EP1034272A1

EP1034272A1 - A cDNA ENCODING C6 $g(b)-CHEMOKINE LEUKOTACTIN-1(Lkn-1) ISOLATED FROM HUMAN

Info

Publication number: EP1034272A1
Application number: EP98959235A
Authority: EP
Inventors: Byoung S. Kwon; Byung S. Youn; Soo-Il Chung; Doo-Hong Park; Seung Jae Baek; Eun-Kyoung Lee
Original assignee: Korea Green Cross Corp
Current assignee: Korea Green Cross Corp
Priority date: 1997-11-27
Filing date: 1998-11-27
Publication date: 2000-09-13
Also published as: KR100367213B1; WO1999028472A1; KR20010022352A; WO1999028473A1; CN1282371A; KR19990042713A; JP2002505076A

Abstract

The present invention relates to a cDNA coding for a novel protein which belongs to C6 β-chemokines and attracts subsets of peripheral blood leukocytes, and a process for preparing the said protein by employing expression vector therefor. Open reading frame of the Lkn-1 cDNA encodes 113 amino acids containing a signal peptide of 21 amino acids, and molecular weight of mature protein consisting of 92 amino acids among them is supposed to be 10,162 dalton. A recombinant Lkn-1 which was expressed in E. coli or insect cell employing the said Lkn-1 cDNA and purified, inhibited colony formation and cell proliferation significantly, attracted neutrophils, monocytes and lymphocytes to cause chemotaxis, and bound to CCR1 and CCR3 receptors. Accordingly, it was determined that the recombinant Lkn-1 protein can be used as a potential drug for antibody production, the treatment during HIV-1 infection, the protection of bone marrow stem cells during chemotherapy or radiotherapy and the inhibition of leukemia, etc.

Description

A CDNA ENCODING C6 β -CHEMOKINE LEUKOTACTIN-1 (Lkn-1)

ISOLATED FROM HUMAN

BACKGROUND OF THE INVENTION

Field of the Invention

The present invention relates to a cDNA encoding a novel C6 ,3 -chemokine isolated from human body and a process for preparing the C6 /? -chemokine, more specifically, to a cDNA coding for a novel protein which belongs to C6 β -chemokines and attracts subsets of peripheral blood leukocytes, a process for preparing the said protein by employing expression vector therefor, and pharmaceutical application of the said protein.

Description of the Prior Art

Chemokines are a family of small cytokines consisting of basic proteins of a low molecular weight, have four cysteines commonly, which are classified into four subfamilies of CXC ( a ) , CC ( β ) , C { γ ) and CX₃C depending on the position of the first and the second cysteines, i.e., whether they lie adjacent or an amino acid intervenes between the two cysteines (see: Baggiolini, M. and Dahinden, C.A., Immunol. Today, 15:127(1994); Kelner, S.G. et al., Science, 266:1395(1994); Bazan, J.F. etal., Nature, 385:640(1997)). Genes of chemokine subfamilies locate on a same chromosome in a cluster, for example, a -chemokine genes locate on the human chromosome 4ql2-21 and β -chemokine genes exist on the human chromosome 17qll-32 and the mouse chromosome 11.

Chemokines have biological activities such as HIV- inhibitory action, immunoregulatory action, leukocyte migration and inhibitory action against division of hematopoietic stem cells (see: Cocchi, F. et al., Science, 270:1811(1995); Wolpe, S.D. et al . , J. Exp. Med., 167:570(1988); Graham, G.J. et al . , Nature, 344:442(1990); Broxmeyer, H.E. et al., Blood, 76:1110(1990); Youn, B.-S. et al., J. Immunol., 155:2661-2667(1995)).

Also, chemokines bind to transmembrane domain G protein-coupled receptors to activate leukocytes and some of the receptors are also used as coreceptors during HIV-1 infection (see : Oh, K.-O. et al . , J. Immunol.,

147:2978(1991); Alkabatih, G. et al . , Science,

272:1955(1996)). For example, among 8 subtypes of β - chemokine (CC chemokine) receptor, i.e., CCR1, CCR2, CCR3, CCR4, CCR5, CCRβ, CCR7 andCCR8, four subtypes of CCR4, CCRβ, CCR7 and CCR8 show a high affinity to one substance while CCR1, CCR2, CCR3 and CCR5 have a binding affinity to various chemokines .

So far, nine chemokines belonging to β -chemokine subfamily have been known in the art (see : Wilson, S.D. et al., J. Exp. Med., 171:1301(1990); Modi, W.S. et al . , Hum. Genet., 84:185(1990)). Among them, a murine MRP-l("mMRP- 1" , MIP (macrophage inflammatory protein) -related protein-1 or CIO) (_.se_e: Orlofsky, A. et al . , Cell Regul . , 2:403 (1991) ) and a murine MRP-2 ( "mMRP-2") (see: Youn, B.-S. et al., J. Immunol., 155:2661(1995)) are distinguished from the rest of β -chemokines in that they have two extra cysteine residues, thereby forming the third disulfide bond and their N-terminal regions are very long. Based on the previous findings, they are classified into C6 β -chemokines .

Under the circumstances, there are strong reasons for exploring and developing novel human MRPs, since human MRPs may be employed as a potential drug for the treatment of HIV-1 infection or for the protection of bone marrow stem cells in the course of chemotherapy or radiotherapy.

SUMMARY OF THE INVENTION

The present inventors have made an effort to isolate MRP genes from various human cell lines. As a result, the inventors isolated a novel cDNA of MRP belonging to C6 β -chemokines from a human monocytic cell line and determined its nucleotide sequence and amino acid sequence deduced therefrom. Further, they successfully expressed the said MRP cDNA in recombinant E. coli or insect cell, and discovered that: the expressed recombinant protein inhibited colony formation and proliferation of myeloid stem cell and progenitor cell jLn vivo and _vn_ vitro as well, and attracts subsets of peripheral blood leukocytes (lymphocyte, monocyte and neutrophil) in chemotactic fashion. Hereinafter, the said MRP cDNA isolated from human body and the recombinant MRP are referred to as "Lkn-1 (leukotactin-1) cDNA" and "recombinant Lkn-1", respectively.

The first object of the invention is, therefore, to provide a novel cDNA encoding C6 β -chemokine (Lkn-1 ) and an amino acid sequence deduced therefrom.

The second object of the invention is to provide an expression vector comprising the said Lkn-1 cDNA and a recombinant microorganism transformed with the vector.

The third object of the invention is to provide a process for preparing a recombinant Lkn-1 from the said microorganism. The fourth object of the invention is to provide a method for protecting myeloid cells from cytotoxic anti-cancer drugs or radiation using the recombinant Lkn-1. BRIEF DESCRIPTION OF THE DRAWINGS

The above and the other objects and features of the present invention will become apparent from the following descriptions given in conjunction with the accompanying drawings, in which:

Figure 1(A) shows a nucleotide sequence (SEQ ID N0:1) of a DNA fragment of 202bp and an amino acid sequence deduced therefro (SEQ

ID NO: 2) .

Figure 1 (B) shows a comparison of the amino acid sequence (SEQ ID NO: 2) of Lkn-1 with the 87th to 110th amino acid sequence (SEQ ID NO: 3) of mMRP-2.

Figure 2 is a photograph showing a result of RT-

PCR (reverse transcriptase-polymerase chain reaction) of total RNA isolated from various human cell lines.

Figure 3 shows a nucleotide sequence (SEQ ID NO: 4) of a Lkn-1 cDNA and an amino acid sequence deduced therefrom (SEQ ID

NO: 5) .

Figure 4 shows a comparison of an amino acid sequence of mature Lkn-1(SEQ ID NO: 7) with those of mMRP-l(SEQ ID NO: 8), mMRP-2 (SEQ ID

NO: 9), hMIP-1 a (SEQ ID NO: 10), mMIP- l ff ( SEQ ID NO : I D and hMIP-l β ( SEQ ID

NO: 12 ) . Figure 5(A) is a graph which shows effect of Lkn-1 on the colony formation of myeloid cells in bone marrow.

Figure 5(B) is a graph which shows effect of Lkn-1 on the colony formation of myeloid cells in spleen.

Figure 5(C) is a graph which shows effect of Lkn-1 on the proliferation rate of myeloid cells in bone marrow.

Figure 5(D) is a graph which shows effect of Lkn-1 on the proliferation rate of myeloid cells in spleen.

Figure 6 (A) is a graph which shows dose-dependent suppressive effect of Lkn-1 on the proliferation rate of myeloid cells in bone marrow.

Figure 6(B) is a graph which shows dose-dependent suppressive effect of Lkn-1 on the proliferation rate of myeloid cells in spleen.

Figure 7 (A) is a graph which shows time-dependent suppressive effect of Lkn-1 on the proliferation rate of CFU-GM in bone marrow.

Figure 7 (B) is a graph which shows time-dependent suppressive effect of Lkn-1 on the proliferation rate of BFU-E in bone marrow. Figure 7 (C) is a graph which shows time-dependent suppressive effect of Lkn-1 on the proliferation rate of CFU-GEMM in bone marrow.

Figure 7 (D) is a graph which shows time-dependent suppressive effect of Lkn-1 on the proliferation rate of CFU-GM in spleen.

Figure 7 (E) is a graph which shows time-dependent suppressive effect of Lkn-1 on the proliferation rate of BFU-E in spleen.

Figure 7(F) is a graph which shows time-dependent suppressive effect of Lkn-1 on the proliferation rate of CFU-GEMM in spleen.

Figure 8 (A) is a graph which shows a chemotaxis attracting lymphocytes by a recombinant Lkn-1 and RANTES (regulated on activation, normal T cell expressed and secreted) .

Figure 8 (B) is a graph which shows a chemotaxis attracting monocytes by a recombinant

Lkn-1 and hMIP-lα .

Figure 8 (C) is a graph which shows a chemotaxis attracting neutrophils by a recombinant Lkn-1 and IL-8.

Figure 9 (A) is a graph showing relative fluorescence measured in lymphocytes which are stimulated by a series of addition of RANTES and a recombinant Lkn-1.

Figure 9 (B) is a graph showing relative fluorescence measured in lymphocytes which are stimulated by a series of addition of a recombinant Lkn-1 and RANTES.

Figure 9(C) is a graph showing relative fluorescence measured in monocytes which are stimulated by a series of addition of hMIP-1 a and a recombinant Lkn-1.

Figure 9(D) is a graph showing relative fluorescence measured in monocytes which are stimulated by a series of addition of a recombinant Lkn-1 and hMIP-lα.

Figure 9(E) is a graph showing relative fluorescence measured in neutrophils which are stimulated by a series of addition of IL-8 and a recombinant Lkn-1.

Figure 9(F) is a graph showing relative fluorescence measured in neutrophils which are stimulated by a series of addition of a recombinant Lkn-1 and IL-8.

Figure 10(A) is a graph showing relative fluorescence measured in a HOS cell line expressing CCR1 which are stimulated by a series of addition of various agents. Figure 10(B) is a graph showing relative fluorescence measured in a HOS cell line expressing CCR3 which are stimulated by a series of addition of various agents.

Figure 10 (C) is a graph showing peak amplitude of calcium responses depending on concentrations of Lkn-1 and hMIP-lα.

Figure 10 (D) is a graph showing peak amplitude of calcium responses depending on concentrations of Lkn-1, eotaxin and RANTES.

DETAILED DESCRIPTION OF THE INVENTION

To isolate the Lkn-1 gene from a human cell line, the present inventors first cloned an exon of the Lkn-1 gene, which may be used for the preparation of a probe. That is, human genomic DNA was digested with Hind III, separated on an agarose gel, and analyzed with southern blot by employing the ³²P-labelled mMRP-2 cDNA(_.see: Youn, B.S. et al . , J. Immunol., 155:2661(1995)) as a probe to obtain a DNA fragment of 7. Okb which can be hybridized with mMRP-2. Then, to isolate an exon sequence of Lkn-1 gDNA from the Hindlll- digested DNA fragment of 7. Okb, human genomic DNA was digested with Hindlll completely, and separated on an agarose gel to isolate a DNA fragment of 7. Okb. The fragment thus obtained was inserted into a vector, and the vector thus obtained was introduced into a host cell. After transformation, a colony showing hybridization with the said mMRP-2 cDNA probe was selected. After a DNA fragment of 7. Okb was isolated from the colony and digested with Alul, a DNA fragment of 202bp which can be hybridized with mMRP-2 cDNAwas cloned. And then, the nucleotide sequence of the fragment was determined, which differentiated parts of intron and exon from the DNA fragment. Then, in order to clone a novel Lkn-1 cDNA from a human cell line, Lkn-1 PCR primers permitting amplification of a lOObp-DNA fragment from the Lkn-1 exon sequence confirmed as above were prepared, and RT-PCR was carried out using total RNAs isolated from various human cell lines as a template, respectively, to select cell lines having Lkn-1 mRNA. One of the cell lines thus selected, a human monocytic THP-1 cell line activated by interleukin-4 (IL-4) was chosen and its cDNA library was prepared. On the other hand, RT-PCR was carried out using total RNA of the said THP-1 cell line as a template and the said Lkn-1 PCR primer to prepare a lOObp-DNA fragment of Lkn-1 exon. The cDNA library of a THP-1 cell line constructed as above was hybridized with a probe of the lOObp-DNA fragment of Lkn-1 exon. As a result, a Lkn-1 cDNA showing a positive reaction was obtained.

Determination of the nucleotide sequence of the said Lkn-1 cDNA and the deduced amino acid sequence revealed that the Lkn-1 cDNA is a novel one, open reading frame of the Lkn-1 cDNA encodes 113 amino acids containing a signal peptide of 21 amino acids, and the predicted molecular weight of mature Lkn-1 protein consisting of 92 amino acids is 10,162 dalton. Also, it was found that the Lkn-1 has no potential

N-glycosylation site and belongs to C6 β -chemokine family having two cysteine residues as in mMRP-1 and mMRP-2, in addition to four cysteine residues commonly appeared in β -chemokine family.

The novel Lkn-1 cDNA thus cloned was inserted into an expression vector and introduced into a host cell such as E. coli or insect cell to express the recombinant Lkn-1.

On the other hand, myelosuppressive activity of the recombinant Lkn-1 was investigated for myeloid stem cell and progenitor cell derived from human bone marrow and spleen, which demonstrated that the recombinant Lkn-1 inhibited colony formation and proliferation of the myeloid cells in a concentration- and time-dependent manner in vivo and in vitro as well . Further, it was observed that the recombinant Lkn-1 attracted human peripheral blood neutrophils, monocytes and lymphocytes strongly to cause chemotaxis, and induced calcium flux in the said cells . Particularly, it was found that the recombinant Lkn-1 binds to the RANTES and hMIP-1 a receptors and does not bind to a IL-8 receptor (although it attracts neutrophils strongly to cause chemotaxis, likeIL-8) . Also, it was revealed that receptors of the recombinant Lkn-1 are CC chemokine receptor 1(CCR1) and CCR3. The recombinant Lkn-1 was a stronger agonist at

CCR1 than hMIP-1 a or RANTES which had been already known to bind to CCR1, and it was a stronger agonist at CCR3 receptor than RANTES although it was a weaker agonist the receptor than eotaxin.

The recombinant Lkn-1 of the invention showing the characteristics mentioned above can be used for antibody production, the treatment during HIV-1 infection, the protection of bone marrow stem cells during chemotherapy or radiotherapy and the inhibition of leukemia, etc.

The present invention is further illustrated in the following examples, which should not be taken to limit the scope of the invention.

Example 1: Cloning of exon of Lkn-1 genomic DNA

In order to clone a genomic DNA homologous to mMRP-2 from human, total human genomic DNA was digested with BamHI, EcoRI, Hind III, PstI, Xbal and Xhol, fractionated on a 1.0% agarose gel, and analyzed by southern blot employing a ³²P-labelled mMRP-2 cDNA as a probe to obtain a Hindlll fragment of 7. Okb showing a positive signal. In order to prepare sublibrary of the said HindiII fragment, lOOFg of human genomic DNAwas digested with Hindlll completely, and fractionated on a 1% agarose gel at a voltage of 20V for 16 hours. Then, a DNA fragment near 7. Okb was isolated employing Gene-clean kit (Bio 101, USA) and inserted into a Hindlll-digested dephosphorylated pBluescript SK⁺ vector (Stratagene, USA). Electro-max (GIBCO-BRL, USA) competent cell was transformed with the recombinant vector thus prepared by the aid of electroporation and cultured on a solid medium. About 2xl0⁵ colonies formed was hybridized with the mMRP-2 probe. As a result, it was found that only one colony showed a positive signal of hybridization and contained the insert DNA of 7. Okb.

In order to isolate an exon sequence from the said genomic DNA fragment of 7. Okb, the pBluescript SK⁺ vector containing the insert DNA of 7. Okb was isolated from the said colony showing a positive signal of hybridization, digested with Alul, fractionated on a 1.5% agarose gel, and analyzed by Southern blot to clone a DNA fragment of 202bp which was hybridized with a mMRP-2 cDNA.

A nucleotide sequence (SEQ ID N0:1) of the said DNA fragment of 202bp was determined to find intron and a part of exon translated into amino acids (see: Figure 1(A)) . As a result, it was found that the amino acid sequence (SEQ ID NO: 2) of the part of exon showed homology of 50% to that of mMRP-2 from the 87th to 110th amino acid and the second cysteine (indicated as "*") of two additional cysteines common inmMRP-1 andmMRP-2 was conserved in Lkn-1 (see: Figure 1 (B) ) . In Figure 1 (B) , boxes showed amino acids conserved in Lkn-1 and mMRP-2.

Example 2 : Cloning of a Lkn-1 cDNA

In order to clone a Lkn-1 cDNA from a human cell line, PCR primers permitting amplification of the Lkn-1 exon of lOObp, i.e., a forward primer of 5 '-TTCCTCACCAAGAAGGGG- 3' (SEQ ID NO: 13) and a reverse primer of 5'- CTTTTTCATGCAATCCTG-3' (SEQ ID N0:14) were first prepared based on the amino acid sequence (SEQ ID NO: 2) disclosed in Figure 1(B) . Then, PCR was carried out using the 202bp-DNA fragment prepared in Example 1, total RNA of a human monocytic THP-1 cell line(ATCC TIB202) activated by lOOFg/ml of IL-4 for 24 hours, total RNA of a macrophage cell line U937 (ATCC CTL1593) and total RNA of a HL-60 cell line (ATCC CCL240) as a template, respectively (see: Figure 2).

In Figure 2, lanes 1 to 4 show results of PCR using the 202bp-DNA fragment as a positive control, total RNA of the THP-1 cell line, total RNA of the U937 cell line and total RNA of the HL-60 cell line, respectively; and, lane M shows lOObp-ladder as a DNA size-marker. As shown in Figure 2, the THP-1 cell line activated by IL-4 produced Lkn-1 mRNA.

In order to prepare THP-1 cDNA library, human mRNA was isolated from the THP-1 cell line activated by IL-4, reverse-transcribed employing Time Saver cDNA kit (Pharmacia Biotech, USA) to give double stranded cDNA, and attached with BstXI adapter (Invitrogen, USA). Then, fractionation on an agarose gel was performed to isolate a cDNA of 0.5kb or more, which was inserted into a PRc/CMV( Invitrogen, USA) vector digested with BstXI to prepare cDNA library. The cDNA library was hybridized with the lOObp-DNA fragment of Lkn-1 exon amplified by PCR above as a probe. As a result, one cDNA clone showing a positive signal was isolated.

Example 3 : Nucleotide sequencing of the Lkn-1 cDNA

Figure 3 shows a nucleotide sequence (SEQ ID NO: 4) of the cDNA clone obtained in Example 2 and an amino acid sequence deduced therefrom (SEQ ID NO: 5) . In Figure 3, the underlined is a signal peptide; the sequence in a box is the amino acid sequence(SEQ ID NO:2) of Lkn-1 shown in Figure 1(B) which was used as a probe during the screening of THP-1 cDNA library; and, (***) indicates a translation stop codon.

As shown in Figure 3, open reading frame of the Lkn-1 cDNA(SEQ ID NO: 6) encodes 113 amino acids consisting of 21 amino acids forming a signal peptide and 92 amino acids forming a mature protein whose molecular weight is supposed to be 10,162 dalton. Also, it was found that there is no N-glycosylation site in the deduced amino acid sequence (SEQ ID NO: 5) of Lkn-1. Figure 4 shows comparison of an amino acid sequence of mature Lkn-1 (SEQ ID NO: 7) with those of mMRP-1 (SEQ ID NO: 8) , mMRP-2 (SEQ ID NO: 9) , hMIP-1 a (SEQ ID NO: 10) (see: Youn, B.S. et al., J. Immunol., 155:2661(1995)), mMIP-1 a (SEQ ID NO:ll) (see: Kwon, B.S. and Weissman, S.M., Proc. Natl. Acad. Sci., USA, 86:1963(1989)) and hMIP-1 β (SEQ ID NO:12) (see: Sherry, B. et al . , J. Exp. Med., 168 : 2251 (1988) ) . In Figure 4, boxes and (•) indicate four conserved cysteine residues and two additional cysteine residues, respectively. As shown in Figure 4, it was revealed that: (l)Lkn-l has a long N-terminal region before the first two cysteine residues of four cysteine residues common in β -chemokine family; and,

(2)Lkn-2 belongs to C6 -chemokine family having two cysteine residues further as in mMRP-1 and mMRP-2 besides four cysteine residues common in β -chemokine family. Moreover, it was found that the amino acid sequence (SEQ ID NO: 7) of mature Lkn-1 shows homology of only about 43% to that of mMRP-1 (SEQ ID NO: 8) or mMRP-2 (SEQ ID NO: 9) and homology of 40% to that of hMIP-ltf (SEQ ID NO: 10) although Lkn-1 was isolated as a human counterpart of mMRP-2. Example 4: Expression of a recombinant Lkn-1

Example 4-1: Expression of a recombinant Lkn-1 in E. coli

In order to express only a mature Lkn-1 protein without the putative signal peptide as a recombinant protein, PCR amplification of the open reading frame of the mature Lkn-1 was carried out using the Lkn-1 cDNA cloned in Example 2 as a template, the following PCR primers and Pfu polymerase (Stratagene, USA):

forward primer:

5 ' -CGAATTCCATATGCAGTTCACAAATGATGCAGAG-3 ' (SEQ ID NO: 15)

reverse primer:

5 ' -CGCCGCTCGAGTTGAGTAGGGCTTCAGC-3 ' (SEQ ID NO:16)

The DNA fragment thus amplified was digested with Ndel/Xhol and cloned into a plasmid pET21a (Novagen, USA). The recombinant plasmid thus constructed was designated as pET21a-Lkn-l and introduced into E. coli XL-1 Blue. The recombinant plasmid pET21a-Lkn-l permits expression of a recombinant Lkn-1 which has one additional methionine residue in N-terminus and six additional histidines at C-terminus of a mature Lkn-1.

Transformant thus prepared was designated as 'Escherichia coli (XL-1 Blue) hMRP-2 ' , and deposited with American Type Culture Collection (ATCC, Rockville, MD 20852, USA) , an international depositary authority as deposition No. ATCC 98166 on September 10, 1996.

The said transformant was cultured to express Lkn-1, and inclusion bodies were obtained, dissolved in 20ml of denaturation buffer (6M guanidine-HCl, 20mM Tris-HCl, pH7.9, 500mM NaCl, 4mM n-octylglucopyranoside) and centrifuged to obtain a supernatant. Then, chromatography using an activated Ni-column (Novagen, USA) and a heparin-agarose column (Pharmacia Fine Chemicals, USA) was carried out to purify a His-tagged recombinant Lkn-1. Electrophoresis revealed that a molecular weight of a recombinant Lkn-1 is about 12kDa.

Example 4-2: Expression of a recombinant Lkn-1 in an insect cell

In order to express a recombinant Lkn-1 containing a signal peptide, a Lkn-1 cDNA containing a signal peptide sequence was inserted with a PstI restriction site at N- terminus and a Xbal restriction site at C-terminus, and amplified by PCR. Then, the PCR product thus amplified was isolated and inserted into a PVL 1392 vector (Invitrogen, USA) digested with Pstl/Xbal to construct a recombinant plasmid PVL 1392-Lkn-l. And then, a Sf-21 insect cell was transfected with both the said recombinant plasmid and AcNPV (Autoαrapha California nuclear polyhedrosis baculovirus) to transfer the Lkn-1 cDNA in the said recombinant plasmid to the AcNPV. AcNPV-Lkn-1 viral plaque was isolated based on the characteristics that the virion is occlusion-negative, and grown in the Sf-21 insect cell in a serum-free Ex-Cell 400 medium (JRH Biosciences, USA) for later use.

A recombinant Lkn-1 was expressed in a High five cell line (Invitrogen, USA) cultured in a Ex-cell 400 medium, and the Lkn-1 thus expressed was purified using a HiTrap-Heparin column (Pharmacia Biotech., USA) and a HiTrap-SP column (Pharmacia Biotech., USA). Western blot analysis revealed that a molecular weight of the recombinant Lkn-1 analyzed immediately after purification is about 12kDa. Example 5: Myelosuppressive activity of recombinant Lkn-1

Example 5-1: Inhibition of colony formation of bone marrow cells by the recombinant Lkn-1 in vitro

Since some of the β -chemokines have a myelosuppressive activity in vitro, an effect of the recombinant Lkn-1 purified in Example 4-1 on the colony formation by myeloid progenitor cell present in human bone marrow was investigated. That is, in order to form colony of bone marrow cells by CFU-GM (colony forming unit-granulocyte-macrophage) , human bone marrow cells of low density obtained by centrifugation employing Ficoll-Hypaque gradient (1.070gm/cm³, Sigma Chemical Co., USA) were plated on a 0.3% agar culture medium containing 10% FBS in a concentration of 5xl0⁴cells/ml, and stimulated by rhGM-CSF (recombinant human granulocyte-macrophage- colony stimulating factor, lOOU/ml, Immunex Corporation, USA) + rhSLF(recombinant human steel factor, 50ng/ml, Immunex Corporation, USA) . On the other hand, in order to form colony of bone marrow cells by CFU-GM, BFU- E (burstforming unit-erythroid) and CFU-GEMM (colony forming unit-granulocyte-erythroid-macrophage-megakaryocyte) , the said bone marrow cells were plated on a 1% methylcellulose culture medium containing 30% FBS in a concentration of 5xl0⁴ cells/ml, and stimulated by rhEPO (recombinant human erythropoietin, lU/ml, Amgen Corporation, USA) , rhlL- 3 (recombinant human interleukin-3, lOOU/ml, Immunex Corporation, USA) or rhSLF.

After the stimulation, the cells were cultured in a BNP-210 incubator (Tabai ESPEC Corp., USA) under an environment of 5% C0₂ and 5% 0₂, and number of colonies was counted after 14 days (see: Table 1) . In this connection, the recombinant Lkn-1 was added in a concentration of 3-50ng/ml to a plate. Table 1 Effect of the recombinant Lkn-1 on the colony formation of human bone marrow cells of low density

a: Experimental group without addition of the recombinant L n-1 to the reaction solution b: Growth factor used for the stimulation of colony formation c: Level of change compared with the control (%) d: Level of change compared with the control is significant (p<0.001 ) .

As can be seen in Table 1 above, the recombinant Lkn-1 inhibited colony formation by CFU-GM, BFU-E and CFU-GEMM significantly in a concentration-dependent manner . Level of the inhibition of colony formation was 22-63% compared with the control. On the other hand, it was found that the recombinant Lkn-1 cannot inhibit colony formation by CFU-GM stimulated by GM-CSF alone or by BFU-E stimulated by EPO alone, which demonstrates that the recombinant Lkn-1 has an inhibitory effect on immature progenitor cells which can be stimulated by various growth factors. Example 5-2 : Inhibition of proliferation of myeloid cells by the recombinant Lkn-1 n vivo

The biological activities of Lkn-1 were evaluated in vivo . That is, the purified Lkn-1 was intravenously injected into C3H/HeJ mice and absolute numbers of granulocyte macrophage (CFU-GM) , erythroid (BFU-E) and multipotential progenitor cells (CFU-GEMM) and their proliferation rate in bone marrow and spleen were determined, respectively: C3H/HeJ mice were injected through the tail vein with either

0.1ml of sterile pyrogen-free saline or 8 μ g of purified Lkn-1 diluted in sterile pyrogen-free saline. After 24h from the injection, myeloid cells of low density were prepared from bone marrow of femur and spleen of the mice analogously as in Example 5-1. Then, CFU-GM was plated on 0.3% agar culture medium, and stimulated by 10% PWM mouse spleen cell- conditioned medium. Similarly, BFU-E and CFU-GEMM were plated on 0.9% methylcellulose culture medium, respectively, and stimulated by 1 uint of rhEPO, O.lmmole/L hemin and 1% PWM mouse spleen cell-conditioned medium. In this connection, the bone marrow and spleen cells were plated at respective concentrations of 7.5xl0 and l.OxlO⁶ cells/ml.

After the stimulation, the cells were cultured under the same condition described in Example 5-1, and number of colonies was counted after 5 to 7 days of incubation (see : Figures. 5(A) and 5(B)) . On the other hand, proliferation rates, i.e., cycling rates of CFU-GM, BFU-E and CFU-GEMM were determined as percentage of the cells in S-phase of cell cycle by mesuring the proportion of progenitors in DNA synthesis (i .e. , S-phase of cell cycle) by the aid of specific activity (20Ci/mmol) , tritiated thymidine (50 μ Ci/ml) kill technique, which is based on in vitro calculation of reduction in the number of colonies formed after pulse exposure of cells to hot tritiated thymidine for 20min as compared with a comparable amount of cold thymidine (see: Figures. 5(C) and 5(D)). Figures 5(A) to 5(D) revealed that Lkn-1 rapidly decreased numbers of colonies by myeloid stem/progenitor cells and their proliferation rate (i.e., cell cycling rate) in the bone marrow and spleen. On the other hand, the nucleated cellularity in bone marrow, spleen and blood were assessed, and found to be not significantly affected as compared with control.

In order to investigate dose-dependency of these suppressive effects of Lkn-1, cycling rates of CFU-GM, BFU-E and CFU-GEMM were determined analogously as described above except for varying concentration of Lkn-1 from 0.1 to

20 μ g (see: Figures 6 (A) and 6(B)). As can be seen in Figures 6(A) and 6(B), it was demonstrated that: CFU-GM, BFU-E and CFU-GEMM from the mice receiving 3 to 10 g of Lkn-1 were in a noncycling or slow cycling state in both marrow and spleen, while the cells from the mice receiving 0.1 to I μ q of Lkn-1 showed no change in their cycling rate except for splenic CFU-GEMM. The overall cell cycling rate was 80 to 90% decreased by Lkn-1, and BFU-E and CFU-GEMM appeared to be more sensitive to Lkn-1 than CFU-GM.

Further, in order to investigate time-dependency of these suppressive effects of Lkn-1, C3H/HeJ mice were injected with 8/ig of Lkn-1, and the cell cycling status of CFU-GM, BFU-E and CFU-GEMM from bone marrow and spleen were examined respectively, at different time points (see: Figures 7(A) to 7(F)). As shown in Figures 7(A) to 7(F), the suppressive effect of Lkn-1 was time-dependent and reversible in bone marrow and spleen as well. That is, Lkn-1 placed the CFU-GM, BFU-E and CFU-GEMM in a noncycling or a slow cycling state within 12h and returned them to control level after 72h.

These dose- and time-dependent, and reversible suppressive effects of Lkn-1 in vivo on proliferation of myeloid cells strongly suggested that: Lkn-1 has potential clinical use in protecting normal hematopoietic cells from cytotoxic anti-cancer drugs or radiation.

Example 6: Investigation of the recombinant Lkn-1 as a chemokine

In order to investigate whether the recombinant Lkn-1 may cause chemotaxis, peripheral blood mononuclear cells (PBMC) of a healthy man were obtained by centrifugation employing Ficoll-Hypaque gradient (1.077gm/cm³) . And then, monocytes were isolated from the PBMC thus obtained based on their attached activity onto the surface of plastic, and the said isolation step was repeated twice. In this connection, cells remained after the step for the monocyte isolation were obtained as lymphocytes. Purity of the monocytes and lymphocytes thus obtained was determined by microscopic examination of cytospin dyed with Diff- Quick (Baxter Scientific, USA). As a result, it was found that purity of the monocytes and lymphocytes was 90% and 88%, respectively.

Also, red blood cells were precipitated by using 3% Dextran T 500 (Pharmacia Fine Chemicals, USA) and obtained by centrifugation employing Ficoll-Hypaque gradient. The red blood cells thus obtained were dissolved in a hypotonic solution to obtain human neutrophils. Purity of the neutrophils thus obtained was determined by morphology. As a result, it was found that the purity was 95%.

The monocytes and lymphocytes isolated above were suspended in RPMI 1640 (Gibco, USA) containing 0.5% low endotoxin BSA(Sigma Chemical Co., USA) and 20mM Hepes in concentrations of 2xl0⁶ cells/ml and 8xl0⁶ cells/ml, respectively. The neutrophils were suspended in HBSS in a concentration of lxlO⁶ cells/ml. Then, level of cell migration in a 48-well microchamber (Neuroprobe, USA) was determined as followings. Lower wells of the microchamber were filled with only a buffer solution (control) or a buffer containing the recombinant

Lkn-1, hMIP-lα (PeproTech., USA), RANTES (PeproTech. , USA), IL-8 (PeproTech. , USA) or eotaxin (PeproTech. , USA), and upper wells were filled with 50F1 of the said cell suspensions. A well was partitioned by a suitable filter without polyvinylpyrolidine to give a lower well and an upper well. When neutrophils and lymphocytes were used, a diameter of the filter pore was 3Fm. When monocytes were used, it was 5Fm. After the said microchamber was incubated at 37°C for 1 hour (for neutrophils), 2 hours (for monocytes) or 4 hours (for lymphocytes), the filter was separated from the chamber and washed. The cells on the filter were fixed and dyed with Diff-Quick. Thus, number of the cells was counted (see: Figures 8(A) to 8(C)).

In Figures 8 (A) to 8 (C) , a value obtained by dividing number of migrating cells in an experimental group treated with a chemokine by number of migrating cells in a control was represented as a chemotactic index. Figure 8(A) is a graph which shows chemotaxis attracting lymphocytes by the recombinant Lkn-1 and RANTES. Figure 8(B) is a graph which shows chemotaxis attracting monocytes by the recombinant

Lkn-1 and hMIP-1 a . Figure 8(C) is a graph which shows chemotaxis attracting neutrophils by the recombinant Lkn-1 and IL-8.

As shown in Figures 8 (A) to 8 (C) , it was revealed that the recombinant Lkn-1 is a strong chemokine attracting human peripheral blood neutrophils, monocytes and lymphocytes. Also, the recombinant Lkn-1 showed chemotaxis attracting neutrophils in a similar manner to IL-8 (see : Figure 8(C)) and chemotaxis attracting monocytes in a similar manner to hMIP-1 a (see: Figure 8 (B) ) ; and, it showed an improved chemotaxis attracting lymphocytes in a concentration- dependent manner up to a concentration of lOFg/ml of Lkn-1 although it showed lower level of chemotaxis than RANTES (see: Figure 8 (A) ) .

Example 7 : Analysis of calcium flux by the recombinant Lkn-1

Example 7-1: Analysis of calcium flux in lymphocytes, monocytes and neutrophils

It was investigated whether the recombinant Lkn-1 may bind to a receptor for activation of lymphocytes, monocytes and neutrophils to induce calcium efflux. Its competitive relationships with other agonists against the receptor was also examined. Receptor activation was determined by measuring a change in [Ca²⁺] in subsets (lymphocytes, monocytes and neutrophils) of peripheral blood leukocytes isolated using a MSIII fluorimeter (Photon Technology Internationl, USA) . That is, cells were reacted with 2FM fura-2AM (Sigma Chemical Co., USA) at 37°C for 45 minutes, washed twice, and resuspended in HBSS (pH 7.4) containing 0.05% BSA in a concentration of lxlO⁷ cells/ml. 2ml of the cell suspension was added into a stirred, water-jacketed cuvette, and activated continuously at 340nm and 380nm at 37°C. Emitted fluorescence was measured at 510nm before and after addition of 25nM agonist (the recombinant Lkn-1, hMIP-lα, RANTES, IL-8 or eotaxin) (see: Figures 9(A) to 9(F) ) .

In Figures 9(A) to 9(F), relative fluorescence was expressed as a relative ratio of fluorescence activated at 340nm and 380nm. Figure 9(A) shows relative fluorescence measured in lymphocytes which are stimulated by a series of addition of RANTES and the recombinant Lkn-1; Figure 9(B) shows relative fluorescence measured in lymphocytes which are stimulated by a series of addition of the recombinant Lkn-1 and RANTES; Figure 9(C) shows relative fluorescence measured in monocytes which are stimulated by a series of addition of hMIP-lα and the recombinant Lkn-1; Figure 9(D) shows relative fluorescence measured in monocytes which are stimulated by a series of addition of the recombinant Lkn-1 and hMIP-1 a ; Figure 9(E) shows relative fluorescence measured in neutrophils which are stimulated by a series of addition of IL-8 and the recombinant Lkn-1; Figure 9 (F) shows relative fluorescence measured in neutrophils which are stimulated by a series of addition of the recombinant Lkn-1 and IL-8. As shown in Figures 9 (A) to 9 (F) , it was found that the recombinant Lkn-1 induced calcium influx in lymphocytes, monocytes and neutrophils.

On the other hand, when G protein-coupled receptors were continuously exposed to ligands having the same binding site as the receptor signal within a short time, the said receptors were desensitized. Such a phenomenon occurred when the said cells expressing receptors were stimulated by the recombinant Lkn-1. As shown in Figures 9(A) to 9(F) , it was found that the recombinant Lkn-1 desensitized lymphocytes and monocytes completely when stimulation by RANTES and hMIP-1 a followed (_.see: Figures 9(B) and 9(D)). Also, RANTES or hMIP-lff did not desensitize lymphocytes and monocytes completely when stimulation by the recombinant Lkn-1 followed (see: Figures 9(A) and 9(C)), which demonstrated that the recombinant Lkn-1 shares receptors of RANTES and hMIP-lff, and induces more calcium flux than RANTES and hMIP-1 a . In addition, neutrophils were not desensitized by the recombinant Lkn-1 during a further stimulation by IL-8 although the recombinant Lkn-1 induced calcium flux in neutrophils (see: Figure 9(F)). Moreover, IL-8 did not desensitize neutrophils during a further stimulation by the recombinant Lkn-1 (see: Figure 9(E)). Therefore, it was clearly demonstrated that the recombinant Lkn-1 does not share IL-8 receptors although it is a strong factor to induce calcium flux in neutrophils like IL-8.

Example 7-2: Analysis of calcium flux in a cell line expressing CC chemokine receptors

It was investigated whether the recombinant Lkn-1 can activate a cell line expressing CC or CXC chemokine receptors because it was found in Example 7-1 that the recombinant Lkn-1 activated receptors which can be activated by RANTES and hMIP-lα . Analysis was carried out in the same manner as in Example 7-1 except for the use of a HOS cell line expressing recombinant CCR1, CCR2B, CCR3, CCR4, CCR5 or CXCR4 (s^e: AIDS Research and Reference Reagent Program of NIH, USA) instead of the use of subsets of leukocytes isolated. It has been known that hMIP-ltf binds to both CCR1 and CCR5, and RANTES binds to CCR1, CCR3 and CCR5. Figure 10(A) shows relative fluorescence measured in a HOS cell line expressing CCR1 which are stimulated by a series of addition of various agents, and Figure 10 (B) shows relative fluorescence measured in a HOS cell line expressing CCR3 which are stimulated by a series of addition of various agents.

As shown in Figures 10(A) and 10(B), it was revealed that the recombinant Lkn-1 strongly induced calcium flux in a HOS cell line expressing CCR1 or CCR3. However, calcium flux by the recombinant Lkn-1 in HOS cell lines expressing other receptors was not observed.

Also, as shown in Figure 10 (A) , CCR1-H0S cells were not stimulated further by hMIP-1 a or RANTES after stimulation by the recombinant Lkn-1 while further stimulation of CCR1-H0S cells by the recombinant Lkn-1 was not affected after stimulation by hMIP-1 a or RANTES.

Figure 10(A) revealed that the recombinant Lkn-1 is a stronger agonist against CCR1 than hMIP-lα or RANTES, which was clearly confirmed from calcium responses depending on concentrations of the recombinant Lkn-1 and hMIP-1 a as shown in Figure 10 (C) .

Moreover, as shown in Figure 10 (B) , CCR3-HOS cells were desensitized almost completely during further stimulation by eotaxin or the recombinant Lkn-1 after the cells were stimulated by eotaxin; CCR3-HOS cells were desensitized almost completely during further stimulation by RANTES after the cells were stimulated by the recombinant Lkn-1; and, further stimulation of CCR3-HOS cells by eotaxin was, however, not affected after the cells were stimulated by the recombinant Lkn-1 or RANTES. Also, calcium responses depending on concentrations of eotaxin, the recombinant Lkn-1 and RANTES showed that the recombinant Lkn-1 is a stronger agonist against CCR3 than RANTES although it is not a stronger one than eotaxin (see: Figure 10(D)).

As clearly illustrated and demonstrated as aboves, the present invention provides a cDNA coding for a novel Lkn-1 protein which belongs to C6 β -chemokines, and an amino acid sequence deduced therefrom. Open reading frame of the Lkn-1 cDNA encodes 113 amino acids containing a signal peptide of 21 amino acids, and molecular weight of mature protein consisting of 92 amino acids among them is supposed to be 10,162 dalton. A recombinant Lkn-1 which was expressed in E. coli or insect cell employing the said Lkn-1 cDNA and purified, inhibited colony formation and cell proliferation significantly, attracted neutrophils, monocytes and lymphocytes to cause chemotaxis, and bound to CCR1 and CCR3 receptors. Accordingly, it was determined that the recombinant Lkn-1 protein can be used as a potential drug for antibody production, the treatment during HIV-1 infection, the protection of bone marrow stem cells during chemotherapy or radiotherapy and the inhibition of leukemia, etc.

SEQUENCE LISTING

(1) GENERAL INFORMATION: (i) APPLICANT:

(A) NAME: KOREA GREEN CROSS CORPORATION et al .

(B) STREET: 227, Gugal-Ri, Kiheung-Eup

(C) CITY: Yongin, Kyonggi-Do

(D) STATE: not applicable (E) COUNTRY: Korea

(F) POSTAL CODE (ZIP) : 449-900

(G) TELEPHONE: 02-584-0131 (H) TELEFAX: 02-582-6331

(ii) TITLE OF INVENTION: A cDNA ENCODING C6 β -CHEMOKINE

LEUKOTACTIN-1 (Lkn-1) ISOLATED FROM HUMAN

(iii) NUMBER OF SEQUENCES: 16

(iv) COMPUTER READABLE FORM:

(A) MEDIUM TYPE: Floppy disk

(B) COMPUTER: IBM PC compatible

(C) OPERATING SYSTEM: PC-DOS/MS-DOS (D) SOFTWARE: Patentln Release #1.0, Version #1.30

(EPO)

(2) INFORMATION FOR SEQ ID NO:l: (i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 202 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: DNA

(iv) ANTI-SENSE: NO (vi) ORIGINAL SOURCE:

(A) ORGANISM: Human

(vii) IMMEDIATE SOURCE:

(A) LIBRARY: genomic DNA library from human

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:l:

CTGCCATCAG CAGAGAAAGG AAAAAACAGG CTGTGTTGAC TGGGAAATCT 50

GAGGAGCAGG GAGGATGGGG CCCCCTGTCT CCATCTGCCC ACACCTCAGT 100 TTGTAATCTT TCTCTCCCTT GTTCCCCAGA TTCCTCACCA AGAAGGGGCG 150 GCAAGTCTGT GCCAAACCCA GTGGTCCGGG AGTTCAGGAT TGCATGAAAA 200 AG 202

(2) INFORMATION FOR SEQ ID NO: 2:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 24 amino acids

(B) TYPE: amino acid (D) TOPOLOGY: linear

(ii) MOLECULE TYPE: protein

(vi) ORIGINAL SOURCE: (A) ORGANISM: Human

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 2:

Phe Leu Thr Lys Lys Gly Arg Gin Val Cys Ala Lys Pro Ser 1 5 10

Gly Pro Gly Val Gin Asp Cys Met Lys Lys 15 20

(2) INFORMATION FOR SEQ ID NO: 3:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 24 amino acids

(B) TYPE: amino acid (D) TOPOLOGY: linear

(ii) MOLECULE TYPE: protein

(vi) ORIGINAL SOURCE: (A) ORGANISM: Murine

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 3:

Phe lie Ser Lys Arg Gly Phe Gin Val Cys Ala Asn Pro Ser 1 5 10 sp Arg Arg Val Gin Arg Cys lie Glu Arg 15 20

(2) INFORMATION FOR SEQ ID NO: 4:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 582 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single (D) TOPOLOGY: linear

(ii) MOLECULE TYPE: DNA (iv) ANTI-SENSE: NO

(vi) ORIGINAL SOURCE: (A) ORGANISM: Human

(vii) IMMEDIATE SOURCE:

(A) LIBRARY: DNA library from human (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 4:

CGGAGCCAGG AAGCAGTGAG CCCAGGAGTC CTCGGCCAGC CCTGCCTGCC 50 CACCAGGAGG ATGAAGGTCT CCGTGGCTGC CCTCTCCTGC CTCATGCTTA 100 TTGCTGTCCT TGGATCCCAG GCCCAGTTCA CAAATGATGC AGAGACAGAG 150 TTAATGATGT CAAAGCTTCC ACTGGAAAAT CCAGTAGTTC TGAACAGCTT 200 TCACTTTGCT GCTGACTGCT GCACCTCCTA CATCTCACAA AGCATCCCGT 250 GTTCACTCAT GAAAAGTTAT TTTGAAACGA GCAGCGAGTG CTCCAAGCCA 300 GGTGTCATAT TCCTCACCAA GAAGGGGCGG CAAGTCTGTG CCAAACCCAG 350 TGGTCCGGGA GTTCAGGATT GCATGAAAAA GCTGAAGCCC TACTCAATAT 400 AATAATAAAC AGACAAAAGA GGCCAGCCAC CCACCTCCAA CACCTCCTGT 450 GAGTTTCTTG GTCTGAAATA CTTAAAAAAT ATATATATTG TTGTGTCTGG 500 TAATGAAAGT AATGCATCTA ATAAAGGTAT TCAATTTTTT AACTTTGCTT 550 GAGTTTTAAG AGGAAATAAA CTAATATAAA AC 582 (2) INFORMATION FOR SEQ ID NO: 5:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 113 amino acids

(B) TYPE: amino acid (D) TOPOLOGY: linear

(ii) MOLECULE TYPE: protein

(vi) ORIGINAL SOURCE: (A) ORGANISM: Human

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 5:

Met Lys Val Ser Val Ala Ala Leu Ser Cys Leu Met Leu lie 1 5 10

Ala Val Leu Gly Ser Gin Ala Gin Phe Thr Asn Asp Ala Glu 15 20 25 Thr Glu Leu Met Met Ser Lys Leu Pro Leu Glu Asn Pro Val 30 35 40

Val Leu Asn Ser Phe His Phe Ala Ala Asp Cys Cys Thr Ser 45 50 55

Tyr lie Ser Gin Ser lie Pro Cys Ser Leu Met Lys Ser Tyr 60 65 70 Phe Glu Thr Ser Ser Glu Cys Ser Lys Pro Gly Val He Phe

75 80

Leu Thr Lys Lys Gly Arg Gin Val Cys Ala Lys Pro Ser Gly

85 90 95

Pro Gly Val Gin Asp Cys Met Lys Lys Leu Lys Pro Tyr Ser 100 105 110

He

(2) INFORMATION FOR SEQ ID NO: 6: (i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 342 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS : single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: DNA

(iv) ANTI-SENSE: NO (vi) ORIGINAL SOURCE:

(A) ORGANISM: Human (vii) IMMEDIATE SOURCE:

(A) LIBRARY: DNA library from human (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 6:

ATGAAGGTCT CCGTGGCTGC CCTCTCCTGC CTCATGCTTA TTGCTGTCCT 50 TGGATCCCAG GCCCAGTTCA CAAATGATGC AGAGACAGAG TTAATGATGT 100 CAAAGCTTCC ACTGGAAAAT CCAGTAGTTC TGAACAGCTT TCACTTTGCT 150 GCTGACTGCT GCACCTCCTA CATCTCACAA AGCATCCCGT GTTCACTCAT 200 GAAAAGTTAT TTTGAAACGA GCAGCGAGTG CTCCAAGCCA GGTGTCATAT 250 TCCTCACCAA GAAGGGGCGG CAAGTCTGTG CCAAACCCAG TGGTCCGGGA 300 GTTCAGGATT GCATGAAAAA GCTGAAGCCC TACTCAATAT AA 342 (2) INFORMATION FOR SEQ ID NO: 7:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 92 amino acids

(B) TYPE: amino acid (D) TOPOLOGY: linear

(ii) MOLECULE TYPE: protein

(vi) ORIGINAL SOURCE: (A) ORGANISM: Human (xi) SEQUENCE DESCRIPTION: SEQ ID NO: :

Gin Phe Thr Asn Asp Ala Glu Thr Glu Leu Met Met Ser Lys 1 5 10

Leu Pro Leu Glu Asn Pro Val Val Leu Asn Ser Phe His Phe 15 20 25

Ala Ala Asp Cys Cys Thr Ser Tyr He Ser Gin Ser He Pro 30 35 40

Cys Ser Leu Met Lys Ser Tyr Phe Glu Thr Ser Ser Glu Cys 45 50 55 Ser Lys Pro Gly Val He Phe Leu Thr Lys Lys Gly Arg Gin

60 65 70

Val Cys Ala Lys Pro Ser Gly Pro Gly Val Gin Asp Cys Met

75 80

Lys Lys Leu Lys Pro Tyr Ser He 85 90

(2) INFORMATION FOR SEQ ID NO: 8:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 95 amino acids

(B) TYPE: amino acid (D) TOPOLOGY: linear

(ii) MOLECULE TYPE: protein

(vi) ORIGINAL SOURCE: (A) ORGANISM: Murine

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 8:

Gly Leu He Gin He Met Glu Lys Glu Asp Arg Arg Tyr Asn 1 5 10

Pro Pro He He His Gin Gly Phe Gin Asp Thr Ser Ser Asp 15 20 25

Cys Cys Phe Ser Tyr Ala Thr Gin He Pro Cys Lys Arg Phe 30 35 40

He Tyr Tyr Phe Pro Thr Ser Gly Gly Cys He Lys Pro Gly 45 50 55 He He Phe He Ser Arg Arg Gly Thr Gin Val Cys Ala Asp

60 65 70 Pro Ser Asp Arg Arg Val Gin Arg Cys Leu Ser Thr Leu Lys

75 80

Gin Gly Pro Arg Ser Gly Asn Lys Val He Ala 85 90 95

(2) INFORMATION FOR SEQ ID NO: 9:

(i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 101 amino acids (B) TYPE: amino acid (D) TOPOLOGY: linear

(ii) MOLECULE TYPE: protein

(vi) ORIGINAL SOURCE: (A) ORGANISM: Murine

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 9:

Gin He Thr His Ala Thr Glu Thr Lys Glu Val Gin Ser Ser 1 5 10

Leu Lys Ala Gin Gin Gly Leu Glu He Glu Met Phe His Met 15 20 25

Gly Phe Gin Asp Ser Ser Asp Cys Cys Leu Ser Tyr Asn Ser

30 35 40 Arg He Gin Cys Ser Arg Phe He Gly Tyr Phe Pro Thr Ser

45 50 55

Gly Gly Cys Thr Arg Pro Gly He He Phe He Ser Lys Arg 60 65 70

Gly Phe Gin Val Cys Ala Asn Pro Ser Asp Arg Arg Val Gin

75 80

Arg Cys He Glu Arg Leu Glu Gin Asn Ser Gin Pro Arg Thr 85 90 95

Tyr Tyr Lys 100

(2) INFORMATION FOR SEQ ID NO: 10:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 67 amino acids

(B) TYPE: amino acid (D) TOPOLOGY: linear

(ii) MOLECULE TYPE: protein (vi) ORIGINAL SOURCE: (A) ORGANISM: Human

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 10:

Ala Ser Leu Ala Ala Asp Thr Pro Thr Ala Cys Cys Phe Ser 1 5 10

Tyr Thr Ser Arg Gin He Pro Gin Asn Phe He Ala Asp Tyr 15 20 25

Phe Glu Thr Ser Ser Gin Cys Ser Lys Pro Gly Val He Phe 30 35 40 Leu Thr Lys Arg Ser Arg Gin Val Cys Ala Asp Pro Ser Glu 45 50 55

Glu Trp Val Gin Lys Tyr Val Ser Asp Leu Glu 60 65

(2) INFORMATION FOR SEQ ID NO: 11

(i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 66 amino acids (B) TYPE: amino acid (D) TOPOLOGY: linear

(ii) MOLECULE TYPE: protein (vi) ORIGINAL SOURCE:

(A) ORGANISM: Murine

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 11: Ala Pro Tyr Gly Ala Asp Thr Pro Thr Ala Cys Cys Phe Ser 1 5 10

Tyr Ser Arg Lys He Pro Arg Gin Phe He Val Glu Val Phe 15 20 25

Glu Thr Ser Ser Leu Cys Ser Gin Pro Gly Val He Phe Leu 30 35 40

Thr Lys Arg Asn Arg Gin He Cys Ala Asp Ser Lys Glu Thr 45 50 55

Trp Val Gin Glu Tyr He Thr Asp Leu Glu 60 65 (2) INFORMATION FOR SEQ ID NO: 12:

(i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 67 amino acids

(B) TYPE: amino acid (D) TOPOLOGY: linear (ii) MOLECULE TYPE: protein

(vi) ORIGINAL SOURCE: (A) ORGANISM: Human (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 12:

Ala Pro Met Gly Ser Asp Pro Pro Thr Ala Cys Cys Phe Ser 1 5 10 Tyr Thr Ala Arg Lys Leu Pro Arg Asn Phe Val Val Asp Tyr 15 20 25

Tyr Glu Thr Ser Ser Leu Cys Ser Gin Pro Ala Val Val Phe 30 35 40

Gin Thr Lys Arg Ser Lys Gin Val Cys Ala Asp Pro Ser Glu 45 50 55

Ser Trp Val Gin Glu Val Tyr Tyr Asp Leu Glu 60 65

(2) INFORMATION FOR SEQ ID NO: 13: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 18 base pairs (B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: DNA

(vii) IMMEDIATE SOURCE: (B) CLONE: primer

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 13;

TTCCTCACCA AGAAGGGG

(2) INFORMATION FOR SEQ ID NO: 14 (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 18 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (vii) IMMEDIATE SOURCE: (B) CLONE: primer

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 14: CTTTTTCATG CAATCCTG 18

(2) INFORMATION FOR SEQ ID NO: 15: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 34 base pairs (B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: DNA

(vii) IMMEDIATE SOURCE: (B) CLONE: primer

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 15:

CGAATTCCAT ATGCAGTTCA CAAATGATGC AGAG 34

(2) INFORMATION FOR SEQ ID NO: 16: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 28 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA

(vii) IMMEDIATE SOURCE: (B) CLONE: primer (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 16:

CGCCGCTCGA GTTGAGTAGG GCTTCAGC 28

Claims

WHAT IS CLAIMED IS:

1. A cDNA of human C6 3-chemokine Lkn-1 (leukotactin- 1) whose nucleotide sequence is represented as following (SEQ ID NO: 6) :

ATG AAG GTC TCC GTG GCT GCC CTC TCC TGC CTC ATG CTT 39 ATT GCT GTC CTT GGA TCC CAG GCC CAG TTC ACA AAT GAT 78 GCA GAG ACA GAG TTA ATG ATG TCA AAG CTT CCA CTG GAA 117 AAT CCA GTA GTT CTG AAC AGC TTT CAC TTT GCT GCT GAC 156 TGC TGC ACC TCC TAC ATC TCA CAA AGC ATC CCG TGT TCA 195 CTC ATG AAA AGT TAT TTT GAA ACG AGC AGC GAG TGC TCC 234 AAG CCA GGT GTC ATA TTC CTC ACC AAG AAG GGG CGG CAA 273 GTC TGT GCC AAA CCC AGT GGT CCG GGA GTT CAG GAT TGC 312 ATG AAA AAG CTG AAG CCC TAC TCA ATA TAA 342

2. Human C6 ╬▓-chemokine Lkn-1 (leukotactin-1) whose amino acid sequence is represented as following (SEQ ID NO: 5) :

Met Lys Val Ser Val Ala Ala Leu Ser Cys Leu Met Leu He 1 5 10

Ala Val Leu Gly Ser Gin Ala Gin Phe Thr Asn Asp Ala Glu 15 20 25

Thr Glu Leu Met Met Ser Lys Leu Pro Leu Glu Asn Pro Val 30 35 40

Val Leu Asn Ser Phe His Phe Ala Ala Asp Cys Cys Thr Ser 45 50 55

Tyr He Ser Gin Ser He Pro Cys Ser Leu Met Lys Ser Tyr 60 65 70 Phe Glu Thr Ser Ser Glu Cys Ser Lys Pro Gly Val He Phe

75 80

Leu Thr Lys Lys Gly Arg Gin Val Cys Ala Lys Pro Ser Gly 85 90 95

Pro Gly Val Gin Asp Cys Met Lys Lys Leu Lys Pro Tyr Ser 100 105 110 He

3. A cDNA of human C6 ╬▓ -chemokine Lkn-1 (leukotactin- 1) from which the 1st to 63th nucleotide sequences are deleted.

4. An expression vector which comprises the cDNA of claim 1.

5. An expression vector PVL 1392-Lkn-l which comprises the cDNA of claim 1.

6. A process for preparing a recombinant leukotactin-1 which comprises the steps of transforming a host cell with the expression vector of claim 4, culturing the transformant, and obtaining the recombinant Lkn-1 therefrom.

7. The process of claim 6 wherein the host cell is E. coli or insect cell.

8. A recombinant leukotactin-1 prepared by a process which comprises the steps of transforming a host cell with the expression vector of claim 4, culturing the transformant, and obtaining the recombinant Lkn-1 from the culture.

9. The recombinant leukotactin-1 of claim 8 which inhibits colony formation.

10. The recombinant leukotactin-1 of claim 8 which attracts human peripheral blood neutrophils, monocytes and lymphocytes strongly to cause chemotaxis.

11. The recombinant leukotactin-1 of claim 8 which binds to CCR1 and CCR3 receptors.

12. The recombinant leukotactin-1 of claim 11 which shares receptors with RANTES (regulated activated normal T cell expressed sequence) and hMIP-1 a (human macrophage inflammatory protein-1 a ) and does not share receptors with IL-8 (interleukin-8) .

13. An expression vector which comprises the cDNA of claim 3.

14. An expression vector pET21a-Lkn-l which comprises the cDNA of claim 3.

15. Escherichia coli (XL-1 Blue) hMRP-2 transformed with the expression vector of claim 14 (ATCC 98166).

16. A process for preparing a recombinant leukotactin-1 which comprises the steps of transforming a host cell with the expression vector of claim 13, culturing the transformant, and obtaining the recombinant Lkn-1 therefrom.

17. The process of claim 16 wherein the host cell is E. coli or insect cell.

18. A recombinant leukotactin-1 prepared by a process which comprises the steps of transforming a host cell with the expression vector of claim 13, culturing the transformant, and obtaining the recombinant Lkn-1 therefrom.

19. The recombinant leukotactin-1 of claim 18 which inhibits colony formation.

20. The recombinant leukotactin-1 of claim 18 which attracts human peripheral blood neutrophils, monocytes and lymphocytes strongly to cause chemotaxis.

21. The recombinant leukotactin-1 of claim 18 which binds to CCR1 and CCR3 receptors.

22. The recombinant leukotactin-1 of claim 21 which shares receptors with RANTES (regulated activated normal T cell expressed sequence) and hMIP-1 a (human macrophage inflammatory protein-1 a ) and does not share receptors with IL-8 (interleukin-8) .

23. A method for protecting myeloid cells from cytotoxic anti-cancer drugs or radiation which comprises a step of administrating human C╬▓ _/S-chemokine Lkn-1 (leukotactin-1) into a subject.

24. The method of claim 23, wherein the human C6 ╬▓ - chemokine Lkn-1 (leukotactin-1) is the recombinant leukotactin-1 of claim 8.

25. The method of claim 23, wherein the human C╬▓ ╬▓ - chemokine Lkn-1 (leukotactin-1) is the recombinant leukotactin-1 of claim 18.