Drug – bio-affecting and body treating compositions – Immunoglobulin – antiserum – antibody – or antibody fragment,... – Monoclonal antibody or fragment thereof
Reexamination Certificate
1997-08-25
2002-01-29
Nolan, Patrick J. (Department: 1644)
Drug, bio-affecting and body treating compositions
Immunoglobulin, antiserum, antibody, or antibody fragment,...
Monoclonal antibody or fragment thereof
C424S133100, C424S135100, C530S388700, C530S387100
Reexamination Certificate
active
06342220
ABSTRACT:
FIELD OF THE INVENTION
This invention relates to the recombinant synthesis and purification of protein antibodies that influence survival, proliferation, differentiation or maturation of hematopoietic cells, especially platelet progenitor cells and to antibodies that influence the growth and differentiation of cells expressing a protein kinase receptor. This invention also relates to the cloning and expression of nucleic acids encoding antibody ligands (thrombopoietin receptor agonist antibodies) capable of binding to and activating a thrombopoietin receptor such as c-mpl, a member of the cytokine receptor superfamily. This invention further relates to the use of these antibodies alone or in combination with other cytokines to treat immune or hematopoietic disorders including thrombocytopenia and to uses in assays.
BACKGROUND OF THE INVENTION
In 1994 several groups reported the isolation and cloning of thrombopoietin (F. de Sauvage et al.,
Nature
369:533 (1994); S. Lok et al.,
Nature
369:565 (1994); T D. Bartley et al.,
Cell
77:1117 (1994); Y. Sohma et al.,
FEBS Letters
353:57 (1994); D J. Kuteret al.,
Proc. Natl. Acad. Sci.
91:11104 (1994)). This was the culmination of more than 30 years of research initiated in the late 50's when Yamamoto (S. Yamamoto,
Acta Haematol Jpn.
20:163-178. (1957)) and Kelemen (E. Kelemen et al.,
Acta Haematol
(Basel). 20:350-355 (1958)) proposed that physiological platelet production is controlled by a humoral factor termed “thrombopoietin” (TPO). Although routinely detected in urine, plasma and serum from thrombocytopenic animals and patients, as well as kidney cell conditioned media, purification of TPO proved to be a daunting task (for a review see MS. Gordon et al.,
Blood
80:302 (1992); W. Vainchenker et al.,
Critical Rev. Oncology/Hematology
20:165 (1995)). In the absence of purified TPO and the apparent fact that numerous plieotrophic cytokines affected megakaryocytopoiesis (M S. Gordon et al.,
Blood
80:302 (1992); W. Vainchenker et al.,
Critical Rev. Oncology/Hematology
20:165 (1995)), the existence of a lineage specific factor that regulated platelet production was doubted until the discovery of the orphan cytokine receptor c-Mpl in 1990 (M. Souyri et al.,
Cell
63:1137 (1990); I. Vigon et al.,
Proc. Natl Acad. Sci.
89:5640 (1992)). The expression of c-Mpl was found to be restricted to progenitor cells, megakaryocytes and platelets, and c-Mpl antisense oligonucleotides selectively inhibited in vitro megakaryocytopoiesis (M. Methia et al.,
Blood
82:1395 (1993)). From this it was postulated that c-Mpl played a critical role in regulating megakaryocytopoiesis and that its putative ligand may be the long sought TPO (M. Methia et al., supra). Following this discovery several groups utilizing c-Mpl ligand specific cell proliferation assays and c-Mpl as a purification tool isolated and cloned the ligand for c-Mpl (F. de Sauvage et al., supra; S. Lok et al., supra; T D. Bartley et al., supra). In addition two other groups independently reported the purification of the Mpl-ligand using standard chromatography techniques and megakaryocyte assays (Y. Sohma et al., supra; D J. Kuteret al., supra). In the years since its reported discovery numerous studies clearly indicate that the Mpl-ligand possess all the characteristics that have long been attributed to the purported regulator of megakaryocytopoiesis and thrombopoiesis and consequently, is now referred to as TPO. The Mpl ligand is currently referred to as either TPO or as megakaryocyte growth and differentiation factor (MGDF).
Human TPO consists of 332 amino acids that can be divided into 2 domains; an amino terminal domain of 153 amino acids showing 23% identity (50% similarity) to erythropoietin (EPO) and a unique 181 amino acid C-terminal domain that is highly glycosylated ((F. de Sauvage et al., supra; S. Lok et al., supra; T D. Bartley et al., supra). The EPO-like domain of TPO contains 4 cysteines, 3 of which are conserved with EPO. The first and last and the two middle cysteines form two disulfide bridges, respectively, which are both required for activity (T. Kato et al.,
Blood
86 (suppl 1):365 (1995)). None of the Asn-linked glycosylation sites present in EPO are conserved in the EPO-like domain of TPO, however, the EPO-like domain of recombinant TPO (rTPO) contains 2-3O-linked glycosylations (M. Eng et al.,
Protein Science
5(suppl 1): 105 (1996)). A recombinant truncated form of TPO (rTPO153), consisting of only the EPO-like domain, is fully functional in vitro, indicating that this domain contains all the required structural elements to bind and activate Mpl (F. de Sauvage et al., supra; D L. Eaton et al.,
Blood
84(suppl 1):241 (1994)). The carboxy terminal domain of TPO contains 6 N-linked and 18 O-linked glycosylate sites and is rich in proline, serine and threonine (M. Eng et al., supra). The function of this domain remains to be elucidated. However, because of its high degree of glycosylation this region may act to stabilize and increase the half life of circulating TPO. This is supported by the observation that rTPO153 has a half life of 1.5 hours compared to 18-24 hours for full length glycosylated rTPO (GR. Thomas et al.,
Stem Cells
14(suppl 1) (1996).
The two domains of TPO are separated by a potential dibasic proteolytic cleavage site that is conserved among the various species examined. Processing at this site could be responsible for releasing the C-terminal region from the EPO domain in vivo. The physiological relevance of this potential cleavage site is unclear at this time. Whether TPO circulates as an intact full length molecule or as a truncated form is also equivocal. When aplastic porcine plasma was subjected to gel filtration chromatography, TPO activity present in this plasma resolved with a Mr. of ~150,000 ((F. de Sauvage et al., supra). Purified full length rTPO also resolves at this Mr., whereas the truncated forms resolve with Mr. ranging from 18,000-30,000. Using TPO ELISAs that selectively detect either full length or truncated TPO it has also been shown that full length TPO is the predominant form in the plasma of marrow transplant patients (Y G. Meng et al.,
Blood
86(suppl. 1):313 (1995)).
Prior to the discovery of c-Mpl and the isolation of TPO, it was thought that megakaryocytopoiesis was regulated at multiple cellular levels (M S. Gordon et al., supra; W. Vainchenker et al., supra; Y G. Meng et al., supra). This hypothesis was based on the observation that certain hematopoietic growth factors stimulated proliferation of megakaryocyte progenitors while others primarily affected maturation (M S. Gordon et al., supra; W. Vainchenker et al., supra; Y G. Meng et al., supra). Other data indicated that plasma from thrombocytopenic animals contained distinct activities that either affected proliferation (meg-CSF) or maturation (TPO) of megakaryocytes (R J. Hill et al.,
Exp.Hematol
20:354 (1992)). Wendling and her colleagues (F. Wendling et al.,
Nature
369:571 (1994)) initially dispelled this theory by demonstrating that all the megakaryocyte colony-stimulating and thrombopoietic activities in thrombocytopenic plasma could be neutralized by soluble Mpl. This indicated that these activities are due to a single factor, the Mpl-ligand. Numerous studies have now shown that recombinant forms of TPO not only induce proliferation of progenitor megakaryocytes but also their maturation (K. Kaushansky et al.,
Nature
369:568 (1994); F C. Zeigler et al.,
Blood
84:4045 (1994); V C. Broudy et al.,
Blood
85:1719 (1995); J L. Nichol et al.,
J. Clin. Invest.
95:2973 (1995); N. Banu et al.,
Blood
86:1331 (1995); N. Debili et al.,
Blood
86:2516 (1995); P. Angchaisuksiri et al.,
Br. J. Haematol.
93:13 (1996); E S. Choi et al.,
Blood
85:402 (1995)). Human CD34+, CD34+CD41+cells (F C. Zeigler et al., supra; V C. Broudy et al., supra; J L. Nichol et al., supra; N. Banu et al., supra;) or purified murine stem cells (sca+,lin−, kit+) (K. Kaushansky et al., supra; F C. Zeigler et al., supra) cultured
Adams Camellia W.
Carter Paul J.
Fendly Brian M.
Gurney Austin L.
Genentech Inc.
Kelber Steven B.
Nolan Patrick J.
Piper Marbury Rudnick & Wolfe LLP
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