antibody responses. Therefore, cTfh2 cells might possibly influence humoral immunity by inducing expansion of activated and atypical MBCs, and by generating P. vivax-specific MBCs and antibody responses following natural infection.

Mathematical Modeling Unveils Optimization Strategies for Targeted Radionuclide Therapy of Blood Cancers.

Targeted radionuclide therapy is based on injections of cancer-specific molecules conjugated with radioactive nuclides. Despite the specificity of this treatment, it is not devoid of side-effects limiting its use and is especially harmful for rapidly proliferating organs well perfused by blood, like bone marrow. Optimization of radioconjugates administration accounting for toxicity constraints can increase treatment efficacy. Based on our experiments on disseminated multiple myeloma mouse model treated by 225Ac-DOTA-daratumumab, we developed a mathematical model which investigation highlighted the following principles for optimization of targeted radionuclide therapy. 1) Nuclide to antibody ratio importance. The density of radioconjugates on cancer cells determines the density of radiation energy deposited in them. Low labeling ratio as well as accumulation of unlabeled antibodies and antibodies attached to decay products in the bloodstream can mitigate cancer radiation damage due to excessive occupation of specific receptors by antibodies devoid of radioactive nuclides. 2) Cancer binding capacity-based dosing. The total number of specific receptors on cancer cells is a critical factor for treatment optimization, which estimation may allow increasing treatment efficacy close to its theoretical limit. Injection of doses significantly exceeding cancer binding capacity should be avoided since radioconjugates remaining in the bloodstream have negligible efficacy to toxicity ratio. 3) Particle range-guided multi-dosing. The use of short-range particle emitters and high-affinity antibodies can allow for robust treatment optimization via initial saturation of cancer binding capacity, enabling redistribution of further injected radioconjugates and deposited dose towards still viable cells that continue expressing specific receptors.

Preparation and Construction of Chimeric Humanized Broadly Reactive Antibody 10H10 to Protein E of Tick-Borne Encephalitis Virus.

A full-length humanized chimeric antibody 10H10ch that specifically interacts with the surface glycoprotein E of flaviviruses was obtained. To construct it, we used variable fragments of the heavy and light chains of the monoclonal antibody 10H10 that form the active center of the antibody and a fragment of the constant part of the heavy chain of the human IgG1 antibody. The resulting full-length chimeric humanized antibody 10H10ch specifically interacted with the E protein of flaviviruses pathogenic to humans, such as tick-borne encephalitis, Zika, West Nile, and dengue viruses. An immunochemical assessment of the interaction constants of the 10H10ch antibody with a panel of native and recombinant flavivirus antigens by ELISA and biolayer interferometry showed that the dissociation constant (Kd) of the chimeric antibody is in the nanomolar region and is comparable to that of the high-affinity mouse monoclonal antibody 10H10. The possibility of using the resulting chimeric humanized antibody 10H10ch for the diagnosis, prevention, and treatment of various flavivirus infections is discussed.

Monoclonal antibody targeting CEACAM1 enhanced the response to anti-PD1 immunotherapy in non-small cell lung cancer

Carcinoembryonic antigen-related cellular adhesion molecule 1 (CEACAM1), an extensively studied cell surface molecule, mainly expressed by certain epithelial, endothelial, lymphoid and myeloid cells, and is an attractive target for cancer immunotherapy. Here, to investigate the anti-tumor effects and mechanisms of CEACAM1 antibody, we prepared the antibody and explored its anti-tumor effects on Non-small Cell Lung Cancer (NSCLC) in vitro and in vivo. Firstly, antigen of human CEACAM1 recombinant protein was immunized on BALB/c mice and the high-affinity mouse anti-human monoclonal antibody 3C11 was selected by hybridoma technique. Next, ELISA was applied to detect the blocking effects of 3C11 on CEACAM1-CEACAM1 and CEACAM1-CEACAM5. Then, cell assays and ELISA were used to evaluate the role of 3C11 in blocking CEACAM1-CEACAM1 immunosuppressive signal transduction between dendritic cells (DCs) and T cells or natural killer cells (NK) and tumor cells. Finally, the synergistic anti-tumor effect of 3C11 combined with anti-PD-1 antibody was evaluated through cell stimulation assays and NCI-H358-induced tumor models in mice. The results showed the EC50 of 3C11 binding to NCI-H358 or exhausted T cells were 0.04971 μg/mL and 0.03475 μg/mL, respectively. 3C11 activated the exhausted T cells and enhanced the killing effect of NK by blocking CEACAM1-CEACAM1. In addition, the combination of 3C11 and anti-PD1 antibody produced synergistic anti-tumor effect on NSCLC. Its improved tumor growth inhibition value (TGI) of anti-PD-1 from 18 % to 85 % in vivo. These findings suggest that 3C11 can be considered an effective immunotherapy drug for NSCLC.

A mathematical model simulating the adaptive immune response in various vaccines and vaccination strategies

Vaccination has been widely recognized as an effective measure for preventing infectious diseases. To facilitate quantitative research into the activation of adaptive immune responses in the human body by vaccines, it is important to develop an appropriate mathematical model, which can provide valuable guidance for vaccine development. In this study, we constructed a novel mathematical model to simulate the dynamics of antibody levels following vaccination, based on principles from immunology. Our model offers a concise and accurate representation of the kinetics of antibody response. We conducted a comparative analysis of antibody dynamics within the body after administering several common vaccines, including traditional inactivated vaccines, mRNA vaccines, and future attenuated vaccines based on defective interfering viral particles (DVG). Our findings suggest that booster shots play a crucial role in enhancing Immunoglobulin G (IgG) antibody levels, and we provide a detailed discussion on the advantages and disadvantages of different vaccine types. From a mathematical standpoint, our model proposes four essential approaches to guide vaccine design: enhancing antigenic T-cell immunogenicity, directing the production of high-affinity antibodies, reducing the rate of IgG decay, and lowering the peak level of vaccine antigen-antibody complexes. Our study contributes to the understanding of vaccine design and its application by explaining various phenomena and providing guidance in comprehending the interactions between antibodies and antigens during the immune process.

MRNA-based influenza vaccine expands breadth of B cell response in humans.

Eliciting broad and durable antibody responses against rapidly evolving pathogens like influenza viruses remains a formidable challenge. The germinal center (GC) reaction enables the immune system to generate broad, high-affinity, and durable antibody responses to vaccination. mRNA-based severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) vaccines induce persistent GC B cell responses in humans. Whether an mRNA-based influenza vaccine could induce a superior GC response in humans compared to the conventional inactivated influenza virus vaccine remains unclear. We assessed B cell responses in peripheral blood and draining lymph nodes in cohorts receiving the inactivated or mRNA-based quadrivalent seasonal influenza vaccine. Participants receiving the mRNA-based vaccine produced more robust plasmablast responses and higher antibody titers to H1N1 and H3N2 influenza A viruses and comparable antibody titers against influenza B virus strains. Importantly, mRNA-based vaccination stimulated robust recall B cell responses characterized by sustained GC reactions that lasted at least 26 weeks post-vaccination in three of six participants analyzed. In addition to promoting the maturation of responding B cell clones, these sustained GC reactions resulted in enhanced engagement of low-frequency pre-existing memory B cells, expanding the landscape of vaccine-elicited B cell clones. This translated to expansion of the serological repertoire and increased breadth of serum antibody responses. These findings reveal an important role for the induction of persistent GC responses to influenza vaccination in humans to broaden the repertoire of vaccine-induced antibodies.

Humanized Recombinant Immunotoxin Targeting hCG Demonstrates Therapeutic Potential for Advanced Stage Difficult to Treat Cancers

We report the development of an immunotherapeutic molecule, a humanized immunotoxin, for treating hCG-expressing advanced-stage cancers. PiPP, a high-affinity anti-hCG monoclonal antibody, is used in the immunotoxin for ‘homing’ hCG-positive cancer cells. The deimmunized (DI) form of α-Sarcin, a fungal-origin toxin that lacks functional T-cell epitopes, is used in the design to ensure minimal immunogenicity of the immunotoxin for repetitive use in humans. A single-chain variable fragment (scFv) of PiPP was constructed by linking the humanized VH and VL regions of the antibody. The scFv part of the antibody was further linked to the toxin α-Sarcin (DI) at the gene level and expressed as a recombinant protein in E. coli. The immunotoxin was purified from the bacterial cell lysate and analyzed for binding and cytotoxicity to hCG-secreting colorectal and pancreatic cancer cells. The results showed that the scFv(PiPP)-Sarcin immunotoxin was able to bind to colorectal and pancreatic cancer cells and killed approximately 85% of the cells. In vivo testing of the immunotoxin produced results similar to those of in vitro testing against colorectal adenocarcinoma-induced tumors. This immunotoxin could be a promising immunotherapeutic agent for treating colorectal, pancreatic and other terminal-stage hCG-expressing cancers.

Denosumab for Management of Hypercalcemia in Primary Hyperparathyroidism.

Denosumab is a completely human monoclonal high-affinity antibody that binds to the nuclear factor kappa-B ligand (RANKL) and is widely used to treat osteoporosis. Furthermore, it can potentially lower serum calcium levels by inhibiting osteoclast activation and preventing bone calcium from being released into the blood. This review aimed to provide evidence of the efficacy and safety of denosumab in treating hypercalcemia in primary hyperparathyroidism (PHPT). PubMed and the Cochrane Library were searched for published studies that described denosumab for hypercalcemia management in PHPT. Data were extracted by two independent investigators and analyzed using SPSS 23. The risk of bias was assessed by NIH Quality Assessment Tool. In total, 161 patients with PHPT from 18 studies were included in this review. The average age was 61 (47-72) years and the highest serum calcium was 3.76 (3.11-4.20) mmol/l. We found that denosumab can effectively reduce the serum calcium level by a median reduction of 0.5 mmol/l within 3 days. Significant reduction was maintained for 14 days. The serum calcium-lowering effect weakened after one month. In conclusion, denosumab has a potential clinical value in treating hypercalcemia in patients with PHPT.

B Cell Lymphoma 6 (BCL6): A Conserved Regulator of Immunity and Beyond

B cell lymphoma 6 (BCL6) is a conserved multi-domain protein that functions principally as a transcriptional repressor. This protein regulates many pivotal aspects of immune cell development and function. BCL6 is critical for germinal center (GC) formation and the development of high-affinity antibodies, with key roles in the generation and function of GC B cells, follicular helper T (Tfh) cells, follicular regulatory T (Tfr) cells, and various immune memory cells. BCL6 also controls macrophage production and function as well as performing a myriad of additional roles outside of the immune system. Many of these regulatory functions are conserved throughout evolution. The BCL6 gene is also important in human oncology, particularly in diffuse large B cell lymphoma (DLBCL) and follicular lymphoma (FL), but also extending to many in other cancers, including a unique role in resistance to a variety of therapies, which collectively make BCL6 inhibitors highly sought-after.

Biochemical rationale for transfusion of high titre COVID-19 convalescent plasma

We aimed to model binding of donor antibodies to virus that infects COVID-19 patients following transfusion of convalescent plasma (CCP). An immunosorbent assay was developed to determine apparent affinity (Kd, app). Antibody binding to virus was modelled using antibody concentration and estimations of viral load. Assay and model were validated using reference antibodies and clinical data of monoclonal antibody therapy. A single Kd, app or two resolvable Kd, app were found for IgG in 11% or 89% of CCP donations, respectively. For IgA this was 50%-50%. Median IgG Kd, app was 0.8nM and 3.6nM for IgA, ranging from 0.1-14.7nM and 0.2-156.0nM respectively. The median concentration of IgG was 44.0nM (range 8.4-269.0nM) and significantly higher than IgA at 2.0nM (range 0.4-11.4nM). The model suggested that a double CCP transfusion (i.e. 500 mL) allows for > 80% binding of antibody to virus provided Kd, app was < 1nM and concentration > 150nM. In our cohort from the pre-vaccination era, 4% of donations fulfilled these criteria. Low and mid-range viral loads are found early post exposure, suggesting that convalescent plasma will be most effective then. This study provides a biochemical rationale for selecting high affinity and high antibody concentration CCP transfused early in the disease course.

T follicular regulatory cells in food allergy promote IgE via IL-4.

T follicular regulatory (TFR) cells are found in the germinal center (GC) response and, along with T follicular helper (TFH) cells, help to control the development of high-affinity antibodies (Ab). Although TFR cells are generally thought to repress GC B cells and the Ab response, we have previously shown that in a mouse food allergy model, TFR cells produce IL-10 and play an essential helper role such that in the absence of TFR cells, IgE responses are diminished. Here we show that in this food allergy response, TFR cells produced IL-4 that promotes the generation of antigen-specific IgE. We show that food allergy-primed TFR cells specifically upregulate IL-4 gene transcription and produce functional IL-4 that promoted IgE responses both in vitro and in vivo. We determined that IgE responses are dependent on a high level of IL-4 produced by follicular T cells in the GC, explaining the need for IL-4 produced by TFR cells in the food allergy response. Overall, our findings have demonstrated that in food allergy, TFR cells can produce IL-4 and regulate IgE in a manner that augments the role of TFH cells in IgE responses.

High-affinity antibodies specific to the core region of the tau protein exhibit diagnostic and therapeutic potential for Alzheimer’s disease

BackgroundRecent advances in blood-based biomarker discovery are paving the way for simpler, more accessible diagnostic tools that can detect early signs of Alzheimer’s disease (AD). Recent successes in the development of amyloid-targeting immunotherapy approaches mark an important advancement in providing new options for the treatment of AD. We have developed a set of high-affinity monoclonal antibodies (mAbs) to tau protein that have the potential as tools for diagnosis and treatment of AD.MethodsSheep were immunised with either full-length tau (1-441) or truncated paired helical filament (PHF)-core tau (297–391). A stringent bio-panning and epitope selection strategy, with a particular focus directed to epitopes within the disease-relevant PHF-core tau, was used to identify single-chain antibodies (scAbs). These scAbs were ranked by affinity for each epitope class, with leads converted to high-affinity mAbs. These antibodies and their potential utility were assessed by their performance in tau immunoassays, as well as their ability to prevent tau aggregation and propagation. Further characterisation of these antibodies was performed by immunohistochemical staining of brain sections and immuno-gold electronmicroscopy of isolated PHFs.ResultsOur work resulted in a set of high-affinity antibodies reacting with multiple epitopes spanning the entire tau protein molecule. The tau antibodies directed against the core tau unit of the PHF inhibited pathological aggregation and seeding using several biochemical and cell assay systems. Through staining of brain sections and PHFs, the panel of antibodies revealed which tau epitopes were available, truncated, or occluded. In addition, highly sensitive immunoassays were developed with the ability to distinguish between and quantify various tau fragments.ConclusionThis article introduces an alternative immunodiagnostic approach based on the concept of a “tauosome” – the diverse set of tau fragments present within biological fluids. The development of an antibody panel that can distinguish a range of different tau fragments provides the basis for a novel approach to potential diagnosis and monitoring of disease progression. Our results further support the notion that tau immunotherapy targeting the PHF-core needs to combine appropriate selection of both the target epitope and antibody affinity to optimise therapeutic potential.

ELISA-R: an R-based method for robust ELISA data analysis.

Enzyme-linked immunosorbent assay (ELISA) is a technique to detect the presence of an antigen or antibody in a sample. ELISA is a simple and cost-effective method that has been used for evaluating vaccine efficacy by detecting the presence of antibodies against viral/bacterial antigens and diagnosis of disease stages. Traditional ELISA data analysis utilizes a standard curve of known analyte, and the concentration of the unknown sample is determined by comparing its observed optical density against the standard curve. However, in the case of vaccine research for complicated bacteria such as Mycobacterium tuberculosis (Mtb), there is no prior information regarding the antigen against which high-affinity antibodies are generated and therefore plotting a standard curve is not feasible. Consequently, the analysis of ELISA data in this instance is based on a comparison between vaccinated and unvaccinated groups. However, to the best of our knowledge, no robust data analysis method exists for "non-standard curve" ELISA. In this paper, we provide a straightforward R-based ELISA data analysis method with open access that incorporates end-point titer determination and curve-fitting models. Our modified method allows for direct measurement data input from the instrument, cleaning and arranging the dataset in the required format, and preparing the final report with calculations while leaving the raw data file unchanged. As an illustration of our method, we provide an example from our published data in which we successfully used our method to compare anti-Mtb antibodies in vaccinated vs non-vaccinated mice.

B-069 Characterization of a panel of antibodies as diagnostics for NASH and NAFL

Abstract Background Nonalcoholic fatty liver disease (NAFLD) is on the rise in Western industrialized countries and associated comorbidities like e.g. cirrhosis are a major cause of liver transplantations in the USA. In NAFLD, excess fat is accumulated in the liver whereas this build-up is not caused by alcohol. NASH (nonalcoholic steatohepatitis) and NAFL (nonalcoholic fatty liver) are two forms of NAFLD. NAFL represents a stage of the disease in which the liver does not exhibit major signs of inflammation or damage, while NASH does. NAFL does not necessarily progress to cause liver damage. NASH on the other hand may cause fibrosis and lead to cirrhosis with a risk of progression into liver cancer. Since NAFLD shows a prevalence of over 24-25% in both the USA and Europe, diagnosis moves more and more into focus. The current gold standard for NASH diagnosis is liver biopsy, and NAFLD as such can also be diagnosed by ultrasound and MRI, among others. A quest for biomarkers is ongoing to develop blood tests that allow NAFLD diagnosis by immunoassays for example. We have developed high affinity and specificity antibodies against four highly discussed marker proteins for NAFLD: RBP4 (Retinol Binding Protein 4), FGF21 (Fibroblast Growth factor 21), CHI3L1 (Chitinase-3-like protein 1) and PAI-1 (Plasminogen activator inhibitor-1). All four markers are discussed to be elevated in NAFLD patient serum. Methods We characterized the developed anti-RBP4 and anti-FGF21 antibodies via an in-house developed Chemiluminescence Immunoassay (CLIA). Results We have determined the limit of quantification (LoQ), limit of detection (LoD) and linear range of measurable concentrations for our antibody pairs. We further conducted spike and recovery experiments showing the precision of the developed antibodies against RBP4 and FGF21. Conclusions Together, we present antibody pairs for RBP4 and FGF21 suitable as a part of a diagnostic panel that has high potential to be applied in the diagnosis of NAFLD.

B-036 Systematic Evaluation of Antibody-Antigen Interactions for Suitability in Immunoassays Using Parallel Microarray ELISA

Abstract Background A critical step in immunoassay development is the selection of the appropriate capture and detection antibodies. The use of suboptimal antibodies can drastically affect the quality of the final assay. Traditional selection methods involve a direct testing of potential antibodies in the final assay setting; a time-consuming and material-intensive exercise that is often too complex to test all possibilities. Alternatives, such as abstract K-on/K-off determinations via SPR or BLI can be costly and often show limited translatability to the ultimate assay format. Here, we describe a novel systematic approach to rapidly screen antibody-antigen interactions for key assay specifications and test for translatability of the findings. Methods Nanoliter volumes of potential capture antibodies were spotted onto the bottom of ELISA wells, generating a microarray of antibodies in each well (&amp;gt;11,000 spots per plate). For the analysis of capture antibody-antigen interactions, a titration series of biotinylated analyte in negative sample matrix was applied. Binding was visualized using strep-polyHRP, precipitating TMB and high resolution scanning of each ELISA well. Antibody pair performance was investigated using unlabeled analyte and biotinylated detection antibody options. Sensitivity, specificity, dynamic range, binding speed, binding strength and matrix compatibility were investigated using time courses, concentration ranges, dilutions, interferent spikes and pH/salt/detergent challenges. Signals were quantified to calculate and statistically compare each possible interaction. Results Antibody-antigen interactions were investigated via parallel microarray ELISA for two example immunoassays: the HBsAg and cTnI test. For both analytes, a panel of antibodies was screened revealing antibodies with diverse characteristics. Striking differences were observed depending on the sample matrix composition. Without optimization, antibodies could be identified showing dynamic assay ranges of 3-4 log orders. Time course experiments revealed high-affinity antibodies suitable for rapid diagnostic tests. For HBsAg, levels down to 0.01 IU/mL (∼40 pg/mL) could be reproducibly detected, which is well below the minimal requirement of 0.13 IU/mL. Reactivity against multiple virus serotypes could be confirmed. Interestingly, for the cTnI test, a strong synergistic effect was observed when specific monoclonal antibodies were combined in the capture mix, resulting in a 6-fold sensitivity enhancement compared to their separate performances. Finally, we show that the results obtained with the parallel microarray ELISA approach are highly translatable to the final assay format, ranging from bead-based dry-chemistry assays to chemiluminescent immunoassays. Conclusions The parallel microarray ELISA evaluation presents a robust and reliable method for identifying the best antibodies for an immunoassay. It allows for a systematic and quantitative screen of numerous antibody options within 1-2 weeks under real immunoassay conditions with minimal antibody amounts. This method has shown to be highly translatable and is customizable to accommodate required assay specifications.

ANTIPASTI: Interpretable prediction of antibody binding affinity exploiting normal modes and deep learning.

The high binding affinity of antibodies toward their cognate targets is key to eliciting effective immune responses, as well as to the use of antibodies as research and therapeutic tools. Here, we propose ANTIPASTI, a convolutional neural network model that achieves state-of-the-art performance in the prediction of antibody binding affinity using as input a representation of antibody-antigen structures in terms of normal mode correlation maps derived from elastic network models. This representation captures not only structural features but energetic patterns of local and global residue fluctuations. The learnt representations are interpretable: they reveal similarities of binding patterns among antibodies targeting the same antigen type, and can be used to quantify the importance of antibody regions contributing to binding affinity. Our results show the importance of the antigen imprint in the normal mode landscape, and the dominance of cooperative effects and long-range correlations between antibody regions to determine binding affinity.

Interleukin 9 mediates T follicular helper cell activation to promote antibody responses.

Antigen-specific humoral responses are orchestrated through complex interactions among immune cells in lymphoid tissues, including the collaboration between B cells and T follicular helper (Tfh) cells. Accumulating evidence indicates a crucial role for interleukin-9 (IL-9) in the formation of germinal centers (GCs), enhancing the generation of class-switched high-affinity antibodies. However, the exact function of IL-9 in Tfh cell regulation remains unclear. In this study, we examined the humoral immune responses of CD4Cre/+Il9rafl/fl mice, which lack an IL-9-specific receptor in Tfh cells. Upon intraperitoneal immunization with sheep red blood cells (SRBCs), CD4Cre/+Il9rafl/fl mice displayed diminished levels of SRBC-specific IgG antibodies in their sera, along with reduced levels of GC B cells and plasma cells. Notably, Il9ra-deficient Tfh cells in the spleen exhibited decreased expression of their signature molecules such as B-cell lymphoma 6, C-X-C chemokine receptor 5, IL-4, and IL-21 compared to control mice. In models of allergic asthma induced by house dust mite (HDM) inhalation, CD4Cre/+Il9rafl/fl mice failed to elevate serum levels of HDM-specific IgE and IgG. This was accompanied by reductions in Tfh cells, GC B cells, and plasma cells in mediastinal lymph nodes. Furthermore, group 2 innate lymphoid cells (ILC2s) were identified as producers of IL-9 under immunizing conditions, possibly induced by leukotrienes released by activated IgD+ B cells around the T-B border. These observations may indicate the critical role of IL-9 receptor signaling in the activation of Tfh cells, with ILC2s potentially capable of supplying IL-9 in organized lymphoid tissues.

Rapid affinity optimization of an anti-TREM2 clinical lead antibody by cross-lineage immune repertoire mining

We describe a process for rapid antibody affinity optimization by repertoire mining to identify clones across B cell clonal lineages based on convergent immune responses where antigen-specific clones with the same heavy (VH) and light chain germline segment pairs, or parallel lineages, bind a single epitope on the antigen. We use this convergence framework to mine unique and distinct VH lineages from rat anti-triggering receptor on myeloid cells 2 (TREM2) antibody repertoire datasets with high diversity in the third complementarity-determining loop region (CDR H3) to further affinity-optimize a high-affinity agonistic anti-TREM2 antibody while retaining critical functional properties. Structural analyses confirm a nearly identical binding mode of anti-TREM2 variants with subtle but significant structural differences in the binding interface. Parallel lineage repertoire mining is uniquely tailored to rationally explore the large CDR H3 sequence space in antibody repertoires and can be easily and generally applied to antibodies discovered in vivo.

Differential detection of SARS-CoV-2 variants and influenza A viruses utilizing a dual lateral flow strip based on colloidal gold-labeled monoclonal antibodies

The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and the seasonal influenza virus are spreading among humans concurrently, especially with the ongoing replacement of mutant strains. It is challenging to differentiate between symptoms for therapy due to the comparable symptoms following infection with the SARS-CoV-2 variants and influenza viruses. Meanwhile, in order to achieve rapid point-of-care testing (POCT) to manage the spread of the disease, we developed a dual lateral flow strip based on colloidal gold-labeled monoclonal antibodies that can perform differential detection of SARS-CoV-2 variants and influenza A viruses (IAV) in this study. High-affinity monoclonal antibodies (mAbs) targeting SARS-CoV-2 and IAV were prepared to capture antigens and labeled with colloidal gold nanoparticles (AuNPs). Based on high-affinity mAbs, two targets were immobilized on one nitrocellulose (NC) membrane to establish the lateral flow strip (LFS) for differential diagnosis of SARS-CoV-2 and IAV. With no reactivity to other viruses, this LFS is extremely specific and can only identify SARS-CoV-2 and IAV. The LFS showed a limit of detection (LOD) of 4.88 ng/mL for the Omicron BA.2 RBD protein and 2.44 ng/mL for the nucleoprotein (NP) protein of H1N1. When analyzing 16 SARS-CoV-2 positive clinical samples, eight IAV positive clinical samples, and six negative samples that had already been pre-confirmed by commercial kits, its clinical application is effectively and accurately proven. These results demonstrated that the LFS integrated with AuNPs has great potential to facilitate quick, easy, and reliable POCT diagnosis for promoting the control of infectious diseases.

MEDI1814 selectively reduces free Aβ42 in cerebrospinal fluid of non-clinical species and Alzheimer's disease patients.

Small molecules and antibodies are being developed to lower amyloid beta (Aβ) peptides. We describe MEDI1814, a fully human high-affinity monoclonal antibody selective for Aβ42, the pathogenic self-aggregating species of Aβ. MEDI1814 reduces free Aβ42 without impacting Aβ40 in the cerebrospinal fluid of rats and cynomolgus monkeys after systemic administration. MEDI1814 administration to patients with Alzheimer's disease (AD; n=57) in single or repeat doses up to 1800mg intravenously or 200mg subcutaneously was associated with a favorable safety and tolerability profile. No cases of amyloid-related imaging abnormalities were observed. Predictable dose-proportional changes in serum exposures for MEDI1814 were observed across cohorts. Cerebrospinal fluid (CSF) analysis demonstrated central nervous system penetration of MEDI1814. Pharmacodynamic data showed dose-dependent suppression of free Aβ42, increases in total (bound and free) Aβ42, but no change in total Aβ40 in CSF across doses. MEDI1814 offers a differentiated approach to impacting Aβ in AD via selective reduction of free Aβ42.