Selection, characterization, and CDR shuffling of naive llama single-domain antibodies selected against auxin and their cross-reactivity with auxinic herbicides from four chemical families.

Indoleacetic acid (IAA)-binding single-domain antibodies (sdAbs) were isolated from a naive phage-display library constructed from the heavy chain antibody repertoire of a Ilama. The highest-affinity sdAb isolated (CSF2A) had a K(D) of 5-20 microM for two IAA-protein conjugates and a K(D) of 20 microM for free IAA. This sdAb also bound to a synthetic auxin analogue, 1-naphthaleneacetic acid (NAA), and to six auxinic herbicides (K(D) values of 0.5-2 mM), but not to serotonin and tryptophan, which are structurally similar to IAA but have no auxinic activity. To understand how sdAb CSF2A binds IAA and to determine which complementary-determining region(s) (CDR) participate(s) most in binding IAA, CSF2A was shuffled with four other sdAb clones by staggered extension process (StEP). After panning against IAA, two shuffled sdAbs were found: sdAb CSB1A, which originated from three different parental clones, and sdAb CSE8A, derived from two parental clones. These shuffled sdAbs and CSF2A were each fused to the B subunit of the Escherichia coli verotoxin, resulting in the formation of the pentamerized sdAbs V2NCSB1A, V2NCSE8A, and V2NCSF2A, which were analyzed by surface plasmon resonance (SPR) along with the sdAbs previously isolated. The shuffled clones had affinity for IAA (20 microM) similar to that of the highest affinity parental clone CSF2A, but much lower affinity for the auxinic herbicides. CDR2 was instrumental in binding IAA, whereas hydrophobic CDR3 was important for binding the auxinic herbicides. A novel SPR methodology is also described for specific immobilization of pentamerized sdAbs, allowing determination of K(D) values of Ab interaction with underivatized, low molecular weight haptens.

Affinity maturation of a V(H)H by mutational hotspot randomization.

V(H)Hs from naive libraries have dissociation constants (K(D)s) in the low micromolar range and thus, for most antibody applications, their intrinsic affinities need to be improved significantly. Non-targeted in vitro affinity maturation approaches based on indiscriminate randomization of complementarity-determining region (CDR) residues or random mutagenesis of conventional antibody variable domains have been shown to improve the affinity of recombinant antibodies by 450- to over 6000-fold. A different, targeted approach based on selective randomization of CDR codons containing AGY/RGYW nucleotide mutational hotspots i.e., "hotspot codons", also promises to be very efficient for improving antibody affinities. Here we employed the latter approach for improving the affinity of PTH22, a parathyroid hormone (PTH)-derived peptide-specific V(H)H that was isolated from a naive llama phage display library. A PTH22 mutant ribosome display library was constructed by randomizing nine CDR2 and CDR3 hotspot codons. The affinity improvement of the lead binder was 30-fold, which seems somewhat low in view of the large number of randomized hotspot codons. Nucleotide sequence analyses of PTH22 and 23 naive V(H)Hs suggested that many AGY/RGYW mutational hotspots are not affinity mutational hotspots but play a role in V(H)H solubility, structure, and deletion/insertion events. Our results indicate that the mutagenesis approach described here is beneficial in terms of yielding moderate increases in affinity while fine-tuning physical properties of an antibody.

Selection of hapten-specific single-domain antibodies from a non-immunized llama ribosome display library.

Picloram-specific variable fragments (V(HH)s) of heavy chain antibodies (HCAbs) were selected from a nai;ve-llama library using ribosome display technology. A cDNA library of V(HH)s was constructed from lymphocytes of a non-immunized llama and engineered to allow in vitro transcription and translation. With no stop codons present on the transcripts, trimeric complexes of ribosomes, mRNAs and nascent peptides were produced for affinity selection, i.e. panning. After three cycles of panning, seven different V(HH)s all belonging to the V(HH) subfamily 1 were isolated. Following another three cycles of selection, only two of the seven V(HH)s persisted. A comparison of these two sequences with known sequences in the literature suggests that point mutations may have been introduced into the DNA pool during PCR amplification steps of library construction, panning and/or cloning. Three separate point mutations causing three independent amino acid changes (nonsynonomous mutations) accumulated in the same sequence and enriched throughout the selection protocol, suggesting that these changes confer binding advantages. Surface plasmon resonance (SPR) analysis was used to determine binding kinetics of the two clones (3-1D2 and 3-1F6) representing the two different sets of isolated complementarity determining region (CDR)3s. Measured K(D)s were 3 and 254 muM, respectively. The results indicate that ribosome display technology can be used to efficiently isolate hapten-specific antibody (Ab) fragments from a nai;ve library and concurrently introduce diversity to the selected pool thereby facilitating molecular evolution. Ribosome display technology can compensate for the limited diversity of a V(HH) nai;ve library and provide an unlimited source of affinity-matured immunoactive reagents in vitro.

Immunomodulation confers herbicide resistance in plants.

In order to create a novel mechanism for herbicide resistance in plants, we expressed a single-chain antibody fragment (scFv) in tobacco with specific affinity to the auxinic herbicide picloram. Transgenic tobacco plants and seedlings expressing this scFv against picloram were protected from its effect in a dose-dependent manner. This is the first successful use of an antibody to confer in vivo resistance to a low molecular weight xenobiotic (i.e. < 1000 Da). Our results suggest the possibility for a generic antibody-based approach to create crops resistant to low molecular weight xenobiotics for subsequent use in the bioremediation of contaminated soils, crop protection and as novel selectable markers.

Emerging trends in the synthesis and improvement of hapten-specific recombinant antibodies.

A key requirement for successful immunotherapeutic and immunodiagnostic applications is the availability of antibodies with high affinity and specificity. In the past, polyclonal antibodies from hyperimmunized animals or monoclonal antibodies from hybridoma cell lines were used extensively and profitably in medicine and immunotechnology. Antibody-based diagnostics, such as immunoassays, are also widely accepted because of their high sensitivity and ease of use as compared to conventional chromatographic techniques. While immunoassays have been used to monitor organic chemical contaminants such as pesticides, food preservatives, antibiotics in agricultural and food industries, hapten-specific antibodies with the desired affinity and specificity are generally difficult to obtain. With the advent of recombinant DNA technology, antibody genes can be amplified and selected through phage display, cell surface display, or cell-free display systems. A particularly useful feature common to all these display systems is the linking of the phenotype and genotype of antibodies during selection. This allows easy co-selection of the desired antibodies and their encoding genes based on the binding characteristics of the displayed antibodies. The selected antibody DNA can be further manipulated for high-level expression, post-translation modification, and/or affinity and specificity improvement to suit their particular applications. Several hapten-specific antibodies, which were successfully selected and engineered to high specificity and affinity using display technologies, have been found to be amenable to conventional immunoassay development. In this review, we will examine different formats of immunoassays designed for hapten identification and various display technologies available for antibody selection and improvement.

Green fluorescent protein-labeled recombinant fluobody for detecting the picloram herbicide.

A green fluorescent protein-labeled fluobody was designed to develop a simple immunoassay method for detecting picloram herbicide in an environmental sample. The gfp gene was successfully inserted into the pSJF2 vector harboring the picloram-specific antibody fragment to yield pSJF2GFP. Picloram spiking in an environmental river sample could be indirectly detected by observing the fluorescence intensity value of the gfp-fluobody, exhibiting specific sensitivity to free picloram with an IC50 value of 50 ppb. Using the gfp-fluobody immunoassay avoids the enzyme-substrate reaction for calorimetric detection that is required in an enzyme-linked immunosorbent assay (ELISA).