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
1999-04-29
2002-09-24
Huff, Sheela (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, C530S391300
Reexamination Certificate
active
06455677
ABSTRACT:
BACKGROUND OF THE INVENTION
1. Field of the Invention
The present invention relates to antibody proteins that specifically bind fibroblast activation protein alpha (FAP&agr;). The invention also relates to the use of said antibodies for diagnostic and therapeutic purposes and methods of producing said antibodies.
2. Related Art
The invasive growth of epithelial cancers is associated with a number of characteristic cellular and molecular changes in the supporting stroma. A highly consistent molecular trait of the reactive stroma of many types of epithelial cancer is induction of the fibroblast activation protein alpha (from now on referred to as FAP), a cell surface molecule of reactive stromal fibroblasts originally identified with monoclonal antibody F 19 (Garin-Chesa, P., et al., “Cell surface glycoprotein of reactive stromal fibroblasts as a potential antibody target in human epithelial cancers,” Proc.
Natl. Acad. Sci
. 87:7235 (1990)). Since the FAP antigen is selectively expressed in the stroma of a range of epithelial carcinomas, independent of location and histological type, a FAP-targeting concept has been developed for imaging, diagnosis and treatment of epithelial cancers and certain other conditions. For this purpose a monoclonal antibody termed F19 that specifically binds to FAP was developed and described in U.S. Pat. No. 5,059,523 and WO 93/05804, which are hereby incorporated by reference in their entirety.
One serious problem that arises when using non-human antibodies for applications in vivo in humans is that they quickly raise a human anti-non-human response that reduces the efficacy of the antibody in patients and impairs continued administration. Humanization of non-human antibodies is commonly achieved in one of two ways: (1) by constructing non-human/human chimeric antibodies, wherein the non-human variable regions are joined to human constant regions (Boulianne, G. L., et al., “Production of functional chimeric mouse/human antibody,”
Nature
312:643 (1984)) or (2) by grafting the complementarity determining regions (CDRs) from the non-human variable regions to human variable regions and then joining these “reshaped human” variable regions to human constant regions (Riechmann L., et al., “Reshaping human antibodies for therapy,”
Nature
332:323 (1988)). Chimeric antibodies, although significantly better than mouse antibodies, can still elicit an anti-mouse response in humans (LoBuglio, A. F., et al., “Mouse/human chimeric monoclonal antibody in man: Kinetics and immune response,”
Proc. Natl. Acad. Sci
. 86:4220 (1989)). CDR-grafted or reshaped human antibodies contain little or no protein sequences that can be identified as being derived from mouse antibodies. Although an antibody humanized by CDR-grafting may still be able to elicit some immune reactions, such as an anti-allotype or an anti-idiotypic response, as seen even with natural human antibodies, the CDR-grafted antibody will be significantly less immunogenic than a mouse antibody thus enabling a more prolonged treatment of patients.
Another serious limitation relating to the commercial use of antibodies for diagnosis, imaging and therapy is their producibility in large amounts. In many instances recombinant expression of native, chimeric and/or CDR-grafted antibodies in cell culture systems is poor. Factors contributing to poor producibility may include the choice of leader sequences and the choice of host cells for production as well as improper folding and reduced secretion. Improper folding can lead to poor assembly of heavy and light chains or a transport incompetent conformation that forbids secretion of one or both chains. It is generally accepted that the L-chain confers the ability of secretion of the assembled protein. In some instances multiple or even single substitutions can result in the increased producibility of antibodies.
Because of the clinical importance of specific immunological targeting in vitro and in vivo of specific disease-related antigens for diagnosis and therapy in humans, there is a growing need for antibodies that combine the features of antigen specificity, low immunogenicity and high producibility.
Therefore, the problem underlying the present invention was to provide antibody proteins that combine the properties of specific binding to FAP, low immunogenicity in humans, and high producibility in recombinant systems.
SUMMARY OF THE INVENTION
The technical problem is solved by the embodiments characterized in the claims.
The present invention provides new antibody proteins having the complementary determining regions of the monoclonal antibody F19 (ATCC Accession No. HB 8269), said new antibody proteins specifically binding to fibroblast activation protein (FAP), characterized in that they have framework modifications resulting in the improved producibility in host cells as compared to a chimeric antibody having the variable regions of F19 and foreign constant regions.
As used herein, an “antibody protein” is a protein with the antigen binding specificity of a monoclonal antibody.
“Complementarity determining regions of a monoclonal antibody” are understood to be those amino acid sequences involved in specific antigen binding according to Kabat (Kabat, E. A., et al.,
Sequences of Proteins of Immunological Interest
, 5th Ed., NIH Publication No. 91-3242. U.S. Department of Health and Human Services, Public Health Service, National Institutes of Health, Bethesda, Md. (1991)) in connection with Chothia and Lesk (Chothia and Lesk,
J. Mol. Biol
., 196:901-917 (1987)).
As used herein, the term “framework modifications” refers to the exchange, deletion or addition of single or multiple amino acids in the variable regions surrounding the individual complementarity determining regions. Framework modifications may have an impact on the immunogenicity, producibility or binding specificity of an antibody protein. “Fibroblast activation protein (FAP)”, also designated fibroblast activation protein alpha (FAP&agr;), is a membrane-bound glycoprotein belonging to the serine protease gene family (WO 97/34927). No shed or secreted form of FAP is known. FAP can be characterized by its binding to the monoclonal antibody F19 (F19 is obtainable from the hybridoma cell line with the accession No. HB 8269 deposited at the ATCC).
The term “fibroblast activation protein specific binding” of an antibody protein is defined herein by its ability to specifically recognize and bind FAP-expressing human cells. The binding specificity of the proteins of the invention can be determined by standard methods for the evaluation of binding specificity such as described in an exemplary fashion in examples 6, 8 and 12.
The term “chimeric antibody” refers to an antibody protein having the light and heavy chain variable regions as described in
FIGS. 17 and 18
and foreign constant regions. “Foreign constant regions” as defined herein are constant regions which are different from the constant regions of F19. For comparing an antibody protein of the invention to a chimeric antibody it is to be understood that such a chimeric antibody must contain the same constant regions as said antibody protein. For the purpose of demonstration and comparison alone the human constant heavy and light chains as described in
FIGS. 19
to
22
are used in an exemplary fashion.
To provide the antibody proteins of the present invention, the nucleic acid sequences of the heavy and light chain genes of the murine antibody designated F19 were determined from RNA extracted from F19 hybridoma cells (ATCC Accession No. HB 8269).
In one embodiment the present invention relates to antibody proteins having the complementary determining regions of the monoclonal antibody F19 (ATCC Accession No. HB 8269), said new antibody proteins specifically binding to fibroblast activation protein (FAP), characterized in that they have framework modifications resulting in the improved producibility in host cells as compared to a chimeric antibody having the variable regions of F19 and foreign constant regions, wherein said antibody protein is d
Bamberger Uwe
Garin-Chesa Pilar
Leger Olivier
Park John Edward
Rettig Wolfgang J.
Boehringer Ingelheim International GmbH
Helms Larry R.
Huff Sheela
Sterne, Kessler, Goldstei & Fox P.L.L.C.
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