Chemistry: natural resins or derivatives; peptides or proteins; – Proteins – i.e. – more than 100 amino acid residues – Blood proteins or globulins – e.g. – proteoglycans – platelet...
Reexamination Certificate
1999-08-23
2004-02-10
Gambel, Phillip (Department: 1644)
Chemistry: natural resins or derivatives; peptides or proteins;
Proteins, i.e., more than 100 amino acid residues
Blood proteins or globulins, e.g., proteoglycans, platelet...
C530S387100, C530S387300, C530S388100, C530S388200, C530S388220, C530S388700, C530S388730, C530S391100, C530S391300, C435S007100, C435S810000, C424S130100, C424S133100, C424S141100, C424S143100, C424S144100, C424S153100, C424S154100, C424S173100, C424S178100
Reexamination Certificate
active
06689869
ABSTRACT:
BACKGROUND OF THE INVENTION
Antibodies typically comprise two heavy chains linked together by disulphide bonds and two light chains. Each light chain is linked to a respective heavy chain by disulphide bonds. 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 is aligned with the variable domain of the heavy chain. The light chain constant domain is 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 a framework of four regions, whose sequences are relatively conserved, connected by three complementarity determining regions (CDRs). 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. CDRs and framework regions of antibodies may be determined by reference to Kabat et al. (“Sequences of proteins of immunological interest” US Dept. of Health and Human Services, US Government Printing Office, 1987).
The preparation of an altered antibody in which the CDRs are derived from a different species than the framework of the antibody's variable domains is disclosed in EP-A-0239400. The CDRs may be derived from a rat or mouse monoclonal antibody. The framework of the variable domains, and the constant domains, of the altered antibody may be derived from a human antibody. Such a humanized antibody elicits a negligible immune response when administered to a human compared to the immune response mounted by a human against a rat or mouse antibody. Humanized CAMPATH-1 antibody is disclosed in EP-A-0328404 (Campath® antibody is a registered trademark).
SUMMARY OF THE INVENTION
The present invention relates to an antibody which binds to the CD18 antigen, to the preparation of such an antibody and to a pharmaceutical composition which contains the antibody. The invention also relates to methods of using the anti-CD18 antibody.
DETAILED DESCRIPTION OF THE INVENTION
According to one aspect of the present invention, there is provided a humanized antibody in which sufficient of the amino acid sequence of each CDR shown below is provided such that the antibody is capable of binding to the human CD18 antigen:
light chain: CDR1 (SEQ ID NOS: 3 and 4)
CDR2 (SEQ ID NOS: 5 and 6)
CDR3 (SEQ ID NOS: 7 and 8)
heavy chain: CDR1 (SEQ ID NOS: 11 and 12)
CDR2 (SEQ ID NOS: 13 and 14)
CDR3 (SEQ ID NOS: 15 and 16).
According to another aspect, the invention provides a DNA molecule encoding a humanized antibody in which sufficient of the amino acid sequence of each CDR shown above is provided such that the antibody is capable of binding to the human CD18 antigen.
The antibody preferably has the structure of a natural antibody or a fragment thereof. The antibody may therefore comprise a complete antibody, a (Fab′)
2
fragment, a Fab fragment, a light chain dimer or a heavy chain dimer. The antibody may be an IgG such as IgG1, IgG2, IgG3 or IgG4; or IgM, IgA, IgE or IgD. The constant domain of the antibody heavy chain may be selected accordingly. The light chain constant domain may be a kappa or lambda constant domain.
The antibody may be a chimeric antibody of the type described in WO 86/01533. A chimeric antibody according to WO 86/01533 comprises an antigen binding region and a non-immunoglobulin region. The antigen binding region is an antibody light chain variable domain and/or heavy chain variable domain. Typically the chimeric antibody comprises both light and heavy chain variable domains. The non-immunoglobulin region is fused to the C-terminus of the antigen binding region. The non-immunoglobulin region is typically a non-immunoglobulin protein and may be an enzyme region, a region derived from a protein having known binding specificity, from a protein toxin or indeed from any protein expressed by a gene. The non-immunoglobulin region may be a carbohydrate region. The two regions of the chimeric antibody may be connected via a cleavable linker sequence.
The light chain CDRs 1 to 3 and heavy chain CDRs 1 to 3 of SEQ ID NOS: 3 to 8 and SEQ ID NOS: 11 to 16, respectively, are the CDRs of the YFC51.1.1 rat antibody which is a CD18 antibody. The specificity of a humanized antibody for the human CD18 antigen can be determined by flow cytometry, monocyte adhesion and/or by T-cell proliferation assays as follows:
Monocyte (MNC) Adhesion
MNC's are treated with the phorbol diester PDBu (10
−9
M) in the presence and absence of antibody (20 &mgr;l) for 5 minutes. These cells are then transferred to bovine aortic endothelial cell (BAEC) monolayers and incubated for 30 minutes in a humidified atmosphere of 95% air, 5% CO
2
at 37° C. Non-adherent cells are removed by washing in phosphate buffered saline (PBS) three times. The adherent cells are then lysed in situ with 50 &mgr;l of 0.5% hexadecyltrimethyl ammonium bromide. Dianisidine dihydrochloride (0.63 mM) containing 0.4 mM hydrogen peroxide is added (250 &mgr;l) to each well and incubated for a further 10 minutes. Enzyme activity is then assessed using the presence of monocyte-specific myeloperoxidase, recorded as an increase in absorbance. The optical density of the samples can then be recorded at 450 nm using a multi-well plate reader (Anthos series, Lab Teck instruments). Comparisons can then be made between treated and untreated samples (Bath et al.,
J. Immunol Meth
., 118: 59-65 (1989)).
Flow Cytometry
Surface labeling of rat, rabbit, guinea-pig and human monocytes with antibody is carried out according to the method of Gladwin et al., (
Biochim. Biophys. Acta
, 1052: 166-172 (1990)). Briefly, 1 ml aliquots of a cell suspension (5×10
6
) are incubated with the appropriate antibody, monodispersed and incubated on melting ice for 30 minutes. The cells are twice washed in PBS and incubated for a further 30 minutes with a 1:200 dilution of rabbit anti-rat F(ab′)
2
FITC conjugate on melting ice. The cells are finally washed three times in PBS and fixed in 0.1% para-formaldehyde. Analysis of surface labeling can be performed using an Epics Elite flow cytometer (Coulter cytometry, Hialeah, Fla.) using standard computer, electronics and optics. The Elite is configured with a 15 mW 488 nm Argon-ion laser (Cyonics model 2201, San Jose, Calif.). Monocyte and lymphocyte populations are separated by forward angle light scatter and side scatter. Green fluorescence data for 2×10
4
monocytes is collected using bit-map gating and collected on a three decade log scale. Green fluorescence data for 2×10
4
neutrophils is collected in a similar manner. For each sample, mean fluorescence intensity in the presence of the primary mAb is compared with cells incubated with rabbit anti-rat F(ab′)
2
FITC fragments alone and the percentage labeling of the cells determined. Samples can be labeled in triplicate and repeat experiments can be performed on three separate occasions.
T-cell Proliferation Assay
Human mononuclear cells are prepared from defibrinated blood using density gradient separation over Ficoll-paque® solution. Lymphocytes (2×10
5
cells) are cultured in each well of a flat bottomed 96-well microtitre plate (Nunclon, Roskild, Denmark), in RPMI 1640 supplemented with 10% autologous serum, 2 mM glutamine and 100 iU penicillin/100 &mgr;g ml
−1
streptomycin. Triplicate cultures are set up with the medium alone or with antigen (Tetanus Toxoid, 3 &mgr;g ml
−1
) or mitogen (PHA, 1 &mgr;g ml
−1
), in the presence or absence of different concentrations of monoclonal antibodies.
Crowe J. Scott
Sims Martin J.
Waldmann Herman
Cambridge University Technical Services Limited
Gambel Phillip
Hamilton Brook Smith & Reynolds P.C.
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