Human antibodies that bind human TNF&agr;

Details Human antibodies that bind human TNF&agr; Human antibodies that bind human TNF&agr;

1999-03-16

2001-07-10

Saunders, David (Department: 1642)

Chemistry: molecular biology and microbiology

Micro-organism, tissue cell culture or enzyme using process...

Recombinant dna technique included in method of making a...

C435S335000, C435S320100, C536S023530

Reexamination Certificate

active

06258562

ABSTRACT:

BACKGROUND OF THE INVENTION
Tumor necrosis factor &agr; (TNF&agr;) is a cytokine produced by numerous cell types, including monocytes and macrophages, that was originally identified based on its capacity to induce the necrosis of certain mouse tumors (see e.g., Old, L. (1985)
Science
230:630-632). Subsequently, a factor termed cachectin, associated with cachexia, was shown to be the same molecule as TNF&agr;. TNF&agr; has been implicated in mediating shock (see e.g., Beutler, B. and Cerami, A. (1988)
Annu. Rev. Biochem.
57:505-518; Beutler, B. and Cerami, A. (1989)
Annu. Rev. Immunol.
7:625-655). Furthermore, TNF&agr; has been implicated in the pathophysiology of a variety of other human diseases and disorders, including sepsis, infections, autoimmune diseases, transplant rejection and graft-versus-host disease (see e.g., Moeller, A., et al. (1990)
Cytokine
2:162-169; U.S. Pat. No. 5,231,024 to Moeller et al.; European Patent Publication No. 260 610 B1 by Moeller, A., et al. Vasilli, P. (1992)
Annu. Rev. Immunol.
10:411-452; Tracey, K. J. and Cerami, A. (1994)
Annu. Rev. Med.
45:491-503).
Because of the harmful role of human TNF&agr; (hTNF&agr;) in a variety of human disorders, therapeutic strategies have been designed to inhibit or counteract hTNF&agr; activity. In particular, antibodies that bind to, and neutralize, hTNF&agr; have been sought as a means to inhibit hTNF&agr; activity. Some of the earliest of such antibodies were mouse monoclonal antibodies (mAbs), secreted by hybridomas prepared from lymphocytes of mice immunized with hTNF&agr; (see e.g., Hahn T; et al., (1985)
Proc Natl Acad Sci USA
82: 3814-3818; Liang, C-M., et al. (1986)
Biochem. Biophys. Res. Commun.
137:847-854; Hirai, M., et al. (1987)
J Immunol. Methods
96:57-62; Fendly, B. M., et al. (1987)
Hybridoma
6:359-370; Moeller, A.. et al. (1990)
Cytokine
2:162-169; U.S. Pat. No. 5,231,024 to Moeller et al.; European Patent Publication No. 186 833 B1 by Wallach, D.; European Patent Application Publication No. 218 868 A1 by Old et al.; European Patent Publication No. 260 610 B1 by Moeller, A., et al.). While these mouse anti-hTNF&agr; antibodies often displayed high affinity for hTNF&agr; (e.g., Kd≦10
−9
M) and were able to neutralize hTNF&agr; activity, their use in vivo may be limited by problems associated with administration of mouse antibodies to humans, such as short a serum half life, an inability to trigger certain human effector functions and elicitation of an unwanted immune response against the mouse antibody in a human (the “human anti-mouse antibody” (HAMA) reaction).
In an attempt to overcome the problems associated with use of fully-murine antibodies in humans, murine anti-hTNF&agr; antibodies have been genetically engineered to be more “human-like.” For example, chimeric antibodies, in which the variable regions of the antibody chains are murine-derived and the constant regions of the antibody chains are human-derived, have been prepared (Knight, D. M, et a. (1993)
Mol. Immunol.
30:1443-1453; PCT Publication No. WO 92/16553 by Daddona, P. E., et al.). Additionally, humanized antibodies, in which the hypervariable domains of the antibody variable regions are murine-derived but the remainder of the variable regions and the antibody constant regions are human-derived, have also been prepared (PCT Publication No. WO 92/11383 by Adair, J. R., et al.). However, because these chimeric and humanized antibodies still retain some murine sequences, they still may elicit an unwanted immune reaction, the human anti-chimeric antibody (HACA) reaction, especially when administered for prolonged periods, e.g., for chronic indications, such as rheumatoid arthritis (see e.g., Elliott, M. J., et al. (1994)
Lancet
344:1125-1127; Elliot, M. J., et al., (1994)
Lancet
344:1105-1110).
A preferred hTNF&agr; inhibitory agent to murine mAbs or derivatives thereof (e.g., chimeric or humanized antibodies) would be an entirely human anti-hTNF&agr; antibody, since such an agent should not elicit the HAMA reaction, even if used for prolonged periods. Human monoclonal autoantibodies against hTNF&agr; have been prepared using human hybridoma techniques (Boyle, P., et al. (1993)
Cell. Immunol.
152:556-568; Boyle, P., et al. (1993)
Cell. Immunol.
152:569-581; European Patent Application Publication No. 614 984 A2 by Boyle, et al.). However, these hybridoma-derived monoclonal autoantibodies were reported to have an affinity for hTNF&agr; that was too low to calculate by conventional methods, were unable to bind soluble hTNF&agr; and were unable to neutralize hTNF&agr;-induced cytotoxicity (see Boyle, et al.; supra). Moreover, the success of the human hybridoma technique depends upon the natural presence in human peripheral blood of lymphocytes producing autoantibodies specific for hTNF&agr;. Certain studies have detected serum autoantibodies against hTNF&agr; in human subjects (Fomsgaard, A., et al. (1989)
Scand. J Immunol.
30:219-223; Bendtzen, K., et al. (1990)
Prog. Leukocyte Biol
10B:447-452), whereas others have not (Leusch, H-G., et al. (1991)
J. Immunol. Methods
139:145-147).
Alternative to naturally-occurring human anti-hTNF&agr; antibodies would be a recombinant hTNF&agr; antibody. Recombinant human antibodies that bind hTNF&agr; with relatively low affinity (i.e., K
d
~10
−7
M) and a fast off rate (i.e., K
off
~10
−2
sec
−1
) have been described (Griffiths, A. D., et al. (1993)
EMBO J.
12:725-734). However, because of their relatively fast dissociation kinetics, these antibodies may not be suitable for therapeutic use. Additionally, a recombinant human anti-hTNF&agr; has been described that does not neutralize hTNF&agr; activity, but rather enhances binding of hTNF&agr; to the surface of cells and enhances internalization of hTNF&agr; (Lidbury, A., et a. (1994)
Biotechnol. Ther.
5:27-45; PCT Publication No. WO 92103145 by Aston, R. et al)
Accordingly, human antibodies, such as recombinant human antibodies, that bind soluble hTNF&agr; with high affinity and slow dissociation kinetics and that have the capacity to neutralize hTNF&agr; activity, including hTNF&agr;-induced cytotoxicity (in vitro and in vivo) and hTNF&agr;-induced cell activation, are still needed.
SUMMARY OF THE INVENTION
This invention provides human antibodies, preferably recombinant human antibodies, that specifically bind to human TNF&agr;. The antibodies of the invention are characterized by binding to hTNF&agr; with high affinity and slow dissociation kinetics and by neutralizing hTNF&agr; activity, including hTNF&agr;-induced cytotoxicity (in vitro and in vivo) and hTNF&agr;-induced cellular activation. Antibodies of the invention are further characterized by binding to hTNF&agr; but not hTNF&bgr; (lymphotoxin) and by having the ability to bind to other primate TNF&agr;s and non-primate TNF&agr;s in addition to human TNF&agr;.
The antibodies of the invention can be fill-length (e.g., an IgG1 or IgG4 antibody) or can comprise only an antigen-binding portion (e.g., a Fab, F(ab′)
2
or scFv fragment). The most preferred recombinant antibody of the invention, termed D2E7, has a light chain CDR3 domain comprising the amino acid sequence of SEQ ID NO: 3 and a heavy chain CDR3 domain comprising the amino acid sequence of SEQ ID NO: 4. Preferably, the D2E7 antibody has a light chain variable region (LCVR) comprising the amino acid sequence of SEQ ID NO: 1 and a heavy chain variable region (HCVR) comprising the amino acid sequence of SEQ ID NO: 2.
In one embodiment, the invention provides an isolated human antibody, or an antigen-binding portion thereof, that dissociates from human TNF&agr; with a K
d
of 1×10
−8
M or less and a K
off
rate constant of 1×10
−3
s
−1
or less, both determined by surface plasmon resonance, and neutralizes human TNF&agr; cytotoxicity in a standard in vitro L929 assay with an IC
50
of 1×10
−7
M or less. More preferably, the isolated human antibody, or antigen-binding portion thereof, dissociates from human TNF&

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