Generation of native bovine mAbs by phage display

Abstract

Modeling of disease pathogenesis and immunity often is carried out in large animals that are natural targets for pathogens of human or economic relevance. Although murine mAbs are a valuable tool in identifying certain host/pathogen interactions, progress in comparative immunology would be enhanced by the use of mAbs isolated from the host species. Such antibodies would reflect an authentic host immune response to infection or vaccination, and as they are host derived, would allow the application of in vivo experiments that previously have been unrealizable in large animals because of induction of an antispecies immune response. The advent of antibody phage display technology provides a way of producing host-derived mAbs in animals where the molecular genetics of Ig formation are known. Exploiting recent advances in the molecular immunology of cattle, we report here the design of an optimized phage display vector, pComBov, for the construction of combinatorial libraries of bovine Ig antigen-binding fragments (Fab) of native sequence. By using this system, we initially have generated and characterized a panel of bovine mAbs against a model antigen glutathione S-transferase. The isolated mAbs showed features typical of bovine Igs and recognized glutathione S-transferase with high specificity in ELISA and by Western blotting. The pComBov expression system can be readily adapted for the preparation of libraries from related ruminant species and advances the use of monoclonal reagents derived in this way for comparative studies in animals of economic importance.

Figure 1

Oligonucleotide primers used in expression vector and library construction. Primer sequences are written 5′ to 3′. Restriction endonuclease recognition sites are underlined. Degenerate nucleotides are indicated as follows: S = C or G; W = A or T; M = A or C; Y = C or T.

Figure 2

pComBov expression cassette. Modified from pComb3H phagemid (19) on a pBluescript background (23). The CL was cloned from bovine genomic DNA. Digestion with SpeI and NheI removes the geneIII sequence, allowing expression of soluble Fab. Arrows indicate the position of cleavage of secretory sequences (pelB or ompA) during periplasmic expression of Fab. Amino acid resides (single-letter code) immediately after the cleavage site are the optimized bovine amino-terminal Ig sequences. RBS, ribosome binding site.

Figure 3

(A) Alignment of predicted amino acid sequences for the selected anti-GST clones. The nucleotide sequences of the V_H and V_L inserts of eight dominant and three minority clones were determined and translated into protein sequence (single-letter code). Unique V_L (i–iii) and V_H (i–iv) are shown for the dominant clones. Each minority clone expressed unique V_L and V_H sequence patterns. The sequences are segregated into CDR and FR regions based on homology to known bovine sequences (–18). A dash (–) indicates identity to the uppermost sequence; ∗ indicates no amino acid at this position. (B) V_L and V_H chain usage in the dominant selected anti-GST clones. Three V_L (i–iii) and four V_H (i–iv) sequences were found in various combinations in the eight dominant clones. Clones G67, G72, G74, and G76 expressed the same V_L and V_H combination and are therefore identical. Clone G63 differs by only two V_H FR residues from clone G77.

Figure 4

Antigen specificity of anti-GST Fab. (A) ELISA. Fab supernatants were diluted 1:2 and tested for reactivity against a range of protein antigens. Optical densities were read at 630 nm after 15 min; data are results from one of three similar experiments. Reactivity against GST is indicated in bold. WT, pComBov (no V_H or V_L sequences); HEL, hen egg lysozyme; KLH, keyhole limpet hemocyanin; FITC, fluorescein isothiocyanate-BSA conjugate; OVA, chicken ovalbumin; NO, no coating antigen. (B) Western blot GST and BSA were separated by SDS/PAGE and transferred to nitrocellulose, then incubated with supernatants containing anti-GST Fab. Fab recognized GST (26 kDa) but not BSA (not shown). WT, as in A; POS, positive control anti-GST mouse mAb; NEG, anti-bovine Ig-horseradish peroxidase only; MW, molecular weight (kDa).