Combinatorial Events in Generation of Antibody Diversity Pp. 377-383
Recognition of Peptides by Antibodies and Investigations of Affinity Using Biosensor Technology Pp. 385-395
Immunoglobulin Cross-Reactivity Examined by Library Screening, Crystallography and Docking Studies Pp. 397-408
The Immune Diversity in a Test Tube – Non–Immunised Antibody Libraries and Functional Variability in Defined Protein Scaffolds Pp. 409-416
Of Minibody, Camel and Bacteriophage Pp. 417-430
Antibody-based Fluorescence Polarization Assay to Screen Combinatorial Libraries for Sweet Taste Compounds Pp. 431-438
Antibody-Ligand Interactions: Computational Modeling and Correlation with Biophysical Measurements Pp. 439-449
[Back
to top] Combinatorial
Events in Generation of Antibody Diversity
Combinatorial
association of immunoglobulin gene elements is the most important process in
the creation of extreme diversity of antibody molecules. The recombination of
germ-line variable gene elements V, D, and J can potentially generate approximately
6000 variable genes of human heavy chains. As joining of these elements is
imprecise and is occurring with nucleotide additions or deletions, the created
diversity is in fact much higher. The assembled variable genes can be revised
and edited resulting in a change of their affinity and even specificity. Due to
somatic hypermutation, the affinity of synthesized antibody increases even
more. Another variant of combinatorial recombination is joining of complete
variable genes with one of the several constant genes and the formation of
various immunoglobulin isotypes with different effector functions but with the
same antibody specificity. Consequently, these processes not only develop the
antibody repertoire but also solve some other problems of the adaptive immune
response.
[Back to top] Recognition of Peptides by Antibodies and Investigations of Affinity Using Biosensor Technology
The study of peptide-antibody interactions has many applications in biology and medicine. Synthetic peptides corresponding to single protein epitopes are used instead of intact proteins as reagents for the diagnosis of viral and autoimmune diseases. Furthermore, antibodies raised against peptides are useful reagents for isolating and characterizing gene products. In this review, methods for analysing the molecular basis of peptide-antibody interactions are described, such as amino acid replacement studies, X-ray crystallography of peptide-antibody complexes and biosensor technology based on surface plasmon resonance. The importance of peptide conformation in antibody recognition is discussed, and the antigenic reactivity of epitopes in synthetic peptides and in cognate, intact proteins is compared.
[Back to top] Immunoglobulin Cross-Reactivity Examined by Library Screening, Crystallography and Docking Studies
Antibodies
are extremely diverse with respect to their specificities and affinities for
target molecules. Despite rigorous selection, some antibodies are
cross-reactive whereby they recognize their natural antigens along with other
molecules. In this review, we discuss our efforts toward understanding the
cross-reactivity of selected immunoglobulins. Investigations that are discussed
employed screens of combinatorial peptide libraries, crystallography of
ligand-protein complexes, and computer-based peptide docking simulations. In the
first example, two different antibodies (NC6.8 and NC10.14) bound the same
trisubstituted guanidine (NC174) with similar affinities, but utilized
predominantly dissimilar binding strategies. However, there was one common
binding strategy, in which the cyanophenyl portion of NC174 was inserted end-on
into the binding crevices of the NC6.8 and NC10.14 antibodies. In the second
example, scanning of peptide libraries and X-ray crystallography were used to
design and test synthetic peptides for binding to the Mcg L chain dimer. Again,
end-on insertion was favored for all peptides larger than dipeptides in the
voluminous Mcg binding cavity. Finally, automated docking was used for rapid
predictions of complexes for the Fv molecule from a broadly cross-reactive human
IgM (Mez) and nearly two thousand peptides. Certain amino acids, including the
aromatic residues Trp and Phe, functioned as anchoring groups in automated
docking. Anchoring groups acted in most of the peptides that were otherwise
accommodated by a variety of binding strategies in the docked complexes. We
suggest that anchoring of at least a portion of a ligand in a binding site is a
common mechanism for antibody recognition.
[Back to top] The Immune Diversity in a Test Tube – Non–Immunised Antibody Libraries and Functional Variability in Defined Protein Scaffolds
Technologies to develop and evolve the function of proteins and, in particular, antibodies have developed rapidly since the introduction of phage display. Importantly, it has become possible to identify molecules with binding properties that cannot be found by other means. A range of different approaches to create general libraries that are useful for the selection of such molecules specific for essentially any kind of target have emerged. We herein review some of the most prominent approaches in the field and in particular discuss specific features related to the development of antibody libraries based on single antibody framework scaffolds. This approach not only permits identification of a range of specific binders, but also facilitates further evolution of initially derived molecules into molecules with optimised functions.
[Back to top] Of Minibody, Camel and Bacteriophage
This review describes the design process from conception through realisation and optimisation of a ‘minibody’ - a minimised antibody. The result was a proteinaceous molecule of novel fold and metal binding activity. We explain how combinatorial approaches, using phage display libraries, were used to randomise loop regions of the minibody. Variants were then selected for desired activities including in vitro inhibition of human interleukin-6 and the protease of the non-structural protein, NS3, of the hepatitis C virus. One such variant was successfully minimised further to produce a cyclic peptide with similar inhibition properties. Thus the work reviewed provides examples of two important processes in protein design and protein minimisation. We conclude by discussing the role of such studies in medical applications and small molecule drug discovery. We also highlight the potential of our work and similar techniques in the post-genomic era.
[Back to top] Antibody-based Fluorescence Polarization Assay to Screen Combinatorial Libraries for Sweet Taste Compounds
D. S.
Linthicum, J. Patel, and N. Cairns
Technological advances in instrumentation, chemical synthesis methods, molecular biology and biochemistry have fueled the recent growth in high throughput screening. Assays are available in a vast range for formats, including fluorescence, luminescence, absorbance, and scintillation detection. Antibodies represent a powerful tool for novel compound discovery and their utility in this regard should not be underestimated. We have designed a fluorescence polarization immunoassay for the identification of novel sweeteners. The assay is based on monoclonal antibodies that bind superpotent sweet taste compounds and libraries of suitable test compounds can be rapidly screened using these antibodies as “artificial taste receptors.”
[Back to top] Antibody-Ligand Interactions: Computational Modeli ng and Correlation with Biophysical Measurements
Several new aspects of computer-assisted molecular modeling strategies and biophysical techniques, such as fluorescence spectroscopy, circular dichroism, and absorption spectroscopy, have proved useful in the analysis and description of antibody-ligand interactions. The molecular features involved in determining the specificity of antibody-ligand interactions, such as electrostatics (e.g. partial charges, salt bridges, p-cation motifs), hydrogen-bonds, polarization, hydrophobic interactions, hydration and solvation effects, entropy, and kinetics can be identified using a battery of biophysical techniques. An understanding of these parameters is essential to our use of antibodies as tools in high throughput screening of chemical libraries for the discovery of novel compounds.