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
1995-05-25
2003-01-14
Caputa, Anthony C. (Department: 1642)
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, C530S387700, C530S388800, C530S388850
Reexamination Certificate
active
06506881
ABSTRACT:
FIELD OF THE INVENTION
The present invention relates to humanised antibody molecules (HAMs) having specificity for human milk fat globule (HMFG) and to processes for their production using recombinant DNA technology.
BACKGROUND TO THE INVENTION
The term “humanised antibody molecule” (HAM) is used to describe a molecule having an antigen binding site derived from an immunoglobulin from a non-human species, the remaining immunoglobulin-derived parts of the molecule being derived from a human immunoglobulin. The antigen binding site may comprise: either a complete variable domain from the non-human immunoglobulin fused onto one or more human constant domains; or one or more of the complementarity determining regions (CDRs) grafted onto appropriate human framework regions in the variable domain. The abbreviation “MAb” is used to indicate a monoclonal antibody.
In the description, reference is made to publications by number. These numbers are placed in square brackets [ ]. The publications are listed in numerical order at the end of the description.
Natural immunoglobulins have been known for many years, as have the various fragments thereof, such as the Fab, Fab′ (Fab′)
2
and Fc fragments, which can be derived by enzymatic cleavage. Natural immunoglobulins comprise a generally Y-shaped molecule having an antigen-binding site towards the outer end of each arm. The remainder of the structure, and particularly the stem of the Y, mediates the effector functions associated with immunoglobulins.
Natural immunoglobulins have been used in assay, diagnosis and, to a more limited extent, therapy. However, such uses, especially in therapy, have been hindered by the polyclonal nature of natural immunoglobulins. A significant step towards the realisation of the potential of immunoglobulins as therapeutic agents was the discovery of procedures for the production of monoclonal antibodies of defined specificity [1]. However, most MAbs are produced by hybridomas which are fusions of rodent spleen cells with rodent myeloma cells. The resultant MAbs are therefore essentially rodent proteins. There are few reports of the production of human MAbs.
Since most available MAbs are of rodent origin, they are naturally antigenic in humans and thus can give rise to an undesirable immune response termed the HAMA (Human Anti-Mouse Antibody) response. Therefore, the use of rodent MAbs as therapeutic agents in humans is inherently limited by the fact that the human subject will mount an immunological response to the MAb and will either remove it entirely or at least reduce its effectiveness.
Therefore proposals have been made for making non-human MAbs less antigenic in humans. Such techniques can be generically termed “humanisation” techniques. These techniques generally involve the use of recombinant DNA technology to manipulate DNA sequences encoding the polypeptide chains of the antibody molecule.
Early methods for humanising MAbs related to production of chimeric antibodies in which an antigen binding site comprising the complete variable domains of one antibody are fused to constant domains derived from a second antibody. Methods for carrying out such chimerisation procedures are described in EP-A-0 120 694 (Celltech Limited), EP-A-0 125 023 (Genentech Inc.), EP-A-0 171 496 (Res. Dev. Corp. Japan), EP-A-0173494 (Stanford University) and EP-A-0 194 276 (Celltech Limited).
EP-A-0 194 276 discloses a process for preparing an antibody molecule having the variable domains from a mouse MAb and the constant domains from a human immunoglobulin. It also describes the production of an antibody molecule comprising the variable domains of a mouse MAb, the CH1 and CL domains of a human immunoglobulin and a non-immunoglobulin-derived protein in place of the Fc portion of the human immunoglobulin.
Subsequently, a number of further patent applications have been published relating to chimeric antibodies, including tumour specific chimeric antibodies. Among these applications are WO-A-87/02671 (Int. Gen. Eng. Inc.), EP-A-0 256 654 (Centocor), EP-A-0 266 663 (Int. Gen. Eng. Inc. & Oncogen), WO-A-89/00999 (Int. Gen. Eng. Inc.) and EP-A-0 332 424 (Hybritech Inc.).
Such humanised chimeric antibodies, however, still contain a significant proportion of non-human amino acid sequence, i.e. the complete variable domains. Thus, such humanised antibodies may elicit some HAMA response, particularly if administered over a prolonged period [2].
In an alternative approach, described in EP-A-0 239 400 (Winter), the complementarity determining regions (CDRs) of a mouse MAb have been grafted onto the framework regions of the variable domains of a human immunoglobulin by site directed mutagenesis using long oligonucleotides. Such CDR-grafted humanised antibodies are less likely to give rise to a HAMA response than humanised chimeric antibodies in view of the lower proportion of non-human amino acid sequence which they contain. There are three CDRs (CDR1, CDR2 and CDR3) in each of the heavy and light chain variable domains.
The earliest work on CDR-grafted humanised MAbs was carried out on a MAb recognising the synthetic antigen NP or NIP. However, subsequently, examples in which a mouse MAb recognising lysozyme and a rat MAb recognising an antigen on human T cells respectively were humanised have been described [3, 4]. The preparation of the CDR-grafted antibody to the antigen on human T cells is also described in WO-A-89/07452 (Medical Research Council). Recently the preparation of a humanised CDR-grafted antibody that binds to the interleukin 2 receptor has been described [5]. Further examples of humanised CDR-grated antibodies having specificity for anti-viral [6, 7], anti-tumour [8] and anti-T cell [9 and EP-A-0 403 156] antigens have been described more recently.
Our copending International Patent Specification No. WO-A-91/09967 relates to the CDR grafting of antibodies in general.
It has been widely suggested that immunoglobulins, and in particular MAbs, could potentially be very useful in the diagnosis and treatment of cancer [10, 11]. There has therefore been much activity in trying to produce immunoglobulins or MAbs directed against tumour-specific antigens. So far, over one hundred MAbs directed against a variety of human carcinomas have been used in various aspects of tumour diagnosis or treatment [12].
There have been a number of papers published concerning the production of chimeric monoclonal antibodies recognising cell surface antigens. For instance, genetically engineered murine/human chimeric antibodies which retain specificity for tumour-associated antigens have been described [13 and WO-A-89/01783]. Also, a recombinant murine/human chimeric monoclonal antibody specific for common acute lymphocytic leukaemia antigen has been described [14].
SUMMARY OF THE INVENTION
We have now prepared humanised antibodies to human milk fat globule (HMFG) derived from the anti-HMFG mouse MAb CTMO1 [15].
According to the present invention, there is provided a humanised antibody molecule (HAM) having specificity for human milk fat globule (HMFG) and having an antigen binding site wherein at least one of the complementarity determining regions (CDRs) of the variable domain is derived from the mouse monoclonal antibody CTMO1 (CTMO1 MAb) and the remaining immunoglobulin-derived parts of the HAM are derived from a human immunoglobulin or an analogue thereof.
The HAM may comprise a chimeric humanised antibody or a CDR-grafted humanised antibody. When the HAM comprises a CDR-grafted humanised antibody, each heavy or light chain variable domain may comprise only one or two CTMO1-derived CDRs. Preferably, however, all three heavy and light chain CDRs are derived from CTMO1.
The CTMO1 MAb is a mouse MAb of the type IgG1-kappa raised against the membrane-associated antigen of HMFG and has been extensively studied [15]. The CTMO1 MAb has been shown to recognise breast, ovarian and non-small cell lung can
Adair John Robert
Baker Terence Seward
Lyons Alan Howard
Owens Raymond John
Caputa Anthony C.
Celltech R&D Limited
Holleran Anne L.
Woodcock & Washburn LLP
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