A novel synthetic naïve human antibody library allows the isolation of antibodies against a new epitope of oncofetal fibronectin

Abstract

Human monoclonal antibodies (mAbs) can routinely be isolated from phage display libraries against virtually any protein available in sufficient purity and quantity, but library design can influence epitope coverage on the target antigen. Here we describe the construction of a novel synthetic human antibody phage display library that incorporates hydrophilic or charged residues at position 52 of the CDR2 loop of the variable heavy chain domain, instead of the serine residue found in the corresponding germline gene. The novel library was used to isolate human mAbs to various antigens, including the alternatively-spliced EDA domain of fibronectin, a marker of tumor angiogenesis. In particular, the mAb 2H7 was proven to bind to a novel epitope on EDA, which does not overlap with the one recognized by the clinical-stage F8 antibody. F8 and 2H7 were used for the construction of chelating recombinant antibodies (CRAbs), whose tumor-targeting properties were assessed in vivo in biodistribution studies in mice bearing F9 teratocarcinoma, revealing a preferential accumulation at the tumor site.

Acknowledgments

Financial contributions from the Swiss National Science Foundation, the ETH Zürich, the European Union (ADAMANT and IMMUNO-PDT Projects), the Swiss Cancer League, the Swiss-Bridge Foundation and the Stammbach Foundation are gratefully acknowledged.

Figures and Tables

Figure 1 Design and cloning strategy for the PHILO antibody library. (A) Three-dimensional structure of a scFv antibody fragment. Heavy chain and light chain backbone are represented in dark grey and light grey, respectively. Residues subject to random mutation are DP47 CDR3 position 95–100 (dark grey space-fill representation), light chain position 91 to 96 (light grey space-fill representation) (in more details, for DPK22 CDR3 residue 91–96, and for DPL16 CDR3 position 92–95 and 95b). The scFv structure was displayed using the program PyMol, based on the protein data base (Brookhaven Protein Data Bank) file 1igm. The residue numbers were defined as previously published in reference 23 and 63. (B) Library cloning strategy. A point mutation was introduced, converting residue S52 of VH to D, K, N or Y. Mutagenesis in the CDR3 regions was generated by PCR using partially degenerate primers. Genes are indicated as rectangles and CDRs as numbered boxes. The VH and VL segment were then assembled by PCR and cloned into the pHEN1 vector.64 Primers used in the amplification and assembly are listed in Table 1.

Figure 2 Characterization of the PHILO library. (A) PCR colony screening of 12 clones of each sub-library. As negative control a BirA insert (1,200 bp) of a pHEN1 vector was amplified and a scFv in pHEN1 was amplified as a positive control. All the tested clones showed an insert with the correct size of approximately 1,000 bp. (B) Dot blot analysis of 752 induced supernatants of individual library clones. The soluble scFv fragments were detected with the anti-myc-tag mAb 9E10. More than 90% of the clones express a detectable amount of soluble scFv fragment.

Figure 3 Experimental evidence of non-overlapping epitopes on EDA, recognized by the scFv(F8) and scFv(2H7) antibodies. (A) ELISA and sandwich ELISA results are depicted in dark grey and light grey column, respectively, using microtiter plates coated with the recombinant 11A12 fragment of fibronectin (ELISA) or the F8 antibody (sandwich ELISA). (B) BIAcore experiments were performed on 11A12-coated CM5 chips. The F8 antibody in the small immunoprotein (SIP) format15 was injected at saturation, followed by injection of either scFv(F10) (black line) or the negative control scFv(B7) antibody (light grey). The EDA epitope recognized by scFv(B7) is identical to the one recognized by the F8 antibody. (C) Triple complex formation of SIP(F8), 11A12 and scFv(F10) in size exclusion chromatography on Superdex 200. Peak 1 corresponds to the triple complex made by SIP(F8) + 11A12 + scFv(F10); peak 2 corresponds to SIP(F8) + 11A12; peak 3 corresponds to scFv(F10) + 11A12; peak 4 corresponds to 11A12 antigen alone.

Figure 4 Biodistribution studies in tumor bearing mice of scFv(F8) and scFv(A9) in diabody format (A) and CRAbs (B). Both parts report the schematic representation of antibody structure, the size exclusion analysis of antibody fragments on Superdex75 (Amersham, GE) (all antibodies have a main elution peak around 10 mL, which corresponds to the expected size of 52–54 kDa) and the biodistribution analysis displayed as percent injected dose per gram (%ID/g) in tumor (Tu) and in measured mice organs: liver (Li), lung (Lu), spleen (Sp), heart (He), kidney (Ki), intestine (Int), blood (Blo). Radioactivity in tumor and organs have been counted at 24 (dark grey) and 48 (light grey) hours post intravenous injection of radiolabeled antibodies. In (B), in the SEC profile of F8-10aa linker-2H7, the peak corresponding to the monomeric CRAb is marked. Small grey arrows indicate the retention volumes of the standard proteins (from left to right: blue dextran, bovine serum albumin, ovalbumin, scFv(F8), ribonuclease A).

Table 1 Sequence of synthetic primers

Table 2 Summary of test selections with PHILO and ETH2-GOLD libraries

Table 3 Sequences of the scFv CDRs specific to the extra-domain A of fibronectin