Distinct in vitro binding properties of the anti-CD20 small modular immunopharmaceutical 2LM20-4 result in profound and sustained in vivo potency in cynomolgus monkeys.
OBJECTIVE
To characterize the in vitro binding and effector function properties of CD20-directed small modular immunopharmaceutical (SMIP) 2LM20-4, and to compare its in vivo B-cell depletion activity with the mutated 2LM20-4 P331S [no in vitro complement-dependent cytotoxicity (CDC)] and rituximab in cynomolgus monkeys.


METHODS
Direct binding is examined in flow cytometry, confocal microscopy, scatchard and lipid raft assays. Effector function assays include CDC and Fc-mediated cellular toxicity. In the 6-month-long in vivo B-cell depletion study, single i.v. dosages of 1 or 10 mg/kg of anti-CD20 proteins were administered to monkeys and B-cell counts were monitored in peripheral blood, bone marrow and lymph nodes.


RESULTS
2LM20-4 has lower saturation binding to human primary B cells and recruits fewer CD20 molecules into lipid rafts compared with rituximab; however, it induces higher in vitro CDC. In competitive binding, 2LM20-4 only partially displaces rituximab, suggesting that it binds to a fraction of CD20 molecules within certain locations of the plasma membrane as compared with rituximab. In monkeys, 2LM20-4 had more sustained B-cell depletion activity than rituximab in peripheral blood and had significantly more profound and sustained activity than 2LM20-4 P331S and rituximab in the lymph nodes.


CONCLUSIONS
SMIP 2LM20-4, which binds to a fraction of CD20 molecules as compared with rituximab, has more potent in vitro CDC, and more potent and sustained B-cell depletion activity in cynomolgus monkeys. Our work has considerable clinical relevance since it provides novel insights related to the emerging B-cell depletion therapies in autoimmune diseases.
Inflammation and Immunology, Pfizer Biotherapeutics Research and Development, Cambridge, MA 02140, USA.
Cheryl
C
Nickerson-Nutter
Lioudmila
L
Tchistiakova
Nilufer P
NP
Seth
Marion
M
Kasaian
Barbara
B
Sibley
Stephane
S
Olland
Richard
R
Zollner
William A
WA
Brady
Kendall M
KM
Mohler
Peter
P
Baum
Alan
A
Wahl
Deborah
D
Herber
Yulia
Y
Vugmeyster
David
D
Wensel
Neil M
NM
Wolfman
Davinder
D
Gill
Mary
M
Collins
Kyri
K
Dunussi-Joannopoulos
D000818:Animals; D000911:Antibodies, Monoclonal / Q000494:pharmacology; D058846:Antibodies, Monoclonal, Murine-Derived / Q000494:pharmacology; D000920:Antibody-Dependent Cell Cytotoxicity / Q000187:drug effects* / Q000276:immunology; D018951:Antigens, CD20 / Q000187:drug effects* / Q000276:immunology*; D001402:B-Lymphocytes / Q000276:immunology / Q000378:metabolism; D001665:Binding Sites; D002478:Cells, Cultured; D004195:Disease Models, Animal; D006801:Humans; D007155:Immunologic Factors / Q000494:pharmacology; D066298:In Vitro Techniques; D016014:Linear Models; D008252:Macaca fascicularis; D011897:Random Allocation; D000069283:Rituximab; D012680:Sensitivity and Specificity; D057127:Single-Chain Antibodies / Q000494:pharmacology*
D000911:Antibodies, Monoclonal; D058846:Antibodies, Monoclonal, Murine-Derived; D018951:Antigens, CD20; D007155:Immunologic Factors; D057127:Single-Chain Antibodies; D000069283:Rituximab
Blood. 2002 May 1;99(9):3256-62
11964291
J Biol Chem. 1998 Jan 2;273(1):344-8
9417086
J Exp Med. 1988 Jul 1;168(1):127-42
3260935
Curr Opin Immunol. 2008 Dec;20(6):639-45
18775493
Blood. 2008 Nov 15;112(10):4170-7
18583569
N Engl J Med. 2008 Feb 14;358(7):676-88
18272891
J Autoimmun. 2007 Mar-May;28(2-3):62-8
17391915
Nat Rev Immunol. 2006 May;6(5):394-403
16622478
Clin Exp Immunol. 2005 Mar;139(3):439-46
15730389
Mol Immunol. 2007 Sep;44(16):3823-37
17768100
Blood. 2004 Sep 15;104(6):1793-800
15172969
Arthritis Rheum. 2006 Sep;54(9):2793-806
16947627
J Immunol. 2005 Jan 15;174(2):817-26
15634903
Arch Neurol. 2009 Feb;66(2):259-61
19204165
Proc Natl Acad Sci U S A. 1987 May;84(10):3439-43
3106970
Mol Immunol. 1993 Aug;30(12):1089-96
8366859
Haematologica. 2006 Feb;91(2):176-83
16461301
Arthritis Res Ther. 2009;11(6):134
20017888
Nat Med. 2000 Apr;6(4):443-6
10742152
Annu Rev Immunol. 1987;5:21-42
3036181
J Immunol. 2006 Jul 1;177(1):362-71
16785532
Nat Rev Rheumatol. 2009 Aug;5(8):433-41
19581902
Nat Rev Rheumatol. 2009 Dec;5(12):711-6
19946298
J Clin Invest. 2009 Aug;119(8):2143-59
19620786
Ann Rheum Dis. 2007 Sep;66(9):1259-62
17412738
Blood. 2001 Mar 1;97(5):1392-8
11222385
N Engl J Med. 2009 Nov 26;361(22):2143-52
19940299
Ann Rheum Dis. 2008 Mar;67(3):330-4
17827182
Clin Immunol. 2005 Dec;117(3):207-13
16169773
Arthritis Rheum. 2007 Sep;56(9):3044-56
17763423
J Rheumatol. 2008 Jul;35(7):1245-55
18609733
J Biol Chem. 1994 Feb 4;269(5):3469-74
8106388
Blood. 2004 Apr 1;103(7):2738-43
14551143
Immunol Rev. 2008 Jun;223:284-99
18613843
J Immunol. 2003 Aug 1;171(3):1581-7
12874252