Selection of antibodies based on antibody kinetic binding properties.

Molecular evolution approaches to developing molecules with characteristics particularly suited for specific applications have become important tools in biomedicine and biotechnology. Not only is it possible to identify molecules with specificities that cannot easily be obtained by other means, but it is also possible to fine-tune in an efficient manner the properties for, in principle, any specified application. Attention has particularly been put into identifying molecules with specific reaction-rate and affinity properties. Depending on the intended application, the binding of a molecule to its target is desired to be long-lived or short-lived. In biosensors, it will generally be appropriate for the association between the ligand and its receptor to be rapid. However, the dissociation of the complex should also be fast to ensure a rapid response of the sensor to a changing environment, particularly in on-line systems. In contrast, stable, nondissociating interactions are favored when, for example, an antibody (Ab) is used for tumor imaging or tumor therapy. In conventional immunoassays, high affinity (and specificity) is often sought to ensure a high sensitivity of the assay. However, under conditions in which a high throughput rather than a highly sensitive format is necessary, it may be more important to have a rapid association rate and a rapid establishment of equilibrium of the assay system than simply to have an assay based on high affinity alone

Is tailored treatment superior to non-tailored treatment for pain and disability in women with non-specific neck pain? A randomized controlled trial

The evidence for the effect of treatments of neck pain is modest. In the absence of causal treatments, a possibility is to tailor the treatment to the individuals' functional limitations and symptoms. The aim was to evaluate treatment effects of a tailored treatment versus a non-tailored treatment. Our hypothesis was that tailored treatment (TT) would have better effect on pain intensity and disability than either non-tailored treatment (NTT) (same treatment components but applied quasi-randomly) or treatment-as-usual (TAU) (no treatment from the study, no restrictions). We further hypothesized that TT and NTT would both have better effect than TAU.One hundred twenty working women with subacute and chronic non-specific neck pain were allocated to 11\ua0weeks of either TT, NTT or TAU in a randomized controlled trial with follow-ups at 3, 9 and 15\ua0months. The TT was designed from a decision model based on assessment of function and symptoms with defined cut-off levels for the following categories: reduced cervical mobility, impaired neck-shoulder strength and motor control, impaired eye-head-neck control, trapezius myalgia and cervicogenic headache. Primary outcomes were pain and disability. Secondary outcomes were symptoms, general improvement, work productivity, and pressure pain threshold of m. trapezius.Linear mixed models analysis showed no differences between TT and NTT besides work productivity favoring TT at 9- and 15-months follow-ups. TT and NTT improved significantly more than TAU on pain, disability and symptoms at 3-month follow-up. General improvement also favored TT and NTT over TAU at all follow-ups.Tailored treatment according to our proposed decision model was not more effective than non-tailored treatment in women with subacute and chronic neck pain. Both tailored and non-tailored treatments had better short-term effects than treatment-as-usual, supporting active and specific exercise therapy, although therapist-patient interaction was not controlled for. Better understanding of the importance of functional impairments for pain and disability, in combination with a more precise tailoring of specific treatment components, is needed to progress.Current Controlled Trials ISRCTN 49348025. Registered 2 August 2011

Real time analysis of antibody-antigen reaction kinetics.

Surface plasmon resonance, i.e. detection of changes in refractive index on a surface, was used in a biosensor to evaluate the dissociation/association rate and affinity constants of human monoclonal IgG and IgM antibodies and Tab fragments. The results showed that an observed difference in affinity constants between intact and fragmented IgG anti-tetanus antibody was related to approximately 10-fold differences in dissociation rate constants, since the association rate constants were in the same range, i.e. 2–3×105 (m-1s-1). Affinity constants, as determined by conventional solid phase enzyme immunoassays, were substantially higher than the constants produced by the biosensor. Human monoclonal IgM anti-Tnα antibodies showed, furthermore, one order of magnitude higher association rate constants, as compared with the IgG antibodies, but since the dissociation rate constants were more than ten times higher, the resulting affinity constants of the anti-carbohydrate IgM antibodies were still somewhat lower than those of the IgG antibodies

In vitro molecular evolution of antibody genes mimicking receptor revision

Antibody evolution in vivo proceeds mainly by stepwise improvements, accomplished by single base pair substitutions. Lately, receptor revision, i.e. exchange of large parts of the V gene for another sequence, has been suggested to provide a complementary route for affinity maturation. By employing a receptor revision like evolution process in vitro using combinatorial libraries and phage display selection, we demonstrate here that maturation of a clone may preferentially proceed through exchange of a large gene segment rather than via minor sequence changes. These modifications of a CD40-specific human antibody fragment outline how receptor revision like events may provide an advantage to a particular clonotype put under selective pressure. (C) 2002 Elsevier Science Ltd. All rights reserved

Limited mutagenesis increases the stability of human carboxypeptidase U (TAFIa) and demonstrates the importance of CPU stability over proCPU concentration in down-regulating fibrinolysis

Procarboxypeptidase U [proCPU, thrombin-activatable fibrinolysis inhibitor (TAFI), EC 3.4.17.20] belongs to the metallocarboxypeptidase family and is a zymogen found in human plasma. ProCPU has been proposed to be a molecular link between coagulation and fibrinolysis. Upon activation of proCPU, the active enzyme (CPU) rapidly becomes inactive due to its intrinsic instability. The inherent instability of CPU is likely to be of major importance for the in vivo down-regulation of its activity, but the underlying structural mechanisms of this fast and spontaneous loss of activity of CPU have not yet been explained, and they severely inhibit the structural characterization of CPU. In this study, we screened for more thermostable versions of CPU to increase our understanding of the mechanism underlying the instability of CPU's activity. We have shown that single as well as a few 2-4 mutations in human CPU can prolong the half-life of CPU's activity at 37 degrees C from 0.2 h of wild-type CPU to 0.5-5.5 h for the mutants. We provide evidence that the gain in stable activity is accompanied by a gain in thermostability of the enzyme and increased resistance to proteolytic digest by trypsin. Using one of the stable mutants, we demonstrate the importance of CPU stability over proCPU concentration in down-regulating fibrinolysis

Development of interleukin-1 receptor antagonist mutants with enhanced antagonistic activity in vitro and improved therapeutic efficacy in collagen-induced arthritis.

Interleukin-1 receptor antagonist (IL-1Ra) is a naturally occurring inhibitor of the pro-inflammatory interleukin-1-mediated activation of the interleukin-1 receptor (IL-1R). Although wild-type IL-1Ra is used for treatment of inflammatory diseases, its effect is moderate and/or short-lived. The objective of this study was to generate IL-1Ra mutants with enhanced antagonistic activity for potential therapeutic use. Using a directed evolution approach in which libraries of IL-1Ra gene mutants were generated and screened in functional assays, mutants with desired properties were identified. Initially, diversity was introduced into the IL-1Ra using random mutagenesis. Mutations resulting in enhanced antagonistic activity were identified by screening in a reporter cell assay. To further enhance the antagonistic activity, selected mutations were recombined using the DNA recombination technology Fragment-INduced Diversity (FIND). Following three rounds of FIND recombination, several mutants with up to nine times enhanced antagonistic activity (mean IC50 +/- SEM value: 0.78 +/- 0.050 vs. 6.8 +/- 1.1 ng/ml for mutant and wild-type, respectively) were identified. Sequence analysis identified the mutations D47N, E52R and E90Y as being most important for this effect, however, the mutations P38Y, H54R, Q129L and M136N further enhanced the antagonistic function. Analysis of identified mutations in protein models based on the crystal structure of the IL-1Ra/IL-1R complex suggested that mutations found to enhance the antagonistic activity had a stabilizing effect on the IL-1Ra mutants or increased the affinity for the IL-1R. Finally, the therapeutic effect of one mutant was compared to that of wild-type IL-1Ra in collagen-induced arthritis in mice. Indeed, the enhanced antagonistic effect of the mutants observed in vitro was also seen in vivo. In conclusion, these results demonstrate that directed evolution of IL-1Ra is an effective means of generating highly potent therapeutic proteins