Approximately half of the clones expressed protein in the — nM range in microtiter plate cultures Table S1 in Supplementary Material. A limitation of the dissociation rate screening with the Biacore instrument is that single antigen concentrations are utilized for screening and clones must be expressed as pure sdAbs to generate full kinetic information. In view of this, we explored two approaches for obtaining full kinetic data using microtiter plate cultures containing sdAb-ABTAG fusions. Microtiter plate periplasmic extracts from the 48 clones from round 2 subjected to off-rate screening were screened by SCK to determine k a , k d , and K D Table S2 in Supplementary Material.
As expected, the concentration range was not ideal for individual clones but in most instances two or three of the concentrations gave acceptable responses Figure 4 A. Fitting the sensorgrams to a interaction model gave a surprisingly good approximation of the rate constants and K D s Figure 4 B; Table S2 in Supplementary Material , as indicated by the rate constants and affinities of some of the clones determined by more rigorous analyses Table 1.
The median K D for these analyzed clones was 7. Figure 4. Fitting of representative sensorgrams to a interaction model A. Black lines represent raw data; red lines represent fits. To take full advantage of these higher throughput approaches, we made a second, more diverse, sublibrary by cloning phage-displayed sdAbs eluted from round 1 panning into the ABTAG-fusion protein expression vector.
Of the randomly picked clones from the round 1 sublibrary, 52 were positive for antigen binding by ELISA data not shown. No antigen binding was observed for several of the clones—low capture levels and low affinities may account for this observation. Three of the five clones identified by conventional panning 2A7, 2G9, and 1F6 were found in this screen. Eleven of the 52 clones were 2G9, three or four were 2A7, and one was 1F6. Of the remaining 36 clones, 32 were unique; four clones were found twice.
The sensorgram data were generally of high quality, fitting well to a interaction model Figure 5 A. The method gave excellent K D approximations Figure 5 B , as evidenced by the values obtained for clones that were subjected to a more stringent SPR analysis, i. The median K D for these analyzed clones was 0. Values are not given for clones with capture levels of less than RUs. Several of the clones had higher affinities, attributable to slower off-rates, than those of clones from the round 2 library.
While the off-rates determined by the capture method were derived using double referencing reference cell and zero-concentration subtraction , accurate determination of very slow off-rates can be challenging, even in the best of circumstances.
The affinities of these four clones, which ranged from 4 to pM Table 1 , were all higher than the affinities of the top round 2 clones Figure 6 ; Table S2 in Supplementary Material with clone exhibiting an exceedingly slow off-rate Table 1. Figure 5.
Figure 6. Iso-affinity plots of the highest affinity single-domain antibodies sdAb clones obtained using conventional panning or sdAb-ABTAG screening. To better understand why some of the highest-affinity sdAbs against CEACAM6 were not recovered through conventional panning of the phage-displayed V H H library, we interrogated the library to moderate depth approximately 2.
Both the medium-affinity sdAbs recovered via conventional panning as well as the extremely high-affinity ones recovered only via ABTAG screening were observed in the library at roughly similar frequencies.
However, several medium-affinity sdAbs in particular 2A7 and 2G9 rapidly outcompeted the higher-affinity sdAbs, becoming enriched 50—fold in the first round of panning and making up the majority of CEACAM6-specific sdAbs by round 2. Thus, utilizing a high-throughput screening technology was necessary in this case to recover the most desirable antibody specificities.
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Table 2. The present study has shown that ABTAG can be a useful addition to the toolbox for antibody discovery by in vitro display methods. Compared to conventional phage display approaches to antibody isolation, ABTAG technology yielded a much higher number of unique sequences while simultaneously giving detailed antigen binding information.
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With the rapid rise in antibody drugs in recent years, the number of different applications for this class of biologics is ever increasing. Along with each application is also a desirable set of kinetic parameters and functional properties For instance, naked inhibitory mAbs targeting a surface receptor would preferentially have tight-binding with slow off-rates.
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Conversely, tumor-imaging molecules, or those carrying a toxic payload, require shorter half-lives and faster off-rates. Selection of antibodies based on kinetics using a full kinetics screening method, such as the ProteOn methods described here, is an obvious advantage, and affords a moderate throughput of up to clones per day, extrapolating from the time required to screen the 52 round 1 clones described here, with generation of high quality kinetic and affinity data.
Maximizing the number of clones available for functional screening is also clearly a key aspect of the search for antibodies with desired functionalities such as cross-species reactivity and binding to specific epitopes, for example, epitopes that deliver activation, inhibitory, internalization or no biological activity. The ABTAG technology supports the growing trend away from largely blind selection of limited numbers of binders as evidenced, for example, by the use of next generation sequencing to more effectively capture the full diversity of the immune response and bypass B-cell or antibody library screening 1 , Other affinity tags are available for use in purification and in screening technologies such as SPR; however, none of these tags are ideal.
The use of streptavidin requires chemical or enzymatic biotinylation of the proteins being screened; SPR screening of biotinylated proteins is not always straightforward and rapid. His-tagged proteins can be captured by anti-His-tag antibodies but commercial sources of these antibodies can be costly and the antibodies can have variable binding affinity depending on the presentation of the His-tag in the context of the protein. Monoclonal antibodies can be captured on anti-Fc surfaces but the use of Fcs as fusion tags generates homodimeric fusion proteins which are undesirable for some assays.
The remaining seven were characterized by longer CDR3s, up to 24 residues; one of these clones was , which was the round 2 clone with the highest affinity and slowest off-rate Table 1. The remaining 31 were characterized by CDR3 lengths of 6—25 residues. The CDR3 lengths of clones , , , and were 12, 11, 16, and 12 residues, respectively. Clones , , , and had off-rates in the 6. We speculate that this observation may relate to the very slow off-rates of the former group of sdAbs which may translate into less efficient elution of these sdAb-phages by high pH; however, a stochastic element to the panning process cannot be ruled out.
Clearly, as has been the experience of many groups, phage display selection is ineffective in discriminating antibodies based on affinity and probably depends instead on other factors e. However, it is unknown whether the display efficiency of these sdAbs on the surface of phage is comparable to their soluble expression. Another benefit of the screening method described here is that both E.
Culture supernatants were used directly for off-rate screening and periplasmic extracts were used for SCK screening of the 48 round 2 clones. The screening of up to 10 4 clones in a week by the approach described here is realistic, provided that antigen cost is not prohibitive. This represents very good sampling of the phage clones eluted after a single round of panning, typically 10 5 —10 6.
Of the methods described here, the ProteOn method has the highest throughput, followed by the off-rate screening method and the SCK method, although the throughput potential of the latter two methods is not that different. Analysis of about 30 clones a day is possible with the SCK method. The ProteOn instrument used here has recently been discontinued and will be supported only until However, as newer higher throughput instruments become available, ABTAG screening throughput equaling or exceeding that achieved here with the ProteOn should not be an issue Both scFv and Fab libraries, including humanization and affinity maturation libraries, can be screened in a manner similar to that described here for sdAbs.
In addition to screening for affinity, ABTAG technology can be applied to screening for protein stability, an unmet need in the field of protein engineering.
When the efficacy of a protein needs to be evaluated in vivo , the protein-ABTAG can be mixed with BSA to overcome the limitation of short serum half-life of the protein. Finally, screening libraries constructed from other proteins, receptors, ligands or enzymes, for which binding partners are available, could also be exploited with ABTAG. In summary, ABTAG technology provides a powerful and versatile tool for antibody discovery and protein engineering applications.
GH and CRM conceived experiments and planned and wrote the manuscript. GH supervised the Biacore analyses and created the figures.
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TB constructed the libraries, conducted the panning and coordinated the off-rate and ProteOn screening of the round 1 and round 2 library clones. JB devised and conducted the ProteOn analyses. SR conducted the SCK screening of the round 2 clones. KH performed and analyzed NGS experiments. JZ is an inventor on US patent 9,, B2.
All other authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. We thank Yanal Murad and Shenghua Li for help with the round 1 and round 2 library screening. Identification of cross-reactive single-domain antibodies against serum albumin using next-generation DNA sequencing. Protein Eng Des Sel — Beyond natural antibodies: the power if in vitro display technologies. Nat Biotechnol — Development trends for therapeutic antibody fragments.
Nat Biotechnol —7. Naturally-occurring antibodies devoid of light chains. Nature —8. Selection and identification of single domain antibodies from camelid heavy-chain antibodies. FEBS Lett —6. Shark variable new antigen receptor biologics — a novel technology platform for therapeutic drug development. Expert Opin Biol Ther — Isolation of monomeric V H s by a phage selection.
J Biol Chem — Disulfide linkage engineering for improving biophysical properties of V H domains. Protein Eng Des Sel —9.
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A V L single-domain antibody library shows a high-propensity to yield non-aggregating binders. Protein Eng Des Sel —8. Pentabody-mediated antigen delivery induces antigen-specific mucosal immune response. Mol Immunol — BSA-Specific Antibodies. United States Patent No. Google Scholar. Zhang J, Hirama T. Screening of Protein Candidates. Furthermore, the processing of gp is critical for the production of infectious HIV viral particles [ 2 , 12 , 18 , 19 ]. As a trimer, Env is post-translationally cleaved by Furin into gp and gp41, which remain non-covalently associated [ 20 , 21 ].
Receptor binding of gp causes a conformational change which exposes the fusion peptide of gp41 necessary for fusion with the cell membrane, and allows entry of the viral particle into the cell [ 23 ].