New Anti-CD20 Monoclonals
Pr Guillaume CARTRON, MD Ph-D
Head of Hematology Department, UMR-CNRS 5235
CHRU Montpellier, University of Montpellier Conflicts of interest
Active patents for new monoclonal antibodies – N° WO 03/035904, N° WO2005/118854, GB0822345.5 : Licensed to Clinical Data – PCT/EP2013/050787 With companies developping monoclonal antibodies – Takeda Millenium: Honorarium – Roche/Genentech: Honorarium/consultancy – GSK: Honoraires – Celgene: Honorarium/consultancy – Sanofi: Honorarium Aims of this talk
To understand the different mechanisms of action of anti-CD20 mAbs
To understand different ways to improve anti-CD20 mAbs activity
To know differences between the « new anti-CD20 mAbs » (obinutuzumab, ublituximab and ofatumumab) compared to rituximab Therapeutic antibodies: a very old story
1891. Emil von Behring infused and cured a child with anti-diphteria sera
1893. Paul Gibier « take the juice of tumor, infuse in a horse and then infuse the serum of the horse to the patient » 1895. Jules Héricourt and Charles Richet infused the first patient with sarcoma How rituximab works in vivo?
Apoptosis
Complement-dependent Antibody-dependent cytotoxicity (CDC) cellular cytotoxicity (ADCC
Targeted CR3 Death antigen signal C1qR FcR Effector C1q Effector cell cell Tumour cell
CR3 MAb Membrane attack complex
Phagocytosis
Lysis Cheson BD, Leonard JP. New Engl J Med 2008; 359: 613–626. Ways to improve rituximab efficacy
Complement
** FcgRs 142 C1q 184 Effector cells
CD20 219- 225 1 KISH FLKM ESLN FIRAH TPY IN IYN CEPA* N*P SEKN SPST QYCY homme TLSH FLKM RRLE LIQTS KPY VD IYDCEPS NS SEKN SPST QYCN souris Epitope/function Fc/function Type 1 or Type 2 mAb
Type I mAbs Type II mAbs Rituximab, Ofatumumab, Veltuzumab, Tositumomab, Obinutuzumab Ublituximab Localize CD20 into lipid rafts Do not lo localize CD20 into lipid rafts CDC No CDC No homotypic adhesion Homotypic adhesion
ADCC
Phagocytosis Type 1 or Type 2 mAb
Type I mAbs Type II mAbs Rituximab, Ofatumumab, Veltuzumab, Tositumomab, Obinutuzumab Ublituximab Localize CD20 into lipid rafts Do not lo localize CD20 into lipid rafts CDC No CDC No homotypic adhesion Homotypic adhesion Caspase dependant cell death Non-caspase dependant cell death Bind CD20 molecules Bind half of CD20 molecules/type I CD20 modulation No CD20 modulation Trogocytosis ADCC
Phagocytosis CD20 structure and organisation
Large loops: Lipid raft Small loops: 142-184 monomer 72-80
219-225
Tetramer COOH NH2 Membran cell organisation Proteins associated with CD20: MHC I, MHC II, CD53, CD81, CD82, CD40, BCR,… CD20 migration into lipid rafts induces CDC
Phospholipids
Sphyngolipids
Cholesterol Translocation Lipid raft
Cbp CD20 CD20
Lyn Lyn P CD20 type I epitope
142 170 173 184
KISH FLKM ESLN FIRAH TPY IN IYN CEPA NP SEKN SPST QYCY S-S 167 183
NH2 COOH
Polyack MJ, et al. Blood 2002; 99:3256. Ofatumumab induces higher CDC
▼: ofatumumab ▼: ofatumumab ■: rituximab ■: rituximab ▲: 7D8▲: 7D8 ▲: 7D8 ●: 11B8●: 11B8
CDC CD20 binding
Teeling JL, et al. Blood 2004; 104: 1793-1800. Ofatumumab, a discontinuous epitope
Type I mAbs
142 170 173 184 Rituximab, 2H7, KISH FLKM ESLN FIRAH TPY IN IYN CEPA NP SEKN SPST QYCY LT20
142 159 166 184 Ofatumumab KISH FLKM ESLN FIRAH TPY IN IYN CEPA NP SEKN SPST QYCY 72 80 IPA GI YAPI
Teeling JL, et al. Blood 2006; 177: 362-371 Ofatumumab binding site
A ring of hydrophobic residues surrounding a deep positively charged pocket
Du J, et al. Mol Immunol 2009; 46: 2419-2423. Ofatumumab CD20 epitope
Du J, et al. Mol Immunol 2009; 46: 2419-2423. Ofatumumab exhibits higher CDC against CD20low cells
rituximab ofatumumab
CEM cells + anti-CD20 antibodies at 10µg/mL
Teeling JL, et al. J Immunol 2006; 177: 362-371. Ofatumumab concentration-effect relationship
ofatumumab ofatumumab
rituximab rituximab
Daudi + CMN DHL4 Daudi
Teeling JL, et al. Blood 2004; 104: 1793-1800.
Bleeker WK, et al. Br J Haematol 2008; 140: 303-312. Anti-CD20 mAbs induces homotypic adhesion differently
Type 2 Type 1
Bologna L, et al. J Immunol 2011; 186: 3762-3769. Homotypic adhesion induces peripheral localisation of actin and mitochondria
Raji cells expressing Ac-GFP-labelling actin + Tos
JC-1 labeled cells Ivanov A, et al. J Clin Invest 2009; 119: 2143-2159. Cell death induces by type II mAbs differs from apoptosis
Type 2 Type 1
Tos Apoptosis CtrlCtrl
Ivanov A, et al. J Clin Invest 2009; 119: 2143-2159. Involvment of lysosomes in type II mAbs cell death
Type II Type II Type II
Cathepsin B staining
Type II
LAMP-1Lysosomal associated membran protein Ivanov A, et al. J Clin Invest 2009; 119: 2143-2159. Type II mAbs bind half of CD20 molecules compared to type I mAbs
Cragg MS, et al. Blood 2003; 101: 2738-2743. Comparison of the obinutuzumab-CD20 epitope- binding with other CD20 mAbs
70°
90° Niederfellner G, et al. Blood 2011; 118:358. CD20 Type II epitope
Type I mAbs
142 170 173 184 Rituximab, 2H7, LT20 KISH FLKM ESLN FIRAH TPY IN IYN CEPA NP SEKN SPST QYCY
Type II mAbs
142 172 178 184 Obinutuzumab, tositumomab KISH FLKM ESLN FIRAH TPY IN IYN CEPA NP SEKN SPST QYCY
Niederfellner G, et al. Blood 2011; 118:358. Elbow hinge substitution
167°
140°
Niederfellner G, et al. Blood 2011; 118:358. Model of type I or type II mAbs-CD20 interactions
Type I - Inter tetramer binding
CD20 - More CD20 bound - Translocation into lipid rafts - CDC - « open » configuration of CD20 Lipid rafts - Caspase dependant cell-death
- Intra tetramer binding Type II - Half CD20 bound - No translocation into lipid rafts CD20 - No CDC - « closed » configuration of CD20 - No caspase dependant cell-death - Homotypic adhesion Ways to improve rituximab efficacy
Complement
** FcgRs 142 C1q 184 Effector cells
CD20 219- 225 1 KISH FLKM ESLN FIRAH TPY IN IYN CEPA* N*P SEKN SPST QYCY homme TLSH FLKM RRLE LIQTS KPY VD IYDCEPS NS SEKN SPST QYCN souris Epitope/function Fc/function Type I and type II mAbs exhibit similar Fc-mediated functions
Antibody FcgRs B-lymphoma cell
Antibody
FcgRs
CD20 NK cell
Granzyme Perforine Macrophage B-lymphoma cell ADCC. CD20 ADPC. FcgRs expression
FcgRI FcgRIIa FcgRIIb FcgRIIc FcgRIIIa FcgRIIIb
Monocytes/ + + + + macrophages
NK cells + +
Neutrophils +/- + +
B lymphocytes +
Dendritic cells + + + +
Mastocytes +/- + +
Platelets + FCGR3A polymorphism influences IgG1 binding to NK-cells
IgG1
IgG1
FcgRIIIa-158F FcgRIIIa-158V
NK NK Human IgG1 have a better affinity for FcgRIIIa-158VV NK-cells than FcgRIIIa-158FF NK cells
Koene HR, et al. Blood 1997: 90: 1109-1114. Dall’Ozzo S, et al. Cancer Research 2004; 64: 4664-4669. Amino acid 158 interacts with Fc arm FcgRIIIa-158VF influences rituximab response in human
100% 100 90% P = 0.03
80 67% P = 0.03
60 51%
40
20 Objective Response (%) Response Objective FCGR3A-158VV 0 FCGR3A-158F carriers M2 M12 Cartron G, et al. Blood 2002; 99:754–758. Is FcgRIIIa-158VF influences all IgG1 mAbs response?
Trastuzumab
Musolino A, et al. J Clin Oncol 2007; 26: 1789-1796. Ways to improve ADCC
Mutation Fc
Low fucose Effets of glycoengiennering and Fc mutations
Teneur en fucose : Teneur en fucose : Teneur en fucose : Target cells : WIL2-S
sFcγRIIIaV binding98 % sFcγRIIIa4 % F binding 98 % Cytotoxicité (%) Cytotoxicité
Cellules cible : Raji Unités de résonancedeUnités Unités de résonancedeUnités Temps (s) Temps (s) Okazaki A, et al. J Mol Biol 2004; 336: 1239-1249. Superior ADCC: The effect of glycoengineering
Obinutuzumab Rituximab Ofatumumab Ublituximab Rituximab Z138 (PBMC: V/V) 100
80
60 dependent dependent
- 40
killing (%) killing 20
Antibody 0
–20 0.064 0.32 1.6 8 40 200 1000 Antibody concentration (ng/mL)
PBMC: V/V = peripheral blood mononuclear cells expressing human FcγRIIIa V/V. Herter S, et al. ASH 2010. Abstract 3925 (Poster presentation) De Romeuf C et al. Br J Haematol 2008; 140: 635-643 Conclusion
ADCC
Direct ADPC effect
CDC Rituximab Ofatumumab Obinutuzumab Ublituximab
Arbitrary scale: Rituximab referenced with 3 for all in vitro activities New Anti-CD20 Monoclonals
Pr Guillaume CARTRON, MD Ph-D
Head of Hematology Department, UMR-CNRS 5235
CHRU Montpellier, University of Montpellier