<article> The iGEM parts reg is a core part of the iGEM engineering biology approach. Proper documentation to describe the DNA parts functionality is crucial to enable future teams to find and choose the parts which are best suited for their project. During our project we constructed synthetic nanobodies by so called “nanobody grafting”. For grafting, a nanobody scaffold is needed on which the complementarity-determining regions (CDRs) 1 to 3 of a donor antibody, suitable for ones purpose. The nanobody scaffold we chose, cAbBCII-10, is already submitted to the iGEM parts reg. However,the description showed a lack of background information and therefore was not suitable to enable teams to identify the nanobody and lacked background information on the nanobody. We added the corresponding background information as well as the description how future iGEM teams can use this part as grafting scaffold.
</br> As part of our project, we grafted the CDRs of mAbs (monoclonal antibodies) against the sex hormones estradiol (E2) and progesterone onto the 3DWT scaffold to obtain nanobodies. As CDR acceptor, also called "scaffold", we used the nanobody cAbBCII-10 [1] (or also NbBcII10 [2] or 3DWT [3], from here on called "3DWT"). This nanobody is also available as part of a composite part in the iGEM parts reg (BBa_K2082001). However, the nanobody is not available as basic part, so we contributed to future iGEM teams in submitting the basic part of cAbBCII-10 (BBa_K3410001). We used the sequence deposited in the PDB by Vincke et al. [2], [3]. Originally, 3DWT was isolated as an antibody capturing and neutralizing enzymes of the β-lactamase B class [1]. β-lactamases are enzymes that hydrolyses β-lactam rings, thus mediating resistance to β-lactam antibiotics and are produced by numerous bacteria. β-lactamases can be divided into classes A, C and D due to differences in their amino acid sequence. Furthermore, they are assigned to the class B of metalloenzymes [4]. 3DWT is suitable as a scaffold, both as an acceptor of CDRs of antibodies from the same taxonomy [5], [6] as well as from other taxonomies [7]. Furthermore, 3DWT has been humanized, meaning that 3DWT is suitable for the use, for example, for human treatment without causing an immune reaction [2].
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</br> The amino acid sequence of 3DWT is as follows:
</br>QVQLVESGGGSVQAGGSLRLSCTASGGSEYSYSTFSLGWFRQAPGQEREAVAAIASMGGLTYYAD
</br>SVKGRFTISRDNAKNTVTLQMNNLKPEDTAIYYCAAVRGYFMRLPSSHNFRYWGQGTQVTVSS
</br> The successful grafting of a new nanobody requires to find an antibody against your antigen of interest (AOI) with an openly available sequence, in order to use its complementarity-determining regions (CDRs), positioned on its variable heavy chain (VH). In a first step of in silico nanobody grafting, the amino acid sequences of both donor (e.g. your antibody against your AOI) and acceptor (e.g. 3DWT) are numbered. There are different numbering schemes for immunoglobulin variable domains available [8]. We decided to use the AHo numbering scheme, because placement of gaps is based on the spatial alignment of known three dimensional structures of immunoglobulin domains and not on sequence variability [9]. The numbering of the VH can be performed using online available tools, we used the tool ANARCI (SAbPred) [10]. For further work, the numbered residues were transferred to an excel sheet. For the grafting process, it is important to consider a series of structurally important framework residues next to the CDRs: These core framework residues have to be grafted from the donor to the acceptor as well [11], [12]. Both, the CDRs and the functional important framework residues of the donor were grafted onto the sequence of the acceptor (3DWT) and a new nanobody is created [7].
</br> Here you can see how the in silico graft looked like in our project for the estradiol nanobody:
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We used 3DWT as a scaffold for our grafting approach for the generation of new and synthetic nanobodies against progesterone and estradiol. We chose the same approach for both hormones with an identical workflow. Shown here are the results for our estradiol nanobody.
</br> Using ChimeraX [13], we optically verified the 3D structure of the grafted nanobodies for steric obstacles to antigen binding: For example, we checked, if the grafted residues sterically impaired residues from the framework:
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</br> Cloning and transformation of the initial grafts into the E. coli strain ER2738 was tested by colony PCR and did work. In a next step, the via epPCR generated library will be cloned and transformed into ER2738.
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</br> By grafting the CDRs from a monoclonal antibody against Estradiol or Progesterone onto the CDRs from 3DWT, a new and synthetic nanobody is generated. But not only the CDRs are grafted, some for the stability important core framework residues from the donor antibody´s VH chain are grafted too. Through affinity maturation measures like Phage Display Technology, a strong binder can be established. In our project we could not obtain strong binding nanobodies with our initial grafted nanobodies, which was to be expected. Consequently, their binding affinity and specifity has to be improved using suitable affinity maturation methods like phage display [7].
</br> We have confirmed that 3DWT can be used as scaffold for a grafting approach and provided its amino acid sequence as a basic part for future iGEM generations. </article>

References

<article> [1] K. E. Conrath et al., “β-Lactamase Inhibitors Derived from Single-Domain Antibody Fragments Elicited in the Camelidae,” Antimicrobial Agents and Chemotherapy, vol. 45, no. 10, pp. 2807–2812, 2001, doi: 10.1128/AAC.45.10.2807-2812.2001.
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</br> [7] H. J. Wagner, S. Wehrle, E. Weiss, M. Cavallari, and W. Weber, “A Two-Step Approach for the Design and Generation of Nanobodies,” International journal of molecular sciences, vol. 19, no. 11, 2018, doi: 10.3390/ijms19113444.
</br> [8] M. Dondelinger et al., “Understanding the Significance and Implications of Antibody Numbering and Antigen-Binding Surface/Residue Definition,” Frontiers in immunology, vol. 9, p. 2278, 2018, doi: 10.3389/fimmu.2018.02278.
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</br> [13] E. F. Pettersen et al., “UCSF Chimera--a visualization system for exploratory research and analysis,” Journal of computational chemistry, vol. 25, no. 13, 2004, doi: 10.1002/jcc.20084. </article>