CONCLUSIONES Y RECOMENDACIONES

Full-length R6.5 and YN1 IgG antibodies show specific binding to human ICAM-1 or mouse ICAM-1, respectively, in ELISA experiments (C. Greineder, personal communication), demonstrating that the parental antibodies are capable of ICAM-1 binding and that the biotinylated human ICAM-1 and mouse ICAM-1 are functional. R6.5 and YN1 scFvs are in a VH-linker-VL orientation, where the linker consists of the 16

amino acids AGGGGSGGGGSGGGGS. These scFvs have been successfully expressed in insect cells (C. Greineder, personal communication). Surprisingly, when tested in ribosome display, no specific binding to human or mouse ICAM-1 can be observed with either scFv (Fig. 5.5A, B). Different variations to the display protocol were tested, including the addition of disulfide bond enhancers, different blocking agents, varying concentration of receptors and ternary complexes, and different elution methods, but all without success. Since full-length R6.5 and YN1 produced in insect cells are clearly functional, we hypothesized that the apparent lack of binding in ribosome display stems from the poor stability or folding of R6.5 and YN1 scFvs during in vitro synthesis. We therefore decided to graft the CDRs of these antibodies onto more stable scFv frameworks.

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Figure 5.5. Ribosome display of anti-ICAM-1 scFvs against ICAM-1. Biotinylated human or mouse ICAM-1 was immobilized on neutravidin-coated wells and blocked with (A, B) BSA or (C, D) BSA or casein. The binding of (A) R6.5, (B) YN1, and (C) hR6.5 scFv with ICAM-1 was shown by ribosome display after 23 cycles of PCR. (D) The binding of the stabilized and humanized hYN1 with ICAM-1 was shown by ribosome display. Reverse primer 18 was used for RT and products after 25 cycles of PCR are shown.

Consensus human heavy- and light-chain frameworks from the HuCal library35 were chosen. VH3 was chosen as the new heavy-chain framework for both R6.5 and YN1

because it is most commonly seen in nature35, has exceptional stability33,45, and was also the most frequently selected heavy-chain framework in previous ribosome display experiments8. Vκ2 and Vκ4 were chosen as the light-chain frameworks for R6.5 and YN1,

respectively, since they most closely resemble their original light-chain frameworks, and because Vκ domains are more stable than Vλ33. The heavy and light chains were

connected in the same VH-linker-VL orientation, but this time with the 20-amino-acid

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connect scFvs for selection in ribosome display8. Since the new frameworks were chosen from human consensus sequences, the stability-enhanced scFvs hR6.5 and hYN1 were also simultaneously humanized. Although ribosome display of hR6.5 showed no specific binding to human ICAM-1 (Fig. 5.5C), ribosome display of hYN1 showed strong and specific binding to mouse ICAM-1 (Fig. 5.5D). Moreover, the choice of blocking agent did not affect binding, although casein blocking showed a lower background than BSA blocking. For RT, tolAk (primer 18) was used as the reverse primer instead of the usual inner reverse primer pRDV_BbsI_r (primer 16) since preliminary RT experiments showed the strongest specific bands when tolAk was used. Our CDR grafting strategy has enabled successful display of hYN1 scFv in ribosome display format, as assessed by specific mouse ICAM-1 binding.

5.4 Discussion

The Off7 truncation study revealed the importance of shielding the hydrophobic core of ankyrin repeats with the capping repeats. The removal of both N- and C-terminal capping repeats (Off7I3) showed no specific MBP binding (Fig. 5.3). Based on the co-crystal structure9, the Off7-MBP interaction interface involves residues from the three internal repeats, but not the capping repeats (the single amino acid mutation in the N-cap is not responsible for binding)9. Therefore, the lack of Off7I3-MBP binding cannot be attributed to disruptions of the binding interface, but instead can be explained by the instability of the ankyrin repeat protein without the two capping repeats. With the

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removal of either the N- or C-cap (Off7I3C or Off7NI3), no noticeable effect in binding to MBP was observed. However, our experimental conditions might not be stringent enough to explore any destabilization effect of a missing N- or C-cap, particularly since the tethered ribosome enhances the solubility of the displayed protein44. Nonetheless, our results underscore the important stabilizing effect of the hydrophilic caps in sealing the hydrophobic core of ankyrin repeat proteins13.

On the other hand, the lack of specific binding by Off7NI2 and Off7I2C can be explained by disruptions to the Off7-MBP binding interface. Eight of the 12 potential interaction residues are located within the first and second internal repeats, while 10 of the 12 potential interaction residues are located within the second and third internal repeats9. It would be interesting to parse the energetic contributions of residues in each internal repeat by comparing MBP binding of Off7 with alanine substitutions for the interaction residues in the different internal repeats with ribosome display. Moreover, additional randomized repeats could be added to these fragments and selected for MBP binding to investigate if this step-wise approach could provide more stable and higher- affinity binders than wild-type Off7.

Ribosome display of SCI-57 showed specific binding to the insulin receptor (Fig. 5.4A), although the signal-to-background ratio could be further improved. One improvement could be replacing PDI with other chaperones or isomerases to achieve a translation reaction condition for optimal disulfide bonds formation. In particular, the disulfide bond enhancers from NEB could be tested, since they were specifically designed to supplement the PURExpress translation system to enhance proper folding of

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disulfide-bonded proteins. Another improvement would be to optimize the buffer pH for better insulin folding. Previous work indicates that a basic pH (between 9.5 and 11) facilitates proper folding of insulin46,47, and our work in engineering Lactococcus lactis to secrete SCI-57 (Chapter 6) also suggests that environmental pH plays an important role in obtaining bioactive molecules.

The role of the intrachain disulfide bond on the biological activity of leptin remains unclear. While Imagawa et al.43 reported that eliminating the disulfide bond has no effect on human leptin signaling in mice, Müller et al.40 and Giese et al.41 demonstrated the opposite is true in rat adipocytes in vitro and in mice with mouse leptin, respectively. Our results suggest the disulfide bond is dispensable for specific binding of human leptin to leptin receptors, but the stability of the leptin double mutant (C96S/C146S) is likely to be lower. It would be interesting to investigate if the importance of the disulfide bond is species-specific. Future work could include engineering leptin(C96S/C146S) to increase its stability. A ribosome display screen could be set up under high salt conditions, where ternary complexes would first be incubated with agarose beads displaying different hydrophobic ligands to remove poorly folded proteins44. The remaining unbound complexes could then be selected for binding against leptin, achieving the dual engineering objective of increasing stability while maintaining (or enhancing) affinity.

The apparent lack of binding of hR6.5 to human ICAM-1 may be due to several reasons. It is possible that the VH3-Vκ2 framework is less stable than the VH3-Vκ4

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Hanes et al.8 selected 12 useful binders with the VH3-Vκ2 framework, but only 1 with the

VH3-Vκ4 framework. VH3-Vκ2 is thus clearly able to produce scFvs that are functional and

stable enough for ribosome display. Since full-length R6.5 IgG exhibited specific binding to human ICAM-1, one possibility is that the VH3-Vκ2 framework cannot position the

R6.5 CDRs in a conformation necessary for human ICAM-1 binding. Although the consensus human Vκ2 most closely resembles the wild-type R6.5 murine Vκ framework,

other Vκ frameworks could be tested. Point mutations could be introduced into the

framework of hR6.5 and screened for human ICAM-1 binding using ribosome display.

In summary, we have refined the ribosome display protocol, and we have demonstrated that Off7 fragments, SCI-57, leptin, and hYN1 can be successfully displayed in this format to achieve specific binding to their respective targets. Using ribosome display, we have elucidated some of the stability determinants of these molecules, including the importance of capping repeats in DARPins, the role of disulfide bonds in agonist folding, and the choice of antibody framework for functional display in vitro. Furthermore, these proteins are ready to be further engineered by constructing DNA libraries from these starting templates and employing in vitro evolution to quickly identify mutants with new and improved properties.

5.5 References

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