Screening for Nanobodies via Phage Display

A common way for the affinity selection of nanobodies is based on the absorption of antigens to the plastic surfaces of tubes or plates based on polar or hydrophobic interactions. Yet, the absorption of the protein to the plastic might disrupt its molecular fold and thus the epitope conformation. This method is nevertheless commonly used for ELISA assays, which were also used for validation of positive binders. However the aim of this project was to select for nanobodies to be used in IF studies. Therefore, a more comprehensive validation protocol eliminating affinity molecules, which do not bind to the native epitope, needed to be established.

My first screens of the libraries were entirely carried out in tubes coated with the target antigens as described above. I found that the first round of panning is most crucial for the outcome of the screen as that step comprises the highest level of competition among displayed nanobodies. To reduce stringency in the first panning round, I used hyperphages to infect the initial library [157]. This leads to polyvalent display of nanobodies, thus facilitating the binding of phages present in lower copy number or having a lower affinity as illustrated in Figure 14. The titer of purified phages was measured during panning rounds to monitor the progress of affinity selection.

As expected, the amount of phages decreased after the first panning but increased again as specific binders were selected (see Figure 14). Due to amplification of only few genetically different phage populations during the panning rounds, the number of new candidates after each screen is typically limited, depending on the selection conditions in the panning. To increase the number and also select for alternative probes, the conditions of the pannings were modified along with new screenings. Particularly, I modified the amount of antigen to bind more phages during the affinity selection step. The amount of antigen was still reduced in successive panning rounds to enforce the competitive selection procedure as summarized in Table 22. In consecutive screens, the washing steps were adjusted to be less stringent allowing also the selection of molecules with lower affinities.

The time of individual washing steps was further reduced from overnight in the initial screens to 10 minutes in the latest screens resulting in identification of yet new nanobody families.

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After up to three rounds of phage display, 80 clones were picked and tested in a monoclonal phage-ELISA assay. A HRP-coupled antibody directed against the major coat protein of the phage was used for detection of bound candidates. When the fluorescent readout of the subsequent enzymatic reaction described in section 2.2.16 was at least 10-fold over the background signal, binding of tested candidates was considered positive. Those positive clones were grown in liquid culture for plasmid isolation followed by sequencing. As expected, several redundant sequences were identified, which indicates the enrichment of particular phage populations due to specific antigen binding. The obtained sequences were grouped into nanobody families according to the composition of their CDR regions. If only a single amino acid in CDR1 or CDR2 was different, two nanobody sequences were still considered to belong to the same family. However, differences in the composition of the CDR3 were generally considered a separate family as the CDR3 is known to be an important factor for epitope binding [51].

Each family contained from 4 to 33 clones indicating the amplification of particular sequences. To map the CDR regions, the conserved framework sequences of the nanobodies were aligned according to Maass et al. [155]. Figure 15 shows exemplary sequences marking the complementary regions determining the nanobody specificity. As expected, the framework region is highly conserved among all selected nanobodies, regardless of the antigen used for screening. This is in line with observations from Maass et al. [155]. Notably, the selected nanobody candidates possess either two or four cysteine residues promoting the formation of one or two disulfide bridges. Nanobodies requiring the formation of two disulfide bridges were occasionally reported to show low expression yields in bacteria and impaired functionality. However these candidates were not discarded at this point as I was following a dedicated strategy to validate the nanobody function.

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Figure 14: Titer of amplified phages during subsequent panning rounds. A: Schematic illustration of phages produced upon infection with M13 K07ΔpIII hyperphages for polyvalent display in the first panning round and classical MK13K07 helper phages in the subsequent panning rounds. Each phage expresses 5 copies of the phage minor coat protein pIII (black ellipse). The nanobody (green triangle) is expressed from a phagemid (circle) as a fusion construct together with the pIII protein. Depending on the phage type used, either one or five copies of the nanobody are displayed by the phage mediating the affinity selections. B: Phage titer for exemplary panning rounds against SNAP-25, syntaxin 1A and VAMP2. An initial decrease of the phage titer indicates the elimination on unspecific phages. In the third panning for syntaxin 1A and SNAP-25, the phage concentration considerably increased due to selection and amplification of specific phage candidates. Notably, the phage titer for panning against VAMP2 did not rise significantly, which indicates that no positive candidate was selected and amplified.

Figure 15: Sequence alignment of selected nanobody candidates. Three randomly picked sequences per antigen are shown out of several hundred. After alignment, the conserved framework as well as the three CDR regions can be identified. CDR1 and CDR2 were found to show only little variation in both length and composition. In contrast, the CDR3 region typically playing the major part in epitope detection exhibits high diversity in different nanobodies.

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