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
1995-12-18
2001-03-27
Scheiner, Laurie (Department: 1648)
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...
C530S387900, C530S388100
Reexamination Certificate
active
06207804
ABSTRACT:
BACKGROUND OF THE INVENTION
This invention relates to novel compositions of matter, hereinafter called biosynthetic antibody binding sites or BABS, useful, for example, in specific binding assays, affinity purification, biocatalysis, drug targeting, imaging, immunological treatment of various oncogenic and infectious diseases, and in other contexts. More particularly, this invention relates to biosynthetic polypeptides having a structure similar to native antibody binding sites, DNAs encoding the polypeptides prepared by recombinant DNA techniques, vectors comprising these DNAs, and methods for the design and production of these polypeptides.
Antibodies are proteins belonging to a group of immunoglobulins elicited by the immune system in response to a specific antigen or substance which the body deems foreign. Antibodies can both recognize and bind that antigen, and are involved in a number of effector reactions such as complement fixation and allergic responses.
There are five classes of human antibodies which have the ability to selectively recognize and preferentially bind a specific antigen. Each antibody class has the same basic structure (see FIG.
1
), or multiples thereof, consisting of two identical polypeptides called heavy or H chains (molecular weight in IgG approximately 50,000 d each) and two identical polypeptides called light or L chains (molecular weight approximately 25,000 d each). Each of the five antibody classes has a similar set of light chains and a distinct set of heavy chains. A light chain is composed of one variable and one constant domain, while a heavy chain is composed of one variable and three or more constant domains. The variable domains determine the specificity of the immunoglobulin, the constant regions have other functions.
Amino acid sequence data indicate that each variable domain comprises three hypervariable regions flanked by four relatively conserved framework regions (Kabat et. al.,
Sequences of Proteins of Immunological Interest
[U.S. Department of Health and Human Services, third edition 1983, fourth edition, 1987]). The hypervariable regions have been assumed to be responsible for the binding specificity of individual antibodies and to account for the diversity of binding of antibodies as a protein class.
Monoclonal antibodies, or homogeneous antibodies of identical genetic parentage and binding specificity, have been useful both as diagnostic and therapeutic agents. They are routinely produced according to established procedures by hybridomas generated by fusion of mouse lymphoid cells with an appropriate mouse myeloma cell line. Human monoclonal antibodies are difficult to produce by cell fusion techniques since, among other problems, human hybridomas are notably unstable, and removal of immunized spleen cells from humans is not feasible as it is for rodents. Monoclonals which have specificities of significant therapeutic value are generally of murine or rat origin, and are therefore immunogenic to the human immune system.
Chimeric antibodies composed of human and non-human amino acid sequences potentially have improved therapeutic value as they presumably would elicit less circulating human antibody against the non-human immunoglobulin sequences. Accordingly, hybrid antibody molecules have been proposed which consist of immunoglobulin light and heavy chain amino acid sequences from different mammalian sources. The chimeric antibodies designed thus far comprise variable regions from one mammalian source, and constant regions from human or another mammalian source (Morrison et al., 1984, Proc. Natl. Acad. Sci. U.S.A., 81:5851-6855; Neuberger et al., 1984, Nature 312:604-608; Sahagan et al., 1986, J. Immunol. 137:1066-1074; EPO application nos. 84302368.0, Genentech; 85102665.8, Research Development Corporation of Japan; 85305604.2, Stanford; P.C.T. application no. PCT/GB85/00392, Celltech Limited).
It has been reported that constant regions are not required for antigen recognition or binding; these properties have been localized to the variable domains of the antibody molecule located at the amino terminal end of both the heavy and light chains. The variable regions remain noncovalently associated (as V
H
V
L
dimers, termed Fv regions) even after proteolytic cleavage from the native antibody molecule, and retain much of their antigen recognition and binding capabilities (Inbar et al., Proc. Natl. Acad. Sci. U.S.A., 1972, 69:2659-2662; Hochman et. al., 1973, Biochem. 12:1130-1135 and 1976, Biochem. 15:2706-2710; Sharon and Givol, 1976, Biochem. 15:1591-1594; Rosenblatt and Haber, 1978, Biochem. 17:3877-3882; Ehrlich et al., 1980, Biochem. 19:4091-40996).
SUMMARY OF THE INVENTION
A class of novel biosynthetic polypeptides has now been designed and engineered which comprise biosynthetic antibody binding sites, that is, “BABS” or chimeric polypeptides defining stucture capable of selective antigen recognition and preferential antigen binding.
In its broadest aspects, this invention features polypeptides comprising biosynthetic antibody binding sites, DNA encoding these polypeptides prepared by recombinant DNA techniques, vectors comprising these DNAs, and methods for the production of these polypeptides.
In one aspect, the invention is based on the observation that three subregions of the variable domain of each of the heavy and light chains of native immunoglobulin molecules collectively are responsible for antigen recognition and binding. Each of these subregions, called herein “complementarity determining regions” or CDRs, consists of one of the hypervariable regions or loops and of selected amino acids or amino acid sequences disposed in the framework regions which flank that particular hypervariable region. It has now been discovered that framework regions from diverse species are effective to maintain CDRs from diverse other species in proper conformation so as to achieve true immunochemical binding properties in a biosynthetic protein. Thus, BABS produced in accordance with the invention comprise biosynthetically produced novel sequences of amino acids defining polypeptides designed to bind with a preselected antigenic material. The structure of these synthetic polypeptides is unlike that of naturally occurring antibodies, fragments thereof, or known synthetic polypeptides or “chimeric antibodies” in that the regions of the BABS responsible for specificity and affinity of binding, (analogous to native antibody variable regions) are themselves chimeric, e.g., comprise amino acid sequences homologous to portions of at least two different antibody molecules.
The invention thus provides a chimeric polypeptide defining a region capable of selective antigen binding and recognition. This chimeric polypeptide comprises amino acid sequences homologous to portions of the CDRs of the variable domain of one immunoglobulin light or heavy chain, and other sequences homologous to the framework regions, or FRs, of the variable domain of a second, different immunoglobulin light or heavy chain. Polypeptides so constructed bind a specific preselected antigen determined by the CDRs. Preferably, the chimeric polypeptides comprise an amino acid sequence homologous to at least a portion of the variable regions of a mammalian immunoglobulin, such as those of mouse, rat, or human origin. In one preferred embodiment, the biosynthetic antibody binding site comprises FRs homologous with a portion of the FRs of a human immunoglobulin and CDRs homologous with CDRs from a mouse immunoglobulin. This type of chimeric polypeptide displays the antigen binding specificity of the mouse immunoglobulin, while its human framework minimizes human immune reactions. In addition, the chimeric polypeptide may comprise other amino acid sequences. It may comprise, for example, a sequence homologous to a portion of the constant domain of an immunoglobulin, but preferably is free of constant regions (other than FRs).
The invention also provides a single chain composite polypeptide having antigen binding abilities, and comprising a pair of amino acid sequences
Huston James S.
Oppermann Hermann
Curis, Inc.
Elrifi Ivor R.
Mintz Levin Cohn Ferris Glovsky & Popeo P.C.
Morency Michel
Parkin Jeffrey S.
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