<<Seriously..Miljenko, what do you mean by TRAP vs. fragment?>>
Lucentis (Y0317, RhuFab V2) is “smaller” (yes, right! little!) version of the anti-VEGF antibody (Avastin, antibody with intact human Fc region), and some property (like binding affinity and plasma half-life) were changed into desired direction.
J Mol Biol. 1999 Nov 5;293(4):865-81.
Selection and analysis of an optimized anti-VEGF antibody: crystal structure of an affinity-matured Fab in complex with antigen.
Chen Y, Wiesmann C, Fuh G, Li B, Christinger HW, McKay P, de Vos AM, Lowman HB.
Department of Protein Engineering, Genentech, Inc., 1 DNA Way, South San Francisco, CA 94080, USA.
The Fab portion of a humanized antibody (Fab-12; IgG form known as rhuMAb VEGF) to vascular endothelial growth factor (VEGF) has been affinity-matured through complementarity-determining region (CDR) mutation, followed by affinity selection using monovalent phage display. After stringent binding selections at 37 degrees C, with dissociation (off-rate) selection periods of several days, high affinity variants were isolated from CDR-H1, H2, and H3 libraries. Mutations were combined to obtain cumulatively tighter-binding variants. The final variant identified here, Y0317, contained six mutations from the parental antibody. In vitro cell-based assays show that four mutations yielded an improvement of about 100-fold in potency for inhibition of VEGF-dependent cell proliferation by this variant, consistent with the equilibrium binding constant determined from kinetics experiments at 37 degrees C. Using X-ray crystallography, we determined a high-resolution structure of the complex between VEGF and the affinity-matured Fab fragment. The overall features of the binding interface seen previously with wild-type are preserved, and many contact residues are maintained in precise alignment in the superimposed structures. However, locally, we see evidence for improved contacts between antibody and antigen, and two mutations result in increased van der Waals contact and improved hydrogen bonding. Site-directed mutants confirm that the most favorable improvements as judged by examination of the complex structure, in fact, have the greatest impact on free energy of binding. In general, the final antibody has improved affinity for several VEGF variants as compared with the parental antibody; however, some contact residues on VEGF differ in their contribution to the energetics of Fab binding. The results show that small changes even in a large protein-protein binding interface can have significant effects on the energetics of interaction. Copyright 1999 Academic Press.
From Patent:
<<RhuFab V2 Y0317 was created by humanization of the murine A.4.6.1 (Presta et al., Cancer Res., 57: 4593-4599 (1997) monoclonal antibody using a process previously described for other antibodies (Carter et al., Proc. Natl. Acad. Sci. USA. 89: 4285-4289 (1992); Presta et al., J. Immunol., 151: 2623-2632 (1993); Werther et al., J. Immunol. 157: 4986-4995 (1996)). Briefly, cDNAs encoding the muMAb A.4.6.1 variable light and variable heavy chains were isolated using RT-PCR from hybridoma cells producing the murine monoclonal antibody. These cDNAs were cloned and fused to human CL and human CH1 domains (Werther et al., J. Immunol., 157: 4986-4995 (1996)), generating a mouse-human chimeric Fab. The six complement-determining regions (CDRs) (denoted in FIG. 1 in bold type) were transplanted into a previously humanized antibody vector encoding a consensus human .kappa. subgroup I light chain and a consensus human subgroup III heavy chain (Carter et al., Proc. Natl. Acad. Sci. USA, 89: 4285-4289 (1992)). Transferring just the CDR residues into the human framework caused a 1000-fold reduction in binding to the VEGF antigen. Several framework residues near the CDRs (denoted in FIG. 1 in italicized and underlined type) also were changed to improve binding to the target (Presta et al., Cancer Res. 57: 4593-4599 (1997)). In all, seven heavy-chain residues and one light-chain residue were changed outside of the CDRs. The heavy and light chains were then moved into a phage-display vector (Baca et al., J. Biol. Chem. 272: 10678-10684 (1997)), replacing the hGH gene of phGHam-g3 (Bass et al., Proteins, 8: 309-314 (1990)). Site-directed mutagenesis was used to change VL Met4Leu to preclude methionine oxidation and VH Thr231Leu for ease of cloning to the geneIII fusion. This vector is termed Y0101 and was used as the starting point for optimization of the CDRs in binding to the VEGF antigen (Muller et al., Structure, 6: 1153-1167 (1998)). Only mutations in CDRs H1 and H3 were found to improved binding and were incorporated into the final version pY0317. The changes from the pY0101 plasmid to the pY0317 plasmid are: Thr28Asp, Asn31His, His101Tyr, Ser105Thr. All these changes are in the variable heavy-chain region. The pY0317 plasmid is a Fab phage display vector. A plasmid diagram of this plasmid appears in FIG. 2A. >>
From PIb safety:
<<The primary safety measures were changes from baseline in VA, intraocular pressure (IOP), intraocular inflammation, and production of antiranibizumab antibody. Dose-limiting toxicity was defined by intraocular inflammation, elevated IOP, reduced VA, or hemorrhage within 90 days after injection. RESULTS: All patients completed this single intravitreal injection study, and 500 mug of ranibizumab was the maximum tolerated dose.>>
So, Lucentis relative to Avastin have shorter half-life (~2-3 days in ayes, 0.5 days in plasma versus two weeks for Avastin), higher VEGF binding affinity (~100 times) and MTD dose of 500 mcg (as active dose). Good property for intravitreal injection? I guess! Have no idea is there selection among different VEGF-A forms??
Now, haw will VEGF-Trap (unchanged, as far as I know????) perform in intravitreal injection with higher half-life and potentially higher IOP? I will need to read about VEGF-Trap pre-clinical experiments before going further with comparison.
Miljenko |
