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Anti-VEGF-C VC2.2.2


Below, you find published and yet unpublished downloads concerning anti-VEGF-C VC2.2.2

PloSONE article about VC2.2.2 and its sister and parent antibody fragments:

Rinderknecht M, Villa A, Ballmer-Hofer K, Neri D, Detmar M (2010) Phage-derived fully human monoclonal antibody fragments to human vascular endothelial growth factor-C block its interaction with VEGF receptor-2 and 3. PLoS One 5: e11941.

To our knowledge, this is the first peer-reviewed report published in Pubmed that characterizes the selection of a potentially function-blocking fully human anti-VEGF-C antibody fragment. There have been other publications reporting alleged function-blocking properties of anti-VEGF-C antibodies but none have been pursued further and none of the published neutralizing anti-VEGF-C antibodies were of human origin (see Previous publications).

Below, you can find the dissertation at the ETH Zurich, that describes the development of VC2.2.2 and the challenges encountered:

Link to the ETH e-collection entry

Researchers interested in further investigating the published anti-VEGF-C antibody fragment are welcome to do so, the amino acid sequences of the antigen-binding regions have been published deliberately in the PLoSONE article. Please note that the antibody fragment has been developed using phage-display technology that was obtained by Material Transfer Agreement from Philochem AG. The vectors used in the study are therefore not necessarily easily available for further research. However, you could synthesize the DNA encoding the relevant CDRs yourself and insert it into an IgG, Fab, scFv or sdAb vector. This might not necessarily result in a VEGF-C binding antibody (fragment), since the framework regions can also be important in positioning the CDRs correctly. Otherwise, get the specifications of the ETH-2 Gold library and use the correct human germ line segment (DP47 for the heavy chain, and DPK-22 for kappa light chain or DPL16 for lambda light chain) in your vector.

As described in Rinderknecht et al, VC2.2.2 binds only via the heavy chain to VEGF-C. Below are some considerations why this might be the case and what implications this has:

With an unfolded VL, the VH is able to insert itself in smaller crevices, a property often seen for camelid VHH or single domain antibodies like those produced by Ablynx. Interestingly, the two epitope regions on VEGF-C discovered with our peptid mapping microarray come together on the extreme ends of the assembled VEGF-C homodimer (each monomer contributing a distinct epitope stretch), with a cleft about 13 nm wide. This is the exact width of the VH when measured across the CDRs and could mean that the VH fits snugly into the VEGF-C cleft (as shown here in a previously unpublished representation). The VL would therefore in the folded state obstruct binding to VEGF-C and only allow binding when unfolded (and "dangling around").

What could be done next?

The incorrect folding of the VL domain is most probably due to mutations in the VH:VL dimerization interface (see publication and dissertation). Possibly, the folding state of either the scFv or the VH could be stabilized by either mutating Glu44 back to glycine (to correct the dimerization interface and promote correct assembly of the scFv, although this might disrupt binding to ∆N∆C- VEGF-C at all) or mutating more residues in the dimerization interface into hydrophilic residues to more closely resemble VHH and to stabilize the VH (Davies et al. 1996). Incorporation of all three observed mutations into one VH might also be an interesting strategy.