Making artificial antibodies: A format for phage display of combinatorial heterodimeric arrays

The fusion proteins were constructed by fusing a Flag peptide to the N or C terminus of pVII and pIX. The constructs were amplified by PCR with single-stranded VCSM13 DNA as template. The primers used for the four permutations were as follows: pVII-Flag, VII-FOR (5′-CTATCCATGGCAATGGAGCAGGTCGCGGATTTC-3′) and VII-fBW (5′-ATTTAGCTAGCTTATTTGTCATCGTCATCTTTGTAGTCTCTTTGACCCCCAGCGATTAT-3′); Flag-pVII, VII-fFOR (5′-CTATCCATGGCAGACTACAAAGATGACGATGACAAAATGGAGCAGGTCGCGGATTTC-3′) and VII-BW (5′-GATTTAGCTAGCTTATTATCTTTGACCCCCAGCGATTAT-3′); pIX-Flag, IX-FOR (5′-CTATCCATGGCAATGAGTGTTTTAGTGTATTCT-3′) and IX-fBW (5′-ATTTAGCTAGCTTATTTGTCATCGTCATCTTTGTAGTCTGAGGAAGTTTCCATTAAACG-3′); Flag-pIX, IX-fFOR (5′-CTATCCATGGCAGACTACAAAGATGACGATGACAAAATGAGTGTTTTAGTGTATTCT-3′) and IX-BW (5′-GATTTAGCTAGCTTATTATGAGGAAGTTTCCATTAAACG-3′).

The PCR products were digested by restriction enzymes NcoI and NheI and inserted into the phagemid vector pCGMT (15).

Two murine catalytic antibodies, 21H3 and 2H6 (15–18), and one murine anti-cocaine antibody, 92H2 (generated by our laboratory), were used to construct V_H-(Gly₄Ser)-pVII and V_L-(Gly₄Ser)-pIX fusion proteins. The V_H sequences in each case were fused to the N terminus of pVII, and the V_L sequences were fused to the N terminus of pIX. The constructs were engineered by inserting a linker sequence, Gly₄Ser, between V_H and pVII and between V_L and pIX. The Fab genes were available from our previous work and were the most readily manipulated sequences for amplification of variable regions. First, the V_H-linker fragments were amplified by PCR by using primers H1 and H2 for Fab 21H3, H1 and H3 for Fab 2H6, and H1 and H4 for Fab 92H2. The V_L-linker fragments were amplified by using primers L1 and L2 for Fab 21H3, L1 and L3 for Fab 2H6, and L1 and L4 for Fab 92H2. Then, linker-pVII and linker-pIX were amplified with VCSM13 as template. The primers VII-F and VII-BW were used for linker-pVII, and primers IX-F and IX-BW were used for the linker-pIX construct. Finally, V_H-(Gly₄Ser)-pVII and V_L-(Gly₄Ser)-pIX were assembled through overlap-extension PCR by mixing equimolar amounts of V_H-linker and linker-pVII, or V_L-linker and linker-pIX. The primers H1 and VII-BW were used for V_H-(Gly₄Ser)-pVII, and the primers L1 and IX-BW were used for V_L-(Gly₄Ser)-pIX. The sequences of primers used were as follows and as shown above: H1 (5′-GCCTACGGCAGCCGCTGGATTGTTATTACT-3′), H2 (5′-GCCAGAACCACCACCAGAGACAGTGACCAG-3′), H3 (5′-GCCAGAACCACCACCAGAGACGGTGACTGAGGTTCC-3′), H4 (5′-GCCAGAACCACCACCAGATGAGGAAACGGTGACCGT-3′), L1 (5′-GCTATCGCGATTGCTGTGGCACTGGCTGGT-3′), L2 (5′-GGAGCCGCCGCCGCCAGAATCAGCCCGTTTGATTTC-3′), L3 (5′-GGAGCCGCCGCCGCCAGAATCAGTCCGTTTCAACTC-3′), L4 (5′-GGAGCCGCCGCCGCCAGAGACCAGGCCCCCGAGGCC-3′), VIIF (5′-TCTGGTGGTGGTTCTGGCATGGAGCAGGTCGCGATTTC-3′), IXF (5′-TCTGGCGGCGGCGGCTCCATGAGTGTTTTAGTGTATTCT-3′).

After digestion with NcoI and NheI, the V_H-(Gly₄Ser)-pVII fusion proteins of 21H3, 2H6, and 92H2 were ligated into phage display vector pCGMT-1b that was derived from pCGMT and contained two ribosome-binding sites and two leader sequences of ompA and pelB (Fig. 2). V_L-(Gly₄Ser-pIX) was digested with SacI and NheI and ligated into pCGMT-1b that had the V_H-(Gly₄Ser)-pVII insert.

Stable complexes of 5-nm gold particles bound to PCP-BSA were prepared according to known methodology (19, 20). The designation PCP referred to the phosphonate hapten originally used to obtain the antibodies 21H3 and 2H6 (16). To ensure the removal of any unbound protein, the complexes were centrifuged through a 7% glycerol cushion as described (20). The pelleted complexes were resuspended in PBS containing 0.1% BSA and stored at 4°C. Bacteriophage were prepared by 1 mM isopropyl β-d-thiogalactopyranoside induction at 30°C overnight, precipitated by polyethylene glycol, and diluted into PBS containing 0.01% BSA to a final concentration of 5 × 10¹⁰ colony-forming units (cfu)/ml. A 2-μl aliquot of the diluted phage was applied to Formvar-coated nickel grids (200 mesh) for 5 min. Unbound phage were removed by washing with PBS and then 1% BSA in PBS for 10 min. PCP-BSA-gold complexes were applied to the grids undiluted. After 30 min, the grids were washed with PBS and then stained with 1% uranyl acetate for visualization by electron microscopy. Randomly selected areas on the grids were photographed to quantify the number of gold particles associated with phage.

A single clone of XL1-Blue cells carrying the phagemid pCGMT (containing Flag-pVII or pVII-Flag or Flag-pIX or pIX-Flag) or pCGMT-1b [containing V_H-(Gly₄Ser)-pVII and V_L-(Gly₄Ser)-pIX] was picked up and grown at 37°C in superbroth medium containing 1% glucose, 10 μg/ml tetracycline, and 100 μg/ml carbenicillin until OD₆₀₀ ≈ 0.1. Helper phage VCSM13 were added, and the cells were grown at 37°C for another 2 h, and then kanamycin and isopropyl β-d-thiogalactopyranoside were added to a final concentration of 70 μg/ml and 1 mM, respectively. The cells were grown at 28–30°C overnight. The supernatant of the phage was used directly for ELISA. The microtiter plates (Corning) were coated with 25 μl per well of 10 μg/ml mouse anti-Flag antibody to assay Flag constructs with pVII/pIX, PCP-BSA to assay 21H3 and 2H6 constructs, and the cocaine conjugate GNC-BSA (21) to assay the 92H2 construct. The phage ELISA was performed as described (15, 22).

The 2H6 phage were diluted into 92H2 phage at a ratio of 1:100 or 1:10⁸, and the mixture was used for selection against PCP-BSA. In another experiment, the 92H2 phage were mixed with 2H6 at a ratio of 1:100 and the mixture was used for selection against GNC-BSA. Microtiter plates were coated overnight at room temperature with 25 μl per well of the PCP-BSA or GNC-BSA conjugate (10 μg/ml in PBS). After washing five times with water, the plates were blocked with Blotto (PBS/4% nonfat dry milk) at 37°C for 1 h. Then, 50 μl (10¹² cfu per well) of phage mixture (prepared as described above) was added and incubated for 1 h at 37°C. The plates were washed 20 times with PBS containing 0.1% Tween-20 and 10 times with PBS. The bound phage then were eluted with 50 μl per well of elution buffer (0.1 M HCl/glycine/0.1% BSA, pH 2.2). After 10 min, the elution buffer was removed and neutralized with 3 μl of 2 M Tris base. Phage eluted from the first round of selection were used to infect Escherichia coli XL1-Blue cells. Phage particles were rescued from the cells and used for the subsequent round of antigen selections.

The reactions were carried out in 100 mM Bicine (N,N-bis(2-hydroxyethyl)glycine), pH 8.5, containing 10% DMSO as cosolvent. Approximately 150 nM 21H3 phage (1 × 10¹⁴ cfu/ml) in 100 mM Bicine, pH 8.5, was mixed with 5 mM (S)-(−)-sec-phenethyl alcohol and 16 mM vinyl 4-acetamidophenylacetate prepared as stock solutions in DMSO. A 50-μl aliquot was removed every 30 min and quenched by adding 4 μl of 10% HClO₄. The formation of the product (S)-(−)-sec-phenethyl 4-acetamidophenylacetate was monitored by using reversed-phase HPLC [C18-Vydac 201TP54 column; isocratic mobile phase (62% water/0.1% trifluoroacetic acid/38% acetonitrile); 1.75 ml/min; 254 nm].

Phage-Fv antibody (5 × 10¹³ cfu) in 1.5 ml was treated with 0.1% Sarkosyl (N-lauroylsarcosine sodium salt; Sigma) in PBS for 20 h and then loaded onto a HIPrep 26/60 Sephacryl S-100HR gel-filtration column (Pharmacia) preequilibrated with PBS containing 0.1% Sarkosyl. The phage fraction was collected, polyethylene glycol-precipitated, and dialyzed into PBS for assay by phage ELISA.

It was essential to know the orientation of pVII or pIX on the surface of phage to correctly display peptides and proteins. We fused a Flag tag, an octapeptide (Asp-Tyr-Lys-Asp-Asp-Asp-Asp-Lys) that has been used widely in protein detection and purification, to the N or C terminus of pVII and pIX and inserted the constructs into a phagemid vector, pCGMT. Phage particles carrying the four different fusion proteins, Flag-pVII and Flag-IX (Flag fused to the N terminus of pVII or pIX, respectively) and pVII-Flag and pIX-Flag (Flag fused to the C terminus of pVII or pIX, respectively), were rescued from XL1-Blue cells containing those phagemid. The binding capacities of the phage particles to the mAb anti-Flag M2 that recognized the Flag tag were tested by ELISA. It was shown that only the Flag fused to the N terminus of pVII or pIX could be detected by phage ELISA (Fig. 3). The results indicated that the Flag peptide in these constructs was exposed on the outside of the phage coat, and, therefore, the N termini of pVII and pIX also must be on the exterior of the phage particle.

Our principal aim was to test the hypothesis of whether a functional heterodimeric protein could be displayed via the pVII and pIX format. Hence, in three separate examples, the heavy chains of the murine mAbs 21H3, 2H6, and 92H2 were constructed as fusion proteins with pVII and the light chains were constructed as fusion proteins with pIX, and the constructs were displayed on the surface of phage particles. For negative controls, we fused only the V_H of these three antibodies to pVII or the V_L to pIX and omitted the other chain. The correct construction of phagemid pCGMT-1b-21H3, pCGMT-1b-2H6, and pCGMT-1b-92H2 was confirmed by DNA sequencing. The phage particles that displayed antibody 21H3, 2H6, or 92H2 were rescued from XL1-Blue cells with helper phage VCSM13.

Phage ELISA showed that 21H3 and 2H6 phage antibodies specifically bound PCP-BSA and 92H2 phage antibody specifically bound GNC-BSA. A typical result for the 21H3 construct is shown (Fig. 4). For the former two antibodies, the binding activity could be inhibited by free hapten PCP, and for 92H2, it could be inhibited by cocaine. There was no binding to BSA itself. No binding activities were detected for the phage displaying only the V_H or V_L chains (Fig. 4). Consequently, it was concluded that when both the V_H and V_L chains were displayed on the surface of phage as pVII and pIX fusion proteins, respectively, interaction of the chains occurred to form a functional Fv antibody motif.

In the case of the 21H3 phage Fv, the catalytic activity also was investigated and compared with our previously studied 21H3 IgG antibody (18). When 150 nM of the phage antibody (1 × 10¹⁴ cfu/ml) was mixed with 5 mM alcohol and 16 mM vinyl ester, the product ester could be detected within 30 min (data not shown). A comparison of time-course curves showed that the catalytic activity from 150 nM phage Fv was roughly equal to 50 nM of the IgG.

The specific binding of phage Fvs to their antigen labeled with 5-nm colloidal gold was visualized directly by electron microscopy. Examination of filamentous phage 21H3 and 2H6 revealed specific labeling by the PCP-BSA-gold complex at one end of the phage (Fig. 5). It was observed that some of the phage were labeled by more than one gold particle (Fig. 5B). The specificity of the labeling to 21H3 and 2H6 phage was indicated by the absence of labeling by using a BSA-gold complex and also by the fact that 92H2 phage could not be labeled by the PCP-BSA-gold complex (data not shown).

We demonstrated the potential for pVII- and pIX-displayed Fv fragments in selection and evolution experiments by enrichment of a mixture of two antibodies based on function. The 2H6 and 92H2 phage Fvs were used for the experiment because they had similar cell densities under the same growth conditions. The 21H3 gene was more toxic to XL1-Blue cells and showed a much lower cell density.

In our first experiment, the enrichment was tested by mixing 2H6 phage Fv and 92H2 phage Fv at a ratio of 1:100 or 100:1 followed by selection against PCP-BSA or GNC-BSA. After one round of panning, 9 of 10 randomly selected clones were 2H6 phage Fv when mixed at a ratio of 1:100 and selected against PCP-BSA, and 6 of 10 clones were found to be 92H2 phage Fv when mixed at a ratio of 1:100 and selected against GNC-BSA. The results showed that at least a 100-fold enrichment could be achieved per round of panning when a functional Fv fragment was displayed on the phage surface as a result of the interaction of pVII and pIX constructs.

In a second experiment, a selection from a highly dilute mixture was examined. The 2H6 phage Fv were mixed with 92H2 phage Fv at a ratio of 1:10⁸ and panned against immobilized PCP-BSA. The panning was performed for a total of two rounds. After each round of panning, the phage were pooled and tested by ELISA for their ability to bind PCP-BSA and GNC-BSA. The phage mixture before panning showed no binding activity to PCP-BSA and strong binding to GNC-BSA. However, after two rounds of selection, phage showed greatly enhanced binding to PCP-BSA and weak binding to GNC-BSA (Fig. 6). To verify the ratio of 2H6 and 92H2 clones before and after each round of panning, phage were rescued from 15 randomly selected clones and tested by ELISA for their ability to bind PCP-BSA and GNC-BSA. Of the 15 clones, the number of 2H6 clones increased from 7 after first round of panning to 15 after the second round of panning. In contrast, of 15 clones randomly selected from the unpanned phage mixture, none were 2H6. The results showed that a 10⁸-fold enrichment was accomplished after only two rounds of panning.

V_H and V_L domains were found to associate and form an antigen-binding site on the phage surface (23). Though unlikely in our experiments, the possibility existed that while one variable domain was displayed on the phage surface as the intended pVII or pIX fusion protein, the other domain existed as a free chain and associated with the first in the periplasmic space. To verify that the Fv on the phage surface resulted through interaction of both pVII and pIX fusion proteins, the 21H3 phage Fv was treated with 0.1% Sarkosyl in PBS at room temperature for 20 h and purified by gel filtration. Sarkosyl, a relatively mild detergent, has been used frequently for dissolving precipitates of phage particles and was used successfully to dissociate Fv fragments (23). The phage fraction was collected and dialyzed against PBS to remove the Sarkosyl. The antigen-binding activity of 21H3 was tested by phage ELISA before and after the Sarkosyl treatment, and no difference was observed (data not shown).