Drug – bio-affecting and body treating compositions – Immunoglobulin – antiserum – antibody – or antibody fragment,... – Structurally-modified antibody – immunoglobulin – or fragment...
Reexamination Certificate
1995-03-21
2003-05-27
Housel, James (Department: 1648)
Drug, bio-affecting and body treating compositions
Immunoglobulin, antiserum, antibody, or antibody fragment,...
Structurally-modified antibody, immunoglobulin, or fragment...
C424S135100, C424S154100, C424S155100, C424S156100, C435S069600, C435S328000, C530S387300, C536S023530
Reexamination Certificate
active
06569430
ABSTRACT:
FIELD OF THE INVENTION
This invention relates to antibodies.
BACKGROUND TO THE INVENTION
Antibodies, or immunoglobulins, comprise two heavy chains linked together by disulphide bonds and two light chains, each light chain being linked to a respective heavy chain by disulphide bonds. The general structure of an antibody of class IgG (i.e. an immunoglobulin (Ig) of class gamma (G)) is shown schematically in
FIG. 1
of the accompanying drawings.
Each heavy chain has at one end a variable domain followed by a number of constant domains. Each light chain has a variable domain at one end and a constant domain at its other end, the light chain variable domain being aligned with the variable domain of the heavy chain and the light chain constant domain being aligned with the first constant domain of the heavy chain. The constant domains in the light and heavy chains are not involved directly in binding the antibody to antigen.
The variable domains of each pair of light and heavy chains form the antigen binding site. The domains on the light and heavy chains have the same general structure and each domain comprises four framework regions, whose sequences are relatively conserved, connected by three complementarity determining regions (CDRs) (see reference 11). The four framework regions largely adopt a beta-sheet conformation and the CDRs form loops connecting, and in some cases forming part of, the beta-sheet structure.
The CDRs are held in close proximity by the framework regions and, with the CDRs from the other domain, contribute to the formation of the antigen binding site.
SUMMARY OF THE INVENTION
According to one aspect of the present invention there is provided an antibody having at least one CDR which is foreign with respect to the constant region of the antibody, said at least one foreign CDR being selected from CDRs substantially as identified in
FIG. 2
, namely residues 31 to 35 (SEQ ID NO:1), 50 to 65 (SEQ ID NO:2) and 95 to 102 (SEQ ID NO:3), of the heavy chain and residues 24 to 34, 50 to 56 and 89 to 97 of the light chain, the antibody being capable of binding effectively to the antigen Campath-1.
The term “foreign” is used in relation to the CDR(s) and constant region to mean of different origin.
In FIG.
2
and elsewhere in the specification amino acid residues are identified by the conventionally used one letter symbols, as follows:
Amino Acid
One-letter symbol
Alanine
A
Arginine
R
Asparagine
N
Aspartic acid
D
Asparagine or aspartic acid
B
Cysteine
C
Glutamine
Q
Glutamic acid
E
Glutamine or glutamic acid
Z
Glycine
G
Histidine
H
Isoleucine
I
Leucine
L
Lysine
K
Methionine
M
Phenylalanine
F
Proline
P
Serine
S
Threonine
T
Tryptophan
W
Tyrosine
Y
Valine
V
In this specification, effective antibody-antigen binding is used to mean that antibody effects 50% binding to antigen at antibody concentrations of less than or equal to 70 ug/ml, preferably at concentrations of less than or equal to 7 ug/ml. Binding affinity may be tested by assay procedures such as are described in Example 1 herein, eg using Campath-1 antigen obtained from a glycolipid extract from human spleen. (ug=microgram)
Thus, a standard procedure for the extraction of glycolipids can be applied to the extraction of the Campath-1 antigen from human spleens. This standard extraction procedure involves the treatment of 1 volume of homogenised human spleen tissue with 3 volumes of water, 11 volumes of methanol and 5.4 volumes of chloroform. After mixing precipitated material is discarded and a further 3.5 volumes of water are added, followed by further mixing. The mixture is then allowed to separate into two phases, the lower chloroform-containing phase is discarded and the upper aqueous phase is concentrated to provide a crude extract of the Campath-1 antigen, which can if desired be purified further by affinity chromatography, for example using the YTH66.9 antibody referred to hereinafter.
The antibody of the present invention desirably has a light chain with at least one CDR selected from CDRs substantially as identified in
FIG. 2 and a
heavy chain with at least one CDR selected from CDRs substantially as identified in FIG.
2
.
As a further possibility, the antibody of the present invention preferably has three heavy chain CDRs substantially as identified in
FIG. 2
, or three light chain CDRs substantially as identified in FIG.
2
. More preferably, the antibody has all six heavy and light chain CDRs substantially as identified in FIG.
2
.
Hence, in a preferred aspect the present invention provides an antibody having heavy and light chain CDRs which are foreign with respect to the constant region of the antibody, said CDRs being substantially as identified in
FIG. 2
, namely residues 31 to 35, 50 to 65 and 95 to 102 of the heavy chain and residues 24 to 34, 50 to 56 and 89 to 97 of the light chain, the antibody being capable of binding effectively to the antigen Campath-1.
The CDRs identified in
FIG. 2
are of rat origin and may be combined with a range of different variable domain framework regions, as desired, including, eg, framework regions of rat or human origin.
In a further aspect the present invention provides an antibody having heavy and light chain variable domains as identified in the lower lines of sequence information in
FIG. 2
, namely residues 1 to 113 (SEQ ID NO:7) of the heavy chain and residues 1 to 108 (SEQ ID NO:8) of the light chain, the CDRs and constant region of the antibody being foreign with respect to one another, the antibody being capable of binding effectively to the antigen Campath-1.
Such an antibody comprises CDRs and framework regions of rat origin.
The invention also provides an antibody having heavy and light chain variable domains as identified in the upper lines of sequence information in
FIG. 2
, namely residues 1 to 113 (SEQ ID NO:9) of the heavy chain and residues 1 to 108 (SEQ ID NO:10) of the light chain, and that will bind effectively to the antigen Campath-1.
Such an antibody comprises CDRs of rat origin in framework regions of human origin.
Such an antibody may be modified by having a phenylalanine group at residue 27 of the heavy chain in place of serine, and possibly also by having a threonine group at residue 30 of the heavy chain in place of serine. A Ser(27) to Phe mutation is found to increase antibody-antigen binding significantly. However, the mutation of Ser (30) to Thr (in the human framework with the Ser (27) to Phe mutation) has little effect on binding affinity. This illustrates that point mutations in the antibody may have a major effect or little effect on the affinity of the antibody for the antigen. Although the two changes Ser (27) to Phe and Ser (30) to Thr are located within the framework region as defined in reference 11, they lie within the hypervariable loop Hl as defined in reference 18. It is accordingly believed that some changes in the CDRs may similarly be made without necessarily having an adverse effect on antibody-antigen affinity. References to CDRs substantially as identified in
FIG. 2
are accordingly intended to include within their scope not only CDRs identical to those identified in
FIG. 2
but also variants of such CDRs, subject to the requirement of the antibody binding effectively to Campath-1.
The antibody is preferably in biologically pure form, desirably being at least 95% (by wt) free of other biological materials.
The remainder of the antibody, namely the heavy and light chain constant domains and possibly also variable domain framework regions and one or more CDRs, can be based on antibodies of various different types as desired including, eg, rat and human antibodies of different classes. Thus, the constant domains can be selected to have desired effector functions appropriate to the intended use of the antibody. For example, for therapeutic purposes, human IgG1 and rat IgG2b are currently favoured isotypes. Further, of the human IgG isotypes, IgG1 and IgG3 appear to be the most effective for complement and cell mediated lysis, and therefore for killing tumour cells. For other purposes other isotypes
Clark Michael R.
Riechmann Lutz
Waldmann Herman
Winter Gregory P.
Brown Stacy S.
BTG International Limited
Housel James
Nixon & Vanderhye
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