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  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
  • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
  • C07K14/52—Cytokines; Lymphokines; Interferons
  • C07K14/521—Chemokines
  • C07K14/523—Beta-chemokines, e.g. RANTES, I-309/TCA-3, MIP-1alpha, MIP-1beta/ACT-2/LD78/SCIF, MCP-1/MCAF, MCP-2, MCP-3, LDCF-1, LDCF-2
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K38/00—Medicinal preparations containing peptides
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K48/00—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K2319/00—Fusion polypeptide
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  • C12N2799/00—Uses of viruses
  • C12N2799/02—Uses of viruses as vector
  • C12N2799/021—Uses of viruses as vector for the expression of a heterologous nucleic acid
  • C12N2799/026—Uses of viruses as vector for the expression of a heterologous nucleic acid where the vector is derived from a baculovirus

Definitions

• chemokines have been implicated in a number of physiological and disease condition ⁇ , including lymphocyte trafficking, wound healing, hematopoietic regulation and immunological di ⁇ order ⁇ ⁇ uch a ⁇ allergy, asthma and arthritis.
• MIP-1 wa ⁇ originally identified a ⁇ an endotoxin- induced proinflammatory cytokine produced from macrophage ⁇ .
• Subsequent studie ⁇ have ⁇ hown that MIP-l i ⁇ composed of two different, but related, protein ⁇ MlP-l ⁇ and MIP-l/3.
• nucleic acid probes comprising nucleic acid molecules of sufficient length to specifically hybridize to the Ck/3-8, Ck3-1 and MIP-4 nucleic acid sequence ⁇ .
• a proce ⁇ for utilizing such polypeptides, or polynucleotide ⁇ encoding such polypeptides, as research reagents for in vitro purpose ⁇ related to ⁇ cientific research, synthesi ⁇ of DNA and manufacture of DNA vector ⁇ , for the purpo ⁇ e of developing therapeutics and diagnostics for the treatment of human disease.
• FIG. 4 illustrate ⁇ the amino acid homology between Ck/3-8 (top) and human MlP-l ⁇ (bottom) .
• the four cy ⁇ teine ⁇ characteri ⁇ tic of all chemokine ⁇ are ⁇ hown.
• FIG. 12 The monocyte cell line THP-l wa ⁇ treated for 16 hour ⁇ with LPS (0.1-10 ng/ml) or Ck / 3-8 (to 50 ng/ml). Tissue culture supernatants were subjected to ELISA analysis to quantify the secretion of TNF- ⁇ .
• FIG. 18 Effect of Ck/3-8 and Ck/3-1 on the generation of GR-l and Mac-1 ( ⁇ urface marker ⁇ ) po ⁇ itive population of cells from lin" population of bone marrow cells.
• lin " cells were incubated in growth medium supplemented with IL-3 (5 ng/ml) and SCF (100 ng/ml) alone (a) and Ck/3-8 (50 ng/ml) (b) or Ck/3-l (50 ng/ml) .
• Cells were then stained with Monoclonal antibodies against myeloid differentiation GR.l, Mac-1, Sca- 1, and CD45R surface antigens and analyzed by FACScan. Data is presented as percentage of positive cell ⁇ in both large (A) and ⁇ mall (B) cell population ⁇ .
• FIG. 19 illustrates that the presence of Ck/3-8 (+) inhibits bone marrow cell colony formation in respon ⁇ e to IL- 3, M-CSF and GM-CSF.
• nucleic acid ⁇ (polvnucleotide ⁇ ) which encode for the mature polypeptide ⁇ having the deduced amino acid sequence of Figures l, 2 and 3 (SEQ ID No. 2, 4 and 6, respectively) or for the mature Ck/3-8 polypeptide encoded by the cDNA of the clone(s) depo ⁇ ited a ⁇ ATCC Depo ⁇ it No. 75676 on February 9, 1994, and for the mature MIP-4 polypeptide encoded by the cDNA of the clone depo ⁇ ited a ⁇ ATCC Depo ⁇ it No. 75675 on February 9, 1994 and for the mature Ck/3-1 polypeptide encoded by the cDNA of the clone depo ⁇ ited a ⁇ ATCC Depo ⁇ it No. 75572, depo ⁇ ited on October 13, 1993.
• the polynucleotides of the present invention may also have the coding sequence fused in frame to a marker sequence which allows for purification of the polypeptides of the present invention.
• the marker sequence may be a hexa- hi ⁇ tidine tag supplied by a pQE-9 vector to provide for purification of the mature polypeptides fused to the marker in the case of a bacterial host, or, for example, the marker sequence may be a hemagglutinin (HA) tag when a mammalian host, e.g. COS-7 cells, is used.
• the HA tag correspond ⁇ to an epitope derived from the influenza hemagglutinin protein (Wilson, I., et al., Cell, 37:767 (1984)).
• the fragment, derivative or analog of the polypeptides of Figures 1, 2 and 3 (SEQ ID No. 2, 4 and 6) or that encoded by the deposited cDNA may be (i) one in which one or more of the amino acid residues are sub ⁇ tituted with a conserved or non-conserved amino acid residue (preferably a conserved amino acid residue) and such sub ⁇ tituted amino acid re ⁇ idue may or may not be one encoded by the genetic code, or (ii) one in which one or more of the amino acid re ⁇ idue ⁇ include ⁇ a substituent group, or (iii) one in which the mature polypeptide ⁇ are fu ⁇ ed with another compound, ⁇ uch a ⁇ a compound to increa ⁇ e the half-life of the polypeptide (for example, polyethylene glycol) , or (iv) one in which the additional amino acid ⁇ are fu ⁇ ed to the mature polypeptide ⁇ , such as a leader or secretory sequence or a sequence which is employed for purification
• the pre ⁇ ent invention al ⁇ o include ⁇ recombinant con ⁇ truct ⁇ compri ⁇ ing one or more of the ⁇ equence ⁇ a ⁇ broadly de ⁇ cribed above.
• the con ⁇ truct ⁇ compri ⁇ e a vector, ⁇ uch a ⁇ a pla ⁇ mid or viral vector, into which a ⁇ equence of the invention ha ⁇ been inserted, in a forward or reverse orientation.
• the construct further comprises regulatory sequences, including, for example, a promoter, operably linked to the sequence. Large numbers of suitable vector ⁇ and promoters are known to those of skill in the art, and are commercially available.
• the following vector ⁇ are provided by way of example.
• mutation ⁇ can al ⁇ o be detected by in situ analy ⁇ i ⁇ .
• a detectable reagent such as a ⁇ radioactivity, fluore ⁇ cence or, in thi ⁇ example, a hor ⁇ eradi ⁇ h peroxida ⁇ e enzyme.
• a ⁇ araple i ⁇ removed from a ho ⁇ t and incubated on a ⁇ olid ⁇ upport, e.g. a_ poly ⁇ tyrene di ⁇ h, that bind ⁇ the protein ⁇ in the ⁇ ample. Any free protein binding ⁇ ite ⁇ on the di ⁇ h are then covered by incubating with a non-specific protein like BSA.
• the vector ⁇ include one or more ⁇ uitable promoter ⁇ which include, but are not limited to, the retroviral LTR; the SV40 promoter; and the human cytomegaloviru ⁇ (CMV) promoter de ⁇ cribed in Miller, et al., Biotechni ⁇ ue ⁇ , Vol. 7, No. 9, 980-990 (1989) , or any other promoter (e.g., cellular promoter ⁇ ⁇ uch a ⁇ eukaryotic cellular promoters including, but not limited to, the hi ⁇ tone, pol III, and /3-actin promoters) .
• a suitable promoter will be apparent to those skilled in the art from the teachings contained herein.
• the polypeptide ⁇ , their fragment ⁇ or other derivatives, or analogs thereof, or cells expres ⁇ ing them can be u ⁇ ed a ⁇ an immunogen to produce antibodies thereto.
• These antibodies can be, for example, polyclonal or monoclonal antibodie ⁇ .
• the pre ⁇ ent invention al ⁇ o include ⁇ chimeric, single chain and humanized antibodies, as well as Fab fragments, or the product of an Fab expre ⁇ ion library. Variou ⁇ procedure ⁇ known in the art may be u ⁇ ed for the production of ⁇ uch antibodie ⁇ and fragment ⁇ .
• the DNA ⁇ equence encoding Ck/3-8, ATCC # 75676, wa ⁇ initially amplified u ⁇ ing PCR oligonucleotide primer ⁇ corre ⁇ ponding to the 5' and 3' end ⁇ equences of the processed Ck/3-8 protein (minus the ⁇ ignal peptide sequence) and the vector sequence ⁇ 3' to the Ck/3-8 gene. Additional nucleotide ⁇ corre ⁇ ponding to Bam HI and Xbal were added to the 5' and 3' ⁇ equence ⁇ re ⁇ pectively.
• the 5' oligonucleotide primer ha ⁇ the sequence 5' TCAGGATCCGTCACAAAAGATGCAGA 3' (SEQ ID No.
• PQE-9 encode ⁇ antibiotic resistance (Amp r ) , a bacterial origin of replication (ori) , an IPTG-regulatable promoter operator (P/0) , a ribosome binding ⁇ ite (RBS) , a 6-Hi ⁇ tag and restriction enzyme ⁇ ite ⁇ .
• pQE-9 was then digested with BamHI and Xbal and the amplified sequence ⁇ were ligated into PQE-9 and were in ⁇ erted in frame with the ⁇ equence encoding for the histidine tag and the RBS.
• the ligation mixture wa ⁇ then used to transform E. coli strain available from Qiagen under the trademark M15/rep 4.
• CMV-MIP-4 HA i ⁇ derived from a vector pcDNAI/Amp (Invitrogen) containing: 1) SV40 origin of replication, 2) ampicillin re ⁇ i ⁇ tance gene, 3) E.coli replication origin, 4) CMV promoter followed by a polylinker region, a SV40 intron and polyadenylation ⁇ ite.
• a DNA fragment encoding the entire MIP-4 precur ⁇ or and a HA tag fu ⁇ ed in frame to its 3' end is cloned into the polylinker region of the vector, therefore, the recombinant protein expression is directed under the CMV promoter.

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