Cell type-specific monoclonal antibody cross-reactivity screening in non-human primates and development of comparative immunophenotyping panels for CyTOF

Abstract

IntroductionNon-human primates (NHPs) are critical components of drug development because of their similarity to humans. Many key immunology assays, such as flow cytometry, Western blots, immunohistochemistry and immunofluorescence microscopy, make use of antibodies to demarcate specific cell types and quantify signaling moieties. Very few antibodies are raised against non-human primate antigens; instead, researchers typically use anti-human antibodies that are cross-reactive with the non-human primate species that they are studying. To help researchers find antibodies for NHP research, the National Institutes of Health supports a highly valuable database of the cross-reactivity of commercially available antibodies with 13 NHP species (http://www.nhpreagents.org). The database is derived from manufacturer and investigator reports, and typically provides a simple yes/no statement about whether a clone stains a species, with occasional comments about staining intensity or specificity. While an invaluable resource, the database is limited in its coverage. For example, prior to this study, only 28 CD markers had been evaluated in African green monkeys.Additionally, with few exceptions, the database lacks information about the cell types bound by cross-reactive antibodies, and there are many known instances of antibody clones binding different cell types in different species. For example, granulocyte and monocyte marker expression is known to be substantially different in humans than in non-human primates. Anti-human CD33 clone AC104.3E3 was reported in the NIH database and manufacturer's datasheet as cross-reactive with rhesus and cynomolgus macaques, but our lab and others determined that in those species, it prominently stains granulocytes (1, 2), while in humans it stains monocytes and classical dendritic cells. As another example, the Fcγ receptor CD16 is found on granulocytes in humans and sooty mangabeys, but not in macaques or baboons (3, 4), which will likely confound animal studies evaluating therapeutic antibodies, which may bind, transduce signals through and mediate internalization via this Fcγ receptor. Yet another example is CD56, which is expressed on monocytes in macaques (5), but is a canonical NK cell marker in humans. Thus, researchers must confirm that each clone they use is staining the cell population of interest through literature review or experimental verification.Here we present an expansion of both the breadth and depth of primate cross-reactivity data. We screened 332 monoclonal antibodies in blood from two individuals of each of four NHP species: rhesus macaque (Macaca mulatta), cynomolgus macaque (Macaca fascicularis), African green monkey (Chlorcebus aethiops) and olive/ yellow baboon (Papio hamadryas anubis x Papio hamadryas cynocephalus hybrid); and found more than 120 clones that stained one or more populations in each species. Furthermore, we included counter-stain antibodies that allowed us to determine staining specificity in at least five major immune cell populations. Data from ...

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“…We aspirated pelleted barcoded cells to a volume of 50 μL and incubated them with 15 μL of Human TruStain FcX (Biolegend, San Diego, CA) for 10 minutes. We stained cells for 30 min with 175 μL of a reconstituted lyophilized cocktail of metal-tagged cell-surface antibodies described previously ( Bjornson-Hooper et al., 2019a , 2019b ) supplemented with two additional antibody channels ( Data S1 ). We washed and permeabilized surface-stained cells with methanol before aspirating down to 50 μL and staining for 60 min with 190 μL of reconstituted lyophilized intracellular staining antibody cocktail ( Data S1 ).…”

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