Modulating the binding properties of an anti‐17β‐estradiol antibody by systematic mutation combinations

Abstract

The anti-17␤-estradiol antibody 57-2 has been a subject for several protein engineering studies that have produced a number of mutants with improved binding properties. Here, we generated a set of 16 antibody 57-2 variants by systematically combining mutations previously identified from phage display-derived improved antibody mutants. These mutations included three point mutations in the variable domain of the light-chain and a heavy-chain variant containing a four-residue random insertion in complementarity determining region CDR-H2. The antibody variants were expressed as Fab fragments, and they were characterized for affinity toward estradiol, for cross-reactivity toward three related steroids, and for dissociation rate of the Fab/estradiol complex by using time-resolved fluorescence based immunoassays. The doublemutant cycle method was used to address the cooperativity effects between the mutations. The experimental data were correlated with structural information by using molecular modeling and visual analysis of the previously solved antibody 57-2 crystal structures. These analyses provided information about the steroidbinding mode of the antibody, the potential mechanisms of individual mutations, and their mutual interactions. Furthermore, several combinatorial mutants with improved affinity and specificity were obtained. The capacity of one of these mutants to detect estradiol concentrations at a clinically relevant range was proved by establishing a time-resolved fluorescence based immunoassay.Keywords: Antibody; estradiol; mutation; site-directed mutagenesis; steroidThe capability of antibodies to specifically recognize an immense variety of other molecules is widely used in life sciences. For example, immunoassays that are commonly used for the quantitation of the concentrations of specific components in complex molecular mixtures such as blood are based on the recognition properties of antibodies. However, the generation of high-quality antibodies for some antigens is challenging. An example of this type of molecule is the steroid hormone 17␤-estradiol (E2), which consists of a hydrophobic steroid skeleton containing only two functional hydroxyl groups (Fig. 1). Concentration of E2 in the blood is low, and a number of steroids with closely related structures are present in the circulation, many of them in much higher concentration than E2. Therefore, the antibody used as a binder in an E2 immunoassay needs to have high affinity and good specificity. Currently, sufficiently good monoclonal antibodies are not available, and the E2 immunoassays are based on polyclonal antibodies.We have previously cloned three murine antibodies raised against 17␤-estradiol-6-CMO conjugate (Pajunen et al. 1997). One of these is known as antibody 57-2, and it has a moderate affinity toward E2, an excellent capacity to disReprint requests to: Urpo Lamminmäki, Department of Biotechnology, University of Turku, Tykistökatu 6A, 20520 Turku, Finland; e-mail: urpo.lamminmaki@utu.fi; fax: 358-2-3338050.Abbreviations: E2, ...

A different ontogenesis for chronic lymphocytic leukemia cases carrying stereotyped antigen receptors: molecular and computational evidence

“…36 Finally, one should not overlook the fact that there are well-documented cases for a particular lightchain sequence restriction among antibodies of a certain specificity, while still allowing for extensive diversity in the HCDR3. 37 In the present study, remarkable HCDR3 length restrictions were identified for the CLL BCR archetypes corresponding to high-level clusters. In particular, although level 3 clusters could include sequences with a range of HCDR3 lengths, collectively, they were 'enriched' in sequences of just four different HCDR3 lengths ( Figure 5).…”

Mutational landscape of antibody variable domains reveals a switch modulating the interdomain conformational dynamics and antigen binding

“…The effect of the LC-83 mutation on antigen affinity may be context-dependent. We note that the F83S mutation reportedly increased the affinity of a V kappa 1-containing anti-estradiol antibody (45). In summary, LC-83 and LC-106 are frequently mutated in human antibodies.…”

Molecular engineering of antibodies for therapeutic and diagnostic purposes

“…74 A second example of double engineering concerned an anti-17βestradiol mAb. 75 Based on the potential additivity of mutations, Lamminmaki and colleagues systematically explored the binding properties of all the possible combinations between three light-chain point mutations and a four-amino acid heavy-chain insertion previously selected by phage display. They selected one triple combined mutant that displayed a 17-fold affinity increase together with 400-fold reduced cross-reactivity with testosterone.…”