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Generation, affinity maturation, and characterization of a human anti-human NKG2D monoclonal antibody with dual antagonistic and agonistic activity.

ABSTRACT: In humans, NKG2D is an activating receptor on natural killer (NK) cells and a costimulatory receptor on certain T cells and plays a central role in mediating immune responses in autoimmune diseases, infectious diseases, and cancer. Monoclonal antibodies that antagonize or agonize immune responses mediated by human NKG2D are considered to be of broad and potent therapeutic utility. Nonetheless, monoclonal antibodies to NKG2D that are suitable for clinical investigations have not been published yet. Here, we describe the generation, affinity maturation, and characterization of a fully human monoclonal antibody to human NKG2D. Using phage display technology based on a newly generated naïve human Fab library in phage display vector pC3C followed by a tandem chain shuffling process designed for minimal deviation from natural human antibody sequences, we selected a human Fab, designated KYK-2.0, with high specificity and affinity to human NKG2D. KYK-2.0 Fab blocked the binding of the natural human NKG2D ligands MICA, MICB, and ULBP2 as potently as a commercially available mouse anti-human NKG2D monoclonal antibody in immunoglobulin G (IgG) format. Conversion of KYK-2.0 Fab to IgG1 resulted in subnanomolar avidity for human NKG2D. KYK-2.0 IgG1 was found to selectively recognize defined subpopulations of human lymphocytes known to express NKG2D, that is, the majority of human CD8+, CD16+, and CD56+ cells as well as a small fraction of human CD4+ cells. In solution, KYK-2.0 IgG1 interfered with the cytolytic activity of ex vivo expanded human NK cells. By contrast, immobilized KYK-2.0 IgG1 was found to strongly induce human NK cell activation. The dual antagonistic and agonistic activity promises a wide range of therapeutic applications for KYK-2.0 IgG1 and its derivatives.

SUBMITTER: Kwong KY

PROVIDER: S-EPMC2659651 | biostudies-literature | 2008 Dec

REPOSITORIES: biostudies-literature

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Generation, affinity maturation, and characterization of a human anti-human NKG2D monoclonal antibody with dual antagonistic and agonistic activity.

Kwong Ka Yin KY Baskar Sivasubramanian S Zhang Hua H Mackall Crystal L CL Rader Christoph C

Journal of molecular biology 20080916 5

In humans, NKG2D is an activating receptor on natural killer (NK) cells and a costimulatory receptor on certain T cells and plays a central role in mediating immune responses in autoimmune diseases, infectious diseases, and cancer. Monoclonal antibodies that antagonize or agonize immune responses mediated by human NKG2D are considered to be of broad and potent therapeutic utility. Nonetheless, monoclonal antibodies to NKG2D that are suitable for clinical investigations have not been published ye ...[more]

PMID: 18809410

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Project description:Antibodies isolated from human donors are increasingly being developed for anti-infective therapeutics. These antibodies undergo affinity maturation in vivo, minimizing the need for engineering of therapeutic leads for affinity. However, the affinities required for some therapeutic applications may be higher than the affinities of the leads obtained, requiring further affinity maturation in vitro. To improve the neutralization potency of natural human antibody MSL-109 targeting human cytomegalovirus (CMV), we affinity matured the antibody against the gH/gL glycoprotein complex. A phage display library where most of the six complementary-determining regions (CDRs) were allowed to vary in only one amino acid residue at a time was used to scan for mutations that improve binding affinity. A T55R mutation and multiple mutations in position 53 of the heavy chain were identified that, when present individually or in combination, resulted in higher apparent affinities to gH/gL and improved CMV neutralization potency of Fab fragments expressed in bacterial cells. Three of these mutations in position 53 introduced glycosylation sites in heavy chain CDR 2 (CDR H2) that impaired binding of antibodies expressed in mammalian cells. One high affinity (KD<10 pM) variant was identified that combined the D53N and T55R mutations while avoiding glycosylation of CDR H2. However, all the amino acid substitutions identified by phage display that improved binding affinity without introducing glycosylation sites required between two and four simultaneous nucleotide mutations to avoid glycosylation. These results indicate that the natural human antibody MSL-109 is close to a local affinity optimum. We show that affinity maturation by phage display can be used to identify and bypass barriers to in vivo affinity maturation of antibodies imposed by glycosylation and codon usage. These constraints may be relatively prevalent in human antibodies due to the codon usage and the amino acid sequence encoded by the natural human repertoire.

Dataset Information

Generation, affinity maturation, and characterization of a human anti-human NKG2D monoclonal antibody with dual antagonistic and agonistic activity.

Publications

Generation, affinity maturation, and characterization of a human anti-human NKG2D monoclonal antibody with dual antagonistic and agonistic activity.

Similar Datasets

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