Drug – bio-affecting and body treating compositions – Immunoglobulin – antiserum – antibody – or antibody fragment,... – Structurally-modified antibody – immunoglobulin – or fragment...
Reexamination Certificate
2000-01-14
2004-05-18
Saunders, David (Department: 1644)
Drug, bio-affecting and body treating compositions
Immunoglobulin, antiserum, antibody, or antibody fragment,...
Structurally-modified antibody, immunoglobulin, or fragment...
C530S387300
Reexamination Certificate
active
06737056
ABSTRACT:
BACKGROUND OF THE INVENTION
1. Field of the Invention
The present invention concerns polypeptides comprising a variant Fc region. More particularly, the present invention concerns Fc region-containing polypeptides that have altered effector function as a consequence of one or more amino acid modifications in the Fc region thereof.
2. Description of Related Art
Antibodies are proteins which exhibit binding specificity to a specific antigen. Native antibodies are usually heterotetrameric glycoproteins of about 150,000 daltons, composed of two identical light (L) chains and two identical heavy (H) chains. Each light chain is linked to a heavy chain by one covalent disulfide bond, while the number of disulfide linkages varies between the heavy chains of different immunoglobulin isotypes. Each heavy and light chain also has regularly spaced intrachain disulfide bridges. Each heavy chain has at one end a variable domain (V
H
) followed by a number of constant domains. Each light chain has a variable domain at one end (V
L
) and a constant domain at its other end; the constant domain of the light chain is aligned with the first constant domain of the heavy chain, and the light chain variable domain is aligned with the variable domain of the heavy chain. Particular amino acid residues are believed to form an interface between the light and heavy chain variable domains.
The term “variable” refers to the fact that certain portions of the variable domains differ extensively in sequence among antibodies and are responsible for the binding specificity of each particular antibody for its particular antigen. However, the variability is not evenly distributed through the variable domains of antibodies. It is concentrated in three segments called complementarity determining regions (CDRs) both in the light chain and the heavy chain variable domains. The more highly conserved portions of the variable domains are called the framework regions (FRs). The variable domains of native heavy and light chains each comprise four FRs, largely adopting a &bgr;-sheet configuration, connected by three CDRs, which form loops connecting, and in some cases forming part of, the &bgr;-sheet structure. The CDRs in each chain are held together in close proximity by the FRs and, with the CDRs from the other chain, contribute to the formation of the antigen binding site of antibodies (see Kabat et al., Sequences of
Proteins of Immunological Interest
, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, Md. (1991)).
The constant domains are not involved directly in binding an antibody to an antigen, but exhibit various effector functions. Depending on the amino acid sequence of the constant region of their heavy chains, antibodies or immunoglobulins can be assigned to different classes. There are five major classes of immunoglobulins: IgA, IgD, IgE, IgG and IgM, and several of these may be further divided into subclasses (isotypes), e.g. IgG1, IgG2, IgG3, and IgG4; IgA1 and IgA2. The heavy chain constant regions that correspond to the different classes of immunoglobulins are called &agr;, &dgr;, &egr;, &ggr;, and &mgr;, respectively. Of the various human immunoglobulin classes, only human IgG1, IgG2, IgG3 and IgM are known to activate complement; and human IgG1 and IgG3 mediate ADCC more effectively than IgG2 and IgG4.
A schematic representation of the native IgG1 structure is shown in
FIG. 1
, where the various portions of the native antibody molecule are indicated. Papain digestion of antibodies produces two identical antigen binding fragments, called Fab fragments, each with a single antigen binding site, and a residual “Fc” fragment, whose name reflects its ability to crystallize readily. The crystal structure of the human IgG Fc region has been determined (Deisenhofer,
Biochemistry
20:2361-2370 (1981)). In human IgG molecules, the Fc region is generated by papain cleavage N-terminal to Cys 226. The Fc region is central to the effector functions of antibodies.
The effector functions mediated by the antibody Fc region can be divided into two categories: (1) effector functions that operate after the binding of antibody to an antigen (these functions involve the participation of the complement cascade or Fc receptor (FcR)-bearing cells); and (2) effector functions that operate independently of antigen binding (these functions confer persistence in the circulation and the ability to be transferred across cellular barriers by transcytosis). Ward and Ghetie,
Therapeutic Immunology
2:77-94 (1995).
While binding of an antibody to the requisite antigen has a neutralizing effect that might prevent the binding of a foreign antigen to its endogenous target (e.g. receptor or ligand), binding alone may not remove the foreign antigen. To be efficient in removing and/or destructing foreign antigens, an antibody should be endowed with both high affinity binding to its antigen, and efficient effector functions.
Fc Receptor (FcR) Binding
The interaction of antibodies and antibody-antigen complexes with cells of the immune system effects a variety of responses, including antibody-dependent cell-mediated cytotoxicity (ADCC) and complement dependent cytotoxicity (CDC) (reviewed in Daëron,
Annu. Rev. Immunol
. 15:203-234 (1997); Ward and Ghetie,
Therapeutic Immunol
. 2:77-94 (1995); as well as Ravetch and Kinet,
Annu. Rev. Immunol
. 9:457-492 (1991)).
Several antibody effector functions are mediated by Fc receptors (FcRs), which bind the Fc region of an antibody. FcRs are defined by their specificity for immunoglobulin isotypes; Fc receptors for IgG antibodies are referred to as Fc&ggr;R, for IgE as Fc&egr;R, for IgA as Fc&agr;R and so on. Three subclasses of Fc&ggr;R have been identified: Fc&ggr;RI (CD64), Fc&ggr;RII (CD32) and Fc&ggr;RIII (CD16). Because each Fc&ggr;R subclass is encoded by two or three genes, and alternative RNA spicing leads to multiple transcripts, a broad diversity in Fc&ggr;R isoforms exists. The three genes encoding the Fc&ggr;RI subclass (Fc&ggr;RIA, Fc&ggr;RIB and Fc&ggr;RIC) are clustered in region 1q21.1 of the long arm of chromosome 1; the genes encoding Fc&ggr;RII isoforms (Fc&ggr;RIIA, Fc&ggr;RIIB and Fc&ggr;RIIC) and the two genes encoding Fc&ggr;RIII (Fc&ggr;RIIIA and Fc&ggr;RIIIB) are all clustered in region 1q22. These different FcR subtypes are expressed on different cell types (reviewed in Ravetch and Kinet,
Annu. Rev. Immunol
. 9:457-492 (1991)). For example, in humans, Fc&ggr;RIIIB is found only on neutrophils, whereas Fc&ggr;RIIIA is found on macrophages, monocytes, natural killer (NK) cells, and a subpopulation of T-cells. Notably, Fc&ggr;RIIIA is the only FcR present on NK cells, one of the cell types implicated in ADCC.
Fc&ggr;RI, Fc&ggr;RII and Fc&ggr;RIII are immunoglobulin superfamily (IgSF) receptors; Fc&ggr;RI has three IgSF domains in its extracellular domain, while Fc&ggr;RII and Fc&ggr;RIII have only two IgSF domains in their extracellular domains.
Another type of Fc receptor is the neonatal Fc receptor (FcRn). FcRn is structurally similar to major histocompatibility complex (MHC) and consists of an &agr;-chain noncovalently bound to &bgr;2-microglobulin.
The binding site on human and murine antibodies for Fc&ggr;R have been previously mapped to the so-called “lower hinge region” consisting of residues 233-239 (EU index numbering as in Kabat et al.,
Sequences of Proteins of Immunological Interest
, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, Md. (1991)). Woof et al.
Molec. Immunol
. 23:319-330 (1986); Duncan et al.
Nature
332:563 (1988); Canfield and Morrison,
J. Exp. Med
. 173:1483-1491 (1991); Chappel et al.,
Proc. Natl. Acad. Sci USA
88:9036-9040 (1991). Of residues 233-239, P238 and S239 have been cited as possibly being involved in binding, but these two residues have never been evaluated by substitution or deletion.
Other previously cited areas possibly involved in binding to Fc&ggr;R are: G316-K338 (human IgG) for human Fc&ggr;RI (by sequence comparison only; no substitution mutants were evaluated) (Woof et al.
M
Genentech Inc.
Lee Wendy M.
Saunders David
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