United States Patent (19)
`Nagata et al.
`
`III IIHIIII
`
`USOO5574136A
`Patent Number:
`11
`(45) Date of Patent:
`
`5,574,136
`Nov. 12, 1996
`
`54) DNA ENCODING GRANULOCYTE
`COLONY-STMULATING FACTOR
`RECEPTOR AND PROTEIN THEREOF
`
`75 Inventors: Shigekazu Nagata, Suita; Rikiro
`Fukunaga, Minoo, both of Japan
`(73) Assignee: Osaka Bioscience Institute, Japan
`21) Appl. No.:
`923,976
`22) PCT Filed:
`Mar. 22, 1991
`86 PCT No.:
`PCT/JP91/00375
`S371 Date:
`Sep. 22, 1992
`S 102(e) Date: Sep. 22, 1992
`(87) PCT Pub. No.: WO91/14776
`PCT Pub. Date: Oct. 3, 1991
`Foreign Application Priority Data
`(30)
`Mar. 23, 1990 (JP)
`Japan ...................................... 2-74539
`Jul. 3, 1990 (JP)
`Japan .................................... 2-176629
`(51) Int. Cl." ........................ C12N 15/12; C07K 14/705;
`CO7K 141715
`52 U.S. C. ........................ 530/350; 536/23.1; 536/23.5;
`435/69.1; 530/351
`58 Field of Search ................................ 435/69.1, 240.2,
`435/252.3, 320.1; 530/350, 351; 536/23.5,
`23.52
`
`56)
`
`References Cited
`U.S. PATENT DOCUMENTS
`4,675,285 6/1987 Clark et al. .......................... 435/1723
`
`5,422,248 6/1995 Smith et al. ........................... 435/69.
`FOREIGN PATENT DOCUMENTS
`WO91/05046 4/1991 European Pat. Off..
`OTHER PUBLICATIONS
`Yamasaki et al, Science, 241, 1988, pp. 825-828.
`Sims et al, Science, 241, 1988, pp. 585-589.
`Uzumaki et al., PNAS, 86, 1989, pp. 9323–9326.
`Cosman et al., 1990, vol 2(1) pp. 1-31.
`Larsen, A. et al., J. Exp. Med., The Rockefeller Univ. Press.
`vol. 172 "Expression cloning of a human granulocyte colo
`ny-stimulating factor receptor: a structural mosaic of
`hematopoietin receptor, immunoglobulin, and fibronectin
`domains", pp. 1559-1570 (Dec. 1990).
`Park, L. S. et al., Blood, vol. 74, No. 1, "Interleukin-3,
`GM-CSF, and G-CSF, receptor expression on cell lines and
`primary leukemia cells: receptor heterogeneity and relation
`ship to growth factor responsiveness' pp. 56-65, (Jul. 1989).
`Primary Examiner-Marianne P. Allen
`(57)
`ABSTRACT
`A murine G-CSF receptor cDNA was cloned from a cDNA
`library prepared from mouse myeloid leukemia cells and the
`structure analyzed. A human G-CSF receptor cDNA was
`then cloned from cDNA libraries constructed from human
`placenta cells or human histocytic lymphoma cells using the
`murine G-CSF receptor cDNA as a probe, the structure
`analyzed and expressed in a host cell transformed with it.
`The stable production of human G-CSF receptor can be
`accomplished by transforming a host cell with the cloned
`G-CSF receptor cDNA and making the transformant express
`the G-CSF receptor.
`
`5 Claims, 21 Drawing Sheets
`
`Bracco Ex. 2009
`Maia v. Bracco
`IPR2019-00345
`
`1
`
`

`

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`Noy. 12, 1996
`
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`

`

`U.S. Patent
`
`Noy. 12, 1996
`
`Sheet 2 of 21
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`5,574,136
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`Nov. 12, 1996
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`U.S. Patent
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`U.S. Patent
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`Nov. 12, 1996
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`Sheet 5 of 21
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`U.S. Patent
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`Nov. 12, 1996
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`Sheet 6 of 21
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`U.S. Patent
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`Nov. 12, 1996
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`Sheet 7 of 21
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`U.S. Patent
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`Nov. 12, 1996
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`Sheet 8 of 21
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`5,574,136
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`U.S. Patent
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`Noy. 12, 1996
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`Sheet 9 of 21
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`U.S. Patent
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`Nov. 12, 1996
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`U.S. Patent
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`Nov. 12, 1996
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`Sheet 11 of 21
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`U.S. Patent
`U.S. Patent
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`Nov. 12, 1996
`Nov. 12, 1996
`
`Sheet 12 of 21
`Sheet 12 of 21
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`5,574,136
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`U.S. Patent
`U.S. Patent
`
`Nov. 12, 1996
`Nov. 12, 1996
`
`Sheet 13 of 21
`Sheet 13 of 21
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`5,574,136
`5,574,136
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`U.S. Patent
`U.S. Patent
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`Nov. 12, 1996
`Nov. 12, 1996
`
`Sheet 14 of 21
`Sheet 14 of 21
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`5,574,136
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`Nov. 12, 1996
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`Sheet 15 of 21
`Sheet 15 of 21
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`5,574,136
`5,574,136
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`Nov. 12, 1996
`Nov. 12, 1996
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`Nov. 12, 1996
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`Nov. 12, 1996
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`US. Patent
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`Nov. 12, 1996
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`U.S. Patent
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`Nov. 12, 1996
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`Sheet 21 of 21
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`5,574,136
`
`1.
`DNA ENCOOING GRANULOCYTE
`COLONY-STMULATING FACTOR
`RECEPTOR AND PROTEIN THEREOF
`
`FIELD OF THE INVENTION
`This invention relates to an isolated DNA encoding granu
`locyte colony-stimulating factor receptor. More particularly,
`it relates to an isolated DNA encoding a receptor peptide
`capable of specifically binding to a colony-stimulating factor
`(hereinafter, referred to as G-CSF), an expression vector
`containing said DNA, a transformant transformed by said
`vector, and a process for the production of said receptor by
`culturing said transformant. The present invention also
`relates to a recombinant G-CSF receptor prepared according
`to the present process.
`
`10
`
`5
`
`2
`Leukemia, 1, 1-8 (1987); and Park et al., Blood 74, 56-65
`(1989)). Human G-CSF is a 174 amino acid polypeptide
`while murine G-CSF consists of 178 amino acids. Human
`and mouse G-CSFs are highly homologous (72.6%) at the
`amino acid sequence level, in agreement with the lack of
`species-specificity between them (Nicola et al, Nature 314,
`626-628 (1985)). What makes the research in G-CSF more
`interesting is that G-CSF receptor has also recently been
`found in non hemopoietic cells such as human endothelial
`cells (Bussolino et al., Nature 337, 471-473 (1989)) and
`placenta (Uzumaki et al., Proc. Natl. Acad. Sci. USA, 86,
`9323-9326 (1989)).
`As can be seen from the above, the elucidation of the
`interaction between G-CSF and its receptor should greatly
`contribute to the development of the treatment or prophy
`laxis of various diseases including hematopoietic disorders
`using G-CSF, whereby providing more effective and proper
`treatments on such diseases. Thus, such elucidation is impor
`tant not only academically but also clinically. On the other
`hand, the receptor itself can be useful. For instance, a soluble
`form of the G-CSF receptor may be useful clinically to
`inhibit the proliferation of some G-CSF-dependent human
`myeloid leukemia cells (Santoli et al., J.Immunol. 139,
`3348-3354 (1987)). The investigation into the expression of
`G-CSF receptor in tumor cells such as myeloid leukemia
`may be beneficial to establish an effective clinical applica
`tion of G-CSF. Accordingly, owing to the various academic
`and practical usefulness, a stable supply of a G-CSF recep
`tor-encoding gene and the G-CSF receptor has been
`demanded.
`Recently, the technology of genetic engineering has been
`used for the production of various physiologically active
`Substances. The production by the genetic engineering is
`generally carried out by cloning DNA encoding desired
`polypeptide, inserting said DNA into a suitable expression
`vector, transforming an appropriate host cell such as micro
`organism or animal cell by the expression vector, and
`making the transformant express the desired polypeptide.
`To apply the genetic engineering technique to the pro
`duction of G-CSF receptor, cloning of DNA encoding
`G-CSF receptor is firstly required. However, cloning cDNA
`encoding G-CSF receptor was hampered by the low number
`of receptors present on the cell surface (at most hundreds to
`2,000 receptor per cell).
`BRIEF DESCRIPTION OF THE DRAWINGS
`FIG. 1 (A-C) depicts the nucleotide sequence and deduced
`amino acid sequence of murine G-CSF receptor (SEQ ID
`NOS:1-2).
`FIG. 2(A-C) depicts a schematic representation and
`restriction map of murine G-CSF receptor cDNAs (p162,
`p17 and pFl), and hydropathy plots thereof.
`FIG. 3a depicts the saturation binding of murine 'I-G-
`CSF to COS cells.
`FIG. 3b depicts scatchard plot of binding data of murine
`'I-G-CSF to COS cells.
`FIG, 3c depicts saturation binding of murine 'I-G-CSF
`to NFS-60 cells.
`FIG. 3d depicts scatchard plot of binding data of murine
`'I-G-CSF to NFS-60 cells.
`FIG. 4 depicts specific binding of murine G-CSF to
`recombinant murine G-CSF receptor expressed by COS
`cells.
`FIG. 5 depicts crosslinking of murine G-CSF receptor
`expressed in COS cells and those expressed by NFS-60 cells
`with
`I-G-CSF.
`
`30
`
`35
`
`BACKGROUND OF THE INVENTION
`Proliferation and differentiation of hematopoietic cells are
`regulated by hormone-like growth and differentiation factors
`designated as colony-stimulating factors (CSF) (Metcalf, D.
`Nature 339, 27-30 (1989)). CSF can be classified into
`several factors according to the stage of the hematopoietic
`cells to be stimulated and the surrounding conditions as
`25
`follows: granulocyte colony-stimulation factor (G-CSF),
`granulocyte-macrophage colony-stimulation factor (GM
`CSF), macrophage colony-stimulation factor (M-CSF), and
`interleukin 3 (IL-3). G-CSF participates greatly in the dif
`ferentiation and growth of neutrophilic granulocytes and
`plays an important role in the regulation or blood levels of
`neutrophils and the activation of mature neutrophils
`(Nagata, S., "Handbook of Experimental Pharmacology',
`volume "Peptide Growth Factors and Their Receptors', eds.
`Sporn, M. B. and Roberts, A. B., Spring-Verlag, Heidelberg,
`Vol.95/1, pp.699-722 (1990); Nicola, N. A. et al.,
`Annu.Rev. Biochem. 58, pp.45-77 (1989)). Thus, G-CSF
`stimulates the growth and differentiation of neutrophilic
`granulocytes through the interaction between cell-surface
`receptors on precursors of neutrophilic granulocytes to give
`40
`mainly the neutrophilic granulocytes (Nicola, N. A. &
`Metcalf, D., Proc. Natl. Acad, Sci. USA, 81, 3765-3769
`(1984)).
`G-CSF has various biological activities in addition to
`those mentioned above. For example, G-CSF prepared by
`recombinant DNA technology has proven to be a potent
`regulator of neutrophils in vivo using animal model systems
`(Tsuchiya et al., EMBO.J. 6 611-616 (1987); and Nicola et
`al., Annu. Rev. Biochem. 58, 45-77 (1989)). Recent clinical
`trials in patients suffering from a variety of hemopoietic
`disorders have shown that the administration of G-CSF is
`beneficial in chemotherapy and bone marrow transplantation
`therapy (Morstyn et al., Trends Pharmacol. Sci. 10, 154-159
`(1989)). It is also reported that G-CSF stimulates the growth
`of tumor cells such as myeloid leukemia cells.
`55
`Despite the biological and clinical importance of G-CSF,
`little is known about the mechanism through which G-CSF
`exerts its effects. Therefore, it has been needed to elucidate
`such mechanism to establish more effective treatment and
`diagnosis for G-CSF-related disorders. For this purpose, the
`biochemical characterization of specific cell-surface recep
`tors for G-CSF and the evaluation of interaction between
`G-CSF and the receptor must be performed.
`Several reports suggested that the target cells of G-CSF is
`restricted to progenitor and mature neutrophils and various
`myeloid leukemia cells (Nicola and Metcalf, Proc. Natl.
`Acad. Sci. USA, 81, 3765-3769 (1984); Begley et al.,
`
`45
`
`50
`
`60
`
`65
`
`23
`
`

`

`5,574,136
`
`3
`FIG. 6 depicts northern hybridization analysis of murine
`G-CSF receptor mRNA.
`FIG.7(A-D) depicts alignment of amino acid sequence of
`murine G-CSF receptor and those of other growth factors
`and schematic representation of murine G-CSF receptor.
`FIG. 8a depicts the nucleotide sequence and deduced
`amino acid sequence of plasmids pHQ3 and pHG12 (SEQ
`ID NOS:3-4).
`FIG. 8b depicts the nucleotide sequence and deduced
`amino acid sequence in plasmid pHQ2, said sequence being
`different from that of pHQ3 and corresponding to the
`sequence downstream from nucleotide 2,034 in said pHQ3
`(SEQ ID NOS:5-6).
`FIG. 8c depicts the nucleotide sequence and deduced
`amino acid sequence of the insertion present in pHG11, said
`sequence being inserted in pHQ3 (SEQ ID NOS:7-8).
`FIG. 9 depicts a schematic representation and restriction
`map of pHQ3, pHG12, pHQ2 and pHG 11 described in FIG.
`8.
`FIG. 10a depicts saturation binding of murine I-CGF to
`COS cells.
`FIG. 10b depicts scatchard plot of G-CSF binding data.
`FIG. 11 depicts northern hydridization analysis of human
`G-CSF receptor mRNA.
`FIG. 12 depicts detection of human G-CSF receptor
`mRNA by PCR.
`FIG. 13 depicts southern hybridization analysis of human
`G-CSF receptor gene.
`FIG. 14 depicts schematic representation map of expres
`sion vector pEF-BOS.
`
`10
`
`15
`
`20
`
`25
`
`30
`
`4.
`properties commonly found in these members (FIG. 7). A
`probe prepared from thus obtained murine G-CSF receptor
`DNA hybridized with human G-CSF receptor, demonstrat
`ing that human and murine G-CSF receptors are highly
`homologous. Therefore, murine G-CSF receptor can serve
`as so-called "an intermediate' for the preparation of human
`G-CSF receptor.
`As the next step, the present inventors studied with a
`purpose of providing sufficient amount of human G-CSF
`receptor and succeeded in the isolation and cloning of cDNA
`encoding human G-CSF receptor by isolating total mRNA
`from human placenta and U937 cells, constructing cDNA
`library, and screening said library using a probe prepared
`from murine G-CSF receptor cDNA.
`When COS cells were transfected with a plasmid encod
`ing human G-CSF receptor of the present invention, said
`cells expressed a receptor which has similar specific binding
`properties to that of the native human G-CSF receptor.
`Accordingly, the present invention provides an isolated
`DNA encoding G-CSF receptor. The present invention fur
`ther provides an expression vector containing G-CSF recep
`tor DNA. The present invention also provides a method for
`producing G-CSF receptor which comprises transforming a
`host cell by the expression vector, growing said transformant
`in a medium, and recovering G-CSF receptor.
`For purposes of the invention, the term "G-CSF receptor
`peptide' herein used refers to both the mature G-CSF
`receptor peptide and peptide fragments thereof, said frag
`ments having an ability to bind specifically to G-CSF.
`The human G-CSF receptor, which has been hardly
`obtained heretofore, can be easily prepared by the genetic
`engineering by virtue of the present invention, and in turn
`used for many studies directed to, for example, the eluci
`dation of the mechanism through which the G-CSF and/or
`G-CSF receptor exerts the effect, the clinical (for diagnosis
`and treatment) application, as well as for the promotion of
`the practical application. Additionally, probes obtained from
`G-CSF receptor cDNA can facilitate the detection of G-CSF
`receptors on tumor cells such as leukemia cells before the
`clinical application of G-CSF during the treatment of
`patients suffering from these diseases. Therefore, said cDNA
`is useful to perform an effective clinical application of
`G-CSF. Furthermore, proteins or compounds capable of
`binding to G-CSF receptors can be developed by investi
`gating into tertiary structure of soluble-form G-CSF receptor
`which are prepared in large scale by the DNA recombinant
`technology.
`Cloning of DNA encoding murine G-CSF receptor was
`carried out as follows. Thus, G-CSF receptor was initially
`purified from mouse myeloid leukemia NFS-60 cells which
`have relatively higher expression of the G-CSF receptor and
`determined the molecular weight of about 100,000 to 130,
`000 dalton. Total RNA was prepared from NFS-60 cells by
`the guanidine isothiocyanate/CsCl method, and poly(A)
`RNA was selected, which was then used for the synthesis of
`double-stranded cDNA using the reverse transcriptase, DNA
`polymerase and the like. A cDNA library was constructed in
`the mammalian expression vector CDM8 (Seed, Nature 329,
`840–842 (9187)), as 884 pools of 60 to 80 clones. Plasmid
`DNAs from each pool were prepared and introduced into
`COS-7 cells. Two positive pools I62 and J17 which showed
`significant binding of radioiodinated G-CSF were selected.
`From these pools were identified plasmids pl62 and p17
`which have higher binding activity with G-CSF. When
`plasmids p162 and p17 were transfected into COS-7 cells,
`resultant cells expressed receptors capable of binding to
`G-CSF.
`
`DISCLOSURE OF THE INVENTION
`The finding that human and mouse G-CSFs are highly
`homologous (72.6%) lacking species specificity lead to an
`presumption that these G-CSFs probably cross-react. Thus,
`present inventors have studied for the purpose of obtaining
`a G-CSF receptor which can be used in the research as well
`as the diagnostic analysis, and succeeded in the purification
`of the receptor as a protein with a molecular weight (M.W.)
`of 100,000 to 130,000 from a solubilized mouse G-CSF
`receptor from mouse myeloid leukemia NFS-60 cells. The
`purification can be carried out by, for example, extracting
`cell membr