Generation of a new antigen-binding site into the vNAR scaffold through diversification of hypervariable loop 2

Within this work, we generated a new antigen-binding site into the vNAR scaffold, solely facilitating antigen binding. Diversification of nine residues within the surface-exposed loop corresponding to hypervariable loop 2 combined with library screening using yeast surface display proved to be a valid strategy for the identification of bi-specific IgNAR V domains targeting two distinct antigens. Hence, we demonstrated that HV2 of the vNAR domain can be functionalized to form an autonomous antigen- binding site, independent from the conventional paratope, consisting of CDR3 and CDR1 (and potentially HV4).

For this, we chose to use well-characterized EpCAM-binding vNAR 5005 as scaffold for randomization. As aforementioned, this antibody domain binds to EpCAM with high affinity. Additionally, selectivity assays against unrelated target proteins demonstrated no unspecific binding against unrelated target proteins and more importantly, also no off-target binding was observed against CD3ε and non-glycosylated human Fcγ. We successfully showed, that through diversification of HV2, binders can be selected against two proof-of-concept antigens. Isolated and characterized clones comprised moderate affinities for their target, while retaining high affinity against EpCAM.

The surface-exposed loop, comprising hypervariable loop 2 is considerably longer (Lys43 – Arg56) than the nine residues, considered for library design (Ala45 – Thr 53). However, sequence comparison between different vNAR types across different species revealed a conserved Gly-Arg-Tyr motif at the end of this surface-exposed loop (Gly55 – Tyr57), as depicted in Appendix B. Consequently, these

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residues were excluded for library design. Interestingly, CD3ε and EpCAM binding molecule B1 contained an Arg56Gln mutation. Peculiarly, this residue was not considered for library design. This molecule displayed moderate affinities against CD3ε, yet, affinities against EpCAM were substantially diminished compared to parental molecule 5005, giving clear evidence that the conserved Gly-Arg-Tyr motif is crucial for the structural integrity of the vNAR scaffold. In this respect, we were able to show that transformation into the original Gly-Arg-Tyr motif entirely restored affinities towards EpCAM without any loss of function against CD3ε.

Characterization of the established library by FACS analysis for staining with EpCAM showed that the vast majority of displayed clones (~ 89.5 %) retained high affinity for the original antigen, indicating that a large portion of library candidates tolerated well the modification of this structural loop. Related to this, isolated bi-specific vNAR clones did not display any significant loss of affinity against EpCAM, demonstrating the versatility of this new strategy.

It is generally known, that the immunoglobulin family evolved a paramount tolerability in loop length as well as sequence variation, which is most evident for CDR regions of variable domains. However, this feature is a general hallmark of the immunoglobulin domains.[69,215] In this respect, surface- exposed loops at the N-terminal tip of CH2 of human IgG have been engineered for antigen binding.[216] In another very elegant approach, established by Rüker and co-workers, a new antigen- binding site was introduced into C-terminal loop regions of the CH3 domain of human IgG.[69] Most importantly, those engineered Fc fragments, named Fcab, retained the ability to elicit effector functions, clearly indicating the structural integrity of the molecule. This study adds the vNAR domain to the growing class of immunoglobulin scaffold proteins that can be manipulated and tailor-made in terms of establishing a new antigen-binding site.

Although this new methodology allows for the identification and isolation of binders comprising a new antigen-binding site, hence, for bi-specific molecules, affinities of the new HV2-facilitated paratope against their target proteins were only moderate. Notwithstanding, there might be several methodologies applicable to optimize affinities. One could think of randomizing residues Lys43 and Gly44 of HV2 to expand the created antigen-binding loop which may contribute to an enhanced affinity i.e. to affinity maturation. Another possibility might be to identify residues of HV2 not involved in antigen binding via alanine-scanning. After identification, those residues could be diversified in a second generation randomization step and a sublibrary could be established. Screening with significantly decreased target concentration, as performed in the aforementioned affinity maturation process of the conventional antigen-binding site, i.e. randomization of CDR1, might pave the way for the identification and isolation of bi-specific vNAR molecules with significantly enhanced affinities. Finally, randomization of the adjacent loop of HV2 (Asp74 – Ser80) would substantially elongate the surface of a potentially antigen-binding site, drastically increasing the chance to isolate high-affinity binders, which would be progenies of HV2-randomized antigen binding, bi-specific

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clones. Accordingly, this antigen-binding site would comprise two surface-exposed loops, akin to the conventional paratope of EpCAM-binding high-affinity vNAR 5005, consisting of CDR3 and CDR1. Such antigen-binding sites were constructed into domain CH3 of the human Fc-fragment of IgG, as described by Rüker and colleagues.[69] The group randomized the AB and EF loop at the C-terminal tip of CH3 and screened for antigen binding Fc fragments using yeast surface display. With this strategy, comprising a two-looped paratope, they were able to isolate Fc fragments targeting HER2/neu with affinities of 69 nM. Interestingly, also insertions into the surface-exposed loops considered for library design did not interfere with the overall structural and functional characteristics of the Fc-part. As a consequence, one could also take insertions of randomized residues into HV2 into account to elongate the respective loop, for the enhancement of affinities.

Essentially, this study demonstrates that the vNAR scaffold can be engineered in a way that HV2 functions as an independent paratope, solely facilitating antigen binding against a target protein. Importantly, the establishment of a new paratope does not impair the conventional antigen-binding site, composed of CDR3 and CDR1 in its affinity, resulting in a bi-specfifc molecule.

In further studies, taking a closer look to bi-specificity might be worth to consider. In this respect, vNAR molecules need to be expressed as soluble proteins and characterized more meticulously via biolayer interferometry to investigate whether the vNAR molecule is capable of targeting both antigens simultaneously. Although HV2 of the IgNAR V domain is situated in relative distance to the conventional paratope, with the data presented herein, it cannot be excluded that both antigen-binding sites compete for antigen binding to their respective target proteins, which would prevent simultaneous targeting.

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