Fine tuning of an anti-testosterone antibody binding site by stepwise optimisation of the CDRs

Hemminki, Ari; Niemi, Seija; Hautoniemi, Lasse; Söderlund, Hans; Takkinen, Kristiina

doi:10.1016/s1380-2933(98)00002-5

Cited by 35 publications

(36 citation statements)

References 27 publications

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“…On the basis of the structures, we are able to describe the details of the testosterone binding and also interpret previous results obtained in the fine-tuning of the anti-testosterone binding site (12,13). When the results were compared with other available structures of anti-steroid antibodies, interesting, similar features in the binding mechanism among these antibodies were observed.…”

supporting

confidence: 62%

“…Tuning the Properties of the Anti-testosterone Fab Fragment-The recent CDR random mutagenesis approach combined with phage display selections led to the isolation of mutants of the anti-testosterone Fab with significantly improved binding properties (12,13). Only one of the important binding site residues has been changed in the isolated mutants, namely the CDR-H3 residue Glu95H, located at the side of the binding site.…”

Section: Discussionmentioning

confidence: 99%

“…In the affinity maturation selection, two different clones having extensive mutations within the CDR-L1 loop were isolated (13). One clone of the mutant CDR-L1 loop, designated A60, contained the sequence RSSEVIVTRNGYTPIE, and the other, designated A58, contained the sequence RSSQRM-VQRNGHTPLE.…”

Section: Discussionmentioning

confidence: 99%

“…The main problem with the 3-C 4 F 5 antibody is its 1% cross-reaction with dehydroepiandrosteronesulphate (DHEAS), due to the high DHEAS concentration in sera, this cross-reaction prevents the diagnostic use of the 3-C 4 F 5 antibody. The binding site of the recombinant (3-C 4 F 5 ) Fab fragment has recently been targeted to random mutagenesis, and mutants with significantly improved affinity and specificity have been isolated by phage display selections (12,13).…”

mentioning

confidence: 99%

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Structural Insights into Steroid Hormone Binding

Valjakka¹,

Takkinenz²,

Teerinen³

et al. 2002

Journal of Biological Chemistry

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The monoclonal anti-testosterone antibody (3-C 4 F 5 ) has a relatively high affinity (3 ؋ 10 8 M ؊1 ) with an overall good specificity profile. However, the earlier characterized binding properties have shown that both the affinity and specificity of this antibody must be improved if it is intended for use in clinical immunoassays. In this paper, the crystal structures of the recombinant antitestosterone (3-C 4 F 5 ) Fab fragment have been determined in the testosterone-bound and free form at resolutions of 2.60 and 2.72 Å, respectively. The high affinity binding of the (3-C 4 F 5 ) Fab is mainly determined by shape complementarity between the protein and testosterone. Only one direct hydrogen bond is formed between the hydroxyl group of the testosterone D-ring and the main-chain oxygen of Gly100 J H. The testosterone is deeply bound in a hydrophobic pocket, and the close shape complementarity is mainly formed by the third complementarity-determining regions ( The number of different, closely related steroid hormones in human serum is high, and their relative concentrations, which usually are very low, can vary greatly even between normal healthy individuals. Testosterone, the main male sex hormone, is one of the structurally similar steroid hormones. Measurement of serum testosterone levels is important in the evaluation of conditions associated with hyperandrogenism in women (1, 2) and to diagnose hypogonadism, impotence, and problems in spermatogenesis and pubertical development in men (3, 4). Development of monoclonal antibodies that would fulfill the high affinity and selectivity requirements needed for the accurate measurement of testosterone levels in serum samples has not been successful with the use of hybridoma technology. Rabbit polyclonal antibodies are used in most current immunoassays of steroid hormones. However, the supply of polyclonal reagents with consistent quality is a severe problem for the immunodiagnostic industry and requires continuous immunization of many animals. The inherent batch-to-batch variation of the polyclonal sera makes it necessary to optimize the assay parameters for each new serum batch.The three-dimensional structures of steroid binding Fab fragments are important for a general understanding of the molecular basis of specific interactions between antibodies and hydrophobic molecules such as steroids. The molecular basis of steroid antibody interactions has been studied in detail by determining the structures of a progesteronebinding antibody (DB3) in its free and bound form and with different high affinity cross-reactive progesterone derivatives (5-7). The binding site of the DB3 antibody is a hydrophobic pocket, and the binding affinity and specificity are determined by van der Waals interactions and a few hydrogen bonds formed with the progesterone hapten (5). The high affinity binding mode of a number of progesterone derivatives is due to two alternate docking orientations for the steroid skeleton (6). In the cases of the anti-digoxin 26-10 and 40-50 antibodies, the h...

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supporting

confidence: 62%

Section: Discussionmentioning

confidence: 99%

Section: Discussionmentioning

confidence: 99%

mentioning

confidence: 99%

See 2 more Smart Citations

Structural Insights into Steroid Hormone Binding

Valjakka¹,

Takkinenz²,

Teerinen³

et al. 2002

Journal of Biological Chemistry

Self Cite

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show abstract

“…Many recent publications describe the affinity and/or specificity improvement of anti-steroid antibodies by random mutagenesis and phage display selections (4 -6). We have previously reported specificity and affinity improvement of the testosterone-binding Fab fragment initially derived from a hybridoma cell line (3-C 4 F 5 ) (7,8). In the absence of structural data of the 3-C 4 F 5 antibody, the optimization has been done by random mutagenesis and phage display selection of individual CDR 1 mutant libraries.…”

mentioning

confidence: 99%

Crystal Structure of an in Vitro Affinity- and Specificity-matured Anti-testosterone Fab in Complex with Testosterone

Valjakka

Hemminki

Niemi

et al. 2002

Journal of Biological Chemistry

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A highly selective, high affinity recombinant anti-testosterone Fab fragment has been generated by stepwise optimization of the complementarity-determining regions (CDRs) by random mutagenesis and phage display selection of a monoclonal antibody (3-C 4 F 5 ). The best mutant (77 Fab) was obtained by evaluating the additivity effects of different independently selected CDR mutations. The 77 Fab contains 20 mutations and has about 40-fold increased affinity (K d ‫؍‬ 3 ؋ 10 ؊10 M) when compared with the wild-type (3-C 4 F 5 ) Fab. To obtain structural insight into factors, which are needed to improve binding properties, we have determined the crystal structures of the mutant 77 Fab fragment with (2.15 Å) and without testosterone (2.10 Å) and compared these with previously determined wild-type structures. The overall testosterone binding of the 77 Fab is similar to that of the wild-type. The improved affinity and specificity of the 77 Fab fragment are due to more comprehensive packing of the testosterone with the protein, which is the result of small structural changes within the variable domains. Only one important binding site residue Glu-95 of the heavy chain CDR3 is mutated to alanine in the 77 Fab fragment. This mutation, originally selected from the phage library based on improved specificity, provides more free space for the testosterone D-ring. The light chain CDR1 of 77 Fab containing eight mutations has the most significant effect on the improved affinity, although it has no direct contact with the testosterone. The mutations of CDR-L1 cause a rearrangement in its conformation, leading to an overall fine reshaping of the binding site.Steroid hormones have remained a great challenge for immunodiagnostics. They are small, rigid, hydrophobic molecules with only a few functional groups capable of specific interactions with antibodies. The number of different closely related steroids in human serum is high, their in vivo concentrations are low (down to a picomolar level), and their relative concentrations can vary greatly, even between normal healthy individuals. Furthermore, steroids are poorly immunogenic in mice and rats; two species from which monoclonal antibodies are usually generated, making it extremely difficult to produce high affinity and specificity anti-steroid monoclonal antibodies. In most diagnostic immunoassays of steroid hormones, rabbit polyclonal antibodies are used, despite the many drawbacks associated with the use of antisera. The supply of good polyclonal antibody reagents of uniform quality is a severe problem for the immunodiagnostic industry and requires continuous immunization of many laboratory animals.Antibody engineering provides excellent tools to tailor the properties of antibodies with respect to affinity, specificity, and performance for different applications. An in vitro process of antigen-driven selection, based on the display of antibody fragments on the surface of a filamentous bacteriophage, has been shown to be a powerful method for the selection of specific or improved...

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Immunosensors for Diagnostics

2001

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Immunosensors can play an important role in the improvement of public health by providing applications for which rapid detection, high sensitivity, and specificity are important, in areas such as clinical chemistry, food quality, and environmental monitoring. The standard immunoassay techniques are the basis for the development of improved automated devices, exploring the accumulated know how of the implementation of indirect detection methods during the last 40 years. Today's systems achieve high accuracy, specificity, and sample throughput. Disposable immunotests (qualitative and quantitative), based on colored detection of an immunoreaction, are continuously expanding its applications for diagnosis of diseases, drug detection, and food quality control.Associated to the increasing demand of immunoapplications and with the new advances produced in the last decades in fields like biochemistry, biotechnology, electronics, and microfabrication, new transduction devices have been developed and applied to monitor immunoreactions. Optimization of the biochemical characteristics of the binding molecules, improved surface stability of the transducer interfaces, and reliable microsystems and integrated devices are the challenge for the next years of development. An overview of the immunoassay techniques used today in standard diagnosis will be presented, including a brief description of the different immunosensor transducers and some examples of the present developments and commercial devices.

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Fine tuning of an anti-testosterone antibody binding site by stepwise optimisation of the CDRs

Cited by 35 publications

References 27 publications

Structural Insights into Steroid Hormone Binding

Structural Insights into Steroid Hormone Binding

Crystal Structure of an in Vitro Affinity- and Specificity-matured Anti-testosterone Fab in Complex with Testosterone

Immunosensors for Diagnostics

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