1. Structural characterization of a potent humanized anti-CD38 antibody in phase I for the treatment of multiple myeloma and other hematologic malignancies
François Vallée1, Stéphanie Pouzieux1, Alexey Rak1, François Michoux1, Marie-Cécile Wetzel1, Jutta Deckert3, Aimo Kannt2, Veronique Blanc1, Vincent Mikol1
1Sanofi R&D 1Vitry-sur-Seine France and 2Frankfurt am Main Germany, 3ImmunoGen, Inc., Waltham, MA, USA
Introduction
CD38 is a type II transmembrane glycoprotein with both ADP-ribosyl cyclase and glycohydrolase activities with potential receptor functions. CD38 is highly expressed at the surface of malignant plasma cells of multiple myeloma. SAR is a humanized IgG1 antibody targeting CD38 in early clinical development. Several potential mechanisms of action of SAR have been identified including ADCC, CDC and pro-apoptotic activity (Wetzel MC, AACR 2013 #4735, and Wetzel MC, IMW 2013 #P228). Here we report further preclinical characterization of SAR with epitope mapping and a high resolution structure of Fab-SAR in complex with CD38.
Antibody‐dependent cellular cytotoxicity (ADCC)
and phagocytosis (ADCP)
SAR650984
NK cell,
Macrophage
cADPR
2. Complement‐dependent
cytotoxicity (CDC)
ADP-Ribosyl Cyclase
3. Direct cellular
signaling (apoptosis)
4. CD38 enzymatic
activity inhibition
Hydrolase
NAD
Glycohydrolase X
Antibody
Fc Receptor
Complement
cADPR
ADPR
ADPR X
Figure 2. Human CD38 is multi-functional ecto-enzyme which carries both ADP-ribosyl cyclase and glycohydrolase activity
Figure 1. Multiple mechanisms for anti-CD38 antibody SAR to kill cancer cells
Crystallization and structure determination
His-tag CD38 was purified from E. coli and incubated with Fab-SAR The complex was further purified by gel filtration to get a 1:1 stochiometry and concentrated to 26 mg/ml. Monocrystals of Fab-SAR650984/CD38 were obtained in presence of trypsin in 45%PEG400 and Mes buffer at pH6. Crystals belongs to space group P and diffract up to 1.53Å on a synchrotron beamline. Free CD38 and a chimeric model of Fab structures (pdb code 1S78 and 2OSL) were used as models for molecular replacement. One Fab and one CD38 were identified with program PHASER. Structure of CD38/Fab-SAR was then refined with the program Autobuster up to Rfac=0.188 and Rfree=0.208 at 1.53Å resolution.
Results A B A B
Figure 5. Paratope characterization and conformational changes in huCD38 upon binding of Fab-SAR (a) Loop H3 protrudes out of the structure of Fab-SAR and appears to be critical for complex stabilization. (b) The presence of the loop H3 in the interface between huCD38 and Fab induces a ~2.5Å shift of the segment Gln115-Thr116-Val117 towards the light-chain (colored in green). C
RWRQQWSGPGTTKRFPETVLARCVKYTEIHPEMRHVDCQSVWDAFKGAFISKHPCDITEEDYQPLMKLGTQTVPCNKILLWSRIKDLAHQFTQVQRDMFTLEDTLLGYLADDLTWCGEFATSKINYQSCPDWRKDCSNNPVSVFWKTVSRRFAEAACDVVHVMLDGSRSKIFDKDSTFGSVEVHNLQPEKVQTLEAWVIHGGREDSRDLCQDPTIKELESIISKRNIQFSCKNIYRPDKFLQCVKNPEDSSCTSEI A B
Figure 3. (a) Crystal structure of Fab-SAR650984/huCD38. (b) Stick and surface representation of the epitope. CD38 epitope residues are defined as residues that lie within 4Å from any atom of SAR light-chain (red), heavy-chain (blue) or both (purple). (c) Sequence of huCD38. Mutations that are introduced to silence the four glycosylation sites of huCD38 are shown in green.
Figure 6. (a) Inhibition of huCD38 ADPRC activity by SAR (86% at 10nM). (b) Paratope characterization and conformational changes in huCD38 upon binding of Fab-SAR The configuration of key residues that are involved in the ADP-ribosyl cyclase (ADPRC) activity is conserved. This suggests that SAR is likely an allosteric antagonist. (Deckert et al. (July 1, 2014) Clinical Cancer Res, / CCR )
Figure 4. The epitope on huCD38 for SAR consists of the face of the CD38 structure with a central cavity that opposes the catalytic site. Residues Trp125, Glu146, Trp189 and Glu226 that are essential for the catalytic activity of CD38 are colored in orange. The Fab/antigen interface is colored as in Figure 3. (CS: catalytic site)
Conclusion
The crystal structure of SAR Fab/huCD38 complex has shown that SAR neither blocks the access nor alters the configuration of the ADPRC catalytic site of CD38. In vitro assays have demonstrated that SAR behaves as a strong inhibitor of the ADPRC activity of CD38. This suggests that SAR is likely an allosteric antagonist of CD38 that alters the dynamics of the enzyme upon binding.