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...
Patent
1994-06-24
1998-11-17
Eisenschenk, Frank C.
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...
5303877, 530866, 530867, 435 697, 4353201, 4352523, 43525233, 435328, 536 2353, C07K 1646, C12N 1510, C07H 2104
Patent
active
058378219
DESCRIPTION:
BRIEF SUMMARY
This application is a National Stage Application of PCT/US92/09347, filed 04 Nov. 1992.
FIELD OF INVENTION
This invention relates to a novel antigen binding protein construct or "minibody" which includes the essential elements of an antibody.
BACKGROUND OF THE INVENTION
Previous workers (1).sup.1/ have demonstrated that single chain antigen-binding proteins can be generated by linking the heavy chain variable domain and light chain variable domain of an antibody into a single protein, using a short linker peptide. The length and composition of the linker can vary (2). Single chain antibodies can be produced in E. coli which retain antigen-binding activity. Milenic (3) provides one recent example of a single chain antibody that binds a tumor antigen. Several other single chain antibodies generated that bind to various haptens and antigens have been reported.
Gillies and Wesolowski (4) demonstrated that the CH2 domain of antibodies can be deleted with retention of the antigen binding function. The CH3 domains of such .DELTA.CH2 antibodies permit dimerization. .DELTA.CH2 antibodies are useful for in vivo diagnostics and potentially therapy (5).
To produce bispecific antibodies, Kostelny et al (6) fused Fab fragments of antibodies to the leucine zipper portions of fos and jun proteins in the absence of a single chain construct for the antigen combining region. Pack and Pluckthun (7), fused a single chain antibody to amphipathic helices from a four helix bundle or from leucine zipper proteins.
SUMMARY OF THE INVENTION
Minibodies are engineered antibody constructs comprised of the variable heavy (VH) and variable light (VL) chain domains of a native antibody fused to the hinge region and to the CH3 domain of the immunoglobulin molecule. Minibodies are thus small versions of whole antibodies encoded in a single protein chain which retain the antigen binding region, the CH3 domain to permit assembly into a bivalent molecule and the antibody hinge to accommodate dimerization by disulfide linkages. In contrast, native antibodies are comprised of four chains, two heavy and two light.
The size, valency and affinity of the minibody is particularly suited for in vivo targeting. Expression in bacterial or mammalian cells is simplified because minibodies are in single chains.
DESCRIPTION OF THE FIGURES
FIG. 1 depicts one form of a minibody in which VL indicates the variable light chain domain and VH indicates the variable heavy chain domain of a native T84.66 antibody. The hinge and CH3 domain are from human IgG.
FIG. 2 depicts a dimerized minibody.
FIG. 3 depicts the construction of a minibody gene (SEQ ID NO:1 and SEQ ID NO:2).
FIG. 4 depicts CEA binding ability of the expression of the product of a T84.66 minibody.
DETAILED DESCRIPTION OF THE INVENTION
A minibody is a chimeric molecule, typically illustrated by FIG. 1, constructed by fusion of a single chain antibody to the hinge region of an antibody followed directly by the CH3 domain of the immunoglobulin molecule. Two minibodies may dimerize through disulfide linkages as shown by FIG. 2.
The Single Chain Antibody
Single chain antibodies are prepared in the manner described by Bird (1), Pantoliano (2), Milenic (3), or Takkinen (8).
EXAMPLE I
A. The Single Chain T84.66 Antibody
This Example demonstrates the production of a single chain construct derived from the T84.66 antibody depicted by FIG. 3A (SEQ ID NO:1) in Neumaier, M., et al. Cancer Research 50:2128-2134 (1990) (10).
The T84.66 antibody domains were assembled in order VL-peptide linker-VH. The 14 amino acid peptide linker was identical to the "212 linker" described in Pantoliano et al. Biochemistry 30:10117 (1991) (2). The 14 amino acid linker is represented by SEQ ID NO:1 (GSTSGSGKSSEGKG).
The construction was accomplished by polymerase chain reaction/splice overlap extension (PCR/SOE). See Horton (9). The heavy and light chain gene segments from T84.66 were amplified separately using long overlapping primers that encoded the desired 14 amino acid linker. In addition, bacterial pelB
REFERENCES:
Colcher et al., Journal of the National Cancer Institute, 82:14, pp. 1191-1197 (1990).
Cumber et al., Journal of Immunology, 149:1, pp. 120-126 (1992).
Hu et al., Cancer Research, 56:13, pp. 3055-3061 (1996).
City of Hope
Eisenschenk Frank C.
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