Synthetic immune libraries

Researchers have expanded upon the natural diversity generated by the immune system. In 1989 a novel vector system based on the bacteriophage lambda was used to express a synthetic combinatorial library of Fab fragments of the mouse antibody repertoire in Escherichia coli [22]. This system allowed rapid and easy identification of monoclonal Fab fragments that bind a given antigen, in a form suitable for genetic manipulation. For example. 

In contrast to human antibodies derived from large naive or synthetic human antibody libraries, antibodies from immune animals were subjected to in vivo selection and are therefore more likely to recognize a given antigen selectively (i.e., without cross-reactivity to another antigen). 

In ribosome display, the physical link between genotype and phenotype is accomplished by mRNA-ribosome—protein complexes, which are directly used for selection. If a library of different mRNA molecules is translated, a protein library results in which each protein is produced from its own mRNA and remains connected to it. Since these complexes of the proteins and their encoding mRNAs are stable for several days under the appropriate conditions, very stringent selections can be performed. As all steps of ribosome display are carried out in vitro, reaction conditions of the individual steps can be tailored to the requirements of the protein species investigated, as well as the objectives of the selection or evolution experiment. Application of ribosome display has produced scFv fragments of antibodies with affinities in the picomolar range from libraries prepared from immunized mice (Hanes et al., 1998) and more recently from a naive, completely synthetic library (Hanes et al., 2000), and has been used to evolve improved off-rates and stability (Jermutus et al., 2000). 

The affinity of antibodies selected from naive, synthetic, or even immune phage libraries is typically sufficient for use as a research reagent, but too low for some specific therapeutic applications such as viral neutralization or tumor imaging. For many applications, affinity maturation can be bypassed completely by the construction of multivalent molecules. If this is not sufficient selected antibody clones may be affinity matured (see Figure 18.13). 

A prerequisite for CTL-mediated immune response is the formation of the MHC-class I-peptide complex and subsequent recognition by the T-cell repertoire, which can be analyzed in cell lysis assays with MCr-loaded target cells. Chromium release is a measure for peptide-induced cell lysis by CTL, and indicates the potency of the peptide to serve as an allele-specific epitope. A synthetic epitope has been identified with the peptide library approach to elucidate the molecular basis for the observed cross-recognition of two ligands by a single receptor [53]. 

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