Efficient generation of human IgA monoclonal antibodies

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Abstract

Immunoglobulin A (IgA) is the most abundant antibody isotype produced in humans. IgA antibodies primarily ensure immune protection of mucosal surfaces against invading pathogens, but also circulate and are present in large quantities in blood. IgAs are heterogeneous at a molecular level, with two IgA subtypes and the capacity to form multimers by interacting with the joining (J) chain. Here, we have developed an efficient strategy to rapidly generate human IgA1 and IgA2 monoclonal antibodies in their monomeric and dimeric forms. Recombinant monomeric and dimeric IgA1/IgA2 counterparts of a prototypical IgG1 monoclonal antibody, 10-1074, targeting the HIV-1 envelope protein, were produced in large amounts after expression cloning and transient transfection of 293-F cells. 10-1074 IgAs were FPLC-purified using a novel affinity-based resin engrafted with anti-IgA chimeric Fabs, followed by a monomers/multimers separation using size exclusion-based FPLC. ELISA binding experiments confirmed that the artificial IgA class switching of 10-1074 did not alter its antigen recognition. In summary, our technical approach allows the very efficient production of various forms of purified recombinant human IgA molecules, which are precious tools in dissecting IgA B-cell responses in physiological and pathophysiological conditions, and studying the biology, function and therapeutic potential of IgAs.

Introduction

Humoral immunity is essential to host protection against human infections, and relies on the phenomenal diversity of antibody molecules, which sense and inactivate pathogens. At mucosal surfaces, antibodies, and more specifically IgA immunoglobulins produced locally by sub-epithelial plasma cells, play a key role in the protection against toxins, bacteria, viruses and protozoa (Kaetzel, 2007). There are two IgA subclasses in humans, IgA1 and IgA2, which can exist as either monomers or as multimers made of IgA monomeric units polymerized by covalent bounds with the J-chain (principally dimers). IgAs are found in diverse mucosal secretions but are also abundant in serum (2–3 mg/ml) predominantly in the monomeric and IgA1 form (Kaetzel, 2007). On the other hand, mucosal IgAs are mainly present as “secretory IgA” (SIgAs) that are IgA polymers covalently associated with the “secretory component”, which corresponds to the extracellular domain of the polymeric immunoglobulin receptor (pIgR) cleaved away from the surface of epithelial cells (Kaetzel, 2007). Apart from their well-documented role in protecting epithelia from invading pathogens, SIgAs possess additional biological activities; they can act as anti-inflammatory and anti-allergenic molecules, maintain mucosal homeostasis, and regulate intestinal microbiota (Mantis et al., 2011). Surprisingly, the role of circulating serum IgAs is less clear, and should be explored more thoroughly.

In the past 15 years, the use of single cell antibody cloning techniques has enabled the rapid and efficient production of human monoclonal antibodies from different B-cell compartments (Sullivan et al., 2011, Tiller, 2011, Wilson and Andrews, 2012). The molecular and functional characterization of antibodies generated from selected single B-cell sub-populations (captured by flow cytometry or derived from screening of cultured B cells) has provided invaluable insight into the physiological and pathophysiological aspects of humoral immunity in humans (Wardemann and Nussenzweig, 2007, Meffre, 2011, Wilson and Andrews, 2012, Mouquet, 2014). Such studies have: (i) defined B-cell tolerance checkpoints at the transitions between different B-cell developmental stages (Wardemann et al., 2003, Tsuiji et al., 2006, Scheid et al., 2011); (ii) uncovered some of the tolerance mechanisms involved by studying these checkpoints in immunodeficient patients (Ng et al., 2004, Herve et al., 2005, Isnardi et al., 2008, Menard et al., 2011a); (iii) revealed defective tolerance checkpoints in autoimmune diseases (Samuels et al., 2005, Yurasov et al., 2005, Menard et al., 2011a, Menard et al., 2011b); (iv) dissected antibody responses to various pathogens (Wrammert et al., 2008, Scheid et al., 2009, Muellenbeck et al., 2013); (v) identified and characterized broadly neutralizing antiviral antibodies particularly, against HIV-1 (Mouquet, 2014). In fact, probing humoral immunity using single B-cell derived monoclonal antibodies, as both effectors of and surrogates for immune responses, has not only provided invaluable insight into the mechanisms that govern adaptive B-cell responses, but has also contributed to the development of therapeutic and/or vaccine strategies to fight human diseases. However, too few studies have investigated the role IgA-mediated humoral immunity in humans (Di Niro et al., 2010, Di Niro et al., 2012, Benckert et al., 2011), and in none of those, recombinant monoclonals were produced as native IgA antibodies, which definitively restricts certain molecular and functional characterizations.

In order to study IgA biology and IgA B-cell responses in physiological and pathophysiological conditions, we designed an expression cloning-based methodological approach to rapidly produce recombinant human IgA monoclonal antibodies. Expression vectors for human IgA1 and IgA2 compatible with the single B-cell antibody cloning method (Tiller et al., 2008) were created, and used to clone the immunoglobulin variable domain genes of a prototypic antigen-specific antibody, 10-1074, a neutralizing HIV-1 IgG1. Efficient production of purified monomeric and dimeric 10-1074 IgA molecules was made possible by combining a transient transfection-based eukaryotic expression system with fast protein liquid chromatography (FPLC) purification. Human IgA1 and IgA2 were FPLC-purified using a new affinity resin engrafted with anti-IgA chimeric Fabs. Finally, IgA monomers and dimers were separated by FPLC-based size exclusion chromatography.

Section snippets

Expression vectors and cloning of human IgA monoclonal antibodies

Total RNAs were previously extracted from human peripheral blood mononuclear cells (PBMCs) of a healthy donor (Scheid et al., 2011), using TRIzol® reagent (Life Technologies) following manufacturer's instructions. cDNAs were obtained by reverse transcription of RNAs primed with random hexamers (Roche) using SuperScript® III reverse transcriptase (Life technologies) according to the manufacturer's protocol. Primers used to amplify the Fc part of IgA1 and IgA2, and J chain gene products were as

Rapid expression cloning of human IgA monoclonal antibodies

To produce human IgA monoclonal antibodies, we first generated J chain-, Igα1- and Igα2-expression vectors by cloning for each specific product (J chain, IgA1 and IgA2 Fc regions), the corresponding nucleotide fragments were amplified from human PBMC cDNAs (Fig. 1A and B). Igα1- and Igα2-expression vectors were derived from an Igγ1-expression vector, allowing the same efficient and rapid cloning strategy using AgeI and SalI restriction sites as previously described (Tiller et al., 2008) (Fig. 1

Discussion

In the past decade, the development of methodologies for producing monoclonal antibodies from human B cells has allowed a rapid rise in the isolation and characterization of human antibodies (Tiller, 2011, Wilson and Andrews, 2012). Expression cloning of recombinant monoclonal antibodies from single B cells was originally developed 15 years ago to study level B-cell tolerance in humans at a molecular (Wardemann et al., 2003), and its breakage in autoimmune diseases (Yurasov et al., 2005). In the

Acknowledgments

We are grateful to Michel C. Nussenzweig (The Rockefeller University) for kindly providing us the cloning vectors for human immunoglobulins and 10-1074 IgG antibody, and to Caroline Eden (Icahn School of Medicine at Mount Sinai) for technical assistance, helpful comments and manuscript editing. We are very grateful to the ANRS (Agence Nationale de Recherche sur le Sida et les hépatites virales) to have supported the purchase of the FPLC instrument and the two PCR thermocyclers used in this

References (41)

  • T. Tiller et al.

    Efficient generation of monoclonal antibodies from single human B cells by single cell RT-PCR and expression vector cloning

    J. Immunol. Methods

    (2008)
  • T. Tiller et al.

    Cloning and expression of murine Ig genes from single B cells

    J. Immunol. Methods

    (2009)
  • H. Wardemann et al.

    B-cell self-tolerance in humans

    Adv. Immunol.

    (2007)
  • L. Baldi et al.

    Large-scale transfection of mammalian cells

    Methods Mol. Biol.

    (2012)
  • J. Benckert et al.

    The majority of intestinal IgA + and IgG + plasmablasts in the human gut are antigen-specific

    J. Clin. Invest.

    (2011)
  • O. Boussif et al.

    A versatile vector for gene and oligonucleotide transfer into cells in culture and in vivo: polyethylenimine

    Proc. Natl. Acad. Sci. U. S. A.

    (1995)
  • R. Di Niro et al.

    Rapid generation of rotavirus-specific human monoclonal antibodies from small-intestinal mucosa

    J. Immunol.

    (2010)
  • R. Di Niro et al.

    High abundance of plasma cells secreting transglutaminase 2-specific IgA autoantibodies with limited somatic hypermutation in celiac disease intestinal lesions

    Nat. Med.

    (2012)
  • J.M. Hayes et al.

    Glycosylation and Fc receptors

    Curr. Top. Microbiol. Immunol.

    (2014)
  • M. Herve et al.

    Unmutated and mutated chronic lymphocytic leukemias derive from self-reactive B cell precursors despite expressing different antibody reactivity

    J. Clin. Invest.

    (2005)
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