Variant of C6 &bgr;-chemokine leukotactin-1(shLkn-1) with...

Drug – bio-affecting and body treating compositions – Lymphokine

Reexamination Certificate

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C530S324000, C435S069500, C435S071100, C435S071200, C435S471000, C435S320100, C435S252300, C435S255500

Reexamination Certificate

active

06692735

ABSTRACT:

BACKGROUND OF THE INVENTION
1. Field of the Invention
The present invention relates to a variant of C6&bgr;-chemokine (shLkn-1) with enhanced biological activity, more specifically, to a variant of Lkn-1 which belongs to C6 &bgr;-chemokine, a process for preparing a recombinant shLkn-1 by employing expression vector therefor, and pharmaceutical application of the said protein.
2. Description of the Prior Art
Chemokines, a family of small cytokines consisting of basic proteins of low molecular weight, have four cysteine residues commonly, which are classified into four subfamilies of CXC(&agr;), CC(&bgr;), C(&ggr;) and CX
3
C 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. et al., Nature, 385:640(1997)). Genes of chemokine subfamilies locate on a same chromosome in a cluster, for example, &agr;-chemokine genes locate on the human chromosome 4q12-21 and &bgr;-chemokine genes exist on the human chromosome 17q11-32 and the mouse chromosome 11.
Some chemokines have biological activities such as HIV-inhibitory action, immunoregulatory action, leukocyte migration or 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 of G protein-coupled receptors to activate leukocytes and some of the receptors are also used as coreceptors in the course of 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 &bgr;-chemokine(CC chemokine) receptor, i.e., CCR1, CCR2, CCR3, CCR4, CCR5, CCR6, CCR7 and CCR8, four subtypes of CCR4, CCR6, 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, more than nine chemokines belonging to &bgr;-chemokine subfamily have been found(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-1 (“mMRP-1”, MIP(macrophage inflammatory protein)-related protein-1 or C10) (see: Orlofsky, A. et al., Cell Regul., 2:403(1991)), a murine MRP-2(“mMRP-2”) (see: Youn, B. -S. et al., J. Immunol., 155:2661(1995)), and a human Lkn-1(Leukotactin-1) (see: Youn, B. -S. et al., J. Immunol., 159:5201 (1997)) are distinguished from the rest of @-chemokines in the senses that they have two extra cysteine residues, thereby forming the third disulfide bond, and their N-terminal regions are very long. Based on the findings, they are classified into C6 &bgr;-chemokines.
Meanwhile, it was observed that expression of Lkn-1 from recombinant yeast cells such as
Pichia pastoris
(“
P. pastoris
”) results in a recombinant protein with a smaller size than expected for intact Lkn-1 of 92 amino acid residues. The N-terminal amino acid sequence of this smaller form is consistent with that of truncation form Lkn-1 in which 26 amino acids of N-terminus are deleted. Similar truncation of N-terminus has been reported for MPIF-1 (Myeloid Progenitor Inhibitory Factor-1) which is closely related to Lkn-1 in terms of the presence of the extra pair of cysteine residues and the unusually long N-terminus(see: Macphee C. H. et al., J. Immunol., 161:6273(1998)). In this connection, it was suggested that the truncation is correlated with the increase of the biological activity.
Under the circumstances, there are strong reasons for exploring and developing the truncated variant of Lkn-1, since the shorter version may have enhanced biological activity as with MPIF-1 and advantages in mass production. When compared with the native molecule, Lkn-1, the truncated variant of Lkn-1 may have improved potential biological activity as a drug for the treatment of HIV-1 infection or for the protection of bone marrow stem cells during chemotherapy or radiotherapy.
SUMMARY OF THE INVENTION
The present inventors isolated a truncated form of Lkn-1 from the culture media of
P. pastoris
harboring the gene for Lkn-1, and discovered that the truncated Lkn-1 protein indeed has enhanced biological activity as compared with the native Lkn-1. Further, they constructed a cDNA for the truncated form, expressed the said mutant cDNA in a recombinant
P. pastoris
, and showed the expressed recombinant protein inhibits colony formation and proliferation of myeloid stem cell and progenitor cell in vivo. Hereinafter, the said truncated form protein and the recombinant protein thereof are referred to as “shLkn-1”(short version of human leukotactin-1) and “recombinant shLkn-1”, respectively.
The first object of the invention is, therefore, to provide shLkn-1 which has an enhanced biological activity as compared with the intact Lkn-1.
The second object of the invention is to provide an expression vector comprising a cDNA for the said shLkn-1 and a recombinant microorganism transformed with the vector.
The third object of the invention is to provide a process for preparing a recombinant shLkn-1 from the said microorganism.
The fourth object of the invention is to provide a pharmaceutical composition for the antibody production, the treatment during HIV-1 infection, the protection of bone marrow stem cells during chemotherapy or radiotherapy, the inhibition of leukemia.


REFERENCES:
Youn BS. et al. 1997, J Immumol. vol. 159, pp. 5201-5205. Molecular cloning of lekotactin-1: Anovel human beta-chemokine, a chemoattractant for neutrophils, monocytes, and lymphocytes, and a potent agonist at CC chemokine receptors 1 and 3.*
Macphee et al. 1998, J Immunol. vol. 161, pp. 6273-6279. Identificationof atruncated form of the CC chemokine CKbeta demonstrating greatly enahnced bioloigcal activity.*
Hromas et al. 2000. Blood. vol. 95, pp. 1506-1508. The exodus subfamily of CC chemokines inhibits the proliferation of chronic myelogenous leukemia progenitors.*
Virelizier JL. 1999. Dev Biol Stand. vol. 97, pp. 105-109. Blocking of HIV co-recepotr by chemokines.*
J. Fernando Bazan et al., A new class of membrane-bound chemokine with a CX 3C motif, Nature, 385(13):640-644, 1997.
Byung-S. Youn et al., A novel chemokine, macrosphage inflammatory protein-related protein-2, inhibits colony formation of bone marrow myeloid progentorors, J. Immunol., 155:26661-2667, 1995.

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