Hundreds Of Antibodies Research Articles

Precise immunofluorescence canceling for highly multiplexed imaging to capture specific cell states

Cell states are regulated by the response of signaling pathways to receptor ligand-binding and intercellular interactions. High-resolution imaging has been attempted to explore the dynamics of these processes and, recently, multiplexed imaging has profiled cell states by achieving a comprehensive acquisition of spatial protein information from cells. However, the specificity of antibodies is still compromised when visualizing activated signals. Here, we develop Precise Emission Canceling Antibodies (PECAbs) that have cleavable fluorescent labeling. PECAbs enable high-specificity sequential imaging using hundreds of antibodies, allowing for reconstruction of the spatiotemporal dynamics of signaling pathways. Additionally, combining this approach with seq-smFISH can effectively classify cells and identify their signal activation states in human tissue. Overall, the PECAb system can serve as a comprehensive platform for analyzing complex cell processes.

Abstract 5633: Analysis of the immune microenvironment and tumor-infiltrating immune cells across different solid tumors by combined spatial transcriptomics and proteomics

Abstract With the advent of immunotherapies such as immune checkpoint inhibitors and CAR T cells, the interest in the tumor microenvironment and tumor-infiltrating lymphocytes (TILs) cells has increased in recent years as these play a critical role in predicting response to therapy and clinical outcomes. We have reported on the identification of potential targets for CAR T cell therapies by analyzing a multitude of tumor samples with hundreds of antibodies by flow cytometry and a new spatial proteomics platform, the MACSima Imaging Platform (Schäfer et al. Nat. Commun., 2021; Kinkhabwala et.al. Sci. Rep., 2022). Here we report on the analysis of tumor microenvironment and tumor infiltrating immune cells using a novel combined spatial transcriptomics and proteomics approach. For protein expression analysis we generated a panel of 60 fluorochrome conjugated antibodies (REAscreen Immuno-oncology, human, FFPE, version 01) selected to identify immune cells, cancer associated fibroblasts, matrix, blood vessels, lymphatics and malignant epithelial cell populations. RNA analysis was based on a novel in situ hybridization and amplification method followed by a cyclic spatial decoding of transcripts (RNAsky). To standardize and further ease the process of protein and RNA detection we generated the antigen and RNA detection probes in a ready-to-use format, i.e., dried and sealed in 96 well plates ready to be inserted into the MACSima. FFPE specimens of different tumor entities reviewed by a pathologist were first hybridized to probes, which were amplified, and spatially decoded by fluorochrome conjugated oligonucleotides. Subsequently the same specimen was exposed to fluorescently labeled antibodies by cycles of antibody reaction, image acquisition and erasure of signal. Finally, specimens were stained with H&E to report on tissue integrity and correlate spatial orientation. The 2D image stacks were analyzed for transcript and antigen quantification and pattern recognition using both, pixel and segmented single-cell data (MACS iQ View Analysis Software). Across the specimens we identified more than 20 cell types including 12 immune cell subsets. The comparison of the tumor microenvironment, tumor-infiltrating immune cells (TILs) and their spatial organization in relation to the tumor cells allowed to identify differences and similarities across the tumor types. In summary we report here on a novel standardized and automated combined spatial RNA and protein expression analysis of the tumor microenvironment and tumor infiltrating immune cells across tumor types. The characterization dataset demonstrates the ability of our platform to perform standardized in-depth phenotyping of sample cohorts. This will enable discovery and further development of predictive and prognostic biomarkers critical to patient stratification for immunotherapy. Citation Format: Julia Femel, Emily Neil, Dongju Park, Fabio El Yassouri, Anijutta Appelshoffer, Erica Lloyd, Michael DiBuono, Henry Sauer, Hanna Lafayette, Hsinyi Smith, Jinling Wang, Dominic Mangiardi, Alex Makrigiorgos, Paurush Praveen, Silvia Rüberg, Werner Müller, Tanya Wantenaar, Robert Pinard, Andreas Bosio. Analysis of the immune microenvironment and tumor-infiltrating immune cells across different solid tumors by combined spatial transcriptomics and proteomics. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 5633.

Computational approach for binding prediction of SARS-CoV-2 with neutralizing antibodies

Coronavirus disease 2019 (COVID-19) caused by a novel severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has spread worldwide as a severe pandemic and caused enormous global health and economical damage. Since December 2019, more than 197 million cases have been reported, causing 4.2 million deaths. In the settings of pandemic it is an urgent necessity for the development of an effective COVID-19 treatment. While in-vitro screening of hundreds of antibodies isolated from convalescent patients is challenging due to its high cost, use of computational methods may provide an attractive solution in selecting the top candidates. Here, we developed a computational approach (SARS-AB) for binding prediction of spike protein SARS-CoV-2 with monoclonal antibodies. We validated our approach using existing structures in the protein data bank (PDB), and demonstrated its prediction power in antibody-spike protein binding prediction. We further tested its performance using antibody sequences from the literature where crystal structure is not available, and observed a high prediction accuracy (AUC = 99.6%). Finally, we demonstrated that SARS-AB can be used to design effective antibodies against novel SARS-CoV-2 mutants that might escape the current antibody protections. We believe that SARS-AB can significantly accelerate the discovery of neutralizing antibodies against SARS-CoV-2 and its mutants.

To bnAb or Not to bnAb: Defining Broadly Neutralising Antibodies Against HIV-1.

Since their discovery, antibodies capable of broad neutralisation have been at the forefront of HIV-1 research and are of particular interest due to in vivo passive transfer studies demonstrating their potential to provide protection. Currently an exact definition of what is required for a monoclonal antibody to be classed as a broadly neutralising antibody (bnAb) has not yet been established. This has led to hundreds of antibodies with varying neutralisation breadth being studied and has given insight into antibody maturation pathways and epitopes targeted. However, even with this knowledge, immunisation studies and vaccination trials to date have had limited success in eliciting antibodies with neutralisation breadth. For this reason there is a growing need to identify factors specifically associated with bnAb development, yet to do this a set of criteria is necessary to distinguish bnAbs from non-bnAbs. This review aims to define what it means to be a HIV-1 bnAb by comparing neutralisation breadth, genetic features and epitopes of bnAbs, and in the process highlights the challenges of comparing the array of antibodies that have been isolated over the years.

Development and application of therapeutic antibodies against COVID-19.

The pandemic of Coronavirus disease 2019 (COVID-19) caused by the severe acute respiratory syndrome 2 coronavirus (SARS-CoV-2) continues to be a global health crisis. Fundamental studies at genome, transcriptome, proteome, and interactome levels have revealed many viral and host targets for therapeutic interventions. Hundreds of antibodies for treating COVID-19 have been developed at preclinical and clinical stages in the format of polyclonal antibodies, monoclonal antibodies, and cocktail antibodies. Four products, i.e., convalescent plasma, bamlanivimab, REGN-Cov2, and the cocktail of bamlanivimab and etesevimab have been authorized by the U.S. Food and Drug Administration (FDA) for emergency use. Hundreds of relevant clinical trials are ongoing worldwide. Therapeutic antibody therapies have been a very active and crucial part of COVID-19 treatment. In this review, we focus on the progress of therapeutic COVID-19 antibody development and application, discuss corresponding problems and challenges, suggesting new strategies and solutions.

Seeking broad protection

Coronavirus As scientists develop therapeutic antibodies and vaccines against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the risk of emergent coronaviruses makes it important to also identify broadly protective antibodies. Wec et al. isolated and characterized hundreds of antibodies against the viral spike protein of SARS-CoV-2 from the memory B cells of a survivor of the 2003 outbreak caused by the related coronavirus, SARS-CoV. In both of these viruses, the spike protein facilitated viral entry by binding to the angiotensin-converting enzyme 2 (ACE2) receptor on human cells. The antibodies targeted multiple sites on the spike protein, but of nine antibodies that showed strong cross-neutralization, eight targeted the domain that binds to ACE2. These eight antibodies also neutralized a bat SARS-related virus. Illuminating the epitopes on the viral spike protein that bind cross-neutralizing antibodies could guide the design of broadly protective vaccines. Science , this issue p. [731][1] [1]: /lookup/doi/10.1126/science.abc7424

Rapid exploration of the epitope coverage produced by an Ebola survivor to guide the discovery of therapeutic antibody cocktails.

ABSTRACTBackgroundDevelopment of successful neutralizing antibodies is dependent upon broad epitope coverage to increase the likelihood of achieving therapeutic function. Recent advances in synthetic biology have allowed us to conduct an epitope binning study on a large panel of antibodies identified to bind to Ebola virus glycoprotein with only published sequences.Methods and ResultsA rapid, first-pass epitope binning experiment revealed seven distinct epitope families that overlapped with known structural epitopes from the literature. A focused set of antibodies was selected from representative clones per bin to guide a second-pass binning that revealed previously unassigned epitopes, confirmed epitopes known to be associated with neutralizing antibodies, and demonstrated asymmetric blocking of EBOV GP from allosteric effectors reported from literature.ConclusionsCritically, this workflow allows us to probe the epitope landscape of EBOV GP without any prior structural knowledge of the antigen or structural benchmark clones. Incorporating epitope binning on hundreds of antibodies during early stage antibody characterization ensures access to a library’s full epitope coverage, aids in the identification of high quality reagents within the library that recapitulate this diversity for use in other studies, and ultimately enables the rational development of therapeutic cocktails that take advantage of multiple mechanisms of action such as cooperative synergistic effects to enhance neutralization function and minimize the risk of mutagenic escape. The use of high-throughput epitope binning during new outbreaks such as the current COVID-19 pandemic is particularly useful in accelerating timelines due to the large amount of information gained in a single experiment.

Broad neutralization of SARS-related viruses by human monoclonal antibodies.

Broadly protective vaccines against known and preemergent human coronaviruses (HCoVs) are urgently needed. To gain a deeper understanding of cross-neutralizing antibody responses, we mined the memory B cell repertoire of a convalescent severe acute respiratory syndrome (SARS) donor and identified 200 SARS coronavirus 2 (SARS-CoV-2) binding antibodies that target multiple conserved sites on the spike (S) protein. A large proportion of the non-neutralizing antibodies display high levels of somatic hypermutation and cross-react with circulating HCoVs, suggesting recall of preexisting memory B cells elicited by prior HCoV infections. Several antibodies potently cross-neutralize SARS-CoV, SARS-CoV-2, and the bat SARS-like virus WIV1 by blocking receptor attachment and inducing S1 shedding. These antibodies represent promising candidates for therapeutic intervention and reveal a target for the rational design of pan-sarbecovirus vaccines.

Peptide barcoding for high‐throughput functional evaluation of antibodies without immobilization

BackgroundDisplay technologies such as phage display and yeast display can evaluate a large number of antibodies in a high‐throughput manner, and have been used to isolate various antibody medicines. In display technologies, the immobilization of antibodies on the cell surface is essential to establish genotype–phenotype linkages, but this immobilization can affect functions of antibodies. Here, we developed a novel methodology, “peptide barcoding”, for high‐throughput quantitative measurement of binding properties of antibodies without immobilization. In the scheme of “peptide barcoding”, unique peptide barcodes are fused with each antibody, and they represent genotype–phenotype linkages. A mixture of different peptide‐barcoded antibodies is reacted with antigens, and peptide barcodes of functional antibodies are identified by mass spectrometry after cleavage by specific proteases. This methodology will enable high‐throughput development of antibodies for experimental reagents, diagnostics, and pharmaceuticals.MethodsUnique peptide barcodes were designed to have high detectability for mass spectrometry. Peptide‐barcoded nanobodies (anti‐CD4 or anti‐GFP) were produced by Pichia pastoris. A mixture of peptide‐barcoded nanobodies were reacted with antigen‐coated magnetic beads. Peptide barcodes of functional nanobodies were cleaved by a specific protease, enterokinase, and released peptides were identified by mass spectrometry. Precise dissociation constants of nanobodies were measured by BIACORE T200.Results & DiscussionWe designed peptide‐barcoded anti‐CD4 nanobody or anti‐GFP nanobody, and successfully produced them using P. pastoris. We measured the values of dissociation constants of the peptide‐barcoded nanobodies using surface plasmon resonance, and confirmed that addition of peptide barcodes didn’t affect affinities of these nanobodies. To demonstrate high‐throughput evaluation of free nanobodies, we produced peptide‐barcoded nanobody mutants, and mixed them with antigen‐coated magnetic beads. Released peptide barcodes from functional nanoboides were quantified by selected reaction monitoring, and we successfully ranked nanobody mutants in order of affinity. This methodology has potential scalability and could evaluate affinities of hundreds of antibodies in one shot [ 1, 2].Support or Funding InformationThis research was supported by JST, CREST (grant number JPMJCR16G2), Japan.

Abstract 245: Characterization and classification of glioblastoma multiforme using the novel multiparametric cyclic immunofluorescence analysis system MACSima

Abstract Glioblastoma multiforme (GBM), a highly malignant, non-curative brain tumor of the primary central nervous system, has been subclassified in the past years in several distinct subtypes using a multitude of analysis methods. Here, we introduce the MACSima™ imaging platform which allows for fully automated, multiparametric, cyclic immunofluorescence analysis of specimens with hundreds of antibodies. We apply this method to characterize and classify glioblastomas according to published classification schemes and identify novel glioblastoma specific markers. Glioblastoma xenografts derived from primary tumors were dissociated to single cells using the Tumor Dissociation Kit, human and the gentleMACS™ Octo Dissociator (Miltenyi Biotec). Single cells were analyzed for cell surface marker expression by flow cytometry (MACSQuant Analyzer 10) including 371 directly conjugated antibodies (MACS Marker Screen, Miltenyi Biotec). A ranking was applied according to the percentage of positive cells and the stain index, leading to a selection of 96 markers for characterization of eight primary glioblastoma using the MACSima™ imaging platform. Cryosections were fixed by acetone and each specimen was exposed to 96 fluorescent labeled antibodies by cycles of antibody reaction, image acquisition and erasure of signal. The 2D image stacks were analyzed for antigen quantification and pattern recognition using both, pixel and segmented single-cell data. The detection of previously published markers, such as PDGFRα, Olig2, p16, p53, Synaptophysin, CD44, Nestin, Podoplanin, GFAP, MET, Hes-1 and EGFR was used to subclassify the glioblastomas according to i) Motomura (Motomura et al., 2012) into Oligodendrocyte Precursor (OPC), Differentiated Oligodendrocyte (DOC), Astrocytic Mesenchymal (AsMes) or Mixed subtype, and ii) Verhaak (Verhaak et al., 2010) into Proneural, Neural, Classical, or Mesenchymal subtype. The analysis of well-established glioblastoma marker partially already used in CAR T cell based clinical trials such as EGFRvIII, HER2, or IL-13Rα2 revealed a broad inter- and intratumor diversity of expression. Infiltrating immune cells were present in most of the tumors, but showed varying percentages. Finally, segmentation, clustering, and correlation analysis allowed for identification of new marker which might be used for a more robust classification of glioblastomas. In summary, our analysis using the MACSima™ imaging platform reveals a high heterogeneity of protein expression in glioblastomas along with the ability to deeper classify the diverse tumors and identify novel markers that allow selective detection of tumor cells for their potential use in immunotherapy. Citation Format: Sandy Reiß, Stefan Tomiuk, Jutta Kollet, Jan Drewes, Wolfgang Brück, Melanie Jungblut, Andreas Bosio. Characterization and classification of glioblastoma multiforme using the novel multiparametric cyclic immunofluorescence analysis system MACSima [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 245.

Large-scale use of knockout validation to confirm antibody specificity

Abstract Antibodies are among the most common tools in basic science and clinical research. However, an increasing number of studies have shown that not all antibodies are specific, leading to a growing issue of experimental irreproducibility. This is in part due to the cross-reactivity between antibodies and off-target proteins, as well as the variability between different antibody batches. These unspecific antibodies result in wasted time and resources and compromise the advancement of science. In response to this, Abcam launched a knockout (KO) validation program in 2015 as one of its antibody quality control protocols. This initiative uses KO cells lines made possible through a partnership with Horizon Discovery. Target genes are mutated via CRISPR-Cas9 within a haploid cell line, which results in a frameshift and a complete loss of gene expression. These KO cell lines function as true negative controls that can be used at a large scale to confirm antibody specificity to the protein of interest. We have KO-validated hundreds of antibodies, including several that are relevant to immunology and immuno-oncology research. Here we present the data related to key targets validated in western blot, flow cytometry and immunocytochemistry. The antibodies have been tested in KO and wild-type cells to confirm their specificity. By providing researchers with reliable and specific antibodies that work first time, we hope to minimize wasted resources and improve antibody reproducibility.

Individual and meta-immune networks

Networks can be found everywhere—in technology, in nature and in our bodies. In this paper we present how antigen networks can be used as a model to study network interaction and architecture. Utilizing antigen microarray data of the reactivity of hundreds of antibodies of sera of ten mothers and their newborns, we reconstruct networks, either isotype specific (IgM or IgG) or person specific—mothers or newborns—and investigate the network properties. Such an approach makes it possible to decipher fundamental information regarding the personal immune network state and its unique characteristics. In the current paper we demonstrate how we are successful in studying the interaction between two dependent networks, the maternal IgG repertoire and the one of the offspring, using the concept of meta-network provides essential information regarding the biological phenomenon of cross placental transfer. Such an approach is useful in the study of coupled networks in variety of scientific fields.

Proteomics-based Dissection of Human Endoderm Progenitors by Differential Cell Capture on Antibody Array

Heterogeneity, shortage of material, and lack of progenitor-specific cell surface markers are major obstacles to elucidating the mechanisms underlying developmental processes. Here we report a proteomics platform that alleviates these difficulties and demonstrate its effectiveness in fractionating heterogeneous cultures of early endoderm derived from human embryonic stem cells. The approach, designated differential cell-capture antibody array, is based on highly parallel, comparative screening of live cell populations using hundreds of antibodies directed against cell-surface antigens. We used this platform to fractionate the hitherto unresolved early endoderm compartment of CXCR4+ cells and identify several endoderm (CD61+ and CD63+) and non-endoderm (CD271+, CD49F+, CD44+ and B2M+) sub-populations. We provide evidence that one of these sub-populations, CD61+, is directly derived from CXCR4+ cells, displays characteristic kinetics of emergence, and exhibits a distinct gene expression profile. The results demonstrate the potential of the cell-capture antibody array as a powerful proteomics tool for detailed dissection of heterogeneous cellular systems.

Quels auto-anticorps pour le diagnostic et le suivi de la néphropathie lupique ?

Summary Glomerulonephritis is the most serious complication of systemic lupus erythematosus (SLE). Indeed, lupus nephritis evolves by flares that must be addressed as soon as possible, or better be prevented. As a consequence, the immunological diagnosis of this autoimmune disease is essential in conjunction with the monitoring of the renal function. Although hundreds of antibodies have been identified in SLE, some of them seem to accompany or announce the renal disease: anti-dsDNA, anti-nucleosome, anti-α-actinin, and anti-C1q antibodies. This issue aims to communicate the reasons for the tropism of such autoantibodies to the kidneys, and present the interest of these autoantibodies for the diagnosis and the monitoring of lupus nephritis.

GFAP, Ki67 and IDH1: perhaps the golden triad of glioma immunohistochemistry

In this issue of Acta Neuropathologica, Capper et al. [2] describe a monoclonal antibody that specifically and sensitively recognizes the IDH1 (isocitrate dehydrogenase 1) protein carrying the R132H mutation in routinely processed (formalin-fixed, paraffin-embedded) tissues. In 2008, hotspot sequence mutations at position 132 of IDH1 were discovered in 12% of glioblastomas [4] and in about 70% of diffuse astrocytomas, oligodendrogliomas, oligoastrocytomas and secondary glioblastomas [1], a finding that has been confirmed by several other studies. Because more than 90% of IDH1 mutations are of the R132H type and mutations are assumed to be present in all tumor cells, this antibody is able to identify virtually all glioma cells in paraffin sections from the majority of grade-II diffuse gliomas and from their more malignant descendants. When compared with a PCRand restriction endonuclease-based assay for different IDH1 mutations recently described by the same group of authors [3], the immunohistochemical technique is easier to perform and is presumably more sensitive, whereas R132H-immunonegative cases will require additional PCR or sequencing analysis to search for other types of IDH1 and IDH2 mutations. Although the antibody still needs to be evaluated by other institutions on a large number of specimens, it may represent one of the seminal achievements in diagnostic neuropathology of the past decades. Hundreds of antibodies and antigens have been proposed as potential diagnostic markers for gliomas, but only a handful have turned out to be reliable and useful, the most important being GFAP for glial differentiation, developed in the seventies and Ki67/MIB1 for assessing proliferation, established in the eighties for frozen sections and in the nineties for paraffin sections. These antibodies, however, are not able to assist in resolving two common differential diagnostic problems: diffuse versus pilocytic astrocytoma; and invasion of diffuse astrocytoma versus reactive gliosis. Although staining for p53 is often performed to detect single-invading astrocytoma cells, it helps in only a minority of cases, in part because reactive astrocytes and other non-neoplastic cells may also show immunoreactivity for p53. In contrast, staining for R132H is expected to be of help in a significant fraction of cases and the diagnostic value of anti-R132H may reach that of anti-GFAP and MIB1. The present work of Capper et al. is important for other reasons as well. First, because brain biopsies may be (rarely) associated with complications such as hemorrhage and infection, and because an ambiguous neuropathological diagnosis (invasive edge or gliosis) may lead to rebiopsy, increased diagnostic accuracy by demonstration of R132H-positive cells may decrease mortality and morbidity. Second, since IDH1 mutations are associated with more favorable prognosis in diffuse astrocytic and oligodendroglial tumors of grades II–IV, staining for R132H (and for other IDH1 mutations using antibodies to be developed) may provide important prognostic information. Third, the antibody will promote the detection of glioma cells in CSF specimens. Fourth, the availability of the mutation-specific antibody will stimulate research using biopsy materials by clearly identifying tumor cells and correlating them with a variety of structural and molecular features. Fifth, anti-R132H will be useful for biological experiments aimed at further elucidating the function of mutated IDH1 protein; and finally, this report illustrates the efficient and beneficial outcome of a close cooperation and W. Paulus (&) Institute of Neuropathology, University Hospital Munster, Domagkstr. 19, 48129 Munster, Germany e-mail: werner.paulus@uni-muenster.de

High-throughput affinity ranking of antibodies using surface plasmon resonance microarrays

A method was developed to rapidly identify high-affinity human antibodies from phage display library selection outputs. It combines high-throughput Fab fragment expression and purification with surface plasmon resonance (SPR) microarrays to determine kinetic constants ( k on and k off) for 96 different Fab fragments in a single experiment. Fabs against human tissue kallikrein 1 (hK1, KLK1 gene product) were discovered by phage display, expressed in Escherichia coli in batches of 96, and purified using protein A PhyTip columns. Kinetic constants were obtained for 191 unique anti-hK1 Fabs using the Flexchip SPR microarray device. The highest affinity Fabs discovered had dissociation constants of less than 1 nM. The described SPR method was also used to categorize Fabs according to their ability to recognize an apparent active site epitope. The ability to rapidly determine the affinities of hundreds of antibodies significantly accelerates the discovery of high-affinity antibody leads.

Mating antibody phage display with proteomics

Antibodies are central to mastering the next main task of ‘postgenomic’ research: that is, to understand gene function by analysing the proteome. This analysis of more than 90 000 human gene products requires high-throughput methods for antibody generation. In contrast to animal-based methods, in vitro selection systems using recombinant antibodies offer this capability. Antibody phage display, the most commonly used contemporary system, has yielded hundreds of antibodies for therapy, research and diagnostics. Numerous cloning and mutagenesis strategies have been developed to create, preserve and exploit maximal antibody diversity, with libraries of different antibody formats constructed from different genetic sources. Evaluating the spectrum of phage-display antibody libraries indicates that the current focus on generating human therapeutic antibodies has yielded methods that are not readily applicable to postgenomic research. A streamlined process must be defined by closely integrating antigen logistics with library and antibody formats, panning procedures and robotic handling. Only by optimizing the interplay of biology, bioinformatics and technology can an affordable high-throughput process be created to meet the demands of proteome analysis and microarray technology.