A Combinatorial Approach for the Design of Complementarity-determining Region-derived Peptidomimetics with in Vitro Anti-tumoral Activity

Abstract

The great success of therapeutic monoclonal antibodies has fueled research toward mimicry of their binding sites and the development of new strategies for peptide-based mimetics production. Here, we describe a new combinatorial approach for the production of peptidomimetics using the complementarity-determining regions (CDRs) from gastrin17 (pyroEGP-WLEEEEEAYGWMDF-NH 2 ) antibodies as starting material for cyclic peptide synthesis in a microarray format. Gastrin17 is a trophic factor in gastrointestinal tumors, including pancreatic cancer, which makes it an interesting target for development of therapeutic antibodies. Screening of microarrays containing bicyclic peptidomimetics identified a high number of gastrin binders. A strong correlation was observed between gastrin binding and overall charge of the peptidomimetic. Most of the best gastrin binders proceeded from CDRs containing charged residues. In contrast, CDRs from high affinity antibodies containing mostly neutral residues failed to yield good binders. Our experiments revealed essential differences in the mode of antigen binding between CDR-derived peptidomimetics (K d values in micromolar range) and the parental monoclonal antibodies (K d values in nanomolar range). However, chemically derived peptidomimetics from gastrin binders were very effective in gastrin neutralization studies using cell-based assays, yielding a neutralizing activity in pancreatic tumoral cell lines comparable with that of gastrin-specific monoclonal antibodies. These data support the use of combinatorial CDR-peptide microarrays as a tool for the development of a new generation of chemically synthesized cyclic peptidomimetics with functional activity.Antibody-based therapeutics have emerged as important components of therapies for an increasing number of debilitating and life-threatening diseases (1-3). The unique properties of antibodies provide a source of inspiration for active research in antibody engineering. Over the years, a wide range of antibody fragments (Fab, scFv) 8 and variants (dia-, tria-, tetra-, mini-bodies, single-domain antibodies, intramers, etc.) have been developed (4 -8), some of which are used today in clinical therapies (9, 10). One step further in downsizing the antibody molecule is to use peptides derived from one or more of the six hypervariable loops, or "complementarity-determining regions" (CDRs; Fig. 1A) (11). Mutational analysis of antibodycombining sites suggests that only a subset of interface contact residues is essential for binding (12, 13). Several publications have appeared since the first report on CDR-derived peptides (14), with bioactivities even approaching those of the parent antibodies in a few cases. Heap et al. (15) reported a cyclic 17-mer peptide derived from the H3 CDR of an anti-gp120 mAb with only 37-fold lower affinity (K D ϭ 7.5 nM versus 0.2 nM for the mAb) and 32-fold lower HIV-1 neutralizing capacity. Some studies also use a rational design-based approach to make antibody-like binders, with remarkably high in vivo activit...

Computational Identification of Antigen-Binding Antibody Fragments

“…In all five Ab-Ag complexes we analyzed, CDRs L2 and L3 did not rank as likely to bind the Ag on their own. Although CDRs L3 and L2 are considered to have average or low contribution to Ag binding (55,58), some studies demonstrated that they can serve as a basis for Ag-binding peptides (30,59,60). Thus, the identification of potent CDR-derived peptide for each Ab-Ag complex should not rely on the concept that some CDRs are more important than others, but on rational ranking of the CDRs in a given Ab-Ag complex.…”

Binding of CDR‐derived peptides is mechanistically different from that of high‐affinity parental antibodies

“…Indeed, negatively charged or neutral peptides derived from high-affinity anti-G17 monoclonal antibody (mAb) 189DB3 (Barderas et al, 2008a) did not bind G17. In contrast to this, positively charged peptides derived from either the low-affinity PAR10C3 and PAR10D10 anti-G17 scFvs (Barderas et al, 2006), or from the high-affinity mAb 189DB3, or from D1.3 anti-HEL mAb (Harper et al, 1987), or those containing random sequences, also displayed significant G17 binding (Timmerman et al, 2009). These observations implied a lack of correlation between G17 binding affinity of CDR-derived peptide binders and parental antibodies, and prompted us to conduct a detailed analysis of SBs binding properties.…”

“…The names and sequences of all CDR-derived SBs used in our studies are listed in Table 1; the synthesis of these was described before (Timmerman et al, 2009). SBs were derived from the CDR sequences of three anti-G17 murine antibodies (Figure 1), of which one, mAb 189DB3, binds G17 with high affinity (K D ¼ 11.3 nM) (Barderas et al, 2008a) and the two others (scFvs PAR10C3, PAR10D10) with lower affinities (K D !1 mM) (Barderas et al, 2006).…”

“…Sequence information and names for G17 binders (Timmerman et al, 2009) used in this study are given in Table 1 (see methods for a summary of design strategy). SBs that represent only a fraction of the paratope with imperfect structural mimicry are expected to display low gastrin binding affinities.…”

“…In an earlier paper, a combinatorial synthetic approach allowed the selection of CDR-derived G17 binders that neutralized G17 in cell-based proliferation assays (Timmerman et al, 2009). Analysis of high-throughput screening data from >10 000 CDR-based binders derived from five anti-G17 antibodies (antibodies with K D 's ranging from 500 pM to >1 mM) indicated that G17 binding activities correlated primarily with the presence of positive charges in the synthetic binders (SBs).…”