Human Immunoglobulin Repertoires against Tetanus Toxoid Contain a Large and Diverse Fraction of High-Affinity Promiscuous VH Genes

Kruif, John de; Kramer, Arjen; Visser, Therèse; Clements, Carina; Nijhuis, Roy; Cox, Freek; Zande, Vanessa van der; Smit, Renate; Pinto, Daniel Damazio; Throsby, Mark; Logtenberg, Ton

doi:10.1016/j.jmb.2009.02.009

Cited by 22 publications

(24 citation statements)

References 31 publications

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“…Previous reports on somatic mutations (8,(18)(19)(20)(21) fall within the presently observed limits, which can thus be regarded representative for a fully matured response.…”

Section: Statistical Analysessupporting

confidence: 71%

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Limits for Antibody Affinity Maturation and Repertoire Diversification in Hypervaccinated Humans

Poulsen

Jensen

Haurum

et al. 2011

The Journal of Immunology

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The immune system is known to generate a diverse panel of high-affinity Abs by adaptively improving the recognition of pathogens during ongoing immune responses. In this study, we report the biological limits for Ag-driven affinity maturation and repertoire diversification by analyzing Ab repertoires in two adult volunteers after each of three consecutive booster vaccinations with tetanus toxoid. Maturation of on-rates and off-rates occurred independently, indicating a kinetically controlled affinity maturation process. The third vaccination induced no significant changes in the distribution of somatic mutations and binding rate constants implying that the limits for affinity maturation and repertoire diversification had been reached. These fully matured Ab repertoires remained similar in size, genetically diverse, and dynamic. Somatic mutations and kinetic rate constants showed normal and log-normal distribution profiles, respectively. Mean values can therefore be considered as biological constants defining the observed boundaries. At physiological temperature, affinity maturation peaked at kon = 1.6 × 104 M−1 s−1 and koff = 1.7 × 10−4 s−1 leading to a maximum mean affinity of KD = 1.0 × 10−9 M. At ambient temperature, the average affinity increased to KD = 3.4 × 10−10 M mainly due to slower off-rates. This experimentally determined set of constants can be used as a benchmark for analysis of the maturation level of human Abs and Ab responses.

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“…Previous reports on somatic mutations (8,(18)(19)(20)(21) fall within the presently observed limits, which can thus be regarded representative for a fully matured response.…”

Section: Statistical Analysessupporting

confidence: 71%

“…To determine the progression in affinity maturation, k on and k off were determined at 25˚C for a genetically diverse set of [19][20][21][22][23][24][25][26][27][28][29][30][31][32][33][34][35] Abs from each repertoire ( Fig. 2A-C).…”

Section: Statistical Analysesmentioning

confidence: 99%

Limits for Antibody Affinity Maturation and Repertoire Diversification in Hypervaccinated Humans

Poulsen

Jensen

Haurum

et al. 2011

The Journal of Immunology

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“…This may be circumvented by transfecting a single cell with genes that encode antibodies that all use a single, identical light chain. We have recently shown that a large fraction of the human antibody immune repertoire readily converts to single light chain use, resulting in diverse collections of high affinity antibodies that can all use an identical light chain (de Kruif et al, 2009). Random combination of IgG heavy chains in Oligoclonic TM cell lines will result in the generation of both mono-and bispecific molecules with fully functional binding sites.…”

Section: Oligoclonicsmentioning

confidence: 99%

Generation of stable cell clones expressing mixtures of human antibodies

Kruif

Kramer

Nijhuis

et al. 2010

Biotech & Bioengineering

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Therapeutic monoclonal antibodies, a highly successful class of biological drugs, are conventionally manufactured in mammalian cell lines. A recent approach to increase the therapeutic effectiveness of monoclonal antibodies has been to combine two or more of them; however this increases the complexity of development and manufacture. To address this issue a method to efficiently express multiple monoclonal antibodies from a single cell has been developed and we describe here the generation of stable cell clones that express high levels of a human monoclonal antibody mixture. PER.C6 cells were transfected with a combination of plasmids containing genes encoding three different antibodies. Clones that express the three corresponding antibody specificities were identified, subcloned, and passaged in the absence of antibiotic selection pressure. At several time points, batch production runs were analyzed for stable growth and IgG production characteristics. The majority (11/12) of subclones analyzed expressed all three antibody specificities in constant ratios with total IgG productivity ranging between 15 and 20 pg/cell/day under suboptimal culture conditions after up to 67 population doublings. The growth and IgG production characteristics of the stable clones reported here resemble those of single monoclonal antibody cell lines from conventional clone generation programs. We conclude that the methodology described here is applicable to the generation of stable PER.C6(R) clones for industrial scale production of mixtures of antibodies.

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“…Bispecific antibodies (bsAbs) 3 combine two different antigen-binding sites in a single molecule. Bispecific antibodies can address multiple antigens in a single molecule resulting in more specific targeting, novel mechanisms of action, and higher clinical efficacies (1).…”

mentioning

confidence: 99%

“…In the production of bispecific IgG, heterodimer formation can thus be induced by engineering the CH3 domains such that homodimer-favoring interactions are replaced by asymmetric interactions. Most bispecific formats based on native IgG benefit from the use of a common light chain (cLC) (3,4), which has the advantage of preventing mispairing of heavy and light chains without the need for engineering.…”

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confidence: 99%

A new approach for generating bispecific antibodies based on a common light chain format and the stable architecture of human immunoglobulin G1

Nardis

Hendriks

Poirier³

et al. 2017

Journal of Biological Chemistry

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Edited by Peter CresswellBispecific antibodies combine two different antigen-binding sites in a single molecule, enabling more specific targeting, novel mechanisms of action, and higher clinical efficacies. Although they have the potential to outperform conventional monoclonal antibodies, many bispecific antibodies have issues regarding production, stability, and pharmacokinetic properties. Here, we describe a new approach for generating bispecific antibodies using a common light chain format and exploiting the stable architecture of human immunoglobulin G 1 . We used iterative experimental validation and computational modeling to identify multiple Fc variant pairs that drive efficient heterodimerization of the antibody heavy chains. Accelerated stability studies enabled selection of one Fc variant pair dubbed "DEKK" consisting of substitutions L351D and L368E in one heavy chain combined with L351K and T366K in the other. Solving the crystal structure of the DEKK Fc region at a resolution of 2.3 Å enabled detailed analysis of the interactions inducing CH3 interface heterodimerization. Local shifts in the IgG backbone accommodate the introduction of lysine side chains that form stabilizing saltbridge interactions with substituted and native residues in the opposite chain. Overall, the CH3 domain adapted to these shifts at the interface, yielding a stable Fc conformation very similar to that in wild-type IgG. Using the DEKK format, we generated the bispecific antibody MCLA-128, targeting human EGF receptors 2 and 3. MCLA-128 could be readily produced and purified at industrial scale with a standard mammalian cell culture platform and a routine purification protocol. Long-term accelerated stability assays confirmed that MCLA-128 is highly stable and has excellent biophysical characteristics.

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Human Immunoglobulin Repertoires against Tetanus Toxoid Contain a Large and Diverse Fraction of High-Affinity Promiscuous VH Genes

Cited by 22 publications

References 31 publications

Limits for Antibody Affinity Maturation and Repertoire Diversification in Hypervaccinated Humans

Limits for Antibody Affinity Maturation and Repertoire Diversification in Hypervaccinated Humans

Generation of stable cell clones expressing mixtures of human antibodies

A new approach for generating bispecific antibodies based on a common light chain format and the stable architecture of human immunoglobulin G1

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