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
1999-11-05
2003-03-25
Eyler, Yvonne (Department: 1646)
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, C530S388150, C435S325000, C435S326000, C435S328000, C435S331000, C435S334000
Reexamination Certificate
active
06538111
ABSTRACT:
FIELD OF THE INVENTION
The present invention relates to monoclonal antibodies and humanized antibodies which bind specifically to a human interleukin-5 receptor &agr; chain and which are therefore useful for diagnosis or treatment of diseases such as chronic bronchial asthma. The invention also relates to hybridomas and transformants which produce the antibodies, a method for detecting an interleukin-5 receptor &agr; chain immunologically by means of the monoclonal antibodies and humanized antibodies, as well as a method for diagnosing and treating diseases such as chronic bronchial asthma by means of the monoclonal antibodies and humanized antibodies.
BACKGROUND OF THE INVENTION
Interleukin-5 (hereinafter referred to a “IL-5”) is a kind of lymphokine which is secreted by T cells, mast cells and other cells. Murine IL-5 is known to act as a differentiation and growth factor for B cells and eosinophils. Human IL-5 is known to act mainly as a differentiation and growth factor for eosinophils (Advances in Immunology, 57, 145 (1994); Blood, 79, 3101 (1992)). IL-5 exhibits its action through a specific receptor (IL-5 receptor) which is expressed on the surface of a cell such as eosinophil. It has been shown that human and murine IL-5 receptors (hereinafter referred to as “IL-5Rs”) are both composed of two different kinds of proteins, an &agr; chain (hereinafter referred to as “IL-5R &agr;”) and a &bgr; chain (hereinafter referred to as “IL-5R &bgr;”). In addition, it is known that the binding of IL-5 to IL-5R is via IL-5R &agr; and that IL-5R &bgr; alone can not bind to IL-5 (EMBO J., 9, 4367 (1990); ibid., 10, 2833 (1991); J. Exp. Med., 177, 1523 (1993); ibid., 175, 341 (1992); Cell, 66, 1175 (1991), Proc. Natl. Acad. Sci., 89, 7041 (1992)). Furthermore, IL-5R &bgr; is known to be a component of receptors for interleukin-3 (hereinafter referred to as “IL-3”), granulocyte macrophage colony-stimulating factor and others (hereinafter referred to as “GM-CSF”) (Proc. Natl. Acad. Sci., 87.9655 (1990); Cell, 66, 1165 (1991)).
Eosinophils are known to increase in allergic diseases represented by chronic bronchial asthma. Significant infiltration of eosinophils is observed in airways of a patient with chronic bronchial asthma. Eosinophil contains a cytotoxic granular proteins whose deposit is observed in airway tissues of a patient with chronic bronchial asthma or at lesion sites of a patient with atopic dermatitis. These facts suggest that eosinophil plays an important role in the pathogenesis of allergic disorders such as chronic bronchial asthma, atopic dermatitis and the like (Adv. Immunol., 39, 177 (1986); Immunol. Today, 13, 501 (1992)). Hence, studying the kinetics of eosinophils is useful for clinical diagnosis. On the other hand, human IL-5 acts specifically on eosinophils, so IL-5R is believed to be expressed specifically in eosinophils and can therefore be used as a marker specific to human eosinophils. Furthermore, IL-5&bgr; is a receptor for cytokines such as IL-3, GM-CSF and others, so IL-5R &agr; is believed to be a marker specific to eosinophils. Hence, eosinophils can be detected specifically by immunocyte staining using an anti-human IL-5R &agr; chain antibody (hereinafter referred to as “anti-hIL-5R&agr; antibody”). However, no anti-hIL-5R &agr; antibody is presently known that is capable of specific detection of eosinophils.
Significant eosinophilia was observed in IL-5 transgenic mice (J. Exp. Med., 172, 1425 (1990); ibid. 173, 429 (1991); Int. Immunol., 2, 965 (1990)). Eosinophil infiltration in tissues was suppressed by the administration of an anti-IL-5 antibody in animal models of asthma (Am. Rev. Resir. 147, 548 (1993); ibid., 148, 1623 (1993)). These phenomena indicate that IL-5 actually plays an important role in eosinophilia and the infiltration of eosinophils in vivo. It is also reported that IL-5 is expressed in airway mucosal tissues of a human patient with chronic bronchial asthma and at lesion sites of a patient with atopic dermatitis (J. Clin. Invest., 87, 1541 (1991); J. Exp. Med., 173, 775 (1991)). Further investigations demonstrate that IL-5 exhibits in vitro viability-enhancing action on human eosinophils (J. Immunol., 143, 2311 (1989)) and that IL-5 is an eosinophil-selective activator (J. Exp. Med.,167, 219 (1988)).
Hence, antibodies that bind to IL-5R and which can inhibit the biological activity of IL-5 are expected to inhibit the activity of eosinophil, thus being useful in the treatment of allergic diseases such as chronic bronchial asthma. Anti-mouse IL-5R &agr; antibodies which can inhibit the biological activity of IL-5 were produced by using as an antigen those IL-5-dependent cells which express a large number of murine IL-5R on their surfaces (Kokai (Japanese published unexamined patent application) No. 108497/91; Int. Immunol., 2, 181 (1990)). However, in the case of humans, no cells are known which express a large number of IL-5R and the expression of IL-5R is reported to be very low in eosinophils (Cell. Immunol., 133, 484 (1991)). Hence, anti-human IL-5R &agr; antibodies having comparable functions to anti-mouse IL-5R &agr; antibodies are difficult to produce by methods similar to those for producing the latter. An antibody designated as “&agr;16” is disclosed as an antibody against human IL-5R &agr; in EMBO J., 14, 3395 (1995) but this antibody does not have any neutralization activity for IL-5R &agr;.
Human IL-5R &agr; gene was obtained by preparing a cDNA library from human eosinophil (J. Exp. Med., 175, 341 (1992)) or a human promyelocytic cell HL-60 (Cell, 66, 1175 (1991); Kokai No. 78772/94) and screening the library using as a probe an oligo DNA which had been synthesized on the basis of cDNA of murine IL-5R &agr; or a partial amino acid sequence of murine IL-5R &agr; (Kokai No. 54690/94, EMBO J., 9, 4367 (1990)). The transfer of the cDNA into a host cell resulted in the creation of a cell having hIL-5R &agr; expressed on its surface but the expression level of hIL-5R in this cell was very low (≦10
4
molecules) (J. Exp. Med., 177, 1523 (1993)). Hence, if one attempts to produce anti-hIL-5R &agr; antibodies by using this cell as an immunogen, he will find that the relative amount of hIL-5R &agr; is very small, compared with those of proteins from a host cell and that the absolute protein amount of hIL-5R &agr; is also very small. In addition, approximately 80% homology at an amino acid level is observed between murine IL-5R &agr; and human IL-5R &agr; and murine IL-5 can bind to human IL-5R with high affinity (J. Exp. Med.,175, 341 (1992)). These facts suggest that human IL-5R &agr; has a lower immunogenicity for mice or rats which are commonly used as animals to be immunized. In fact, almost all of our attempts to prepare anti-hIL-5R &agr; antibodies using hIL-5R &agr;-expressing cells as an immunogen resulted in a failure.
In the cloning of IL-5R cDNA from a cDNA library of human eosinophil, cDNA encoding soluble human IL-5R &agr; (hereinafter referred to as “shIL-5R &agr;”) has been obtained which corresponds to the N-terminal amino acid sequence (1-313) of IL-5R &agr; which is defective in the transmembrane region and onwards (J. Exp. Med.,175, 341 (1992)). When shIL-5R &agr; is used as an immunogen to produce an anti-hIL-5R &agr; antibody, the shIL-5R &agr; should have the same three dimensional conformation as that of IL-5R &agr; expressed on the cell surface and it should be one secreted and produced by a eukaryotic host cell in order to obtain an anti-hIL-5R &agr; antibody which can inhibit the biological activity of IL-5. In addition, it has been found that the production efficiency of a protein varies significantly depending on the signal peptide (Protein, Nucleic Acid and Enzyme, 35, 2584 (1990)), so it is necessary to select an appropriate signal peptide for secretion and production of the protein.
As mentioned above, it has been found that mRNA which is believed to encode only shIL-5R &agr; is expressed in eosinophils. It has been confirmed that murine IL-5R is expressed not only in eosinophils but also in B cell
Anazawa Hideharu
Furuya Akiko
Hanai Nobuo
Iida Akihiro
Koike Masamichi
Eyler Yvonne
Kyowa Hakko Kogyo Co., LTD
Pennie & Edmonds LLP
Prasad Sarada C
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