Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving antigen-antibody binding – specific binding protein...
Reexamination Certificate
1998-08-07
2001-01-16
Navarro, Albert (Department: 1645)
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving antigen-antibody binding, specific binding protein...
C424S001490, C424S130100, C424S137100, C424S141100, C424S152100
Reexamination Certificate
active
06174691
ABSTRACT:
The present invention relates to immunoreactive compounds; more specifically, it relates to genetically engineered antibodies.
Antibodies (Abs) are key molecules of the immune system. They provide defence against infection by microbial agents and are involved in a host of other immune reactions such as autoimmunity, allergies, inflammation, and graft rejection. Abs are unique in their specificity and are able to distinguish between very similar antigenic determinants of antigens. Because of this property, among others, antibodies are invaluable reagents for detecting, localizing and quantifying antigens.
Abs were initially obtained from immunized animals, but since different anti-sera represent different pools of heterogeneous Abs of varying specificities and isotypes, it was difficult to carry out reproducible studies using sera as the source of antibodies. It was then realized that large quantities of homogeneous Abs are produced in multiple myeloma, a tumour of plasma cells. Much of the information about the structure of immunoglobulins (Igs) was derived from studies using myeloma proteins. With the development of hybridoma technology, it became possible to generate an essentially endless supply of monoclonal antibodies (MAbs) of the desired specificities.
Antibodies are formed by polypeptide chains held together by non-covalent forces and disulfide bridges. A pair of identical light (L) chains (214 amino acids long) is linked to two identical heavy (H) chains to form a bilaterally symmetric structure (FIG.
1
).
The polypeptide chains are folded into globular domains separated by short stretches of peptide segments; the H chain has four or five domains, depending on the isotype, and the L chain has two. The N-terminal portion of each chain constitutes the variable region (V
H
, V
L
). A V
H
-V
L
pair carries the antigen combining site and contributes to antibody specificity. The rest of the chain forms the C region, the region of the molecule responsible for effector functions, such as Fc receptor binding, complement fixation, catabolism and placental transport. Igs with different C regions and therefore of differing isotypes (in human they are IgM, IgD, IgG1-4, IgA2, and IgE) exhibit different biological properties. In most isotypes a hinge region separates C
H
1 and C
H
2 and provides the molecule with segmental flexibility. The enzyme papain cleaves near the hinge to generate the F
ab
and F
c
portions of the antibody molecule.
Murine MAbs are invaluable in research, but human Igs would be preferable for many applications, such as diagnosis and immune therapy, as they may interact more effectively with the patient's immune system. Because of the species difference, mouse Abs administered to humans can induce an immune response resulting in allergy, serum sickness or immune complex disease. These effects preclude repeated administration of MAb. It has also been demonstrated in clinical trials that host Abs neutralize the injected mouse MAbs and account for their rapid clearance. While mouse and rat hybridomas are easy to produce, attempts to make human MAbs have met with limited success. Mouse-human hybridomas are frequently genetically unstable, and the production of human/human hybrids has been hampered by the lack of suitable immortalized human cell lines and immunized human B cells. Due to ethical considerations, in vivo immunization of humans is very restricted.
Gene transfection provides an alternative method of producing MAbs. With this method it is now possible to produce not only wild-type Ig chains, but also novel Igs and mutants that have been constructed in vitro. Antibodies of the desired specificities, binding affinities, isotypes and species origin can be obtained by transfecting the appropriate genes into mouse myeloma or hybridoma cells in culture. Gene transfections circumvent many of the problems inherent in the hybridoma methodology. Since human or chimeric antibodies with the human constant (C) regions can be produced, the problem of immunogenicity can be avoided or minimized. Another advantage over mouse-human or human-human hybridomas is that the transfected mouse cells can be injected intra-peritoneally into mice, where they will proliferate and produce ascites from which large quantities of antibodies can be isolated.
The first chimeric mouse-human antibody made use of the rearranged and expressed V region genes from the myeloma S107 specific for phosphocholine. The V
H
was joined to human C
&ggr;
1 or C
&ggr;
2, and V
L
was joined to human C&kgr; [1]. When expressed together in the same cell, the heavy and light chains assembled into H
2
L
2
tetramers that were secreted. This antibody bound antigen, and reaction with three monoclonal anti-idiotope antibodies verified that the polypeptide chains had folded appropriately to reproduce the antigen binding domains.
Experiments from the laboratory of Hozumi and his co-workers demonstrated that transfection of the rearranged murine TNP-specific &mgr; and &kgr; genes into plasmacytoma and hybridoma lines resulted in the production of pentameric IgM that bound hapten and triggered complement-dependent hemolysis [2, 3]. The TNP V
H
and V
L
genes were linked to human C
&mgr;
and C
&kgr;
segments, respectively, to produce chimera IgM that again exhibited the properties of the wild-type mouse antibody [3, 4].
Two mouse-human chimaeric IgEs were produced that could trigger degranulation of mast cells when cross-linked by antigen on the cell membrane [5, 6].
To explore the potential of chimaeric antibodies in cancer therapy, an antibody was made that consists of the V regions derived from the mouse MAb 17-1A, which recognizes a tumour-associated surface Ag, and human C
&ggr;
3 [7]. This chimaeric antibody had the same binding properties as the original mouse antibody.
Another chimaeric antibody with specificity for the surface antigen associated with certain human carcinomas was found to bind to human carcinoma cells [8].
Thus, murine variable regions were combined with human constant regions. A further step in the humanisation of rodent antibodies was the synthesis of hybrid variable regions in which the framework residues are of human origin and the complementarity-determining regions (CDRs) come from a mouse antibody. In the case of an NP-specific antibody, it was shown that the transfer of the CDRs from an anti-NP hybridoma onto the human framework of a human myeloma protein resulted in the transfer of antigen-specificity [9]. This result, obtained with a hapten antigen, was extended to antibodies directed against hen-egg lysozyme as well as a human T cell surface antigen [10, 11]. Thus, CDR grafting not only facilitates the construction of chimaeric antibodies but also allows the production of therapeutic reagents in which only the antibody CDR residues are of non-human origin.
CDR3 appears to be particularly important in determining the specificity of the antibody. Thus, Taub et al [52] showed that the specificity of an antibody to the platelet fibrinogen receptor was determined largely by the RYD sequence within the CDR3 of the antibody heavy chain. Williams et al [53] used a synthetic peptide from the light chain CDR2 of an antibody to inhibit the interaction of the antibody with its receptor. Conceptually, there is a “minimum recognition unit” of any given antibody, as discussed by Winter and Milstein [54].
We have now identified the minimum recognition unit of a murine monoclonal antibody specific for a mucin molecule associated with epithelial tumours, namely the amino acid sequence EPPT (Glu-Pro-Pro-Thr) (SEQ ID No 1).
One aspect of the present invention therefore provides a molecule comprising the amino acid sequence EPPT (SEQ ID No 1).
All peptides herein are written H
2
N . . . COOH and the amino acids are the naturally-occurring L isomers.
The molecule of the invention may consist of the sequence EPPT (SEQ ID No 1). This short peptide may or may not be useful in its own r
Cancer Therapeutics Limited
Navarro Albert
Reed Smith LLP
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