KR20020057976A - Methods of treating or preventing cell, tissue, and organ damage using human myeloid progenitor inhibitory factor-1(mpif-1) - Google Patents

Abstract

Therapeutic compositions using human nucleic acid precursor inhibitor-1 (MPIF-1) polypeptides (conventionally referred to as MIP-3 and zpahzls? 8 (CK? 8 or ckb-8)) and isolated nucleic acid molecules encoding MPIF- Methods and vectors, host cells and recombinant methods for producing them.

Description

FIELD OF THE INVENTION [0001] The present invention relates to a method for treating or preventing cell, tissue and organ damage using human myeloid precursor inhibitor-1 (MPIF-1) }

Chemokines, also called intercrine cytokines, are structurally functionally related cytokine subfamilies. The size of these molecules is 8-14 kd. In general, chemokines are 20% to 75% homologous to amino acid levels and are characterized by four conserved cysteine residues that form two disulfide bonds. Based on the arrangement of the first two cysteine residues, chemokines were divided into two subfamilies, Alpha and Beta. For the alpha subfamily, the first two cysteines are separated by one amino acid and thereby are referred to as the " C-X-C " subfamily. In the case of the beta subfamily, the two cysteines are in adjacent positions and are therefore referred to as the -C-C-subfamily. Up to now, eight or more different members of this family have been identified in humans.

Interleukin cytokines exhibit a wide variety of functions. A salient feature is the ability to induce chemotactic migration of unique cell types including monocytes, neutrophils, T lymphocytes, basophils, and fibroblasts. Many chemokines possess pro-inflammatory activity and are involved in multiple stages during the inflammatory response. These activities include stimulation of histamine release, release of lysosomal enzymes and leukotrienes, enhancement of adhesion of target immune cells to endothelial cells, enhancement of binding of complement proteins, inducible expression of granulocyte adhesion molecules and complement receptors, and respiratory burst. In addition to participating in the inflammatory response, some chemokines exhibit different activities. For example, macrophage inflammatory protein I (MIP-1) may inhibit hematopoietic stem cell proliferation, platelet factor-4 (PF-4) is a potent endothelial cell growth inhibitor, interleukin- ) Promotes the proliferation of keratinocytes, and GRO is an autocrine growth factor of melanoma cells.

From a variety of biological activities, it is not surprising that chemokines are involved in many physiological and disease states, including lymphocyte communication, wound healing, hematopoietic regulation, and immunological disorders (eg, allergies, asthma, arthritis). An example of a hematopoietic direct lineage regulator is MIP-1. MIP-1 was originally identified as an endotoxin-induced proinflammatory cytokine produced in macrophages. Subsequent studies have shown that MIP-1 is composed of two phases, but the associated proteins MIP-1α and MIP-1β. Both MIP-1α and MIP-1β are chemotaxis factors of macrophages, monocytes and T lymphocytes. Interestingly, biochemical purification and sequencing analysis of pluripotent stem cell inhibitor (ICI) revealed that SCI is identical to MIP-1?. In addition, MIP-1 [beta] offsets the ability of MIP-1 alpha to inhibit hematopoietic stem cell proliferation. These findings lead to the hypothesis that the major physiological role of MIP-1 regulates hematopoiesis in the bone marrow, and that the proposed inflammatory function is secondary. The mode of action of MIP-1α as a stem cell inhibitor is related to its ability to block the cell cycle in the G ₂ S liver. In addition, the inhibitory effect of MIP-1 alpha appears to be restricted to immature progenitor cells, and substantially stimulates the late precursor in the presence of granulocyte macrophage-colony stimulating factor (GM-CSF).

Murine MIP-1 is a major secreted protein from RAW 264.7 (murine macrophage tumor cell line) stimulated by a polysaccharide. It was purified and identified as consisting of two related proteins, MIP-1α and MIP-1β.

Several groups have cloned those that are likely to be human homologues of MIP-1 alpha and MIP-1 beta. In all cases, the cDNA was isolated from the library constructed for activated T-cell RNA.

MIP-1 protein can be detected in early wounded inflammatory cells and induces production of IL-1 and IL-6 from syngeneic fibroblasts. In addition, purified natural MIP-1 (including MIP-1, MIP-1α and MIP-1β polypeptides) causes acute inflammation when injected subcutaneously into the mouse's foodpad or intracisternally into the cerebrospinal fluid of rabbits (Wolpe and Cerami, FASEB J. 3: 2565-73 (1989)). In addition to these pro-inflammatory properties of MIP-1, which may be direct or indirect, MIP-1 was recovered during the early inflammatory phase of wound healing in experimental mouse models using sterile wound chambers (Fahey, et al. Cytokine, 2:92 (1990)). For example, PCT application U.S. Pat. 92/05198 discloses a DNA molecule that is active as a template for producing mammalian macrophage inflammatory proteins (MIPs) in yeast.

Murine MIP-1α and MIP-1β are distinct but closely related cytokines. Partially purified mixtures of these two proteins affect neutrophil function and cause local inflammation and fever. MIP-1α was expressed in yeast cells and was uniformly purified. Structural analysis confirmed that MIP-1α had secondary and tertiary structures very similar to platelet factor 4 (PF-4) and interleukin 8 (IL-8), which share limited sequence homology. In addition, MIP-1 alpha has been shown to be in vivo active in protecting mouse stem cells from in vitro death following tri-thymine, thymidine. MIP-1 alpha has also been shown to enhance the proliferation of more precursor granulosa-macrophage colony-forming cells in response to granulocyte macrophage colony-stimulating factors (Clemens, JM et al., Cytokine 4: 76-82 )).

The polypeptide of the present invention, MPIF-1 (often referred to as MIP-3 and Ck beta-8), is a new member of the beta chemokine family based on amino acid sequence homology.

The present invention uses a separate polynucleotide coding for human myeloid precursor inhibitor-1 (MPIF-1) polypeptide (formerly referred to as MIP-3 and chemokine? 8 (CK? 8 or ckb-8) and MPIF-1 Lt; / RTI > The present invention also provides vectors, host cells and recombinant methods for producing MPIF-1.

The following drawings illustrate specific embodiments of the invention and are not intended to limit the scope of the invention as claimed.

Figure 1 shows the cDNA sequence (SEQ ID NO: 1) encoding MPIF-1 and the corresponding deduced amino acid sequence (SEQ ID NO: 2). The initial 21 amino acids represent the putative leader sequence. Both signal sequences were determined by N-terminal peptide sequencing of proteins expressed in baculovirus.

Figure 2 shows the amino acid homology between MPIF-1 (top) (SEQ ID NO: 2) and human MIP-1 alpha (bottom) (SEQ ID NO: 36). Four cysteine features of all the chemokines are shown.

Figure 3 is an SDS-PAGE gel image after three-step purification of MPIF-1 in expression and baculovirus expression systems.

In Figures 4A-4B, the chemotactic activity of MPIF-1 was determined by chemotaxis assay using a 48-well micro chamber device (Neuro Probe, Inc.). The experimental method is as described in the manufacturer's manual. At each concentration of MPIF-1 tested, migration was examined in a 5-high power field. The resulting results represent the average of the results from two independent experiments. Chemotactic activity is shown for THP-1 (A) cells and human PBMC (B).

Figure 5 shows changes in intracellular calcium concentration in response to MPIF-1 using a Hitachi F-2000 fluorescence spectrophotometer. MPIF-1 expressed in bacteria was added to THP-1 bearing Indo-1 to a final concentration of 50 nM and the intracellular level of calcium concentration was monitored.

Figure 6 shows that a low density population of mouse bone marrow stem cells was stimulated with IL-3 (5 ng / ml), SCF (100 ng / ml), IL-1 alpha (10 ng / ml) M-CSF (5 ng / ml). The data presented represent the mean from two independent experiments (each performed twice). Colonies were counted 14 days after plate culture. The number of colonies formed in the presence of chemokines was expressed as an average percentage of those produced without any additional chemokines.

7A-B show the effect of MPIF-1 and M-CIF on mouse marrow colony formation by HPP-CFC (A) and LPP-CFC (B).

Figure 8 shows the effect of MPIF-1 and M-CIF expressed in baculovirus on M-CFS and SCF-stimulating colony formation of newly isolated bone marrow cells.

Figure 9 shows the effect of MPIF-1 and M-CIF on the proliferation and differentiation of the lin ^- population of IL-3 and SCF-stimulated bone marrow cells.

Figures 10A-B show the effect of MPIF-1 and M-CIF on cell formation of Gr.1 and Mac-1 (surface marker) positive populations from myeloid leukemia bone marrow cells. The growth medium supplemented with IL-3 (5 ng / ml) and SCF (100 ng / ml) alone (a), MPIF-1 (50 ng / ml) (b) or M- lin ^- cells were incubated at room temperature. Subsequently, the cells were treated with bone marrow-differentiated Gr. 1, Mac-1, Sca-1 and CD45R surface antigens and analyzed by FACScan. Data are large (Figure 10A) and are expressed as percent of benign cells in both small (Figure 10B) cell populations.

Figure 11 shows that the presence of MPIF-1 protein inhibits bone marrow cell colony formation in response to IL-3, M-CSF and GM-CSF.

Figure 12 shows that the administration of MPIF-1 inhibits bone marrow cell colony formation. The cells were separated and treated as shown in FIG. The treated cells were cultured in the presence of 1, 10, 50, and 100 ng / ml of MPIF-1 in 1,000 cells in an agar colony formation assay in the presence of IL-3, GM-CSF or M-CSF (5 ng / ml) / Plate density. The data showed colony formation as a percentage of the number of colonies formed by a particular factor alone. The data are presented as the mean of the dishes twice done with the error bars representing the standard deviation.

Figure 13 shows the expression of MPIF-1 encoding RNA in human monocytes. Total RNA from freshly washed mononuclear cells was isolated and treated with 100 U / ml hu rIFN-g, 100 ng / ml LPS, or both. RNA (8 μg) from each treatment was separated by electrophoresis on a 1.2% agarose gel and transferred to a nylon membrane. MPIF-1 mRNA was quantitated by probing with ³² P-labeled cDNA and the resulting radiographic band was quantified as a photomicroscope.

Figure 14 shows the analysis of the MPIF-1 amino acid sequence (SEQ ID NO: 2). Alpha, beta, turn-and-coil region; Hydrophilic and hydrophobic; Amphipatic range; A flexible region; The antigenicity indications and surface probabilities are shown. The amino acid residues 21-30, 31-44, 49-55, 59-67, 72-83, 86-103 and 110-120 in Figure 1 (SEQ ID NO: 2) Or SEQ ID NO: 2 corresponds to the high antigenic region shown in the MPIF-1 protein.

15A-B (A) shows the bone marrow-protective effect of MPIF-1 on 5-Fu-induced death of LPP-CFC cells, Lt; RTI ID = 0.0 > MPIF-1 < / RTI >

Figure 16 shows the effect of mouse MPIF-1 pretreatment on the 5-Fu-inducible reduction of circulating WBC counts.

Figure 17 shows an experimental design comprising three groups of mice (six per group) treated as follows: Group 1, Day 1, Day 2 and Day 3 saline infusion; Group-2, 0 and 3 day 5-Fu injection; And Group 3, Day 0 and Day 3, and MPIF-1 on days 1, 2 and 3. Bone marrow was collected on days 6 and 9 and clonality analysis revealed HPP-CFC and LPP-CFC frequencies Respectively.

Figure 18 shows the effect of MPIF-1 administration on the frequency of HPP-CFC and LPP-CFC in bone marrow prior to the second administration of 5-Fu.

Figure 19 shows MPIF-1 variants. The first 80 of the 120 amino acid sequences of MPIF-1 (Figure 1 (SEQ ID NO: 2)) were represented using a single amino acid letter code, the first 21 residues being the signal sequence cleaved to form the mature wild- . Mutants-1 and -6 contain methionine as an N-terminal residue that does not exist in the wild type. In addition, the first four amino acids (HAAG) of mutant-9 are not present in the wild-type MPIF-1 protein. Mutants-1, -6, and -9 correspond to SEQ ID NOS: 3, 4, and 5, respectively. Mutant-2 corresponds to amino acid residues 46-120 in SEQ ID NO: 2. Mutant-3 corresponds to amino acid residues 45-120 in SEQ ID NO: 2. Mutant-4 corresponds to amino acid residues 48-120 in SEQ ID NO: 2. Mutant-5 corresponds to amino acid residues 49-120 in SEQ ID NO: 2. Mutant-7 corresponds to amino acid residues 39-120 in SEQ ID NO: 2. Mutant-8 corresponds to amino acid residues 44-120 in SEQ ID NO: 2.

In Figures 20A-B, Figure 20A shows the nucleotide sequence (SEQ ID NO: 6) of the human MPIF-1 splice variant cDNA. This cDNA sequence was shown with an open reading frame (SEQ ID NO: 7) coding for a protein of 137 amino acids using a single letter amino acid code. The underlined N-terminal 21 amino acids represent the putative leader sequence. The insertion of the unique 18 amino acid sequence in the splice variant, which did not appear in the MPIF-1 sequence, is highlighted in Italic. Figure 20B compares the amino acid sequence of the MPIF-1 variant (SEQ ID NO: 7) with the amino acid sequence of the wild-type MPIF-1 molecule (SEQ ID NO: 2).

Figure 21 shows the concentration of MPIF-1 mutant protein required for 50% of maximal calcium mobilization induced by MIP-1? In human mononuclear cells.

In Figures 22A-22B, changes in intracellular free calcium levels were measured in human mononuclear cells that responded to 100 ng / ml of the proposed protein as described in Fig.

Figure 23 shows the ability of MPIF-1 mutants to desensitize MIP-1 alpha stimulatory calcium mobilization in human mononuclear cells (Summary).

Figure 24 shows the chemotactic response of human peripheral blood mononuclear cells (PBMC) to MPIF-1 mutants. The number in parentheses reflects the chemotactic stimulation (multiple) above the background observed in the indicated concentration range.

Figure 25 shows the effect of MPIF-1 variants on the growth and differentiation of in vitro hypoproliferative potential colony-forming cells (LPP-CFC).

Figure 26 shows stem cell mobilization in normal mice in response to administration of MPIF-1.

Figure 27 shows a comparison of the effects of G-CSF and MPIF-1 on platelet recovery after two 5-Fu treatments as measured by the FACS Vantage method.

Figure 28 shows a comparison of the effects of G-CSF and MPIF-1 on the recovery of Gra.1 and Mac.1 double positive cells in the blood after treatment with 5-Fu twice.

Figure 29 shows a comparison of the effects of G-CSF and MPIF-1 on the recovery of bone marrow Gra.1 and Mac.1 double positive cells after treatment with 5-Fu as measured by the FACS Vantage method.

Figure 30 shows a comparison of the effects of G-CSF and MPIF-1 on the recovery of intramedullary hematopoietic precursors during two rounds of treatment with 5-Fu.

Figure 31 shows a diagrammatic illustration of the pHE4-5 expression vector (SEQ ID NO: 37) and the subcloned MPIF-1Δ23 cDNA coding sequence. The positions of the kanamycin resistance marker gene, MPIF-1? 23 coding sequence, oriC sequence and Iac Iq coding sequence are shown.

32 shows a schematic diagram of a fermentation process for MPIF-1? 23 production.

33 shows a flow chart of the method used to recover MPIF-1? 23 produced by the process shown in FIG.

Fig. 34 shows a purification process of MPIF-1? 23 produced and recovered by the process shown in Figs. 32 and 33. Fig.

Figure 35 shows the nucleotide sequence (SEQ ID NO: 38) of the regulatory element of the pHE promoter. Two lac operator sequences, the Shine-Delano sequence (S / D), and the Hind III and Nde I restriction sites (deoxyribonucleotide) are shown.

Figures 36A-E show the complete nucleotide sequence (SEQ ID NO: 37) of the pHE4-5 vector.

Figure 37 shows MPIF-I protection of the gastrointestinal tract from radiation-induced damage during short-term monitoring. C57Bl / 6 female mice were treated either before or after receiving a dose below the lethal dose (2x4.5gy 4 hours apart from the ¹³⁷ Cs supply). The survival, condition and weight of the mice were monitored on the date shown. The data represent the percentage change in weight in each group based on the individual mouse weights of the day as a percentage of the weight of the mouse at the start of the experiment.

Figure 38 shows MPIF-I protection of the gastrointestinal tract from radiation-induced damage during long-term monitoring. C57Bl / 6 female mice were treated either before or after receiving a dose below the lethal dose (2x4.5gy 4 hours apart from the ¹³⁷ Cs supply). The survival, condition and weight of the mice were monitored on the date shown. The data represent the percentage change in weight in each group based on the individual mouse weights of the day as a percentage of the weight of the mouse at the start of the experiment. The curve is shown in two fractions. The left fraction represents the change within the first 18 days and the right fraction represents the weight of all groups at the end date.

Figure 39 shows MPIF-I protection of the gastrointestinal tract from radiation-induced damage during short-term monitoring. C57Bl / 6 female mice were treated either before or after receiving a dose below the lethal dose (2x5.5gy 4 hours apart from the ¹³⁷ Cs supply). The survival, condition and weight of the mice were monitored on the date shown. The data represent the percentage change in weight in each group based on the individual mouse weights of the day as a percentage of the weight of the mouse at the start of the experiment.

Figure 40 shows MPIF-I protection of the gastrointestinal tract from radiation-induced damage during long-term monitoring. C57Bl / 6 female mice were treated either before or after receiving a dose below the lethal dose (2x5.5gy 4 hours apart from the ¹³⁷ Cs supply). The survival, condition and weight of the mice were monitored on the date shown. The data represent the percentage change in weight in each group based on the individual mouse weights of the day as a percentage of the weight of the mouse at the start of the experiment.

Figure 41 is a schematic representation of the treatment schedule of the in vivo protection model for lethal irradiation in mice.

Figure 42 shows that MPIF-1 increases survival in mice irradiated with a lethal dose. Statistical analysis was performed using a log-rank nonparametric indicator (nonparametric) and the data are presented as Kaplan-Meier survival curves. The experiment was terminated at day 54 after irradiation.

Figure 43 is an illustration of the treatment schedule of the in vivo protection model for radiation exposure below the lethal dose in the mouse.

Figure 44 shows that MPIF increases the recovery of the immediate-onset bone marrow precursor in irradiated mice below the lethal dose.

Figure 45 shows that MPIF increases the recovery of multipotent bone marrow precursors in irradiated mice below the lethal dose.

Figure 46 shows the effect of MPIF on the proliferation of human CD34 + precursor cells in vitro.

Figure 47 shows the effect of MPIF on toxic drug-induced death in vitro.

Figure 48 shows a summary of in vivo studies of MPIF.

Figures 49A-B show the effect of MPIF pretreatment on 5-FU induced toxicity in vivo myeloid precursor cells. (A) MPIF effect on total colony formation. (B) The effect of MPIF on the recovery of WBC. The results are the average of eight experiments.

Figure 50 shows the chemical protective effect of MPIF on multidisciplinary treatment.

Figure 51 shows the effect of the kinetics of bone marrow recovery after 5-FU treatment on the MPIF dosing schedule.

Figure 52 shows a summary of multi-dose toxicity studies.

Figure 53 shows a summary of observations for nonclinical toxicity studies.

Figures 54A-B show a comparison pf of drug body response of MPIF after intravenous or subcutaneous administration. (A) the drug body response profile at 0 to 24 hours. (B) is a profile of 0 to 4 hours. The dose of MPIF was 20 mg / kg.

Figure 55 shows the development of peripheral blood cell composition in human subjects.

Figure 56 shows the evolution of mean absolute monocyte counts in healthy human volunteers as a treatment group.

Figure 57 shows the MPIF concentration (ng / ml) in healthy volunteers.

Figures 58A-E show the structure of MPIF-1. (A) and (B) show the average structure of MPIF-1, superposition of 30 iridescent annealing (SA) structures to residues 1-77. (C) is identical to A and B except that the N-terminal (1-10) and C-terminal (67-77) residues are omitted for clarity. (D) and (E) illustrate the mapping of MPIF-1 in the same orientation as shown in panel C formed using the program MOLMOL (Koradi, R., et al., J. Mol. Graph. 14: 29-42 (1996)).

Figures 59A-59F show atomic rms distributions of thirty irritant annealing structures for an average structure best suited for residues 11-66 for skeletal atoms (A) and all heavy atoms (B). It also shows the angle grade parameter (S) and the fraction solvent access area (F) for φ (C), for φ (D) and for χ1 (E).

Figures 60A-60G show ¹⁵ N dynamics data of MPIF-1 as a function of moiety. ¹⁵ NT ₁ T ₂ , T ₁ / T ₂ ratio, and NOE are shown in panels A, B, C and D, respectively. ¹⁵ NT ₁ T ₂ , the kinetic parameters calculated from the adjustment of NOE data are shown in the remaining panel; Grade parameter, S ² (E); The internal association time, Te (F), and the rate of change of form (G).

Figures 61A-61D show a comparison of MPIF-1 and other CC chemokine structures, i.e., MIP-l [beta], HCC-2, RANTES and MCP-I. Residues 11 to 66 of MPIF-1 overlap residues 11 to 66 of MIP-1?, Residues 6 to 61 of HCC-2, residues 10 to 65 of RANTES and residues 11 to 67 of MCP-1. N-and C-terminal residues were not indicated for clarity.

Figure 62 shows the amino acid sequence alignment of MPIF-1 and other related CC chemokines. Conserved cysteine is shown in bold type. The conserved hydrophobic moiety and the conserved charged moieties are described in practice 36.

Figures 63A-63D show the surface charge distributions of MPIF-1 using the program MOLMOL (Koradi, R., et al., J. Mol. Graph. 14: 29-42 (1996)). Positive and negative charge areas are shown in blue and red, respectively. For clarity, residues 1-10 and 69-77 are not shown.

[Sequence List]

SUMMARY OF THE INVENTION

According to one aspect of the present invention there is provided a method of preventing or treating wounds in cells, tissues and organs using biologically active, diagnostically useful or therapeutically useful fragments, analogs and derivatives as well as full-length or mature MPIF-1 polypeptides Lt; / RTI >

In another aspect, the invention provides a method of treating or preventing using an isolated polynucleotide encoding the MPIF-1 polypeptide. The MPIF-1 of the present invention is preferably derived from an animal, more preferably from a human.

The present invention also relates to isolated MPIF-1 polypeptides and isolated polynucleotides (DNA and RNA) (including mRNA, DNA, cDNA, and genomic DNA) encoding the polypeptides as well as biologically active, diagnostic or therapeutically useful Fragments, analogs and derivatives thereof.

MPIF-1 polynucleotide.

The invention also provides a polynucleotide encoding the MPIF-1 polypeptide having an amino acid sequence encoded by the amino acid sequence shown in Figure 1 (SEQ ID NO: 2) or the cDNA clone deposited with the ATCC Deposit No. 75676 on March 29, 1994 Or provide a separate nucleic acid molecule comprising the same. The nucleotide sequence determined by sequencing the deposited MPIF-1 clone is shown in Figure 1 (SEQ ID NO: 1) and includes an open reading frame encoding a polypeptide of 120 amino acid residues along with a leader sequence of about 21 amino acid residues , And the expected molecular weight of the mature protein is about 11 kDa in the non-glycosylated form and the glycosylated form is about 11-14 kDa depending on the degree of glycosylation. The amino acid sequence of the mature MPIF-1 protein is shown in Figure 1 (SEQ ID NO: 2).

Thus, one aspect of the present invention is a polypeptide comprising: (a) a nucleotide sequence encoding a MPIF-1 polypeptide having the complete amino acid sequence of Figure 1 (SEQ ID NO: 2); (b) a nucleotide sequence encoding the MPIF-1 polypeptide having the complete amino acid sequence of Figure 1 (SEQ ID NO: 2) without an N-terminal methionine residue; (c) a nucleotide sequence encoding a mature MPIF-1 polypeptide having an amino acid sequence at positions 22-120 of Figure 1 (SEQ ID NO: 2); (d) a nucleotide sequence encoding a MPIF-1 polypeptide having the complete amino acid sequence encoded by the cDNA clone contained in ATCC Deposit No. 75676; (e) a nucleotide sequence encoding a mature MPIF-1 polypeptide having an amino acid sequence encoded by a cDNA clone contained in ATCC Deposit No. 75676; And (f) a polynucleotide having a nucleotide sequence selected from the group consisting of a nucleotide sequence complementary to any of the nucleotide sequences of (a), (b), (c), (d) Lt; RTI ID = 0.0 > isolated < / RTI >

MPIF-1 polynucleotide variants.

The present invention also relates to variants of the polynucleotides encoding the fragments, analogs and derivatives of polypeptides encoded by the cDNA of the deduced amino acid sequence of Figure 1 (SEQ ID NO: 2) or the cDNA of the deposited clone (s) will be. Polynucleotide variants may be naturally-occurring allelic variants or non-naturally occurring variants of the polynucleotides.

Homologous MPIF-1 polynucleotide.

An embodiment of the present invention is a nucleic acid molecule comprising 95%, 96%, 97%, 98% or 99% of any nucleotide sequence of any of the above (a), (b), (c), (d), (e) Or a polynucleotide hybridizing under strict hybridization conditions with a polynucleotide having the same nucleotide sequence or any polynucleotide of (a), (b), (c), (d), (e) ≪ / RTI > These hybridizing polynucleotides do not hybridize under stringent hybridization conditions with polynucleotides having nucleotide sequences consisting of only A residues or only T residues.

Nucleic acid probe.

According to another aspect of the present invention, there is provided a nucleic acid probe comprising or consisting of a nucleic acid molecule having a sufficient length to specifically hybridize with an MPIF-1 nucleic acid sequence.

Recombinant vectors, host cells and expression.

The present invention provides recombinant vectors comprising the isolated nucleic acid molecules of the present invention, host cells containing the recombinant vectors, methods of producing the vectors and host cells, and recombinant techniques to produce MPIF-I polypeptides or peptides .

MPIF-1 polypeptide.

(A) the amino acid sequence of an MPIF-1 polypeptide having a complete 120 amino acid sequence including the leader sequence shown in Figure 1 (SEQ ID NO: 2); (b) an amino acid sequence of MPIF-1 polypeptide having a complete 120 amino acid sequence including the leader sequence shown in Figure 1 (SEQ ID NO: 2) without an N-terminal methionine residue; (c) the amino acid sequence of the mature MPIF-1 polypeptide (without leader) having the amino acid sequence at position 22-120 in Figure 1 (SEQ ID NO: 2); (d) an amino acid sequence of an MPIF-1 polypeptide having a complete amino acid sequence encoded by a cDNA clone contained in ATCC Accession No. 75676 and comprising a leader; And (e) an amino acid sequence selected from the group consisting of the amino acid sequence of a mature MPIF-1 polypeptide having an amino acid sequence encoded by a cDNA clone contained in ATCC Deposit No. 75676.

Homologous MPIF-1 polypeptide.

The polypeptide of the present invention may also comprise a homologous polypeptide having an amino acid sequence that is at least 95% identical to the sequences described in (a), (b), (c), (d) or (e) above, And 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence of SEQ ID NO: 1.

MPIF-1 epitope-bearing polypeptides and polynucleotides encoding them.

A further embodiment of this aspect of the invention is the use of an MPIF-1 polypeptide having the amino acid sequence of an epitope-bearing site in an MPIF-1 polypeptide having an amino acid sequence as set forth in (a), (b), (c), (d) Peptide < / RTI > or polypeptide. Peptides or polypeptides having the amino acid sequence of the epitope-bearing site in the MPIF-1 polypeptides of the present invention comprise at least 6 or at least 7, preferably at least 9, more preferably at least about 30 amino acids to at least about 50 amino acids Containing epitope-bearing polypeptides of any length up to and including the full amino acid sequence of the polypeptides of the invention described above, as well as epitope-bearing polypeptides comprising the entire amino acid sequence, are also encompassed by the invention.

An additional nucleic acid embodiment of the invention is a nucleic acid encoding a polypeptide encoding the amino acid sequence of the epitope-bearing site in the MPIF-1 polypeptide having the amino acid sequence of (a), (b), (c), (d) Or to a separate nucleic acid molecule comprising or consisting < RTI ID = 0.0 > of < / RTI >

MPIF-1 antibody.

According to another aspect of the present invention there is provided an antibody against said polypeptide. In another embodiment, the invention provides isolated antibodies that specifically bind to an MPIF-I polypeptide having the amino acid sequence set forth in (a), (b), (c), (d) .

Further, the present invention provides a method for isolating an antibody that specifically binds to an MPIF-I polypeptide having the amino acid sequence described herein. These antibodies are useful diagnostically or therapeutically as described below.

MPIF-1 antagonists and methods.

In accordance with another aspect of the present invention there is provided an antagonist or an inhibitor of said polypeptide, said antagonist or inhibitor being selected from the group consisting of arteriosclerosis, autoimmune and chronic inflammatory and infectious diseases, histamine- mediated allergic reactions, , Pulmonary inflammatory disease, inhibition of IL-1 and TNF, aplastic anemia and myelodysplastic syndrome. Alternatively, the polypeptides may be used to inhibit the production of IL-1 and TNF- [alpha] and to treat aplastic anemia, myelodysplastic syndrome, asthma and arthritis.

Diagnostic analysis.

According to another aspect of the present invention, there is provided a diagnostic assay for detecting a disease associated with under-expression and over-expression of the polypeptide, and a diagnostic assay for detecting a mutation in a nucleic acid sequence encoding the polypeptide.

In accordance with another aspect of the present invention there is provided a method of screening a polypeptide or a polypeptide encoded by the polypeptide as a research reagent for the purpose of developing therapeutic and diagnostic methods for the treatment of human disease for in vitro purposes involving scientific research, A method of using a polynucleotide is provided.

The present invention also provides a screening method for identifying a compound capable of enhancing or inhibiting a cellular response induced by an MPIF-1 polypeptide, the method comprising contacting a cell expressing MPIF-1 polypeptide with a candidate compound Analyzing the cellular response, and comparing the cellular response to a standard cellular response, wherein the standard is analyzed when contacted without the candidate compound; Thus, an increased cellular response than the standard suggests that the compound is an agonist, and a cellular response that is less than the standard suggests that the compound is an antagonist.

Significantly higher or lower levels of MPIF-1 gene expression relative to " standard " MPIF-1 gene expression levels (i. E. MPIF-1 expression levels in tissues or body fluids from unaffected individuals) (E. G., Serum, plasma, urine, synovial fluid, or spinal fluid) taken from an individual suffering from a < / RTI > Accordingly, the present invention provides a diagnostic method useful during diagnosis of a disorder, comprising: (a) analyzing MPIF-1 gene expression levels in a cell or body fluids of an individual; (b) comparing the standard MPIF-1 gene expression level to the MPIF-1 gene expression level, and thus the increase or decrease in MPIF-1 gene expression levels analyzed relative to standard expression levels indicates a disorder . Such disorders include leukemia, chronic inflammation, autoimmune diseases, solid tumors, and toxicity from radiation and chemotherapy.

A pharmaceutical composition.

In another aspect, the present invention provides a pharmaceutical composition comprising MPIF-1 polynucleotide, a probe, a vector, a host cell, a polypeptide, a fragment, a variant, a derivative, an epitope-bearing site, an antibody, an antagonist or an agonist.

Treatment method.

In accordance with another aspect of the present invention, there is provided a method for treating bone marrow stem cells, comprising the steps of protecting bone marrow stem cells from chemotherapeutic agents for therapeutic purposes, such as chemotherapy, removing leukemia cells, stimulating immune responses, regulating hematopoietic and lymphocyte communication, Methods of using such polypeptides or polynucleotides encoding the polypeptides for the purpose of treating tumors and enhancing host defense against resistance, acute, chronic infection, and stimulating wound healing are provided.

An additional aspect of the invention relates to a method of treating a subject suffering from an anxiety disorder having an increased level of MPIF-1 activity in the body, comprising administering to the individual an effective amount of a separate MPIF-I polypeptide of the invention or an agonist thereof And administering the composition to the subject.

Another aspect of the present invention relates to a method of treating a subject having a reduced level of MPIF-1 activity in the body, comprising the step of administering to said subject a composition comprising a therapeutically effective amount of MPIF-1 antagonist .

These and other aspects of the invention will be apparent to those skilled in the art from the teachings herein.

A detailed description of the preferred embodiment

The present invention provides isolated polynucleotide molecules encoding a polypeptide as a human myeloid precursor inhibitor-1 (MPIF-1) polypeptide (formerly referred to as MIP-3 and chemokine? 8 (CK? 8 or ckb-8) Provides a diagnostic or therapeutic composition and method that utilizes itself, provides a vector, host cell, and recombinant or synthetic method of producing it.

MPIF-1 polynucleotide

According to one aspect of the invention, isolated nucleic acids (polynucleotides) encoding full length or mature MPIF-1 polypeptides having the deduced amino acid sequence of Figure 1 (SEQ ID NO: 2) and ATCC Accession No. 75676 To provide isolated nucleic acids of the mature MPIF-1 polypeptide encoded by the cDNA of the deposited clone. The address of the United States Bacteriological Culture Collection is Manassas University, Bullard 10801, Patents Depository, Virginia, USA 20110-2209. The deposited clones are contained in the pBluescriptSK (-) plasmid (Stratagene, LaJolla, Calif.).

The deposit (s) will remain under the Budapest Treaty on the International Recognition of Microorganism Deposit under the Patent Procedure. These deposits are not intended to be a deposit required by 35 USC § 112, merely providing ease to those skilled in the art. The amino acid sequence of the polynucleotide encoded thereby as well as the sequence of the polynucleotide contained in the deposited substance is hereby incorporated and adjusted if contradictory to the sequence description herein. A license is required to manufacture, use or sell the deposited material, and such license is not granted by this application.

Polynucleotides encoding the polypeptides of the invention are structurally related to the proinflammatory super gene " interleukin " in the cytokine or chemokine family. MPIF-1 is homologous to MIP-1 and has greater homology with MIP-1α than MIP-1β. The polynucleotide encoding MPIF-1, derived from the aortic endothelial cDNA library, contains an open reading frame encoding a polypeptide of 120 amino acid residues and exhibits substantial homology with a large number of chemokines. The largest matches are human macrophage inflammatory protein 1 alpha, exhibiting 36% identity and 66% similarity (Figure 2).

The polynucleotide of the present invention may be in an RNA form or a DNA form, and the DNA includes cDNA, genomic DNA, and synthetic DNA. The DNA can be double-stranded or single-stranded, and in the case of a single strand it can be a cryptic strand or a non-cryptic (antisense) strand. The coding sequence coding for the mature polypeptide may be identical to the coding sequence of the coding sequence shown in Fig. 1 or 20A (SEQ ID NO: 1 or 6) or the deposited clone, or the coding sequence resulting from redundancy or degeneracy of the genetic code Or 20A (SEQ ID NO: 1 or 6), or a different coded sequence encoding the same mature polypeptide, such as a deposited cDNA.

The polynucleotide encoding the mature polypeptide of Figure 1 (SEQ ID NO: 2) or the mature polypeptide encoded by the deposited cDNA is only the coding sequence of the mature polypeptide; A secretion sequence of a mature polypeptide, and an additional secretion sequence such as a leader or secretory sequence or a proprotein sequence; (And optionally additional coding sequences) of mature polypeptides and non-coding sequences (e.g., non-coding sequences 5 'and / or 3' of coding sequences of introns or mature polypeptides).

A " polynucleotide encoding a polypeptide " includes a polynucleotide comprising only the coding sequence for the polypeptide, as well as a polynucleotide comprising additional coding and / or non-coding sequences.

In addition, the present invention relates to a cDNA sequence contained in a polynucleotide sequence of SEQ ID NO: 1 or 6, a complementary strand thereof, and / or a clone deposited.

&Quot; Variant " means a polynucleotide or polypeptide that differs from an MPIF-1 polynucleotide or polypeptide, but retains important characteristics thereof. Typically, variants are closely similar to MPIF-I polynucleotides or polypeptides and are identical in many regions.

Unless otherwise indicated, all nucleotide sequences determined by sequencing DNA molecules herein were determined using an automated DNA sequencer (e.g., Model 373 from Applied Biosystems, Inc.), and the DNA determined herein All amino acid sequences of the polypeptide encoded by the molecule were predicted by translation of the determined DNA sequence. Thus, as is known in the art for any DNA sequence determined by the automated approach, any nucleotide sequence determined herein may contain several real numbers. The nucleotide sequence determined by automation has generally at least about 90% identity, more usually at least about 95% identity, and at least about 99.9% identity to the actual nucleotide sequence of the sequenced DNA molecule. The actual sequence may be determined more accurately by other approaches including manual DNA sequencing methods well known in the art. Also, as is known in the art, a single insertion or deletion in a nucleotide sequence determined relative to an actual sequence results in a frame shift in the translation of the nucleotide sequence so that the predicted amino acid sequence encoded by the determined nucleotide sequence is not Will be completely different from the amino acid sequence that is substantially encoded by the DNA molecule sequenced starting from the deletion site.

Unless otherwise indicated, each " nucleotide sequence " described herein is represented by the sequence of deoxyribonucleotides (abbreviated as A, G, C, and T). However, the " nucleotide sequence " of a nucleic acid molecule or polynucleotide refers to a sequence of deoxyribonucleotides in the case of DNA molecules or polynucleotides and corresponding sequences (A, G, C and U) of ribonucleotides in the case of RNA molecules or polynucleotides , Wherein each thymidine deoxyribonucleotide (T) in a particular deoxyribonucleotide sequence is replaced by a ribonucleotide uridine (U). For example, referring to an RNA molecule having the sequence of SEQ ID NO: 1 or 6 described using the abbreviation deoxyribonucleotide, it is preferred that each deoxyribonucleotide A, G, or C of SEQ ID NO: 1 or 6 is the corresponding ribonucleotide A, G, or C, and each deoxyribonucleotide T is intended to point to an RNA molecule having a sequence that has been replaced by a ribonucleotide U.

Using the information provided herein, nucleic acid molecules of the invention encoding the MPIF-1 polypeptide, such as the nucleotide sequence of Figure 1, can be obtained using standard cloning and screening procedures, such as cDNA cloning using mRNA as a starting material have.

The present invention also relates to a variant of the aforementioned polynucleotide encoding a polypeptide having an inferring amino acid sequence of FIG. 1 (SEQ ID NO: 2) or fragments, analogs and derivatives of a polypeptide encoded by the cDNA of a deposited clone. Variants of polynucleotides may be naturally occurring allelic variants of the polynucleotides or non-naturally occurring variants of the nucleotides.

The present invention also encompasses polynucleotides encoding the same mature polypeptide encoded by the same mature polypeptide as shown in Figure 1 (SEQ ID NO: 2) or the cDNA of a deposited clone, as well as variants of said polynucleotides (SEQ ID NO: 2), or fragments, derivatives or analogs of the polypeptide encoded by the cDNA of the deposited clone). Such nucleotide variants include deletion mutants, substitution mutants, and additions or insertion mutants.

As indicated above. The polynucleotide may have the coding sequence shown in Figure 1 (SEQ ID NO: 2) or a coding sequence which is a naturally occurring allelic variant of the coding sequence of the deposited clone. As is known in the art, allelic variants are another form of polynucleotide sequence that may have substitution, deletion, or addition of one or more nucleotides and do not substantially alter the function of the encoded polypeptide.

The present invention also relates to a method of screening a mature polypeptide for which the coding sequence has a polynucleotide sequence that aids in the expression and secretion of the polypeptide from the host cell, such as a leader sequence that functions as a secretory sequence that regulates the transport of the polypeptide from the cell, Lt; RTI ID = 0.0 > polynucleotides < / RTI > The polypeptide having the leader sequence is a pre-protein and the leader sequence can be cleaved by the host cell to produce a polypeptide in a mature form. The polynucleotide can also encode a proprotein that is a mature protein plus an additional N-terminal amino acid residue. A mature protein with a prosequence is a pro-protein and an inactive form of the protein. Thus, for example, the polynucleotide of the present invention may be a mature protein, a protein having a prosequence, or a protein having both a prosequence and a free sequence (leader sequence). Can be encrypted.

The polynucleotides of the present invention may also have a coded sequence fused in the frame to a marker sequence enabling purification of the polypeptide of the present invention. The marker sequence may be a hexa histidine tag provided by the pQE-9 vector providing for purification of the mature polypeptide fused to the marker in the case of a bacterial host, or the marker sequence may be a mammalian host such as a < RTI ID = Tag may be a maglutinin (HA) tag. The HA tag corresponds to an epitope derived from the influenza hemagglutinin protein (Wilson, I., et al., Cell, 37; 767 (1984)).

&Quot; Gene " means a DNA fraction involved in producing a polypeptide chain; As well as intervening sequences (introns) between each coding fragments (exons) as well as before and after coding regions (leader and tailers).

As noted, the nucleic acid molecules of the present invention may be in the form of RNA, such as mRNA, and may be in the form of DNA, including cDNA and genomic DNA, obtained, for example, by cloning or synthetically produced. The DNA may be double-stranded or single-stranded. Single stranded DNA or RNA may be a cryptic strand, also known as a sense strand, or a non-cryptic strand, also referred to as an antisense strand.

"Separated" means that the material is removed from the original environment (eg, the natural environment if it is naturally occurring). For example, the naturally occurring polynucleotide or polypeptide present in a living animal is not segregated, but the same polynucleotide or DNA or polypeptide separated from all or part of the coexisting material in the natural system is isolated. Such polynucleotides may be part of a vector and / or such polynucleotides or polypeptides may be part of a composition and still be isolated because the vector or composition is not part of its natural environment. The isolated RNA molecule comprises an in vivo or in vitro RNA transcript of the DNA molecule of the present invention. Further, the isolated nucleic acid molecule according to the present invention comprises molecules produced synthetically. However, nucleic acids contained in one clone that are members of a library (e. G., A genomic or cDNA library) and not separated from other members of the library (e. G., In the form of a homogeneous solution containing clones and other members of the library) Nucleic acids present in chromosome formulations (e.g., chromosomal spreads), or nucleic acids present in genomic DNA (e.g., intact, sheared and / or truncated on one or more restriction enzymes) that are not separated by other nucleic acids in the formulation Quot; isolated " for purposes of the present invention.

The isolated nucleic acid molecule of the present invention comprises a DNA molecule comprising or consisting of the open reading frame (ORF) of MPIF-1 cDNA; A DNA molecule comprising or consisting of the coding sequence of a mature MPIF-1 protein; And DNA molecules that encode a MPIF-1 polypeptide that still contains a sequence that is substantially different from the DNA molecule described above, but still encodes a gene encoding an MPIF-1 polypeptide. Of course, the genetic code is well known in the art. Thus, it is routine in the art to form the above-described dextrose variants.

Further, the present invention relates to polynucleotides that hybridize to the above sequences when there is at least 95% identity between the sequences. The present invention relates in particular to polynucleotides that hybridize under stringent conditions with the above-mentioned polynucleotides. As used herein, " stringent conditions " means that hybridization will occur only when the identity between sequences is at least 95%, preferably at least 97%. In a preferred embodiment, the polynucleotide hybridizing with the above-described polynucleotide encodes a polypeptide having substantially the same biological function or activity as the mature polypeptide encoded by the cDNA of Fig. 1 (SEQ ID NO: 1) or the deposited cDNA.

Alternatively, the polynucleotide may hybridize with the polynucleotide of the present invention and have 20 or more bases, preferably 30 or more bases, more preferably 50 or more bases, which have the same identity and may or may not maintain activity as follows Base. ≪ / RTI > For example, such polynucleotides can be used as probes for the polynucleotides of SEQ ID NO: 1, for example as polynucleotide recruitment probes or as diagnostic probes or as PCR primers.

In another aspect, the invention includes a polynucleotide that hybridizes under stringent hybridization conditions with a portion of the polynucleotide in the nucleic acid molecule of the invention described above (eg, a cDNA clone (MPIF-1) contained in ATCC Deposit 75676) Or an isolated nucleic acid molecule comprising the same. &Quot; Strictly hybridizing conditions " include 50% formamide, 5xSSC (750mM NaCl, 75mM trisodium citrate), 50mM sodium phosphate (pH 7.6), 5x Denhardt's solution, 10% sulfuric acid dextran, Lt; RTI ID = 0.0 > 65 C < / RTI > 0.1 x SSC.

Polynucleotides that hybridize to a " part " of a polynucleotide have a reference polynucleotide of at least about 15 nucleotides (nt), more preferably at least about 20 nt, even more preferably at least about 30 nt, Refers to a polynucleotide (DNA or RNA) that hybridizes. More preferably at least about 30 nucleotides (nt), more preferably at least about 20 nt, even more preferably at least about 25 nt, even more preferably at least about 30 nt, even more preferably at least about 30-70 , 40, 45, 50, 55, 60, 65 and / or 70 (of course, fragments of lengths other than those mentioned herein are also useful) nt or more polynucleotides that hybridize with reference polynucleotides of nt or more. These are useful as diagnostic probes and primers described above and described below.

Of course, a larger region of the reference polynucleotide, such as the nucleotide sequence of the deposited cDNA or the polynucleotide corresponding to most, but not all, of the nucleotide sequence shown in SEQ ID NO: 1 or 6 (MPIF-1) ), Such as a polynucleotide that hybridizes with a reference polynucleotide having a length of 50-750 nt or a full-length polynucleotide, is also useful as a probe according to the present invention. For example, a nucleotide region of "at least 20 nt in length" refers to at least 20 contiguous nucleotides from the nucleotide sequence of a reference polynucleotide. As pointed out, such sites are diagnostic useful as primers for amplification of target sequences by probe or polymerase chain reaction (PCR) according to conventional DNA hybridization techniques (see, e.g., Molecular Cloning, A Laboratory Manual, 2nd edition , Sambrook, J., Fritsch, EF and Maniatis, T., eds., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY (1989), incorporated herein by reference).

It is routine to those skilled in the art to form polynucleotides that hybridize to a portion of the MPIF-1 cDNA molecule, since the MPIF-1 cDNA clone has been deposited and its nucleotide sequence determined. For example, sheep by restriction digestion or sonication of the MPIF-1 cDNA clone can be used to form DNA sites of various sizes that are easily polynucleotides that hybridize to a portion of the MPIF-1 cDNA molecule.

Alternatively, the hybridized polynucleotides of the present invention may be formed by synthesis according to known techniques. Of course, polynucleotides that hybridize only with a complementary stretch of a polyA sequence (e.g., the 3 'terminal Poly (A) track of the cDNA) or the T (or U) Polynucleotides, because such polynucleotides hybridize with any nucleic acid molecule (e.g., virtually any double-stranded cDNA clone) that contains a poly (A) stretch or a complement thereof.

As indicated, nucleic acid molecules of the invention encoding the MPIF-I polypeptide include, but are not limited to, coding the amino acid sequence of the mature polypeptide itself; Coding sequences (e.g., pre- or pro- or prepro-protein sequences) of mature polypeptides and additional sequences (eg, coding for a leader or secretory sequence); (E.g., without limitation, intron and unencrypted 5 'and 3' sequences (eg, transcription, mRNA processing (eg, splicing and polyadenylation signals) in the presence or absence of the above- The coding sequence of a mature polypeptide having a role (e. G., A transcribed or untranslated sequence that undergoes a ribosome binding and stabilization of mRNA); an additional coding sequence encoding additional amino acids (e. G., Sequences providing additional functionality) The sequence encoding the polypeptide may be fused to a marker sequence such as a sequence encoding a peptide that facilitates purification of the fused polypeptide. In some preferred embodiments of this aspect of the invention, The marker amino acid sequence is a hexa-histidine peptide, for example, a tag provided in a pQE vector (Qiagen, Ing.) Among many commercially available. Hexa-histidine is provided for easy purification of the fusion protein, as described in Gentz et al., Proc Natl Acad Sci USA 86: 821-824 (1984). (Wilson et al., Cell 37: 767 (1984)), which corresponds to an epitope derived from the influenza hemagglutinin protein. As discussed below, other such fusion proteins are N- RTI ID = 0.0 > MPIF-I < / RTI > polypeptide or fragment fused at its C-terminus to Fc.

Further, the present invention relates to variants of the nucleic acid molecules of the present invention that encode a portion, analog or derivative of the MPIF-I polypeptide. Variants can occur naturally, such as natural allelic variants. &Quot; Allelic variant " means one of several alternate forms of a gene that occupy a given position on the chromosome of an organism. Genes V, Lewin, B., ed., Oxford University Press, New York (1994). Non-naturally occurring variants can be generated using mutagenesis techniques known in the art.

Such variants are those produced by nucleotide substitutions, deletions or additions. Substitutions, deletions or additions may include one or more nucleotides. Variants can be modified in the coding domain, non-coding domain, or both. Deformation in the coding region can produce conservative or non-conservative amino acid substitutions, deletions or additions. Particularly preferred among these are silent substitutions, additions and deletions that do not alter the properties and activity of the MPIF-I polypeptide or a portion thereof. Conservative substitution is particularly preferred in this connection. Most preferred is a nucleic acid molecule encoding a mature protein or a mature amino acid sequence encoded by a cDNA clone deposited as described above.

MPIF-1 homologous polynucleotide.

Further, the invention relates to a polynucleotide encoding a polypeptide of SEQ ID NO: 2 and fragments thereof (having at least 30 bases, preferably at least 50 bases), and a polypeptide encoded by said polynucleotide, wherein said polypeptide has 95% Gt; polynucleotide < / RTI >

Another embodiment of the present invention is a nucleic acid molecule comprising (a) a nucleotide sequence encoding an MPIF-1 polypeptide or fragment having the amino acid sequence of SEQ ID NO: 2, including the predicted leader sequence; (b) a nucleotide sequence encoding an MPIF-I polypeptide or fragment having the amino acid sequence of SEQ ID NO: 2, including the predicted leader sequence without an N-terminal methionine residue; (c) a nucleotide sequence encoding a mature MPIF-1 polypeptide (a full-length polypeptide from which the leader sequence has been removed); (d) a nucleotide sequence encoding a full-length polypeptide having a complete amino acid sequence including a leader encoded by the deposited cDNA clone; (e) a nucleotide sequence encoding a mature polypeptide having an amino acid sequence encoded by the deposited cDNA clone; Or (f) a nucleotide sequence complementary to any of the nucleotide sequences of (a), (b), (c), (d) 96%, 97%, 98% or 99% identical or more of a polynucleotide having the same nucleotide sequence.

A polynucleotide having a nucleotide sequence that is " identical " for example at least 95% to a reference nucleotide sequence encoding a MPIF-1 polypeptide comprises a polynucleotide sequence comprising a maximum of 5 point mutations per 100 nucleotides in the reference nucleotide sequence encoding the polypeptide , The nucleotide sequence of the polynucleotide is the same as the reference sequence. That is, in order to obtain a polynucleotide having a nucleotide sequence that is at least 95% identical to the reference nucleotide sequence, up to 5% of the nucleotides in the reference sequence may be substituted or deleted with other nucleotides, May be inserted into the reference sequence. These mutations of the reference sequence can occur anywhere between the 5 ' or 3 ' terminal positions of the reference nucleotide sequence, between their terminal positions, occurring either sporadically in the nucleotide in the reference sequence or in one or more contiguous groups in the reference sequence have.

In reality, it is contemplated that any particular nucleic acid molecule may be present in a nucleotide sequence that is 80%, 85%, 90%, 92%, 95%, 96%, 97%, 98% or 99% identical to the nucleotide sequence shown in Figure 1, Or more may be determined using conventional computer programs commonly used, such as the Bestfit program (Wisconsin Sequence Analysis Package, Version 8 for Unix, Genetics Computer Group, University Research Park, 575 Science Drive, Madison, WI 53711). Bestfit uses a local homology algorithm (Advances in Applied mathematics 2: 482-489 (1981)) to clone the best homologous fractions of two sequences. If Bestfit or any other sequence alignment program is used to determine whether a particular sequence is, for example, 95% identical to the reference sequence according to the present invention, of course, the percent identity is calculated for the reference nucleotide of the total length and the nucleotide in the reference sequence Set parameters so that up to 5% of the total number of homologous differences is allowed.

In fact, whether a particular nucleic acid molecule is 80%, 85%, 90%, 92%, 95%, 96%, 97%, 98% or 99% identical to the nucleotide sequence of the present invention is determined by a known computer program And can be determined conventionally. The preferred method for determining the best overall match (also referred to as full sequence alignment) between the query sequence (sequence of the invention) and the sequence of interest can be determined using the FASTDB computer program based on the algorithm of Brutlag et al. (Comp. App., Biosci., 6: 237-245 (1990)). In the sequence listing, both the query sequence and the target sequence are DNA sequences. The RNA sequences can also be compared by converting U to T. The result of the full-scale sequence alignment appears as percent identity. The preferred parameters used for FASTDB alignment of the DNA sequence to calculate percent identity are as follows: Matrix = Unitary matrix, k-slice = 4, mismatch penalty = 1, linking penalty = 30, random group length = 0, Cutoff score = 1, gap penalty = 5, gap size penalty = 0.05, window size = 500 or the length of the subject nucleotide sequence (or may be shorter).

If the target sequence is shorter than the query sequence due to 5 'or 3' deletion, not due to internal deletion, the result should be manually corrected. This is because the FASTDB program does not compute the 5 'and 3' truncation of the target sequence when calculating percent identity. In the case of the subject sequence in which the 5 'or 3' end is truncated, the percentage identity is the percentage of the total base of the query sequence, as compared to the query sequence, the base number of the query sequence 5 'and 3' ≪ / RTI > Whether the nucleotides are matched / aligned is determined by the result of the FASTDB sequence alignment. This percentage is then subtracted from the percent identity calculated by the FASTDB program using the specific parameters to obtain the final percent identity score. The corrected score is used for the purpose of the present invention. Only bases outside the 5 'and 3' bases of the target sequence developed by the FASTDB alignment that are not matched / aligned with the query sequence are calculated for the purpose of passively adjusting the percent identity score.

For example, the sequence of 90 bases aligns to the base sequence of 100 bases to determine percent identity. Deletions occur at the 5 'end of the target sequence, so that FASTDB alignment does not show match / alignment in the first 10 bases at the 5' end. The 10 unpaired bases represent 10% of the sequence (the number of bases at the 5 'and 3' ends that do not match / the total number of bases in the query sequence), so 10% is the percent identity score calculated by the FASTDB program Subtract. If the remaining 90 bases are completely identical, the final percent identity is 90%. In another example, 90 target sequences are compared to 100 base sequence doubts. This time, the deletion is an internal deletion, so bases that do not match / align with the query sequence are not on the 5 'or 3' of the target sequence. In this case, the percent identity calculated by FASTDB is not passively corrected. Again, only the 5 ' and 3 ' bases that do not match / align with the query sequence of the target sequence are passively corrected. There are no other passive corrections performed for the purposes of the present invention.

Preferably, the polynucleotides of the present invention (reference sequence) and the program described above for aligning the second sequence are used and the percent identity is calculated manually. The percent identity is the number of identical individual nucleotides between two sequences divided by the total number of nucleotide residues in the reference sequence multiplied by 100%. For example, the number of nucleotide misaligned mismatches (i. E., Point mutations: insertions, deletions and substitutions) are counted to determine the percent identity of the 1 to 360 nucleotides of SEQ ID NO: 1 with the second sequence, This number is subtracted from 360 (the number of nucleotides in the reference sequence) to obtain the same number of nucleotides. Divide the obtained number by 360 and multiply by 100%. (I.e., point mutations: insertion, deletion and substitution of individual nucleotides) at positions 1-5, 18, 201-210, 300, 302, 318-328, 330, 336, 341 and 349-352 of SEQ ID NO: The percent identity is 90% (5 + 1 + 10 + 1 + 1 + 11 + 1 + 1 + 1 + 3 = 36, 360-36 = 324, 324/360 = 0.9, ). The percent identity of the polypeptides is calculated in a similar manner.

As will be appreciated by those skilled in the art, due to the possibility of the sequence determination errors described above and the variability of the cleavage site to the known sequence in different known proteins, the mature MPIF-1 polypeptide encoded by the deposited cDNA contains about 99 amino acids, Anywhere within the -120 amino acid range; The leading sequence of the protein includes about 21 amino acids but may be anywhere within about 15 to about 35 amino acids.

The MPIF-1 variant may contain an encoding region, an unencrypted region, or an alteration in an encoded or unencrypted region. Particularly preferred are polynucleotide variants containing alterations that cause silent substitutions, additions or deletions, but do not alter the properties or activity of the encoded polypeptide. Nucleotide variants produced by latent substitution are preferred, due to the degeneracy of the genetic code. Furthermore, variants in which 5-10, 1-5 or 1-2 amino acids are substituted, deleted or added in any combination are also preferable. It is possible to produce MPIF-1 polynucleotides for various purposes (eg, for the purpose of optimizing codon expression for a particular host (for the purpose of changing the codons in human mRNA to the desired codon by a bacterial host such as E. coli) have.

The native MPIF-1 variant is a so-called " alleLic variant ", which refers to one of several alternative forms of a gene that occupies a given locus on the chromosome of an organism (Gene II, Lewin, ., John Wiley & Sons, New York (1985)]. These allelic variants can vary in polynucleotide and / or polypeptide levels and are included in the present invention. Alternatively, non-native variants can be produced by mutagenesis techniques or direct synthesis.

Nucleic acid probe.

The separated molecules, particularly DNA molecules, are useful as probes for gene mapping by in situ hybridization with chromosomes and for detection of MPIF-1 gene expression in human tissues by Northern blot analysis, for example. The invention also relates to fragments of isolated nucleic acid molecules described herein. A fragment of the nucleotide sequence of the deposited MPIF-1 cDNA or of the isolated nucleic acid sequence having the nucleotide sequence or 20A (SEQ ID NO: 1 or 6) shown in Figure 1 can be used as a diagnostic probe and a primer as described herein Quot; means a fragment which is useful and has a length of at least about 15 nt, more preferably at least 20 nt, even more preferably at least 30 nt, even more preferably at least 40 nt. Of course, larger fragments of 50-500 nt in length, such as the deposited MPIF-1 cDNA nucleotide sequence or the fragment corresponding to the majority of the nucleotide sequence or 20A (SEQ ID NO: 1 or 6) shown in Figure 1, It is useful accordingly. For example, a fragment having a length of 20 nt or greater may comprise a nucleotide sequence of the deposited cDNA or a fragment comprising 20 or more bases derived from a nucleotide sequence as shown in Figure 1 or 20A (SEQ ID NO: 1 or 6) it means. It is obvious to those skilled in the art to generate the DNA fragment because the gene is deposited and the nucleotide sequence shown in FIG. 1 or 20A (SEQ ID NO: 1 or 6) is provided. For example, shearing or limiting endonuclease cleavage by ultrasonication can be readily used to produce fragments of various sizes.

The use of the present invention may include nucleic acid molecules that are 90% or more, 95% or more, 96% or more, 97% or more, 98% or more, or 99% or more identical to the nucleic acid sequence described in the present specification (for example, mn Encoding a polypeptide having an amino acid sequence of an N and / or C terminal deletion, which will be described later, as in the case of the above-mentioned nucleic acid molecule, regardless of whether or not the nucleic acid molecule encodes a polypeptide having MPIF-1 functional activity. This is because even if a particular nucleic acid molecule does not encode a polypeptide having MPIF-1 functional activity, one skilled in the art can know how to use the nucleic acid molecule as, for example, a hybridization probe or a polymerase chain reaction (PCR) primer. The use of the nucleic acid molecule of the present invention which does not encode a polypeptide having MPIF-1 functional activity is particularly useful for (1) isolation of the MPIF-1 gene or splice variant thereof in a cDNA library; (2) A mid-term chromosome spreader to provide accurate chromosomal location of the MPIF-1 gene as described in Verma et al., Human Chromosomes: A manual of BasicTechniques, Pergamon Press, New York (E.g., " FISH "); And (3) Northern blot analysis for detection of MPIF-I mRNA expression in specific tissues.

However, sequences having at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to the nucleic acid sequences described herein that encode polypeptides that possess MPIF-1 functional activity Is preferred. The term " polypeptide having MPIF-1 functional activity " refers to a polypeptide of the invention, such as complete (full length) MPIF-1, mature MPIF-1, and soluble MPIF-1 polypeptide as measured by, for example, a specific immunoassay or biological assay. 1 (e. G. Possessing a sequence contained within the extracellular domain of MPIF-1)]. ≪ / RTI > For example, MPIF-1 functional activity can be measured by detecting the ability of MPIF-1 polypeptides to bind MPIF-1 ligand as is customary in the art. In addition, MPIF-1 functional activity can be measured by detecting the ability of a polypeptide (e.g., a cognate ligand) that is free (free) or expressed on the cell surface to induce a cell expressing the polypeptide.

The fragment of the full-length gene of the present invention can be used as a hybrid-forming probe for cDNA library, which isolates full-length cDNA and separates other cDNA having high sequence affinity or similar biological activity with the gene. Probes of this type preferably contain at least 30 bases and may contain, for example, 50 or more bases. The probe can also be used to identify clones containing the complete gene, including cDNA clones and genomic clones, or control and promoter regions, exons and introns, corresponding to full-length transcripts. An example of a screen involves separating the coding region of a gene by using a known DNA sequence that synthesizes an oligonucleotide probe. A labeled oligonucleotide having a sequence complementary to the gene sequence of the present invention is used for screening a library of human cDNA, genomic DNA, or mRNA to determine which component of the library the probe forms as a hybrid.

The present invention also relates to a polynucleotide fragment of a polynucleotide of the present invention. In the present invention, a "polynucleotide fragment" is a portion of a sequence that is contained within a deposited clone or that encodes a polypeptide encoded by the cDNA in the deposited clone; Quot; means a short polynucleotide having a nucleic acid sequence that is part of a sequence shown in SEQ ID NO: 1 or a complementary strand, or a part of a polynucleotide sequence encoding a polypeptide of SEQ ID NO: 2. The length of the nucleotide fragment of the invention is preferably at least about 15 nt, more preferably at least about 20 nt, even more preferably at least about 30 nt, even more preferably at least about 40 nt, at least about 50 nt, Greater than about 75 nt, or greater than about 150 nt. For example, a fragment that is "at least about 20 nt in length" is meant to include a cDNA sequence contained within the deposited clone or 20 or more contiguous bases from the nucleotide sequence shown in SEQ ID NO: 1 or 6. In the context, " about " means in particular the value quoted and a value that is greater or smaller by some (5, 4, 3, 2 or 1) nucleotides at either or both ends. These nucleotide fragments have applications that include, but are not limited to, residual probes and primers as described herein. Of course, more fragments (e.g., 50, 150, 500, 600 or 2000 nucleotides) are preferred.

In addition, the present invention relates to a method for producing a compound having at least one of residues 50-599, 100-599, 200-599, 300-599, 400-599, 600-1800, 50-500, 100-500, 200-500, 300-500, At least about 25 nucleotides of SEQ ID NO: 1 of SEQ ID NO: 1, 50-400, 100-400, 200-400, 300-400, 50-300, 100-300, 200-300, 50-200, 100-200 and 50-100, At least about 30 nucleotides, at least about 35 nucleotides, at least about 40 nucleotides or at least about 45 nucleotides, preferably at least about 50 nucleotides, at least about 60 nucleotides, at least about 70 nucleotides, at least about 80 nucleotides , About 90 or more nucleotides, or about 100 or more nucleotides.

Moreover, representative examples of polynucleotide fragments of the invention include polynucleotide fragments of about 1-50, 51-100, 101-150, 151-200, 201-250, 251-300, 301-350, 351-400, 401-450, 451 A fragment comprising or consisting of the sequence from the nucleotide number of -500, 501-550 or 551 to the end point of SEQ ID NO: 1 or 6, the complementary strand thereof, or the cDNA contained in the deposited cDNA. In this context, " about " means a range specifically cited and a range that is larger or smaller by several (5, 4, 3, 2, or 1) nucleotides at one or both ends. These fragments preferably encode polypeptides having biological activity. It is preferred that these polynucleotides can be used as probes or primers as described herein. Polynucleotides that form hybrids in these nucleic acid molecules under stringent hybridization or less stringent hybridization conditions, such as polypeptides encoded by the polynucleotides, are also encompassed by the present invention. In the present invention, " polypeptide fragment " means an amino acid sequence contained in SEQ ID NO: 2 or a part of a sequence encoded by a cDNA contained in a deposited clone.

Vector, host cell and protein expression.

The invention also relates to the production of MPIF-1 polypeptides or fragments thereof by vectors containing isolated nucleic acid molecules of the invention, genetically engineered host cells containing recombinant vectors, and recombinant techniques. The present invention also relates to novel expression vectors useful for the production of proteins in bacterial systems. The novel vector is described by the vector of the pHE4 series, particularly the pHE4-5 vector (Fig. 31 and 36A-E).

A polynucleotide encoding a protein of the invention can be coupled to a vector containing a selectable marker for proliferation in a host. The plasmid vector is generally introduced into a host cell in a complex with a host cell or charged lipid in a precipitate (such as a calcium phosphate precipitate), as described below. If the vector is a virus, it can be transfected into host cells after being packaged in vitro using an appropriate packaging cell line.

Vectors comprising a cis-acting regulatory region operably linked to an important polypeptide are preferred for use in the practice of the present invention. The cis-acting control region includes an operator and an encoder sequence. As used herein, the term " operator " refers to a nucleotide sequence that is typically comprised of DNA that regulates transcription of adjacent nucleotide sequences. In general, operator sequences are derived from bacterial chromosomes.

The transcription of the nucleotide sequence encoding the polypeptid of the invention by the higher eukaryote biological organism can be increased by inserting an annealing sequence into the vector. An enhancer is a cis-acting element, usually about 10-300 bp, which acts to increase the transcriptional activity of a promoter in a given host cell-type. Examples of encoders include SV40 encoders, cytomegalovirus early promoter encoders located on the posterior occlusion back side of bp 100-270, polyoma engineers on the posterior occlusion rear side, and adenovirus enhancers .

Suitable trans-acting factors may be supplied by the host, provided by the complementing vector, or by the vector itself upon introduction of the vector into the host.

In a preferred embodiment, the vector provides an expression that is inducible and / or inducible or specific for the cell type. A particularly preferred vector among the above vectors is a vector inducible by an easily manipulated environmental factor (e.g., temperature and nutrient additives). In addition, the pHE4-5 vector described in Example 30 is suitable for the expression of MPIF-1.

Additional expression vectors useful in the present invention include chromosomal-, episomal- and virus-derived vectors such as bacterial plasmids, bacteriophage, yeast episomes, yeast chromosomal elements, viruses such as baculovirus, (E.g., vaccinia virus and retrovirus), and vectors derived from combinations thereof (e.g., cosmid and phagemid).

A suitable nucleic acid sequence can be inserted into the vector by various methods. Generally, the nucleic acid sequence is inserted into the appropriate restriction endonuclease site (s) by methods known in the art. These and other methods are considered within the scope of those skilled in the art.

Nucleic acid inserts may be modified by the addition of appropriate promoters such as the phage lambda PL promoter, the lac, trp and tac promoters of E. coli, the promoters of SV40 early and late promoters and retroviral LTRs, and other prokaryotic or eukaryotic cells or genes Lt; RTI ID = 0.0 > expression < / RTI > Other suitable promoters are known to those skilled in the art. As used herein, the term " promoter " means a nucleotide sequence or nucleotide sequence group that minimally provides a binding site or initiation site for RNA polymerase action. In addition, the expression construct may further contain a site for transcription initiation and termination, a transcribed region, and a ribosome binding site for translation. The coding portion of the mature transcript expressed by the construct preferably comprises initiation and translation initiation in the initiation and termination codons (UAA, UGA or UAG) that are appropriately located at the end of the polypeptide being translated. In addition, the vector may comprise a sequence suitable for amplifying the expression.

As used herein, the phrase " operably linked " refers to a linkage in which a nucleotide sequence is linked to another nucleotide sequence (s) in such a way that the function (or sequence) it means. For example, a protein-encoding sequence operably linked to a promoter / operator sequences the expression of the protein-encoding sequence under the influence or dominance of the sequence. Two nucleotide sequences (e. G., A protein coding sequence and a promoter region sequence attached to the 5 ' end of the coding sequence) are used when the induction of the promoter function results in the transcription of the protein coding sequence mRNA and the binding properties between the two nucleotide sequences Is operably linked if it does not (1) cause the introduction of a frame-shift mutation and (2) does not prevent expression control sequences that direct the expression of the mRNA or protein. Thus, if a promoter can affect the transcription of the nucleotide sequence, the promoter region is operably linked to the nucleotide sequence.

As used herein, the phrase " cloning vector " refers to a nucleic acid sequence that is capable of autonomous replication in a host cell, and which can be cleaved in a determinable manner without loss of the essential biological function of the vector, Quot; refers to a nucleic acid or other nucleic acid sequence of a plasmid or phage that is characterized by a small number of endonuclease recognition sites and in which the nucleic acid can be spliced to cause replication and cloning. The cloning vector may further comprise a label suitable for use in the identification of the transformed cell with the cloning vector. For example, the label is resistant to erythromycin and kanamycin. The term " vehicle " is sometimes used for " vectors ".

As used herein, the phrase " expression vector " means a vector similar to a cloning vector capable of expressing the structural gene cloned into an expression vector after transformation of the expression vector into the host. In an expression vector, a cloned structural gene (any critical coding sequence) is placed under (ie, operably linked to) a particular sequence that causes the gene to be expressed in a particular host. For example, in a pHE4-5 vector, the structural gene is operably linked to a T5 phage promoter sequence and two lac operator sequences. The expression control sequences will vary, and may additionally contain transcription factors (e.g., termination sequences) and / or translation elements (e.g., initiation and termination sites).

As indicated above, the expression vector preferably comprises one or more selectable labels. The label includes dihydrofolate reductase or neomycin resistance gene for eukaryotic cell culture, and tetracycline, kanamycin or ampicillin resistance gene for E. coli and other bacterial cultures. Representative examples of suitable hosts include bacterial cells such as E. coli, Streptomyces, Salmonella typhimurium cells; Fungal cells such as yeast cells [e.g., Saccharomyces cerevisiae or Pichia pastoris (ATCC reception number 201178)]; Animal cells such as CHO, COS, 293 and Bowes melanoma cells; And plant cells, but are not limited thereto. Suitable culture media and conditions for the aforementioned host cells are known in the art.

In addition to the use of an expression vector in the practice of the present invention, the invention includes a novel expression vector comprising an operator and a promoter element operably linked to a nucleotide sequence encoding an important protein. An example of such a vector is pHE4-5 (SEQ ID NO: 37) described in both the following and Example 14. The pHE4-5 vector was deposited at the American Type Culture Collection (ATCC), Patent Depository, 10801 University Boulevard, Manassas, Va. 20110-2209 on September 30, 1997, and the grant number was 209311.

As summarized in Figures 31 and 36, the components of the pHE4-5 vector (SEQ ID NO: 37) are: (1) a neomycinphosphotransferase gene as a selectable marker, (2) Phage promoter sequence, (4) two lac operator sequences, (5) MPIF-1 23 (SEQ ID NO: 27), (6) Shine-Delgarno sequence and (7) lactose operon repressor gene (lacIq). The replication origin (oriC) is derived from pUC19 (LTI, Gaithersburg, Md.). Promoter sequences and operator sequences are prepared synthetically. Synthetic production of nucleic acid sequences is well known in the art. See Clontech 95/96 Catalog, pp 215-216, Clontech, 1020 East Meadow Circle, Palo Alto, CA 94303, USA.

As mentioned above, the pHE4-5 vector contains the lacIq gene. lacIq is an allele of the lacI gene that confers tight control of the lac operator. See Amann, E. et al. Gene 69: 301-315 (1988) and Stark, M. Gene 51: 255-267 (1987). The lacIq gene encodes a repressor protein that binds to the lac operator gene and blocks down-stream (ie, 3 ') sequences. However, the lacIq gene product dissociates from the lac operator in the presence of lactose or a specific lactose analog such as isopropyl-BD-thiogalactopyranoside (IPTG). Thus, MPIF-1 23 is poorly produced in non-inoculated host cells containing the pHE4-5 vector. However, the induction of the host cell by addition of a formulation such as IPTG was inhibited by MPIF-1 23 < / RTI > coding sequence.

The promoter / operator sequence (SEQ ID NO: 38) of the pHE4-5 vector contains the T5 phage promoter and two lac operator sequences. One operator is located 5 'to the transcription initiation site and the other operator is 3' to the same site. The operator, when present with the lacIq gene product, confer tight inhibition of the downstream sequence in the absence of the lac operon endorser (e.g., IPTG). Expression of operably linked sequences downstream from the lac operator may be induced by the addition of lac operon endorsers (e.g., IPTG). The binding of the lac enduclease to the lacIq protein results in the release of the lacIq protein from the lac operator and the transcription initiation of the operably linked sequence. The lac operon regulation of gene expression is outlined in Delvin, T., Textbook of Biochemistry with Clinical Correlations, Fourth Edition (1997), pp 802-807.

The pHE4-5 series vector contains MPIF-1 Contains all components of pHE4-5 except for the 23 coding sequence. Features of the pHE4-5 vector include optimized synthetic T5 phage promoter, lac operator, and Shine-Delagar Reno sequence. In addition, the above sequence is spaced at an optimal interval, so that the expression of the inserted gene can be regulated tightly, and high level expression occurs upon induction.

Known lipase promoters suitable for use in the production of the protein of the present invention include the lac I and lac Z promoters of E. coli, the T3 and T7 promoters, the gpt promoter, the lambda PR and PL promoters, and the trp promoter. Suitable eukaryotic promoters include, but are not limited to, CMV extreme early promoters, HSV thymidine kinase promoters, early and late SV40 promoters, promoters of retroviral LTRs such as the promoters of Rous sarcoma virus (RSV), and metallothionein promoters such as the mouse Metallothionein-I promoter).

In addition, the pHE4-5 vector contains a Shine-Delgarno sequence located 5 'to the AUG start codon. The Shine-Delgarno sequence is generally a short sequence located about 10 nucleotides upstream (5 ') from the AUG start codon. The sequence essentially directs the prokaryotic ribosome to the AUG start codon.

Thus, the present invention also relates to an expression vector useful for the production of the protein of the present invention in bacteria. This aspect of the invention is illustrated by the pHE4-5 vector (SEQ ID NO: 37) (ATCC Accession No. 20931) and variations thereof.

Additional vectors suitable for use in the expression of the proteins of the invention in bacteria include pQE70, pQE60 and pQE-9 (the above being available from Qiagen); pBS vector, pD10, Phagescript vector, pBluescript vector, pNH8A, pNH16A, pNH18A, pNH46A (these are commercially available from Stratagene); And ptrc99a, pKK233-3, pDR540, pRIT5 (these are commercially available from Pharmacia). Preferred eukaryotic vectors are pWLNEO, pSV2CAT, pOG44, pXTl and ptrc99a available from Stratagene; And pSVK3, pBPV, pMSG and pSVL available from Pharmacia.

Other suitable vectors will be apparent to those skilled in the art, examples of which include pBR322 (ATCC 37017), pKK233-3 (Pharmacia Fine Chemicals, Suffu) and GEM1 (Promega Biotec, Madison, Wis.). The pBR322 " backbone " section is expressed in association with appropriate promoter and structural sequences. After the appropriate host strain is transformed and grown to a suitable cell density, the selected promoter is induced by appropriate means (such as temperature transfer or chemical induction) and the cells are cultured for an additional period of time.

In another embodiment, the present invention relates to a host cell containing the above-described construct. The host cell may be a higher eukaryotic cell such as a mammalian cell, a lower eukaryotic cell such as a yeast cell, or a prokaryotic cell such as a bacterial cell. Induction of the construct into the host cell may be effected by phosphate incorporation, DEAE-dextran mediation, cationic lipid mediation, electroporation, transduction, infection or other methods. These methods are described in a number of standard laboratory manuals (e.g., Davis et al., Basic Methods In Molecular Biology (1986)).

Recombinant constructs can be introduced into host cells using well-known techniques (e.g., infection, transduction, transfection, transduction, shock and transformation). The vector may be, for example, a phage, a plasmid, a viral vector or a retroviral vector. The retroviral vector may be capable of replication or may be replication defective. In the latter case, virus proliferation can generally only occur within complementary host cells.

The host cell is genetically engineered (transfected or transformed or transfected) with the vector of the invention, which may be, for example, a cloning vector or an expression vector. The vector may be in the form of, for example, plasmid, viral particle, phage or the like. The genetically engineered host cell may be cultured in a conventional nutrient medium modified to be suitable for activating a promoter, selecting a transformant, or amplifying MPIF-1 and a gene. The culture conditions (e.g., temperature, pH, etc.) are those previously used with the host selected for expression and will be apparent to those skilled in the art.

Polynucleotides of the present invention can be used to produce polypeptides by recombinant techniques. Thus, for example, the polynucleotide sequence may be contained in a vector or plasmid that expresses any one of various expression vectors, particularly a polypeptide. Such vectors include chromosomal, non-chromosomal and synthetic nucleic acid sequences (e.g., SV40 derivatives); Bacterial plasmids; Phage nucleic acid; Yeast plasmids; A combination of a plasmid and a phage nucleic acid, and a vector derived from a viral nucleic acid (e.g., vaccinia virus, adenovirus, poultry smallpox virus, alpha virus and rabies rabies virus). However, any other plasmid or vector may be used as long as it is replicable and viable in the host.

Representative examples of suitable hosts include: bacterial cells (e.g., E. coli, Streptomyces, Salmonella typhimurium); Fungal cells such as yeast; Insect cells such as Drosophila and Sf9; Animal cells such as CHO, COS or Bowes melanoma; Plant cells and so on. The choice of a suitable host is considered to be within the purview of those skilled in the art as taught herein.

More specifically, the present invention also includes recombinant constructs comprising one or more sequences as broadly described above. The construct includes a vector (e.g., a plasmid or a viral vector) into which the sequence of the present invention is inserted in a forward or reverse direction. In a preferred aspect of this embodiment, the construct comprises a regulatory sequence, including, for example, a promoter operably linked to a sequence. A number of suitable vectors and promoters are known to those skilled in the art and are commercially available. The following vectors are examples. Bacterial vectors: pQE70, pQE60 and pQE-9 (these are available from Qiagen); pBS, pD10, Phagescript, psiX174, pBluescriptSK, pbsks, pNH8A, pNH16A, pNH18A, pNH46A (these are commercially available from Stratagene). Eukaryotic vectors: pWLNEO, pSV2CAT, pOG44, pXT1, pSG (these are commercially available from Stratagene), pSVK3, pBPV, pMSG, pSVL (these are available from Pharmacia). However, any other plasmid or vector may be used as long as it is replicable and viable in the host.

Conventional methods can be used to produce a gene product that is encoded by a recombination sequence using a construct in a host cell. Alternatively, the polypeptide of the present invention can be synthetically produced by a conventional peptide synthesizing apparatus.

Mature proteins can be expressed in mammalian cells, yeast cells, bacterial cells or other cells under the control of appropriate promoters. In addition, by using a cell-free translation system, the protein can be produced using RNA derived from the nucleic acid construct of the present invention. Suitable cloning and expression vectors for use with prokaryotic and eukaryotic hosts are described in Sambrook et al., Molecular Cloning: A Laboratory Manual, 2nd Ed., Cold Spring Harbor (1989) Lt; / RTI >

Generally, a recombinant expression vector is a selectable marker that permits transformation of the origin of replication and the host cell (e.g., the ampicillin resistance gene of E. coli and Saccharomyces cerevisiae TRP1 gene), and a selectable marker that is derived from a highly expressed gene Including the promoter, to direct transcription of downstream structural sequences. The promoter may be derived from an operon encoding a glycolytic enzyme such as 3-phosphoglycerate kinase (PGK), alpha-factor, acid phosphatase, heat shock protein, and the like. The heterologous structural sequences are assembled in the appropriate phase with the translation initiation and termination sequences, preferably the leader sequence, which can direct the translation of the translated protein into the surrounding cytoplasmic space or into the extracellular medium. Optionally, the heterologous sequence may encode a fusion protein comprising an N-terminal identifying peptide conferring a desired property (e.g., a simplified purification or stabilization of the expressed recombinant product).

An expression vector useful for bacteria can be constructed by inserting a structural nucleic acid sequence encoding a given protein with appropriate translation initiation and termination signals in an operable reading phase carrying a functional promoter. The vector includes one or more phenotypic selectable labels and a cloning start point to ensure retention of the vector and, if desired, to provide amplification in the host. Suitable prokaryotic hosts for transformation include, but are not limited to, E. coli, Bacillus subtilis, Salmonella typhimurium, and various species within the genus Pseudomonas, Streptomyces and Staphylococcus, Other hosts may also be used.

Cells are typically harvested by centrifugation, disrupted by physical or chemical means, and the resulting crude extract is retained for further purification.

The microbial cells used for the expression of the protein can be disrupted by any conventional method (e.g., methods known in the art), including freeze-thaw circulation, ultrasonication, mechanical disruption or use of cytolytic agents .

In addition, recombinant proteins can be expressed using various mammalian cell culture systems. Examples of mammalian expression systems include the COS-7 cell line of monkey kidney fibroblasts described in Gluzman, Cell 23: 175 (1985), and other cell lines capable of expressing a compatible vector : C127, 3T3, CHO, HeLa and BHK cell lines). The mammalian expression vector may comprise a replication origin, a suitable promoter and an encenger, any necessary ribosome binding site, a polyadenylation site, a splice donor and acceptor site, a transcription termination sequence, and a 5 ' flanking non- . Nucleic acid and polyadenylation sequences derived from the SV40 splice can be used to provide the necessary non-nuclear genetic elements.

In addition, MPIF-1 polypeptides (preferably secreted forms) can be recovered from: purified products from natural sources (including body fluids, tissues and cells) that have been isolated or cultured directly; Products of chemical synthesis methods; And products produced by recombinant techniques from prokaryotic or eukaryotic hosts including, for example, bacteria, yeast, higher plants, insect and mammalian cells. Depending on the host used in the recombinant production method, the MPIF-1 polypeptide may be glycosylated or non-glycosylated. In addition, as a result of the host-mediated method, in some cases the MPIF-1 polypeptide may comprise an initially modified methionine residue. Thus, it is well known in the art that N-terminal methionine, generally encoded by the translation initiation codon, is highly efficiently removed from any protein after translation in all eukaryotic cells. Further, in the case of some proteins, since the N-terminal methionine on most proteins is efficiently removed in most of the zirconia, the prokaryotic removal method will depend on the nature of the amino acid to which the N-terminal methionine is covalently bonded, to be.

The invention encompasses host cells containing vector constructs discussed herein, as well as the removal or replacement of endogenous genetic material (e.g., the MPIF-1 coding sequence) and / or; (E.g., MPIF-I encoding sequence) that is operably associated with the MPIF-I polynucleotide of the invention and activates, alters and / or amplifies endogenous MPIF-I polynucleotides, Particularly primary, secondary, and immortalized host cells of mammalian origin. For example, heterologous recombination can operatively associate a heterologous regulatory region (e.g., a promoter and / or an engineer) and an endogenous MPIF-I polynucleotide sequence (see, for example, U.S. Patent No. 5,641,670, issued June 24, 1997, which is incorporated herein by reference in its entirety, both of which are hereby incorporated by reference herein. 1996. 9. 26. Published International Publication No. WO 96/29411; Natl. Acad. Sci. USA 86: 8932-8935 (1989); and Zijlstra et al., Nature 342: 435-438 (1989) ).

MPIF-1 polypeptide

The invention also provides a peptide or polypeptide comprising an amino acid sequence encoded by the deposited cDNA or the MPIF-1 polypeptide having the amino acid sequence of Figure 1 (SEQ ID NO: 2), a portion of said polypeptide, or a portion of said polypeptide . The terms " peptide " and " oligopeptide " are considered synonymous (as commonly recognized), and each term is intended to be interchangeable when the context needs to represent two or more amino acid chains (chains) It can be used as a reference. The term " polypeptide " is used herein to mean a chain containing at least 10 amino acid residues. The expression and sequence of all oligonucleotides and polypeptides herein are written from left to right and from the amino terminus to the carboxy terminus. The invention also provides a protein comprising or comprising a polypeptide sequence encoded by a polynucleotide of the invention, or a sequence consisting of a sequence encoded by a polynucleotide of the invention.

The invention also encompasses variants of the polypeptide sequence encoded by the clone described and / or deposited in SEQ ID NO: 2.

&Quot; Variant " means a polynucleotide or polypeptide that differs from an MPIF-1 polynucleotide or polypeptide but retains the essential characteristics of an MPIF-1 polynucleotide or polypeptide. In general, variants are generally similar to MPIF-I polynucleotide or polypeptide in general, and in many regions are identical to MPIF-I polynucleotide or polypeptide.

The polynucleotide fragment of the invention preferably encodes a polypeptide that demonstrates MPIF-1 functional activity. A polypeptide demonstrating MPIF-I " functional activity " refers to a polypeptide capable of exhibiting one or more known functional activities associated with the full-length (full) MPIF-I protein. The functional activity can be assessed for biological activity, antigenicity (ability to bind anti-MPIF-1 antibody (or ability to compete with MPIF-1 polypeptide to bind anti-MPIF-1 antibody)], immunogenicity The ability to form antibodies that bind to MPIF-1 polypeptides), the ability to form multimers with the MPIF-1 polypeptides of the invention, and the ability to bind receptors or ligands for MPIF-1 polypeptides But is not limited thereto.

The functional activity of the MPIF-1 polypeptide, and fragments, variants and derivatives thereof, can be analyzed by various methods.

For example, in one embodiment for analyzing the ability to bind or compete with the full-length MPIF-1 polypeptide for binding to an anti-MPIF-1 antibody, the assay can be performed by radioimmunoassay, ELISA (enzyme linked immunosorbant assay) (For example, using colloidal gold, an enzyme or a radioactive isotope label), western blotting, and the like, using a sandwich immunoassay, an immuno-radioactivity assay, a gel diffusion precipitation reaction, Competitive assay systems using techniques such as precipitation, precipitation, coagulation assays (eg, gel flocculation assays, erythrocyte flocculation assays), complement fixation assays, immunofluorescence assays, protein A assays and immunoelectrophoresis assays. Various immunoassay methods known in the art, which are not limited, can be used. In one embodiment, antibody binding is detected by detecting a label on the primary antibody. In another embodiment, the primary antibody can be detected by detecting the binding of the secondary antibody or reagent to the primary antibody. In another embodiment, the secondary antibody is labeled. A number of methods for detecting binding in immunoassays are known in the art and are within the scope of this disclosure.

In another embodiment for detecting the performance of a polypeptide fragment, variant or derivative of the invention that can identify and multimerize the MPIF-1 receptor or ligand, for example, reduced and non-reduced gel chromatography, protein affinity chromatography and Binding can be analyzed by methods known in the art such as affinity blotting. In general, Phizicky, E. et al., 1995, Microbiol. Rev. 59: 94-123]. In another embodiment, the physiological correlation of MPIF-1 binding (signal transduction) to the substrate of MPIF-1 can be analyzed.

In addition, the assays described herein (see Examples) and other methods known in the art can be used to measure the performance of MPIF-I polypeptides, and fragments, variant derivatives and analogs thereof, to determine MPIF-1 related biological activity I can apply it to reveal. Other methods are known to those skilled in the art and are within the scope of the present invention.

The MPIF protein or fragment thereof of the present invention may be present as a monomer or a multimer (i.e., a dimer, a trimer, a tetramer, or a higher multimer). Accordingly, the invention relates to monomers and oligomers of the MPIF proteins of the invention, to their preparation and to compositions (preferably pharmaceutical compositions) containing them. In an embodiment, the polypeptide of the invention is a monomer, dimer, trimer or tetramer. In further embodiments, the oligomer of the present invention is at least a dimer, at least a trimer, or at least a tetramer.

The oligomers encompassed by the present invention are homomultimers or heteromers. As used herein, the term " allelic variant " refers to a multimer containing only the MPIF protein of the invention (including MPIF fragments, variants and fusion proteins as used herein). The homomultimeters may contain MPIF proteins having the same or different polypeptide sequences. In an embodiment, the alleles of the present invention are oligomers containing only the MPIF protein having the same polypeptide sequence. In another embodiment, homomultimers of the invention are oligomers containing a MPIF protein having a different polypeptide sequence. In embodiments, the oligomers of the invention may be homodimers (e. G., Homodimers that contain MPIF proteins with the same or different polypeptide sequences) or homotrimers (e. G., MPIF proteins with the same or different polypeptide sequences) Containing homomeric trimer). In a further embodiment, the homomultimer of the invention is at least a homodimer, at least a homomeric trimer, or at least a homomeric tetramer.

As used herein, the term " heterodimer " refers to a multimer containing a heterologous protein (i. E., A protein containing only a polypeptide sequence that does not correspond to a polypeptide sequence encoded by the MPIF gene) . In an embodiment, the oligomer of the present invention is a heterodimer, heterotrimer or heteromer. In a further embodiment, the heterodimers of the invention are at least heterodimers, at least heterodimers, or at least heterodimers.

The oligomers of the present invention may be the result of hydrophobic, hydrophilic, ionic, and / or covalent association and / or may be indirectly conjugated, for example, by liposome formation. Thus, in one embodiment, a multimer of the invention (e. G., A homodimer or homotrimer) is formed when the protein of the present invention contacts another protein in solution. In another embodiment, the heterodimers (e. G., Heterotrimers or heterodimers) of the present invention can be used when the protein of the invention is contacted with the polypeptide of the invention in solution (a heterologous polypeptide in the fusion protein of the invention Such as by contacting the antibody to the sequence. In another embodiment, the oligomers of the invention are formed by a shared association with the MPIF protein of the invention and / or a shared association between the MPIF proteins of the invention. The shared association may comprise one or more amino acid residues contained within a polypeptide sequence in the polypeptide sequence listed in SEQ ID NO: 2 and contained within a polypeptide encoded by a cDNA clone contained in ATCC Deposit No. 75676. In some cases, the covalent association is a bridging between the cysteine residues located within the polypeptide sequence of the interacting protein in the unmodified (i.e. native) polypeptide. In other cases, the shared associate is the result of chemical manipulation or recombinant manipulation. Alternatively, the shared association includes one or more amino acid residues contained within the heterologous polypeptide sequence in the MPIF fusion protein. In one embodiment, the shared association is between the heterologous sequences contained within the fusion proteins of the invention (see, for example, U.S. Patent No. 5,478,925). In certain embodiments, a shared association is between heterologous sequences contained within the MPIF-Fc fusion protein of the invention (as described herein).

In another embodiment, the MPIF polypeptides and epitope-containing fragments thereof of the invention are fused with heterologous antigens (e.g., polypeptides, carbohydrates, phospholipids or nucleic acids).

In an embodiment, the heterologous antigen is cotton. In a more specific embodiment, the heterologous antigen is the gp 120 protein of HIV or a fragment thereof. Polynucleotides encoding the above polypeptides are also encompassed by the present invention.

The chemistries known in the art can be used to produce the oligomers of the present invention. For example, using techniques of optimization of cross-linker molecules and cross-linker molecule lengths known in the art, it is possible to chemically cross-link the desired protein that is contained in the oligomer of the present invention (see, for example, See U.S. Patent No. 5,478,925, which is incorporated herein by reference). In addition, by generating a multimer of the invention using techniques known in the art, one or more intermolecular bridges can be formed between the cysteine residues located within the polypeptide sequence of the desired protein to be contained within the multimer (e.g., See U.S. Patent No. 5,478,925, the entire contents of which are incorporated herein by reference). Also, by adding cysteine or biotin to the C-terminus or N-terminus of the polypeptide sequence of the protein, the protein of the present invention can be modified according to customary methods, and the production of a multimer containing one or more of the above- Known methods (see, for example, U.S. Patent No. 5,478,925, the entire contents of which are incorporated herein by reference). In addition, techniques known in the art can be applied to produce liposomes containing protein components that are preferably contained within the multimers of the present invention (see, for example, U.S. Patent No. 5,478,925, ).

Alternatively, genetic engineering techniques known in the art can be used to generate the oligomers of the present invention. In one embodiment, the fusion protein techniques described herein, or other techniques known in the art, can be used to recombinantly produce proteins contained within the oligomers of the present invention (e.g., U.S. Patent No. 5,478,925, which is incorporated herein by reference). In an embodiment, the polypeptide of the present invention is encoded in a sequence encoding a cross-linker polypeptide followed by a synthetic polynucleotide (lacking the sequence) encoding the translated polypeptide product in the reverse direction from the original C-terminus to the N-terminus (See, for example, U.S. Patent No. 5,478,925, the entire contents of which is incorporated herein by reference). The term " polynucleotide " In other embodiments, the recombinant techniques described herein or other techniques known in the art can be applied to produce recombinant polypeptides of the invention that contain the transmembrane domain and can be incorporated into liposomes by membrane reconstitution techniques (See, for example, U.S. Patent No. 5,478,925, the entire contents of which is incorporated herein by reference).

Proteins of the present invention can also be chemically synthesized using techniques known in the art (see, for example, Creighton, 1983, Proteins: Structures and Molecular Principles, WH Freeman & Co., See Hunkapiller, M. et al., Nature 310: 105-111 (1984)). For example, a peptide corresponding to a fragment of the MPIF polypeptide of the present invention can be synthesized by using a peptide synthesis apparatus. Moreover, if necessary, nonclassical amino acids or chemical amino acid analogues can be introduced as substitutions or additions to the MPIF polypeptide sequence. Non-classical amino acids generally include D-isomers of common amino acids, 2,4-diaminobutyric acid, a-aminoisobutyric acid, 4-aminobutyric acid, Abu, 2-aminoisobutyric acid, g- Tricarboxylic acid, t-butylglycine, t-butylglycine, t-butylglycine, t-butylglycine, t-butylglycine, Designer amino acids such as phenylalanine, phenylglycine, cyclohexylalanine, b-alanine, fluoro-amino acid and b-methyl amino acid, Ca-methyl amino acid, Na-methyl amino acid and amino acid analogues. But is not limited thereto. Furthermore, the amino acid may be D (preferential) or L (left-line).

Acids Res. 13: 4331 (1986), and Zoller et al., Nucl. Acids Res. 10: (See, for example, Wells et al., Gene 34: 315 (1985)), restriction selection mutations (see, for example, Wells et al., Philos. Trans (See, e. G., R. Soc. London SerA 317: 415 (1986)) using mutagenesis techniques known in the art.

In addition, the present invention also provides a method for modulating MPIF, which is differentially modified during or after translation by, for example, glycosylation, acetylation, phosphorylation, amidation, derivatization by known protecting / blocking groups, proteolytic cleavage, ≪ / RTI > Two NIKA including presence under cyanogen bromide, trypsin, chymotrypsin, papain, V8 protease, NaBH ₄ specific chemical cleavage, acetylation, formylation, oxidation, reduction, metabolic synthesis by the azithromycin a known, not one limited to these Technique to perform any one of a number of chemical variations.

The present invention relates to processes for the treatment of N-linked or O-linked carbohydrate chains (N-terminal or C-terminal end), attachment of chemical moieties to amino acid backbones, chemical modification of N-linked or O- And additional post-translational modifications, including the addition or deletion of N-terminal methionine residues as a result of the expression of prokaryotic host cells. In addition, the polypeptide can be modified with a detectable label (e.g., enzyme label, fluorescent label, isotope label or affinity label) to enable detection and isolation of the protein.

In addition, the present invention provides chemically modified MPIF derivatives that can provide additional advantages such as increased solubility, stability and cycle time of the polypeptide, or reduced immunogenicity (see, for example, U.S. Patent No. 4,179,337 ). Chemical moieties for derivatization can be selected from water soluble polymers (e.g., polyethylene glycol, ethylene glycol / propylene glycol copolymer, carboxymethylcellulose, dextran, polyvinyl alcohol, etc.). The polypeptide may be modified at random positions within the molecule or at predetermined positions in the molecule, which polypeptide may comprise one, two, three or more attached chemical moieties.

The polymer may be of any molecular weight and may be branched or unbranched. In the case of polyethylene glycol, the molecular weight may range from about 1 kDa to about 100 kDa for ease of handling and manufacturing (the term " about "Quot; indicates that some molecules are somewhat larger or smaller than the above-mentioned molecular weights). (E. G., The effect of the biological activity (if any), ease of manipulation, degree or lack of antigenicity, and other known < RTI ID = 0.0 > Different sizes can be used depending on the effect. For example, the average molecular weight of polyethylene glycol is about 200, 500, 1000, 1500, 2000, 2500, 3000, 3500, 4000, 4500, 4500, 5000, 5500, 6000, 6500, 7000, 7500, 8000, 8500, 9000 15,000, 15,000, 16,000, 16,500, 17,000, 17,500, 18,000, 18,500, 19,000, 19,500, 20,000, 25,000, 30,000, 35,000, 15,000, 15,000, 15,000, 15,000, 10,500, 11,000, 11,500, 12,000, 12,500, 13,000, 13,500, , 40,000, 50,000, 55,000, 60,000, 65,000, 70,000, 75,000, 80,000, 85,000, 90,000, 95,000 or 100,000 kDa.

As mentioned above, the polyethylene glycol may have a branched structure. Branched polyethylene glycols are described, for example, in U.S. Patent Nos. 5,643,575, each of which is incorporated herein by reference; Morpurgo et al., Appl. Biochem. Biotechnol. 56: 59-72 (1996); Vorobjev et al., Nucleosides Nucleosides 18: 2745-2750 (1999); Caliceti et al., Bioconjug. Chem. 10: 638-646 (1999).

In consideration of the effect on the functional or antigenic domain of the protein, the polyethylene glycol molecule (or other chemical moiety) must be attached to the protein. There are a number of attachment methods available to those of ordinary skill in the art, such as EP 401,384 (a method of coupling PEG to G-CSF), which is incorporated herein by reference, and Malik et al. Hematol. 20: 1028-1035 (1992)) (PEGylation of GM-CSF using tresilyl chloride). For example, polyethylene glycol may be covalently bonded through an amino acid residue by a reactive group (e.g., a free amino group or a carboxyl group). The reactive group is a group to which an activated polyethylene glycol molecule can be bonded. The amino acid residue having a free amino group may comprise a lysine residue and an N-terminal amino acid residue; The amino acid residue having a free carboxyl group may comprise an aspartic acid residue, a glutamic acid residue and a C-terminal amino acid residue. In addition, a sulfhydryl group can be used as a reactive group to attach the polyethylene glycol molecule. Attachment at the amino group (e.g., attachment at the N-terminal or lysine group) is preferred for therapeutic purposes.

As mentioned above, polyethylene glycol can be attached to the protein by binding to any one of a plurality of amino acid residues. For example, polyethylene glycol can be attached to the protein by covalent attachment to lysine, histidine, aspartic acid, glutamic acid, or cysteine residues. One or more amino acid residues of a protein (such as lysine, histidine, aspartic acid, glutamic acid, or cysteine) or one or more types of protein (such as lysine, histidine, aspartic acid , Glutamic acid, cysteine, and combinations thereof) can be attached to polyethylene glycol. Proteins chemically modified at the N-terminus may be particularly preferred. The use of polyethylene glycol as an example of a composition of the present invention can be advantageously used for the production of various types of polyethylene glycol molecules (by molecular weight, branching etc.), the ratio of polyethylene glycol molecules to protein (or peptide) molecules in the reaction mixture, , And methods for obtaining N-terminally PEGylated proteins. The method of obtaining an N-terminally PEGylated preparation (i. E., The method of separating this moiety from other monoPEGylated moieties if necessary) may be accomplished by purifying N-terminal PEGylated material from a plurality of PEGylated protein molecules . Selective proteins that are chemically modified in the N-terminal modification can be achieved by reductive alkylation using different types of differential reactions of different types of primary amino acids (lysine to N-terminal) useful for the derivatization of specific proteins. Under suitable reaction conditions, it is possible to achieve almost selective derivatization of the protein at the N-terminus using a carbonyl-containing polymer.

As mentioned above, PEGylation of the proteins of the present invention can be accomplished by any number of methods. For example, polyethylene glycol can be attached to the protein either directly or through an intervening crosslinking agent. Systems for attaching polyethylene glycols to proteins as a system without a cross-linker are described in Delgado et al., Crit. Rev. Thera. Drug Carrier Sys. 9: 249-304 (1992); Francis et al. Intern. J. of Hematol. 68: 1-18 (1998); U.S. Patent No. 4,002,531; U.S. Patent No. 5,349,052; WO 95/06058; And WO 98/32466.

One system for directly attaching polyethylene glycols to the amino acid residues of proteins without intervening crosslinking agents utilizes trisylated MPEG produced by the modification of monomethoxypolyethylene glycol (MPEG) using trexylyl chloride (ClSO ₂ CH ₂ CF ₃ ) . In the reaction of the trisylated MPEG with the protein, the polyethylene glycol is attached directly to the amino group of the protein. Accordingly, the present invention includes a protein-polyethylene glycol conjugate produced by reacting a protein of the present invention with a polyethylene glycol molecule having a 2,2,2-trifluoroethanesulfonyl group.

In addition, polyethylene glycol can be attached to the protein using a number of different intercalating agents. For example, urethane crosslinking agents linking polyethylene glycols to proteins are described in U.S. Patent No. 5,612,460, the entire disclosure of which is incorporated herein by reference. It is also possible to use MPEG-succinimidyl succinate, MPEG activated with 1,1-carbonyldiimidazole, MPEG-2,4,5-trichloropentyl carbonate, MPEG-p-nitrophenol carbonate, By the reaction of the protein with a compound such as an MPEG-succinate derivative, a polyethylene glycol conjugate can be produced in which the polyethylene glycol is attached to the protein by a crosslinking agent. A number of additional polyethylene glycol derivatives and reaction chemistry for attaching polyethylene glycols to proteins are described in WO 98/32466, the entire contents of which are incorporated herein by reference. The pegylated protein products produced using the reaction chemistry methods detailed herein are within the scope of the present invention.

In addition, the number of polyethylene glycol moieties attached to each protein of the present invention (i.e., degree of substitution) may vary. For example, an average of one, two, three, four, five, six, seven, eight, nine, ten, twelve, fifteen, seventeen, twenty or more The pegylated protein of the invention can be conjugated to a polyethylene glycol molecule. Similarly, the average degree of substitution is 1-3, 2-4, 3-5, 4-6, 5-7, 6-8, 7-9, 8-10, 9 11, 10-12, 11-13, 12-14, 13-15, 14-16, 15-17, 16-18, 17-19 or 18-20 polyethylene glycols And the like. Methods for measuring the degree of substitution are described, for example, in Delgado et al., Crit. Rev. Thera. Drug Carrier Sys. 9: 249-304 (1992).

&Quot; Polypeptide having MPIF-1 activity " refers to a polypeptide that exhibits similar but not necessarily the same activity as the MPIF-1 protein (full-length protein or preferably mature protein) of the present invention when measured by a specific biological assay . Example 9. MPIF-1 protein activity can be measured by the assay described in Example 10 and FIG. For example, MPIF-1 protein activity is measured using in vivo bone marrow protection assay described in Example 9 below.

In short, a number of lineage-depleted cells (Lin cells) are isolated from mouse bone marrow and incubated in the presence of multiple cytokines with or without MPIF-1. After 48 hours, 5-Fu was added to each set of each culture, followed by incubation for an additional 24 hours, at which time low proliferation survived by any suitable clonogenic assay The number of potential colony-forming cells (LPP-CFC) was measured. A low content (less than 5%) of the LPP-CFC is protected against 5-Fu induced cytotoxicity in the presence of MPIF-1, while a high content [e.g., 30-50% CFCs will be observed in the presence of unrelated proteins and in the absence of MPIF-1. In this assay, high proliferation potential colony-forming cells (HPP-CFC) are also protected from 5-Fu induced cytotoxicity in the presence of MPIF-1, but in some cases not. If the LPP-CFC is not protected, the HPP-CFC is generally not protected.

In addition, MPIF-1 protein activity can be measured by the sub-mortality and lethality model described in Examples 16-18 and the cell protection method described in Example 19. [

Thus, polypeptides that possess MPIF-1 protein activity include polypeptides that exhibit MPIF-1 activity in the assay described above. Although it is not necessary for the degree of activity to be equal to the degree of activity of the MPIF-1 protein, it is desirable that " the polypeptide having MPIF-1 protein activity " The candidate polypeptide exhibits relatively greater activity or only about 20-fold less activity, preferably only about 10-fold less activity than the reference MPIF-1 protein).

The present invention also relates to the use of the MPIF-1 polypeptide having the amino acid sequence (SEQ ID NO: 2) deduced in the system of Figure 1 or having an amino acid sequence encoded by the deposited cDNA, as well as fragments, ≪ / RTI >

The term "fragment", "derivative" and "analog" when referring to the polypeptide of FIG. 1 (SEQ ID NO: 2) or the polypeptide encoded by the deposited cDNA refers to a polypeptide that essentially retains the same biological function or activity as the polypeptide . Thus, analogs include pre-proteins that can be activated by cleavage of the former protein moiety to produce an active, mature protein.

The polypeptides of the present invention may be recombinant polypeptides, natural or synthetic polypeptides, preferably recombinant polypeptides.

Fragments, derivatives or analogs of the polypeptide encoded by the polypeptide (SEQ ID NO: 2) or the deposited cDNA of Figure 1 can be prepared by (i) substituting one or more amino acid residues with a conserved or non-conserved amino acid residue (preferably a conserved amino acid residue) Wherein said substituted amino acid residue is not encoded or encoded by a genetic code; (Ii) one or more amino acid residues comprise a substituent; (Iii) a mature polypeptide is fused with another compound such as a compound that increases the half-life of the polypeptide (e.g., polyethylene glycol); Or (iv) a further amino acid residue is fused to a mature polypeptide, such as a sequence used in the purification of a mature polypeptide or a previous protein sequence, or a leading or secretory sequence. Such fragments, derivatives and analogs are considered to be within the purview of those skilled in the art from the teachings herein.

Any MPIF-1 polypeptide can be used to generate a fusion protein. For example, the MPIF-1 polypeptide can be used as an antigenic marker when fused to a second protein. Antibodies grown against the MPIF-1 polypeptide can be used to indirectly detect the second protein by binding to MPIF-1. Moreover, since secreted proteins target cellular sites based on trafficking signals, MPIF-I polypeptides can be used as target molecules only when they are fused to other proteins.

Examples of domains that can be fused to MPIF-1 include heterologous functional regions as well as heterologous signal sequences. Fusion does not necessarily have to be coordinated and can occur through a cross-linker sequence.

The polypeptide of the present invention is preferably provided in a separated form, and it is preferable that the polypeptide is homogeneously purified.

The polypeptide of the present invention comprises not only the polypeptide of SEQ ID NO: 2 (in particular, the mature polypeptide) but also a polypeptide having 95% or more (more preferably 95% or more identity) similarity with the polypeptide of SEQ ID NO: More than 30 amino acids, more than 35 amino acids, more than 40 amino acids, more than 45 amino acids, more than 50 amino acids, more than 55 amino acids, more than 60 amino acids, more than 65 amino acids, more than 70 amino acids, 75 More than 80 amino acids, more than 85 amino acids, more than 90 amino acids, more than 95 amino acids, and more than 100 amino acids. The protein (polypeptide) fragment may be free-standing or may be contained within a larger polypeptide of the fragment as a portion or region (most preferably as a single continuous region). Representative examples of the polypeptide fragments of the present invention include those from amino acid numbers of about 1-20, 21-40, 41-60, 61-80, 81-100, 102-120, 121-140, 141-160 or 161, Or fragments comprising or consisting of up to two fragments. Furthermore, the length of the polypeptide fragment may correspond to about 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140 or 150 amino acids. In this context, " about " includes, in particular, the ranges or values mentioned, and ranges or values that are as large or small as the number of amino acids at either or both ends (5, 4, 3, 2 or 1) do. Also included in the present invention are polypeptides that encode the polypeptide.

Although deletion of one or more amino acids from the N-terminus of a protein results in a modification of the loss of one or more biological functions of the protein, other functional activities (e.g., biological activity, ability to multimerize, bind MPIF-1 ligand Performance) can still be withheld. For example, the ability of a shortened MPIF-1 mutein to induce / induce or recognize / bind an antibody recognizing the complete or mature form of the polypeptide will generally result in a complete or mature polypeptide residue It is suspended when the majority is not being removed. Whether or not a particular polypeptide lacking the N-terminal residue of the complete polypeptide retains the immunological activity can be readily determined by the typical methods described herein and other methods known in the art. MPIF-I muteins bearing multiple deletion N-terminal amino acid residues may retain some biological or immunological activity. In fact, peptides composed of a very small number of six MPIF-1 amino acid residues can often cause an immune response.

Thus, the polypeptide fragment includes a mature form as well as a fused MPIF-protein. Additional preferred polypeptide fragments include a mature form or secreted MPIF-I protein that retains a series of amino, carboxy, or amino acid residues deleted from the carboxy terminus. For example, any number of amino acids in the 1-60 range can be deleted from the secreted MPIF-I polypeptide or amino terminal of the mature form. Likewise, any number of amino acids in the 1-30 range can be deleted from the secreted MPIF-I polypeptide or the carboxy terminal of the mature form. Furthermore, any combination of amino and carboxy terminal deletions is preferred. Likewise, polynucleotides encoding said polypeptide fragments are also preferred.

As is known in the art, the similarity between two polypeptides is determined by comparing the sequence of the second polypeptide with the amino acid sequence and conservative amino acid substitutions of one polypeptide.

Of course, due to the degeneracy of the genetic code, one skilled in the art will appreciate that nucleic acid sequences of the deposited cDNA (ATCC 75676), nucleic acid sequences shown in FIG. 1 or 20A (SEQ ID NOs: 1 or 6) It is immediately recognizable that a plurality of nucleic acid molecules having a sequence of 97% or more, 98% or more or 99% or more identical encodes a polypeptide having " MPIF-1 protein activity ". In fact, it will be apparent to those skilled in the art, even without performing the above comparative analysis, that all of the degenerate variants of the nucleotide sequence encode the same polypeptide. In addition, it is recognized in the art that, in the case of said nucleic acid molecule that is not a degenerate mutant, a reasonable number also encodes a polypeptide having MPIF-1 protein activity. This is because the person skilled in the art is fully aware of the amino acid substituents that may be less or less likely to have a significant effect on protein function (e.g., substituting one aliphatic amino acid with a second aliphatic amino acid), as described below.

As a practical matter, it is preferred that any particular polypeptide has an amino acid sequence which is 80% or more, 85% or more, 90% or more, 92% or more, 95% or more , 96% or more, 97% or more, 98% or more, or 99% or more identical can be conventionally measured using a known computer program. A preferred method for determining the best overall correct match between a query sequence (subject sequence) and a subject sequence is also referred to as global sequence alignment, see Brutlag et al. [Comp. App. Biosci. 6: 237-245 (1990)] using a FASTDB computer program. For sequence alignment, both query and subject sequences are either nucleotide sequences or both amino acid sequences. The result of the global sequence alignment appears as a percent identity. The preferred parameters used in the FASTDB amino acid alignment are as follows: Matrix = PAM 0, k-tuple = 2, mismatch penalty = 20, length of randomizer group = 0 , The cutoff score = 1, the window size = the sequence length, the gap penalty point = 5, the gap size penalty point = 0.05, the window size = 500, or the length of the subject amino acid sequence is shorter.

If the subject sequence is shorter than the query sequence due to deletion of the N- or C-terminus, not due to internal deletion, the result should be corrected manually. This is because the FASTDB program does not take into account the N- and C-terminal cuts of the subject sequence when calculating the percent identity of global sequences. In the case of the subject sequences cut at the N- and C-termini relative to the query sequence, the subject sequence which does not counteract / align with the residues of the corresponding subject sequence as a percentage of the total base of the query sequence is N- The identity percentage is corrected by calculating the number of residues in the terminating quadrature sequence. Whether or not the residues are properly mapped / aligned is measured by the result of the FASTDB sequence alignment. This percentage is then subtracted from the percent identity calculated by the FASTDB program using the parameter to reach the final percent identity score. This final identity percentage score is used for the purposes of the present invention. For purposes of manual adjustment of the percent identity score, only the residues for the N- and C-termini of the subject sequence that are not in proper match / alignment with the query sequence are considered. That is, only the positions of the quaternary residues of the outermost N- and C-terminal residues of the subject sequence are considered.

For example, to determine percent identity, the 90 amino acid residue target sequence is aligned with the 100 residue amino acid sequence. Deletions occur at the N-terminus of the subject sequence, and thus the FASTDB sequence does not show the matching / arrangement of the first 10 residues of the N-terminus. The ten non-paired residues represent 10% of the sequence (the number of unmatched- and C-terminal residues / the total number of residues in the sequence of the query) and therefore 10% of the percent identity score calculated by the FASTDB program . If the remaining 90 residues are perfectly matched, the final percent identity will be 90%. In another example, a target sequence of 90 residues is compared to a query sequence of 100 residues. In this case, deletion is an internal deletion, and thus there are no residues at the - or C-terminus of the subject sequence that do not match / align with the query sequence. In this case, percent identity calculated by FASTDB is not corrected manually. Again, only the - and C-terminal residue positions of the target sequences that are not matched / aligned with the query sequence are corrected manually, as shown in the FASTDB sequence. No other manual correction is made for the purposes of the present invention.

Preferably, the above-described programs are used to align the second sequence with the polypeptide of the invention and calculate the% identity manually. Percent identity is the total number of amino acid residues in the reference sequence, divided by the number of identical amino acid residues between the two sequences and multiplied by 100%. For example, the number of mismatched amino acids (i.e., point mutations: insertion, deletion, and substitution) is counted to determine the% identity from amino acid 1 to 100 of SEQ ID NO: 2, The number of amino acids of the same amino acid). Divide the resulting number by 100 and then multiply by 100%. If there is a mismatch (i.e., point mutation: insertion, deletion and substitution of each amino acid) at positions 1, 5, 21-23, 41 and 96-100 in SEQ ID NO: 2, the% identity will be 90%. 1 + 1 + 3 + 1 + 4 = 10.100 - 10 = 90.90 / 100 = 0.9. 0.9X100 = 90%

For example, in Bowie, JU et al., "Deciphering the Message in Protein Sequences: Tolerance to Amino Acid Substitutions", Science 247: 1306-1310 (1990) for making amino acid substitutions that do not appear in phenotypes, Here, the authors show two major strategies for studying changes in amino acid sequence tolerance.

The first strategy exploits the acceptance of amino acid substitutions by natural selection during the evolutionary process. By comparing the amino acid sequences in different species, the conserved amino acids can be identified. These conserved amino acids are likely to be important for protein function. In contrast, amino acid positions that are permissive for substitution by natural selection indicate that these positions are not important for protein function. Thus, positions that allow for amino acid substitutions can be altered while maintaining the biological activity of the protein.

The second strategy uses genetic manipulations to introduce amino acid changes at specific locations in the cloned gene to identify areas of importance for protein function. For example, site-directed mutagenesis or alanine-scanning mutagenesis (single alanine mutagenesis at every residue in a molecule) can be used. (Cunningham and Wells, Science 244: 1081-1085 (1989)). The resulting mutant molecules can be tested for biological activity.

As these authors disclose, these two strategies have revealed that proteins allow surprising amino acid substitutions. The authors also indicate which amino acid changes are likely to be allowed at which amino acid positions of the protein. For example, amino acid residues that are most deeply located (within the tertiary structure of the protein) require a nonpolar side chain while the characteristics of the side chain are generally not conserved. Moreover, permissible conservative amino acid substitutions include replacement of the aliphatic or hydrophobic amino acids Ala, Val, Leu, and Ile; Replacement of the hydroxyl residues Ser and Thr; Replacement of acidic residues Asp and Glu; Replacement of the amide residues Asn and Gln, replacement of the basic residues Lys, Arg, and His; Substitution of aromatic residues Phe, Tyr, and Trp, and replacement of small-sized amino acids Ala, Ser, Thr, Met, and Gly.

Fragments, or portions, of the polypeptides of the invention may also be used to generate corresponding full-length polypeptides by peptide synthesis. Thus, fragments may be used as intermediates for the generation of full-length polypeptides. Fragments or portions of the polynucleotides of the invention may also be used to synthesize full-length polynucleotides of the invention.

A suitable secretion signal may be incorporated into the expressed polypeptide so that the decrypted protein is secreted into the endoplasmic reticulum, periplasm, or extracellular environment. The signal may be endogenous to the polypeptide or it may be a heterologous signal.

The polypeptide may be expressed in a modified form, such as a fusion protein, and may include additional heterologous functional domains as well as secretory signals. For example, additional amino acid regions, particularly charged amino acids, may be added to the N-terminus of the polypeptide to improve stability and persistence in the host cell during purification or subsequent manipulation and storage. In addition, the peptide moiety may be added to the polypeptide to facilitate purification. Such regions may be removed prior to the final production of the polypeptide. Among other things, it is a familiar and routine technique in the art to add peptide moieties to polypeptides to promote secretion, improve stability, and promote purification. Preferred fusion proteins include heterologous regions from immunoglobulins useful for solubilizing proteins. For example, EP-A-0 464 533 (Canadian counterpart 2045869) discloses a fusion protein comprising a different protein or a portion thereof, along with various portions of the constant region of the immunoglobulin molecule. In many cases, the Fc portion of the fusion protein is beneficial for use in therapy and diagnosis, thus improving for example the pharmacokinetic properties (EP-A 0232 262). On the other hand, for some uses, it may be desirable to be able to delete the Fc portion after the fusion protein has been expressed, detected and purified in the disclosed beneficial manner. This is the case, for example, when the fusion protein is used as an antigen for immunization and the Fc portion is interfering with its use in therapy and diagnosis. In drug discovery, human proteins, such as the hIL5-receptor, were fused with the Fc portion for the purpose of, for example, high throughput screening assays to identify antagonists of hIL-5. (1995) and K. Johanson et al., The Journal of Biological Chemistry, Vol. 270, No. 16: 9459-9471 (1995) Reference.

The MPIF-1 protein can be prepared by a variety of methods including ammonium sulfate or ethanol precipitation, acid extraction, anion or cation exchange chromatography, phosphocellulose chromatography, hydrophobic interaction chromatography, affinity chromatography, hydroxylapatite chromatography and lectin chromatography Can be recovered and purified from the recombinant cell culture by known methods. Most preferably, high performance liquid chromatography (" HPLC ") is used for purification. The polypeptides of the present invention include products produced by recombinant techniques from eukaryotic or prokaryotic cells, including naturally purified products, products of chemical synthesis, and bacterial, yeast, higher plant, insect and mammalian cells. Depending on the host used in the recombinant production process, the polypeptide of the invention may be glycosylated or non-glycosylated. Moreover, the polypeptides of the present invention may also comprise, in some cases, the first modified methionine residue as a result of a host-mediated process.

MPIF-1 polypeptide variant.

It will be appreciated by those skilled in the art that some amino acid sequences of the MPIF-1 polypeptide may be altered without significant effect on the structure or function of the protein. If such changes in sequence are taken into account, remember that there is a critical region of the protein that determines activity. In general, it is possible to substitute a residue forming a tertiary structure if a similar functioning moiety is used. In other cases, the type of residue may not be entirely important if the change occurs in unimportant regions of the protein.

Accordingly, the invention further encompasses variants of MPIF-1 polypeptides that exhibit substantial MPIF-1 polypeptide activity or that comprise a region of the MPIF-1 protein, such as the protein portion disclosed below. Such mutations include deletions, insertions, inversions, repetitions, and type substitutions (e.g., substitution of one residue with one hydrophilic residue, but not strong hydrophilicity to replace strong hydrophobicity). Small changes or such " neutral " amino acid substitutions will generally have little effect on activity.

Conservative replacements are the replacement of the aliphatic amino acids Ala, Val, Leu and Ile with each other; The exchange of hydroxyl residues Ser and Thr, the exchange of acid residues Asp and GIu, the substitution between the amide residues Asn and Gln, the exchange of the basic residues Lys and Arg and the replacement of the aromatic residues Phe and Tyr.

An additional interest is also to replace the charged amino acid with another charged amino acid or neutral amino acid. This can result in proteins with improved properties such as less aggregation. Prevention of agglomeration is highly desirable. Protein aggregation not only causes a decrease in activity, but can also be a problem in the manufacture of pharmaceutical preparations because they can be immunogenic (Pinckard et al., Clin. Exp. Immunol. 2: 331-340 (1967), Robbins et al Diabetes 36: 838-845 (1987), Cleland et al., Crit. Rev. Therapeutic Drug Carrier Systems 10: 307-377 (1993)).

Substitution of amino acids can also alter the selectivity of binding to cell surface receptors. Ostade et al., Nature 361: 266-268 (1993) discloses a TNF alpha mutation that allows only selective binding of TNF alpha to only one of two known TNF receptors.

As further detailed above, additional guidance on what amino acid changes may not appear in the phenotype (i.e., not having a deleterious effect on the function) can be found in Bowie, JU, et al., "Deciphering the Message in Protein Sequences: Tolerance to Amino Acid Substitutions, " Science 247: 1306-1310 (1990) (see Table 1).

As shown, the changes are desirable for non-essential properties such as conservative amino acid substitutions that do not significantly affect protein folding or activity (see Table 1).

Conservative amino acid substitution Aromatic Phenylalanine, tryptophan, tyrosine Hydrophobicity Louis, Isooru, Balin polarity Glutamine, asparagine Basic Arginine, lysine, histidine acid Aspartic acid, glutamic acid Small in size Alanine, serine, threonine, methionine, glycine

Of course, the number of amino acid substitutions to be made by one skilled in the art will depend on many factors, including those described above and below. Generally speaking, the number of substitutions of any given MPIF-I polypeptide or its mutations will not exceed 50, 40, 30, 20, 10, 5, or 3, depending on the purpose. Specific MPIF-1 amino acid substitutions are described below.

Another aspect of the invention also includes amino acid substitutions. Of particular interest are conservative amino acid substitutions that do not significantly affect folding of the protein. Examples of conservative amino acid substitutions well known to those skilled in the art are set forth in Table 1 above.

Of particular interest to additions are the replacement of the charged amino acid with another charged amino acid or neutral amino acid. This can result in proteins with improved properties such as less aggregation. Prevention of agglomeration is highly desirable. Protein aggregation not only causes a decrease in activity, but can also be a problem in the manufacture of pharmaceutical preparations because they can be immunogenic (Pinckard et al., Clin. Exp. Immunol. 2: 331-340 (1967), Robbins et al Diabetes 36: 838-845 (1987), Cleland et al., Crit. Rev. Therapeutic Drug Carrier Systems 10: 307-377 (1993)).

The MPIF-I protein may contain one or several amino acid substitutions, deletions or additions from natural mutations or human manipulations. Some preferred mutations of the amino acid sequence shown in Figure 1 (SEQ ID NO: 2) are described below. (For example, Ala (21) Met indicates that Ala at position 21 in Figure 1 .

Ala (21) Met Asp (53) Ser

Thr (24) Ala Asp (53) Thr

Lys (25) Asn Asp (53) Met

Asp (26) Ala Ser (51) Gly

Asp (45) Ala Ser (34) Gly

Asp (45) Gly Glu (30) Gln

Asp (45) Ser Glu (28) Gln

Asp (45) Thr Pro (60) Thr

Asp (53) Met Ser (70) Ala

Asp (53) Ala

Asp (53) Gly

For example, site-directed changes at the amino acid level of MPIF-1 can be made by replacing a particular amino acid with a conservative amino acid. Preferred conservative mutations include, for example, preferred complementary mutations include the following:

Ml substituted with A, G, I, L, S, T, or V; H, or K substituted with R; V3 substituted with A, G, I, L, S, T, or M; S4 substituted with A, G, I, L, T, M, or V; V5 substituted with A, G, I, L, S, T, or M; A6 substituted with G, I, L, S, T, M, or V; L8 substituted with A, G, I, S, T, M, or V; S9 substituted with A, G, I, L, T, M, or V; Lll substituted with A, G, I, S, T, M, or V; Ml2 substituted with A, G, I, L, S, T, or V; L13 substituted with A, G, I, S, T, M, or V; G, I, L, S, T, M, or V substituted with A, G, I, L, S: T or M; Or A16 substituted with V; L17 substituted with A, G, I, S, T, M, or V; G18 substituted with A, I, L, S, T, M, or V; S19 substituted with A, G, I, L, T, M, or V; Q20 substituted with N; A21 H substituted with G, I, L, S, T, M, or V, or R22 substituted with K; V23 substituted with A, G, I, L, S, T, or M; T24 substituted with A, G, I, L, S, M, or V; H, or K25 substituted with R; D26 substituted with E; G, I, L, S, T, M, or V; E28 substituted with D; T29 substituted with A, G, I, L, S, M, or V; E substituted with D; W, or F; M32 substituted with A, G, I, L, S, T, or V; M33 substituted with A, G, I, L, S, T, or V; S34 substituted with A, G, I, L, T, M, or V; H, or K35 substituted with R; L36 substituted with A, G, I, S, T, M, or V; L38 substituted with A, G, I, S, T, M, or V; E39 substituted with D; N40 substituted with Q; V42 substituted with A, G, I, L, S, T, or M; L43 substituted with A, G, I, S, T, M, or V; L44 substituted with A, G, I, S, T, M, or V; D45 substituted with E; H, or R < 46 > substituted with K; W, or F; K, or H48 substituted with R; G, I, L, S, T, M, or V; T50 substituted with A, G, I, L, S, M, or V; S51 substituted with A, G, I, L, T, M, or V; G, I, L, S, T, M, or V; D53 substituted with E; I56 substituted with A, G, L, S, T, M, or V; S57 substituted with A, G, I, L, T, M, or V; F, or Y58 substituted with W; T59 substituted with A, G, I, L, S, M, or V; H, or R < 61 > S62 substituted with A, G, I, L, T, M, or V; I63 substituted with A, G, L, S, T, M, or V; S66 substituted with A, G, I, L, T, M, or V; L67 substituted with A, G, I, S, T, M, or V; L68 substituted with A, G, I, S, T, M, or V; E69 substituted with D; S70 substituted with A, G, I, L, T, M, or V; F, or Y; W, or Y; E73 substituted with D; T74 substituted with A, G, I, L, S, M, or V; N75 substituted with Q; S76 substituted with A, G, I, L, T, M, or V; E77 substituted with D; S79 substituted with A, G, I, L, T, M, or V; H, or K substituted with R; G82 substituted with A, I, L, S, T, M, or V; V83 substituted with A, G, I, L, S, T, or M; I84 substituted with A, G, L, S, T, M, or V; W, or F; L86 substituted with A, G, I, S, T, M, or V; T87 substituted with A, G, I, L, S, M, or V; H, or K88 substituted with R; H, or K89 substituted with R; G90 substituted with A, I, L, S, T, M, or V; H, or R < 91 > H, or R92 substituted with K; W, or F; A95 substituted with G, I, L, S, T, M, or V; N96 substituted with Q; S98 substituted with A, G, I, L, T, M, or V; D99 substituted with E; H, or Kl00 substituted with R; Q101 substituted with N; V 102 substituted with A, G, I, L, S, T, or M; Q103 substituted with N; V104 substituted with A, G, I, L, S, T, or M; Ml06 substituted with A, G, I, L, S, T, or V; H, or R; Ml08 substituted with A, G, I, L, S, T, or V; L109 substituted with A, G, I, S, T, M, or V; H, or K110 substituted with R; Llll substituted with A, G, I, S, T, M, or V; Dl 12 substituted with E; Tl3 substituted with A, G, I, L, S, M, or V; H, or R < 14 > L15 substituted with A, G, L, S, T, M, or V; H, or K < 116 > substituted with R; Tl 17 substituted with A, G, I, L, S, M, or V; H, or R < 18 > H, or Kl19 substituted with R; Q substituted with N120.

The resulting constructs can be readily selected for activity or function as disclosed herein and known in the art. Preferably, the resulting construct has increased and / or reduced MPIF-1 activity or function, and the remaining MPIF-1 activity or function is maintained. More preferably, the resulting construct has one or more increased and / or reduced MPIF-1 activity or function, and the remaining MPIF-1 activity or function is maintained.

In addition to conservative amino acid substitutions, variants of MPIF-1 may be either (i) substitution with one or more non-conserved amino acid residues, wherein the substituted amino acid residue may or may not be encoded by the genetic code, or (ii) (Iii) fusion of a mature polypeptide with another compound such as a compound (e. G., Polyethylene glycol) to increase the stability and / or solubility of the polypeptide, or (iv) Region peptide, or leader or secretion sequence, or a fusion of an additional amino acid and a polypeptide, such as a purification facilitating sequence. Such variant polypeptides are considered to be within the purview of those skilled in the art from the teachings herein.

For example, an MPIF-1 polypeptide variant containing a charged amino acid substituted with another charged or neutral amino acid may produce a protein with improved properties such as less aggregation. Aggregation of the pharmaceutical preparations reduces activity and increases the clearance rate due to the immunogenic activity of the aggregates (Pinckard et al., Clin. Exp. Immunol. 2: 331-340 (1967), Robbins et al., Diabetes 36: 838 -845 (1987), Cleland et al., Crit. Rev. Therapeutic Drug Carrier Systems 10: 307-377 (1993)).

For example, preferred non-conservative substitutions of MPIF-1 include the following: for example, preferred non-complementary mutations include:

M1 substituted with D, E, H, K, R, N, Q, F, W, Y, P, or C; K2 substituted with D, E, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P or C; V3 substituted with D, E, H, K, R, N, Q, F, W, Y, P or C; S4 substituted with D, E, H, K, R, N, Q, F, W, Y, P or C; V5 substituted with D, E, H, K, R, N, Q, F, W, Y, P or C; A6 substituted with D, E, H, K, R, N, Q, F, W, Y, P, or C; A7 substituted by D, E, H, K, R, N, Q, F, W, Y, P or C; L, substituted by D, E, H, K, R, N, Q, F, W, Y, P, or C; S9 substituted by D, E, H, K, R, N, Q, F, W, Y, P, or C; D, E, H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y or P; L, substituted by D, E, H, K, R, N, Q, F, W, Y, P, or C; M, substituted by D, E, H, K, R, N, Q, F, W, Y, P, or C; L13 substituted with D, E, H, K, R, N, Q, F, W, Y, P, or C; V14 substituted with D, E, H, K, R, N, Q, F, W, Y, P, or C; T, D, E, H, K, R, N, Q, F, W, Y, P substituted with D, E, H, K, R, N, Q, F, W, , Or A16 substituted with C; L17 substituted with D, E, H, K, R, N, Q, F, W, Y, P, or C; G18 substituted with D, E, H, K, R, N, Q, F, W, Y, P, or C; S, substituted with D, E, H, K, R, N, Q, F, W, Y, P, or C; Q20 substituted with D, E, H, K, R, A, G, I, L, S, T, M, V, F, W, Y, P or C; A2l substituted with D, E, H, K, R, N, Q, F, W, Y, P, or C; D, E, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P or C; V23 substituted with D, E, H, K, R, N, Q, F, W, Y, P, or C; T24 substituted with D, E, H, K, R, N, Q, F, W, Y, P, or C; K25 substituted with D, E, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P or C; D26 substituted with H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P or C; A27 substituted with D, E, H, K, R, N, Q, F, W, Y, P, or C; E28 substituted with H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P or C; T29 substituted with D, E, H, K, R, N, Q, F, W, Y, P, or C; E30 substituted with H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P or C; F31 substituted with D, E, H, K, R, N, Q, A, G, I, L, S, T, M, V, P or C; M32 substituted with D, E, H, K, R, N, Q, F, W, Y, P, or C; M33 substituted with D, E, H, K, R, N, Q, F, W, Y, P, or C; S, substituted with D, E, H, K, R, N, Q, F, W, Y, P, or C; K35 substituted with D, E, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P or C; L, substituted by D, E, H, K, R, N, Q, F, W, Y, P or C; P37 substituted with D, E, H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y or C; L, substituted by D, E, H, K, R, N, Q, F, W, Y, P, or C; E39 substituted with H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P or C; N, substituted by D, E, H, K, R, A, G, I, L, S, T, M, V, F, W, Y, P or C; P41 substituted with D, E, H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y or C; V42 substituted with D, E, H, K, R, N, Q, F, W, Y, P, or C; L43 substituted with D, E, H, K, R, N, Q, F, W, Y, P, or C; L44 substituted with D, E, H, K, R, N, Q, F, W, Y, P, or C; D45 substituted with H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P or C; R46 substituted with D, E, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P or C; F47 substituted with D, E, H, K, R, N, Q, A, G, I, L, S, T, M, V, P or C; H48 substituted with D, E, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P or C; A49 substituted with D, E, H, K, R, N, Q, F, W, Y, P, or C; T50 substituted with D, E, H, K, R, N, Q, F, W, Y, P, or C; D, E, H, K, R, N, Q, F, W, Y, P substituted with D, E, H, K, R, N, Q, F, W, , Or A52 substituted with C; D53 substituted with H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P or C; C54 substituted by D, E, H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y or P; C55 substituted with D, E, H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y or P; D, E, H, K, R, N, Q, F, W, Y, P, or C; S57 substituted with D, E, H, K, R, N, Q, F, W, Y, P, or C; Y58 substituted with D, E, H, K, R, N, Q, A, G, I, L, S, T, M, V, P or C; T59 substituted with D, E, H, K, R, N, Q, F, W, Y, P, or C; P60 substituted with D, E, H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y or C; R61 substituted with D, E, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P or C; S62 substituted with D, E, H, K, R, N, Q, F, W, Y, P, or C; I63 substituted with D, E, H, K, R, N, Q, F, W, Y, P, or C; P64 substituted with D, E, H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y or C; C65 substituted with D, E, H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y or P; S66 substituted with D, E, H, K, R, N, Q, F, W, Y, P, or C; L67 substituted with D, E, H, K, R, N, Q, F, W, Y, P, or C; L68 substituted with D, E, H, K, R, N, Q, F, W, Y, P, or C; E69 substituted with H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P or C; S70 substituted with D, E, H, K, R, N, Q, F, W, Y, P, or C; Y71 substituted with D, E, H, K, R, N, Q, A, G, I, L, S, T, M, V, P or C; F72 substituted with D, E, H, K, R, N, Q, A, G, I, L, S, T, M, V, P or C; E73 substituted with H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P or C; T74 substituted with D, E, H, K, R, N, Q, F, W, Y, P, or C; N75 substituted with D, E, H, K, R, A, G, I, L, S, T, M, V, F, W, Y, P or C; S76 substituted with D, E, H, K, R, N, Q, F, W, Y, P or C; E77 substituted with H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P or C; C78 substituted with D, E, H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y or P; E, A, G, I, L, S, T, M, V, N substituted with D, E, H, K, R, N, Q, F, W, Y, P or C , K80 substituted with Q, F, W, Y, P, or C; P81 substituted with D, E, H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y or C; G82 substituted with D, E, H, K, R, N, Q, F, W, Y, P, or C; V83 substituted with D, E, H, K, R, N, Q, F, W, Y, P, or C; I84 substituted with D, E, H, K, R, N, Q, F, W, Y, P, or C; F85 substituted with D, E, H, K, R, N, Q, A, G, I, L, S, T, M, V, P or C; L86 substituted with D, E, H, K, R, N, Q, F, W, Y, P, or C; T87 substituted with D, E, H, K, R, N, Q, F, W, Y, P, or C; K88 substituted with D, E, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P or C; K89 substituted with D, E, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P or C; G90 substituted with D, E, H, K, R, N, Q, F, W, Y, P, or C; D, E, A, G, I, L substituted with R, D, E, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P, or C; , S, T, M, V, N, Q, F, W, Y, P, or C; F93 substituted with D, E, H, K, R, N, Q, A, G, I, L, S, T, M, V, P or C; D, E, H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y or P; , A95 substituted with R, N, Q, F, W, Y, P, or C; N96 substituted with D, E, H, K, R, A, G, I, L, S, T, M, V, F, W, Y, P or C; P97 substituted with D, E, H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y or C; S98 substituted with D, E, H, K, R, N, Q, F, W, Y, P, or C; D, E, A, G, I substituted with H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P or C , Kl00 substituted with L, S, T, M, V, N, Q, F, W, Y, P, or C; Q101 substituted with D, E, H, K, R, A, G, I, L, S, T, M, V, F, W, Y, P or C; V102 substituted with D, E, H, K, R, N, Q, F, W, Y, P, or C; Q103 substituted with D, E, H, K, R, A, G, I, L, S, T, M, V, F, W, Y, P or C; V104 substituted with D, E, H, K, R, N, Q, F, W, Y, P, or C; C105 substituted with D, E, H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y or P; Ml06 substituted with D, E, H, K, R, N, Q, F, W, Y, P, or C; D, E, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P or C; M108 substituted with D, E, H, K, R, N, Q, F, W, Y, P, or C; L109 substituted with D, E, H, K, R, N, Q, F, W, Y, P, or C; Q, substituted with D, E, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P or C; Llll substituted with D, E, H, K, R, N, Q, F, W, Y, P, or C; Dl 12 substituted with H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P or C; Tl3 substituted with D, E, H, K, R, N, Q, F, W, Y, P, or C; R14 is substituted with D, E, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P or C; I115 substituted with D, E, H, K, R, N, Q, F, W, Y, P, or C; Kl 16 substituted with D, E, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P or C; Tl 17 substituted with D, E, H, K, R, N, Q, F, W, Y, P, or C; Rl 18 substituted with D, E, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P or C; Kl19 substituted with D, E, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P or C; D, E, H, K, R, A, G, I, L.

The resulting constructs can be readily selected for activity or function as disclosed herein and known in the art. Preferably, the resulting construct has increased and / or reduced MPIF-1 activity or function, and the remaining MPIF-1 activity or function is maintained. More preferably, the resulting construct has one or more increased and / or reduced MPIF-1 activity or function, and the remaining MPIF-1 activity or function is maintained.

In addition, one or more amino acids (e.g., 2, 3, 4, 5, 6, 7, 8, 9 and 10) may be replaced with substituted amino acids (conservative or non-conservative) as described above. Substituted amino acids may occur in N- and C-terminal deletion mutants with the general formula m-n listed below, as well as the full length, maturation, or proprotein form of the MPIF-I protein.

A further embodiment of the present invention is a pharmaceutical composition comprising one or more but not more than 50 amino acid substitutions, more preferably not more than 40 amino acid substitutions, more preferably not more than 30 amino acid substitutions, even more preferably not more than 20 amino acids 1 < / RTI > polypeptide having an amino acid sequence containing a substitution. Of course, MPIF containing at least one, but no more than 10, no more than 9, no more than 8, no more than 7, no more than 6, no more than 5, no more than 4, no more than 3, no more than 2 or no more than 1 amino acid substitution in increasing order of preference -1 < / RTI > polypeptide of the present invention. In certain embodiments, the number of additions, substitutions, and / or deletions in the amino acid sequence of Figure 1 or a fragment thereof (e.g., mature form and / or other fragment thereof disclosed herein) is 1-5, 5-10, 5- 25, 5-50, 10-50, or 50-150, with conservative amino acid substitutions being preferred.

As described above, the MPIF-1 polypeptide comprises or consists of the amino acid sequence of SEQ ID NO: 2, SEQ ID NO: 7, or the amino acid sequence encoded by the deposited cDNA clone, except for one or more acid substitutions, deletions, It is possible. Also, as described above, the MPIF-1 polypeptide comprises at least 80%, 85%, 90%, 92%, 95%, 96%, 97% 98%, or 99%. In one embodiment, such a polypeptide variant also has the same or nearly the same structure as MPIF-1 or at least one region thereof. For example, the structure may be a solution structure determined by NMR (see Ex. ) Polynucleotides encoding these polypeptides are also encompassed by the present invention.

For example, an MPIF-1 polypeptide may contain one or several amino acid changes (substitution, deletion, or addition), but no change at any particular residue or position. The positions at which substitutions, deletions or additions may be excluded include positions Cys-54 (Cys-11 in Example 37), Cys-55 (Cys-12 in Example 37), Cys-78 Cys-35 at 37), and four Cys residues at Cys-94 (Cys-51 in Example 37). The conserved residues also include two Cys residues at position Cys-65 of SEQ ID NO: 2 (Cys-22 in Example 37), and Cys-105 (Cys-62 in Example 37). Any one or combination of these positions may be excluded from being substituted, deleted, or added. Preferably, the MPIF-1 variant contains substitution, deletion, or addition except in each of the six Cys residues of MPIF-1.

Additional residues that may be excluded from being changed include Ile-56 (Ile-13 of Example 37), Tyr-58 (Tyr-15 of Example 37), Arg-61 Arg-18), and Ile-63 (Ile-20 of Example 37). Any one or combination of these positions may be excluded from being substituted, deleted, or added. Preferably, MPIF-1 variants contain substitutions, deletions, or additions except at each of these residues. Alternatively, such conservatively substituted moieties may be altered by conservative substitution.

Additional residues that may be excluded from being changed include Ile-56 (Ile-13 of Example 37), Tyr-58 (Tyr-15 of Example 37), Arg-61 Arg-18), Thr-74 (Thr-31 of Example 37) and Thr-87 (Thr-44 of Example 37). Any one or combination of these positions may be excluded from being substituted, deleted, or added. Preferably, MPIF-1 variants contain substitutions, deletions, or additions except at each of these residues. Alternatively, such conservatively substituted moieties may be altered by conservative substitution.

Additional residues that may be excluded from being changed include Ile-56 (Ile-13 of Example 37), Tyr-58 (Tyr-15 of Example 37), Arg-61 Arg-18), and conservative substitutions such as Ile-63 (Ile-20 of Example 37), Thr-74 (Thr-31 of Example 37), and Thr-87 (Thr- ≪ / RTI > Any one or combination of these positions may be excluded from being substituted, deleted, or added. Preferably, MPIF-1 variants contain substitutions, deletions, or additions except at each of these residues. Alternatively, such conservatively substituted moieties may be altered by conservative substitution.

Conservatively substituted residues also include Leu-68 (Leu-25 in Example 37), Tyr-71 (Tyr-28 in Example 37), Ile- (Phe-29 in Example 37), Val-83 (Val-40 in Example 37), Ile-84 (Ile-41 in Example 37), Phe- (Phe-50 in Example 37), Ala-95 (Ala-52 in Example 37), Val-102 (Val-59 in Example 37), Met- Met-63) and Leu-109 (Leu-66 in Example 37). Any one or combination of these positions may be excluded from being substituted, deleted, or added. Preferably, MPIF-1 variants contain substitutions, deletions, or additions except at each of these residues. Alternatively, such conservatively substituted moieties may be altered by conservative substitution.

Additional conservatively substituted residues include Ile-56 (Ile-13 in Example 37), Arg-61 (Arg-18 in Example 37), Tyr-58 ), Thr-74 (Thr-31 in Example 37), and Gly-82 (Gly-39 in Example 37). Any one or combination of these positions may be excluded from being substituted, deleted, or added. Preferably, MPIF-1 variants contain substitutions, deletions, or additions except at each of these residues. Alternatively, such conservatively substituted moieties may be altered by conservative substitution.

Additional conservatively substituted moieties include Pro-64 of SEQ ID NO: 2 (Pro-21 of Example 37), and Pro-97 (Pro-54 of Example 37). Any one or combination of these positions may be excluded from being substituted, deleted, or added. Preferably, MPIF-1 variants contain substitutions, deletions, or additions except at each of these residues. Alternatively, such conservatively substituted moieties may be altered by conservative substitution.

Additional conservatively substituted residues include Gln-101 of SEQ ID NO: 2 (Gln-58 of Example 37). Preferably, the moiety is excluded from being substituted, deleted, or added. Preferably, it is conservatively substituted. More preferably a bulky amino acid with no side chain. Therefore, for example, it is preferable to be substituted with an amino acid other than Trp.

Additional conservatively substituted residues include Arg-61 (Arg-18 in Example 37), Lys-88 (Lys-45 in Example 37), and Arg-91 (Arg- 48). Any one or combination of these positions may be excluded from being substituted, deleted, or added. Preferably, the MPIF-1 variant contains substitutions, deletions, or additions, but not all of these residues. Alternatively, such conservatively substituted moieties may be altered by conservative substitution.

Additional conservatively substituted residues include Lys-89 (Lys-46 in Example 37), Lys-100 (Lys-57 in Example 37), Arg-107 ), And Lys-110 (Lys-67 in Example 37). Any one or combination of these positions may be excluded from being substituted, deleted, or added. Preferably, the MPIF-1 variant contains substitutions, deletions, or additions, but not all of these residues. Alternatively, such conservatively substituted moieties may be altered by conservative substitution.

In addition to the preferred combinations described above, preferred combinations of conserved and conservatively substituted residues include Thr-74 (Thr-31 in Example 37), Gly-82 (Gly-39 in Example 37) (Tyr-15 in Example 37), Cys-54 (Cys-11 in Example 37), Cys-55 (Cys-12 in Example 37), and Cys- 51). Preferably, MPIF-1 variants contain substitutions, deletions, or additions except at each of these residues. Alternatively, such a residue may be changed by conservative substitution.

Preferably, the MPIF-1 variant contains an amino acid change except for at least one or both of the conservatively substituted residues. Alternatively, at least one or both of the conservatively substituted moieties may be changed by conservative substitution.

Preferably, the MPIF-1 variant comprises at least one or all of the conserved residues (e.g., one or more Cys residues) and at least one or both conservatively substituted residues (e.g., one or more of the residues) Lt; / RTI > contains amino acid changes. Alternatively, at least one or both of the conservatively substituted moieties may be changed by conservative substitution.

MPIF-1 splice variant.

In addition, mutants of MPIF-1 were identified and characterized. Several of these analogs include amino terminal truncations. In addition, MPIF-1 analogs generated from the substitution splice site were also identified and characterized (Fig. 20 (SEQ ID NO: 7)). Example 11 discloses the biological activity of these MPIF-1 analogs. The sequences of these analogs are shown in Figure 19 (SEQ ID NOS: 3, 4, and 5 and in amino acid residues 46-120, 45-120, 48-120, 49-120, 39-120, and 44-120 in SEQ ID NO: 2) appear.

In another embodiment, the invention includes amino acid substitutions in a 137 amino acid splice variant of MPIF-1. For example, conservative substitutions include:

M1 substituted by A, G, I, L, S, T, or V of SEQ ID NO: 7, H, or K2 substituted with R; V3 substituted with A, G, I, L, S, T, or M; S4 substituted with A, G, I, L, T, M, or V; V5 substituted with A, G, I, L, S, T, or M; G, I, L, S, T, M, or V; G, I, L, S, T, M, or V; L8 substituted with A, G, I, S, T, M, or V; S9 substituted with A, G, I, L, T, M, or V; L11 substituted with A, G, I, S, T, M, or V; M12 substituted with A, G, I, L, S, T, or V; L13 substituted with A, G, I, S, T, M, or V; Vl4 substituted with A, G, I, L, S, T, or M; T15 substituted with A, G, I, L, S, M, or V; G, I, L, S, T, M, or V; L17 substituted with A, G, I, S, T, M, or V; G18 substituted with A, I, L, S, T, M, or V; S19 substituted with A, G, I, L, T, M, or V; Q20 substituted with N; G, I, L, S, T, M, or V; H, or R < 22 > V23 substituted with A, G, I, L, S, T, or M; T24 substituted with A, G, I, L, S, M, or V; H, or K25 substituted with R; D26 substituted with E; G, I, L, S, T, M, or V; E28 substituted with D; T29 substituted with A, G, I, L, S, M, or V; E substituted with D; W, or F; M32 substituted with A, G, I, L, S, T, or V; M33 substituted with A, G, I, L, S, T, or V; S34 substituted with A, G, I, L, T, M, or V; H, or K35 substituted with R; L36 substituted with A, G, I, S, T, M, or V; L38 substituted with A, G, I, S, T, M, or V; E39 substituted with D; N40 substituted with Q; V42 substituted with A, G, I, L, S, T, or M; L43 substituted with A, G, I, S, T, M, or V; L44 substituted with A, G, I, S, T, M, or V; M46 substituted with A, G, I, L, S, T, or V; L47 substituted with A, G, I, S, T, M, or V; F, or W; H, or R < 49 > H, or R < 50 > substituted with K; H, or K51 substituted with R; I52 substituted with A, G, L, S, T, M, or V; G53 substituted with A, I, L, S, T, M, or V; Q55 substituted with N; M56 substituted with A, G, I, L, S, T, or V; T57 substituted with A, G, I, L, S, M, or V; L58 substituted with A, G, I, S, T, M, or V; S59 substituted with A, G, I, L, T, M, or V; K, or H60 substituted with R; A61 substituted with G, I, L, S, T, M, or V; G, I, L, S, T, M, or V; G63 substituted with A, I, L, S, T, M, or V; W, or Y; K, or H65 substituted with R; G, I, L, S, T, M, or V; T67 substituted with A, G, I, L, S, M, or V; S68 substituted with A, G, I, L, T, M, or V; G, I, L, S, T, M, or V; D70 substituted with E; I73 substituted with A, G, L, S, T, M, or V; S74 substituted with A, G, I, L, T, M, or V; F, or Y, substituted with W; T76 substituted with A, G, I, L, S, M, or V; H, or R < 78 > S79 substituted with A, G, I, L, T, M, or V; I80 substituted with A, G, L, S, T, M, or V; S83 substituted with A, G, I, L, T, M, or V; L84 substituted with A, G, I, S, T, M, or V; L85 substituted with A, G, I, S, T, M, or V; E86 substituted with D; S87 substituted with A, G, I, L, T, M, or V; F, or Y88 substituted with W; W, or Y; E90 substituted with D; T91 substituted with A, G, I, L, S, M, or V; N92 substituted with Q; S93 substituted with A, G, I, L, T, M, or V; E94 substituted with D; S96 substituted with A, G, I, L, T, M, or V; H, or G97 substituted with K, A, I, L, S, T, M, or V; VI00 substituted with A, G, I, L, S, T, or M; I101 substituted with A, G, L, S, T, M, or V; W, or Fl02 substituted with Y; L103 substituted with A, G, I, S, T, M, or V; T104 substituted with A, G, I, L, S, M, or V; H, or KlO5 substituted with R; H, or < RTI ID = 0.0 > R < / RTI > H, or R108 substituted with K, H, or R109 substituted with K; W, or F1; G, I, L, S, T, M, or V; N113 substituted with Q; S115 substituted with A, G, I, L, T, M, or V; D116 substituted with E; H, or K117 substituted with R; Q118 substituted with N; V119 substituted with A, G, I, L, S, T, or M; Q120 substituted with N; V121 substituted with A, G, I, L, S, T, or M; M123 substituted with A, G, I, L, S, T, or V; H, or R124 substituted with K; M125 substituted with A, G, I, L, S, T, or V; L126 substituted with A, G, I, S, T, M, or V; H, or K127 substituted with R; L128 substituted with A, G, I, S, T, M, or V; D129 substituted with E; T130 substituted with A, G, I, L, S, M, or V; H, or R < 13 > I132 substituted with A, G, L, S, T, M, or V; H, or K133 substituted with R; T134 substituted with A, G, I, L, S, M, or V; H, or R < 135 > H, or K136 substituted with R; And / or < RTI ID = 0.0 > Q < / RTI >

For example, a non-conservative substitution in a 137 amino acid splice variant includes: D, E, H, K, R, N, Q, F, W, Y, P, or C in SEQ ID NO: Substituted MI; K2 substituted with D, E, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P or C; V3 substituted with D, E, H, K, R, N, Q, F, W, Y, P or C; S4 substituted with D, E, H, K, R, N, Q, F, W, Y, P or C; V5 substituted with D, E, H, K, R, N, Q, F, W, Y, P or C; A6 substituted with D, E, H, K, R, N, Q, F, W, Y, P, or C; A7 substituted by D, E, H, K, R, N, Q, F, W, Y, P or C; L, substituted by D, E, H, K, R, N, Q, F, W, Y, P, or C; S, D, E, H, K, R, A, G, I, L, S, T substituted with D, E, H, K, R, N, Q, F, W, Y, P or C , M, V, N, Q, F, W, Y, or P; L11 substituted with D, E, H, K, R, N, Q, F, W, Y, P, or C; M, substituted by D, E, H, K, R, N, Q, F, W, Y, P, or C; L13 substituted with D, E, H, K, R, N, Q, F, W, Y, P, or C; V14 substituted with D, E, H, K, R, N, Q, F, W, Y, P, or C; T15 substituted with D, E, H, K, R, N, Q, F, W, Y, P, or C; A16 substituted with D, E, H, K, R, N, Q, F, W, Y, P, or C; L17 substituted with D, E, H, K, R, N, Q, F, W, Y, P, or C; G18 substituted with D, E, H, K, R, N, Q, F, W, Y, P, or C; S, substituted with D, E, H, K, R, N, Q, F, W, Y, P, or C; E, H, K, R, A, G, I, L, S, T, M, V, F, W, Y, P or C; , A, G, I, L, S, T, M, V, N, Q, F, W, Y and A21 substituted with N, Q, F, W, Y, P, or C; V23 substituted with D, E, H, K, R, N, Q, F, W, Y, P, or C; T24 substituted with D, E, H, K, R, N, Q, F, W, Y, P, or C; K25 substituted with D, E, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P or C; H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P or C; , A27 substituted with N, Q, F, W, Y, P, or C; E28 substituted with H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P or C; T29 substituted with D, E, H, K, R, N, Q, F, W, Y, P, or C; E30 substituted with H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P or C; F31 substituted with D, E, H, K, R, N, Q, A, G, I, L, S, T, M, V, P or C; M32 substituted with D, E, H, K, R, N, Q, F, W, Y, P, or C; M, D, E, H, K, R, N, Q, F, W, Y, P substituted with D, E, H, K, R, N, Q, F, W, , Or S < 34 > substituted with C; K35 substituted with D, E, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P or C; L, substituted by D, E, H, K, R, N, Q, F, W, Y, P or C; P37 substituted with D, E, H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y or C; L, substituted by D, E, H, K, R, N, Q, F, W, Y, P, or C; E39 substituted with H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P or C; D, E, H, K, R, substituted with D, E, H, K, R, A, G, I, L, S, T, M, V, F, W, Y, P or C P41 substituted with A, G, I, L, S, T, M, V, N, Q, F, W, Y, or C; V42 substituted with D, E, H, K, R, N, Q, F, W, Y, P, or C; L43 substituted with D, E, H, K, R, N, Q, F, W, Y, P, or C; L44 substituted with D, E, H, K, R, N, Q, F, W, Y, P, or C; H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P or C; , M46 substituted with N, Q, F, W, Y, P, or C; L47 substituted with D, E, H, K, R, N, Q, F, W, Y, P, or C; W48 substituted with D, E, H, K, R, N, Q, A, G, I, L, S, T, M, V, P or C; D, E, A, G, I, L substituted with D, E, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P, , R, S, T, M, V, N, Q, F, W, Y, P or C; D, E, A, G, I, L, S, T, K52 substituted with Q, F, W, Y, P or C; l52 substituted with D, E, H, K, R, N, Q, F, W, Y, P or C; G53 substituted with D, E, H, K, R, N, Q, F, W, Y, P, or C; D, E, H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y or C; Q55 substituted with R, A, G, I, L, S, T, M, V, F, W, Y, P or C; M56 substituted with D, E, H, K, R, N, Q, F, W, Y, P, or C; T57 substituted with D, E, H, K, R, N, Q, F, W, Y, P, or C; L58 substituted with D, E, H, K, R, N, Q, F, W, Y, P, or C; S59 substituted with D, E, H, K, R, N, Q, F, W, Y, P, or C; D, E, H, K, R, N substituted with D, E, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P or C , A61 substituted with Q, F, W, Y, P, or C; A62 substituted with D, E, H, K, R, N, Q, F, W, Y, P, or C; G63 substituted with D, E, H, K, R, N, Q, F, W, Y, P, or C; F64 substituted with D, E, H, K, R, N, Q, A, G, I, L, S, T, M, V, P or C; H65 substituted with D, E, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P or C; A66 substituted with D, E, H, K, R, N, Q, F, W, Y, P, or C; T67 substituted with D, E, H, K, R, N, Q, F, W, Y, P, or C; S68 substituted with D, E, H, K, R, N, Q, F, W, Y, P, or C; A69 substituted with D, E, H, K, R, N, Q, F, W, Y, P, or C; D70 substituted with H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P or C; C71 substituted with D, E, H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y or P; C72 substituted with D, E, H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y or P; D, E, H, K, R, N, Q, F, W, Y, and P substituted with D, E, H, K, R, N, Q, F, W, , Or S74 substituted with C; Y, D, E, H, K, R, N substituted with D, E, H, K, R, N, Q, A, G, I, L, S, T, M, V, P, , Q, F, W, Y, P, or C; D, E, H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, or C; , I, L, S, T, M, V, N, Q, F, W, Y, P, or C; S79 substituted with D, E, H, K, R, N, Q, F, W, Y, P, or C; D, E, H, K, R, N, Q, F, W, Y, P, or C; P81 substituted with D, E, H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y or C; C82 substituted with D, E, H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y or P; S83 substituted with D, E, H, K, R, N, Q, F, W, Y, P, or C; L84 substituted with D, E, H, K, R, N, Q, F, W, Y, P, or C; L, H, K, R, A, G, I, L, S, T, M, V substituted with D, E, H, K, R, N, Q, F, W, , E 86 substituted with N, Q, F, W, Y, P, or C; S87 substituted with D, E, H, K, R, N, Q, F, W, Y, P, or C; Y88 substituted with D, E, H, K, R, N, Q, A, G, I, L, S, T, M, V, P or C; F89 substituted with D, E, H, K, R, N, Q, A, G, I, L, S, T, M, V, P or C; E, H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P or C; , T91 substituted with N, Q, F, W, Y, P, or C; N92 substituted with D, E, H, K, R, A, G, I, L, S, T, M, V, F, W, Y, P or C; S93 substituted with D, E, H, K, R, N, Q, F, W, Y, P, or C; E94 substituted with H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P or C; C95 substituted with D, E, H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y or P; S96 substituted with D, E, H, K, R, N, Q, F, W, Y, P, or C; K97 substituted with D, E, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P or C; P98 substituted with D, E, H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y or C; G99 substituted with D, E, H, K, R, N, Q, F, W, Y, P, or C; V100 substituted with D, E, H, K, R, N, Q, F, W, Y, P, or C; I10l substituted with D, E, H, K, R, N, Q, F, W, Y, P, or C; F 102 substituted with D, E, H, K, R, N, Q, A, G, I, L, S, T, M, V, P or C; D, E, H, K, R, N, Q, F, W, Y, and P substituted with D, E, H, K, R, N, Q, F, W, , Or T104 substituted with C; Kl05 substituted with D, E, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P or C; Kl06 substituted with D, E, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P or C; GlO7 substituted with D, E, H, K, R, N, Q, F, W, Y, P, or C; D, E, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P or C; D, E, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P or C; Fl10 substituted with D, E, H, K, R, N, Q, A, G, I, L, S, T, M, V, P or C; C11l substituted with D, E, H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y or P; A112 substituted with D, E, H, K, R, N, Q, F, W, Y, P, or C; N113 substituted with D, E, H, K, R, A, G, I, L, S, T, M, V, F, W, Y, P or C; D, E, H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y or C; , S115 substituted with R, N, Q, F, W, Y, P, or C; D116 substituted with H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P or C; K117 substituted with D, E, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P or C; D, E, H, K, R, substituted with D, E, H, K, R, A, G, I, L, S, T, M, V, F, W, Y, P or C , V119 substituted with N, Q, F, W, Y, P, or C; Q120 substituted with D, E, H, K, R, A, G, I, L, S, T, M, V, F, W, Y, P or C; V121 substituted with D, E, H, K, R, N, Q, F, W, Y, P, or C; C122 substituted with D, E, H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y or P; M123 substituted with D, E, H, K, R, N, Q, F, W, Y, P, or C; D, E, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P or C; M125 substituted with D, E, H, K, R, N, Q, F, W, Y, P, or C; L126 substituted with D, E, H, K, R, N, Q, F, W, Y, P, or C; K, D, E, H, K, R, N substituted with D, E, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P or C , L128 substituted with Q, F, W, Y, P, or C; H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P or C; , T130 substituted with N, Q, F, W, Y, P, or C; R131 substituted with D, E, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P or C; I132 substituted with D, E, H, K, R, N, Q, F, W, Y, P, or C; K133 substituted with D, E, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P or C; T134 substituted with D, E, H, K, R, N, Q, F, W, Y, P, or C; D, E, A, G, I, L substituted with D, E, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P or C , S, T, M, V, N, Q, F, W, Y, P, or C; And / or N137 substituted with D, E, H, K, R, A, G, I, L, S, T, M, V, F, W, Y,

Protein engineering may be used to improve or alter the properties of MPIF-1 polypeptides. Recombinant DNA techniques known in the art can also be used to create new proteins. Muteins and deletion or fusion proteins can show increased activity or increased stability. In addition, they may be purified at higher yields and may exhibit better solubility under at least some purification and storage conditions. Additional examples of mutations that can be made are described below.

MPIF-1 amino terminal and carboxy terminal deletion

Interferon gamma shows 10-fold higher activity by deleting 8-10 amino acid residues from the carboxy terminus of the protein (Dobeli et al., J. of Biotechnology 7: 199-216 (1988)). Ron et al., J. Biol. Chem., 268 (4): 2984-2988 (1993) reported a modified KGF protein with heparin binding activity even though 3, 8 or 27 amino terminal amino acid residues were deleted. Many other examples are known to those skilled in the art.

Specifically, the N-terminal deletion of the MPIF-1 polypeptide can be represented by the general formula m-120, wherein m is an integer from 2 to 115, and m corresponds to the position of the amino acid residue identified in SEQ ID NO: 2. More specifically, the present invention provides polynucleotides encoding a polypeptide comprising or consisting of the amino acid sequence of the following residues: K-2 to N-120 of SEQ ID NO: 2; V-3 to N-120; S-4 to N-120; V-5 to N-120; A-6 to N-120; A-7 to N-120; L-8 to N-120; S-9 to N-120; C-10 to N-120; L-1l to N-120; M-12 to N-120; L-13 to N-120; V-14 to N-120; T-15 to N-120: A-16 to N-120; L-17 to N-120; G-18 to N-120; S-I9 to N-120; Q-20 to N-120; A-21 to N-120; R-22 to N-120; V-23 to N-120; T-24 to N-120; K-25 to N-120; D-26 to N-120; A-27 to N-120; E-28 to N-120; T-29 to N-120; E-30 to N-120; F-31 to N-120; M-32 to N-120; M-33 to N-120; S-34 to N-I20; K-35 to N- l20; L-36 to N-120; P-37 to N-120; L-38 to N-120; E-39 to N-120; N-40 to N-120; P-41 to N-120; V-42 to N-120; L-43 to N-120; L-44 to N-120; D-45 to N-120; R-46 to N-120; F-47 to N-120; H-48 to N-120; A-49 to N-120; T-50 to N-120; S-51 to N-120; A-52 to N-120; D-53 to N-120; C-54 to N-I20; C-55 to N-120; I-56 to N-120; S-57 to N-120; Y-58 to N-120; T-59 to N-120; P-60 to N-120; R-61 to N-120; S-62 to N-120; I-63 to N-120; P-64 to N-120; C-65 to N-120; S-66 to N-120; L-67 to N-120; L-68 to N-120; E-69 to N-120; S-70 to N-120; Y-71 to N-120; F-72 to N-120; E-73 to N-120; T-74 to N-120; N-75 to N-I20; S-76 to N-120; E-77 to N-120; C-78 to N-120; S-79 to N-120; K-80 to N-120; P-81 to N-120; G-82 to N-I20; V-83 to N-120; I-84 to N-I20; F-85 to N-120; L-86 to N-120; T-87 to N-120; K-88 to N-120; K-89 to N-120; G-90 to N-120; R-91 to N-120; R-92 to N-120; F-93 to N-120; C-94 to N- 120; A-95 to N-120; N-96 to N-120; P-97 to N-120; S-98 to N-120; D-99 to N-120; K-100 to N-120; Q-10l to N-120; V-102 to N-120; Q-103 to N-120; V-104 to N-120; C-105 to N-120; M-106 to N-120; R-107 to N-120; M-108 to N-120; L-109 to N-120; K-110 to N-120; L-111 to N-I20; D-112 to N-120; T-113 to N-120; R-114 to N-120; I-115 to N-120. Polynucleotides encoding these polypeptides are also encompassed by the present invention.

Also, as discussed above, although deletion of one or more amino acids from the C-terminus of a protein may result in a modification of the loss of one or more biological functions of the protein, other functional activities (e.g., biological activity, The ability to bind to the receptor) may still be retained. For example, the ability of a shortened MPIF-I mutein to induce and / or bind an antibody that recognizes the complete or mature form of the polypeptide is such that even when less than half of the residues of the complete or mature polypeptide are removed from the C-terminus Will be retained. Whether a particular polypeptide lacking the C-terminal residue of the complete polypeptide retains such immunological activity can be readily determined by routine methods known in the art and known in the art. MPIF-1 mutein with a large number of deleted C-terminal amino acid residues may retain some biological or immunogenic activity. In fact, peptides composed of six MPIF-1 amino acid residues often cause an immune response.

Accordingly, the present invention further provides a polypeptide wherein one or more residues are deleted from the carboxy terminus of the amino acid sequence of the MPIF-1 polypeptide shown in Figure 1 (SEQ ID NO: 2), represented by the general formula 1-n, And is an integer of 6 to 119 and corresponds to the position of the amino acid residue identified in SEQ ID NO: More specifically, the present invention provides polynucleotides encoding polypeptides comprising or consisting of the amino acid sequences of the following residues: A-27 to K-119 of SEQ ID NO: 2; A-27 to R-118; A-27 to T-117; A-27 to K-116; A-27 to I-115; A-27 to R-114; A-27 to T-113; A-27 to D-112; A-27 to L-111; A-27 to K-110; A-27 to L-109; A-27 to M-108; A-27 to R-107; A-27 to M-106; A-27 to C-105; A-27 to V-104; A-27 to Q-103; A-27 to V-102; A-27 to Q-101; A-27 to K-100; A-27 to D-99; A-27 to S-98; A-27 to P-97; A-27 to N-96; A-27 to A-95; A-27 to C-94; A-27 to F-93; A-27 to R-92; A-27 to R-91; A-27 to G-90; A-27 to K-89; A-27 to K-88; A-27 to T-87; A-27 to L-86; A-27 to F-85; A-27 to I-84; A-27 to V-83; A-27 to G-82; A-27 to P-81; A-27 to K-80; A-27 to S-79; A-27 to C-78; A-27 to E-77; A-27 to S-76; A-27 to N-75; A-27 to T-74; A-27 to E-73; A-27 to F-72; A-27 to Y-71; A-27 to S-70; A-27 to E-69; A-27 to L-68; A-27 to L-67; A-27 to S-66; A-27 to C-65; A-27 to P-64; A-27 to I-63; A-27 to S-62; A-27 to R-61; A-27 to P-60; A-27 to T-59; A-27 to Y-58; A-27 to S-57; A-27 to I-56; A-27 to C-55; A-27 to C-54; A-27 to D-53; A-27 to A-52; A-27 to S-51; A-27 to T-50; A-27 to A-49; A-27 to H-48; A-27 to F-47; A-27 to R-46; A-27 to D-45; A-27 to L-44; A-27 to L-43; A-27 to V-42; A-27 to P-41; A-27 to N-40; A-27 to E-39; A-27 to L-38; A-27 to P-37; A-27 to L-36; A-27 to K-35; A-27 to S-34; A-27 to M-33; A-27 to M-32; A-27 to F-31; A-27 to E-30; A-27 to T-29; A-27 to E-28; M-1 to D-26; M-1 to K-25; M-1 to T-24; M-1 to V-23; M-1 to R-22; M-1 to A-21; M-1 to Q-20; M-1 to S-19 M-1 to G-18; M-1 to L-17; M-1 to A-16; M-1 to T-15; M-1 to V-14; M-1 to L-13; M-1 to M-12 M-1 to L-11; M-1 to C-10; M-1 to S-9; M-1 to L-8; M-1 to A-7. Polynucleotides encoding these polypeptides are also included in the present invention.

Moreover, signal sequences may be added to these C-terminal structures. For example, the amino acid 1-26 of SEQ ID NO: 2, the amino acid 2-26 of SEQ ID NO: 2, the amino acid 3-26 of SEQ ID NO: 2, the amino acid 4-26 of SEQ ID NO: 2, the amino acid 5-26 of SEQ ID NO: 2, 2, amino acids 7-26 of SEQ ID NO: 2, amino acids 8-26 of SEQ ID NO: 2, amino acids 9-26 of SEQ ID NO: 2, amino acids 10-26 of SEQ ID NO: 2, amino acids 11 2, the amino acids 13-26 of SEQ ID NO: 2, the amino acids 14-26 of SEQ ID NO: 2, the amino acids 15-26 of SEQ ID NO: 2, the amino acids 16-26 of SEQ ID NO: 2, 2, amino acids 18-26 of SEQ ID NO: 2, amino acids 19-26 of SEQ ID NO: 2, amino acids 20-26 of SEQ ID NO: 2, amino acids 21-26 of SEQ ID NO: 2, amino acids 22-26 of SEQ ID NO: 26, the amino acids 23-26 of SEQ ID NO: 2, the amino acids 24-26 of SEQ ID NO: 2, the amino acids 25-26 of SEQ ID NO: 2, Amino acid 26 may be added to the N-terminus of each C-terminal structure listed above.

In addition, any of the listed N or C-terminal deletions may be combined to produce N and C-terminal deleted MPIF-I polypeptides. The present invention also provides a polypeptide having one or more amino acids deleted from both the amino terminus and the carboxy terminus, which may be represented as having residue m-n of SEQ ID NO: 2, wherein n and m are integers as described above. Polynucleotides encoding these polypeptides are also encompassed by the present invention.

Additional preferred polypeptide fragments comprise or consist of the amino acid sequence of the following residues: M-1 to T-15; K-2 to A-16; V-3 to L-17; S-4 to G-18; V-5 to S-19; A-6 to Q-20; A-7 to A-21; L-8 to R-22; S-9 to V-23; C-10 to T-24; L-11 to K-25; M-12 to D-26; L-13 to A-27; V-14 to E-28; T-15 to T-29; A-16 to E-30; L-17 to F-31; G-18 to M-32; S-19 to M-33; Q-20 to S-34; A-21 to K-35; R-22 to L-36; V-23 to P-37; T-24 to L-38; K-25 to E-39; D-26 to N-40; A-27 to P-41; E-28 to V-42; T-29 to L-43; E-30 to L-44; F-3I to D-45; M-32 to R-46; M-33 to F-47; S-34 to H-48; K-35 to A-49; L-36 to T-50; P-37 to S-51; L-38 to A-52; E-39 to D-53; N-40 to C-54; P-41 to C-55; V-42 to I-56; L-43 to S-57; L-44 to Y-58; D-45 to T-59; R-46 to P-60; F-47 to R-61; H-48 to S-62; A-49 to I-63; T-50 to P-64; S-51 to C-65; A-52 to S-66; D-53 to L-67; C-54 to L-68; C-55 to E-69; I-56 to S-70; S-57 to Y-7l; Y-58 to F-72; T-59 to E-73; P-60 to T-74; R-61 to N-75; S-62 to S-76; I-63 to E-77; P-64 to C-78; C-65 to S-79; S-66 to K-80; L-67 to P-81; L-68 to G-82; E-69 to V-83; S-70 to I-84; Y-7l to F-85; F-72 to L-86; E-73 to T-87; T-74 to K-88; N-75 to K-89; S-76 to G-90; E-77 to R-91; C-78 to R-92; S-79 to F-93; K-80 to C-94; P-81 to A-95; G-82 to N-96; V-83 to P-97; I-84 to S-98; F-85 to D-99; L-86 to K-100; T-87 to Q-101; K-88 to V-102; K-89 to Q-103; G-90 to V-104; R-91 to C-105; R-92 to M-106; F-93 to R-107; C-94 to M-108; A-95 to L-109; N-96 to K-110; P-97 to L-111; S-98 to D-112; D-99 to T-113; K-100 to R-114; Q-10l to I-115; V-102 to K-116; Q-103 to T-117; V-104 to R-118; C-105 to K-119; M-106 to N-120. These polypeptide fragments may retain the biological activity of the MPIF-I polypeptide of the invention and may be useful for antibody production as described below. Polynucleotides encoding these polypeptide fragments are also encompassed by the present invention.

Also included is a nucleotide sequence encoding a polypeptide consisting of a portion of the complete MPIF-I amino acid sequence encoded by a cDNA clone contained in ATCC Deposit No. 75676, wherein the portion is encoded by a cDNA clone contained in ATCC Deposit No. 75676 Any combination of the amino terminal and carboxy terminal deletions of the complete amino acid sequence encoded by the cDNA clone contained in ATCC Deposit No. 75676, or any combination of the amino acid residues of carboxy Excludes any integer number of amino acid residues from 1 to about 110 amino acids from the terminus. Polynucleotides encoding all of the deletion mutant polypeptide forms are also provided.

The present application is directed to proteins that contain at least 90%, 95%, 96%, 97%, 98%, or 99% identical polypeptides to the MPIF-1 polypeptide sequence set forth herein as mn. In a preferred embodiment, the present application is directed to a polypeptide having at least 90%, 95%, 96%, 97%, 98%, or 99% identical polypeptides to a polypeptide having the amino acid sequence of a specific MPIF-1 N and C- ≪ / RTI > Polynucleotides encoding these polypeptides are also encompassed by the present invention.

A particularly preferred MPIF-1 polypeptide of the amino acid sequence shown in Figure 1 (SEQ ID NO: 2) is as follows:

Thus, in one embodiment, a MPIF-I N-terminal deletion mutation is provided by the present invention. Such a mutation has at least a deletion in the first 22 N-terminal amino acid residues (i.e., deletion of at least Met (1) -Arg (22)) of the first 22 N terminal amino acid residues of Figure 1 1 < / RTI > (SEQ ID NO: 2). Alternatively, the deletion will comprise more than the first 22 N-terminal amino acid residues of Figure 1 (SEQ ID NO: 2), and less than the first 53 N-terminal amino acid residues. On the one hand, the deletion will comprise more than the first 33 N-terminal amino acid residues of Figure 1 (SEQ ID NO: 2), and less than the first 53 N-terminal amino acid residues. Alternatively, the deletion comprises at least the first 37 N-terminal amino acid residues (i.e., deletion of at least Met (1) -Pro (37)) of Figure 1 (SEQ ID NO: 2) will be. Alternatively, the deletion will comprise more than the first 48 N-terminal amino acid residues of Figure 1 (SEQ ID NO: 2) and less than the first 53 N-terminal amino acid residues.

In addition to the range of MPIF-1 N-terminal deletion mutations described above, the present invention is also directed to any combination of the above-mentioned ranges, for example over the first 22 N-terminal amino acid residues of Figure 1 (SEQ ID NO: 2) Deletion of the first 48 N-terminal amino acid residues or less; Terminal amino acid residue in Figure 1 (SEQ ID NO: 2), deletion below the first 48 N-terminal amino acid residue; Deletion of the first 22 N-terminal amino acid residues or more of Figure 1 (SEQ ID NO: 2), first 37 N-terminal amino acid residues or less; Deletion of the first 22 N-terminal amino acid residues or more of Figure 1 (SEQ ID NO: 2), first 33 N-terminal amino acid residues or less; Terminal amino acid residue in Figure 1 (SEQ ID NO: 2), deletion below the first 37 N-terminal amino acid residue; Terminal amino acid residue in Figure 1 (SEQ ID NO: 2), and below the first 48 N-terminal amino acid residue.

In another embodiment, the MPIF-I C-terminal deletion mutation is provided by the present invention. Preferably, the N-terminal amino acid residue of the MPIF-1 C-terminal deletion mutation is the amino acid residue 1 (Met) or 22 (Arg) of Figure 1 (SEQ ID NO: 2). Such a mutation may result in deletion of the last C-terminal amino acid residue (Asn (120)), the last 52 C-terminal amino acid residue or less (e.g., the amino acid residue Glu (69) ) Comprising the amino acid sequence of Figure 1 (SEQ ID NO: 2) with deletion of the amino acid sequence of SEQ ID NO: 2. Alternatively, the deletion will comprise more than the last 10 or 15 C-terminal amino acid residue of Figure 1 (SEQ ID NO: 2), or less than the last 52 C-terminal amino acid residue. Alternatively, the deletion will comprise more than the last 20 C-terminal amino acid residue of Figure 1 (SEQ ID NO: 2), or less than the last 52 C-terminal amino acid residue. Alternatively, the deletion will comprise more than the last 30 C-terminal amino acid residue of Figure 1 (SEQ ID NO: 2) and no more than the last 52 C-terminal amino acid residue. Alternatively, the deletion will comprise more than the last 36 C-terminal amino acid residue of Figure 1 (SEQ ID NO: 2), or less than the last 52 C-terminal amino acid residue. Alternatively, the deletion will comprise more than the last 41 C-terminal amino acid residue of Figure 1 (SEQ ID NO: 2), or less than the last 52 C-terminal amino acid residue. Alternatively, the deletion will comprise more than the last 45 C-terminal amino acid residue of Figure 1 (SEQ ID NO: 2) and no more than the last 52 C-terminal amino acid residue. Alternatively, the deletion will comprise more than the last 48 C-terminal amino acid residue of Figure 1 (SEQ ID NO: 2), or less than the last 52 C-terminal amino acid residue.

In addition to the range of C-terminal deletion mutations described above, the present invention is also directed to any combination of the above-mentioned ranges and includes, for example, more than the last C-terminal amino acid residue of Figure 1 (SEQ ID NO: 2) Deletions below the terminal amino acid residue; Terminal C-terminal amino acid residue of Figure 1 (SEQ ID NO: 2), deletion below the last 45 C-terminal amino acid residue; Terminal C-terminal amino acid residue of Figure 1 (SEQ ID NO: 2), deletion below the last 41 C-terminal amino acid residue; Deletion of more than the last C-terminal amino acid residue of Figure 1 (SEQ ID NO: 2), less than the last 36 C-terminal amino acid residue; Terminal C-terminal amino acid residue of Figure 1 (SEQ ID NO: 2), deletion below the last 10 C-terminal amino acid residue; Deletion of more than the last 10 C-terminal amino acid residue of Figure 1 (SEQ ID NO: 2), less than the last 20 C-terminal amino acid residue; A deletion of at least the last 10 C-terminal amino acid residue in Figure 1 (SEQ ID NO: 2), at or below the last 30 C-terminal amino acid residue; Deletion of more than the last 10 C-terminal amino acid residue of Figure 1 (SEQ ID NO: 2), less than the last 36 C-terminal amino acid residue; Deletion of the last 20 C-terminal amino acid residue or more of Figure 1 (SEQ ID NO: 2), or less than the last 30 C-terminal amino acid residue; And so on.

In another embodiment, an MPIF-1 deletion mutant having an amino acid deleted from both the -terminal and the C-terminal residue is also included in the present invention. Such mutations include all combinations of the N-terminal deletion mutants and the C-terminal deletion mutations described above. Such a mutation may result in a deletion of the first 22 N-terminal amino acid residue in Figure 1 (SEQ ID NO: 2), a deletion in the first 52 N-terminal amino acid residue and a last C-terminal amino acid residue in Figure 1 1 (SEQ ID NO: 2) with a deletion below the C-terminal amino acid residue. Alternatively, the deletion may be at least about the first 33,37, or 48 N-terminal amino acid residues in Figure 1 (SEQ ID NO: 2), the first 52 N-terminal amino acid residues below and the last 10,20, 36, 41, 45, or 48 C-terminal amino acid residues or more, and deletions below the last 52 C-terminal amino acid residues. All combinations of the above ranges are also included.

Among the particularly preferred fragments of the present invention are fragments characterized by the structural or functional properties of MPIF-1. Such fragments include the alpha-helix and alpha-helix forming regions (" alpha-region "), beta-sheet and beta-sheet-forming regions (" Forming region (" coil-region "), a hydrophobic region, a hydrophobic region, an alpha amphiphilic region, a beta ampholytic region, a surface forming Region, and an amino acid residue comprising a highly antigenic indicator region (e.g., containing four or more contiguous amino acids with an antigenicity indicator of at least 1.5, as determined using the default parameters of the Jamesson-Wolf program). Some preferred regions are shown in Figure 14 and include, but are not limited to, the regions of the type described above identified by analysis of the amino acid sequence shown in Figure 1 (SEQ ID NO: 2), such preferred regions being the Garniero-Lopson predicted alpha region , A beta-region, a turn-region and a coil-region; Beta-region, turn-region, and coil-region; kite-doline predicted hydrophilic and hydrophobic regions; Eisenberg alpha and beta amphipathic regions; An emissive surface-forming region; And Jameson-Wolf high-water surface area, which are predicted using the default parameters of these computer programs.

Polynucleotides encoding these polypeptides are also encompassed by the present invention.

In a further embodiment, the polynucleotides of the invention encode the functional properties of MPIF-I. In this regard, a preferred embodiment of the present invention is a method of forming a sacrificial layer of MPIF-1 comprising the steps of forming an alpha helical and alpha helical forming region (" alpha region "), a beta sheet and a beta sheet forming region Quot; region "), a coil and coil forming region (" coil region "), a hydrophilic region, a hydrophobic region, an alpha amphiphilic region, a beta amphoteric region, a flexible region, .

Additional preferred regions are those disclosed in Example 37.

As described above, data showing the structural or functional properties of MPIF-1 shown in FIG. 14 were generated using various modules and algorithms of DNA * STAR fixed to the default parameters. In a preferred embodiment, the data shown in Figure 14 can be used to determine the region of MPIF-1 showing a high degree of ability to antigenicity. The region of high antigenicity is determined from data presented by selecting a value representing a polypeptide region that is likely to be exposed to the surface of the polypeptide in an environment where antigen recognition may occur during the initiation of the immune response.

The above-described preferred regions disclosed in Fig. 14 include, but are not limited to, regions of the above-described type identified by analysis of the amino acid sequence disclosed in Fig. 14, such preferred regions include the Garnier-Robson alpha region, the beta-region, the turn-region and the coil-region; Shaw-Parsen alpha-domain, beta-domain, and coil-domain; kite-dolittle hydrophilic and hydrophobic domains; Eisenberg alpha and beta amphipathic regions; CAPlus Schultz muscle sexual domain; An emissive surface-forming region; And Jameson-Wolf highland surface area.

A highly preferred fragment at this point comprises the MPIF-I region which combines structural features as described above or below as several features.

In one embodiment, the MPIF-1 variant and / or fragment has the same or substantially the same structure as MPIF-1 or a portion thereof, for example, a solution structure determined by nuclear magnetic resonance spectroscopy (NMR) (Example 37) Or may have a structure determined by other techniques. Thus, the MPIF-1 variant and / or fragment has an amino acid sequence that is different from the amino acid sequence of SEQ ID NO: 2 or SEQ ID NO: 7 or the polypeptide encoded by the deposited cDNA clone. However, it has the same structure or almost the same structure as MPIF-1 or at least one region thereof. Polynucleotides encoding such polypeptides are encompassed by the present invention.

The MPIF-1 variant and / or fragment will have the same or nearly the same structure as at least one region of MPIF-1. The region of MPIF-1 comprises an N-terminal loop, a 3 ₁₀ loop, a first beta strand, a first type III turn, a second beta strand, a type 1 turn, a third beta strand, a second type III turn and a helix Example 37). That is, amino acids 56-63 of SEQ ID NO: 2 (numbered 13-20 in Example 37), 64-67 (21-24 in Example 37), 70-74 (27-31 in Example 37) (Example 37 to 32-38), 82-87 (39-44 in Example 37), 88-90 (45-47 in Example 36), 91-95 (48-52 in Example 37) , And 96-98 (Example 37 to 53-55), and 99-109 (Example 37 to 56-66). The MPIF-1 region also contains amino acids 44-120 (1-77 in Example 37), 44-53 (1-10 in Example 37), 54-109 (11-66 in Example 37) of SEQ ID NO: 2, (Example 37 to 11-62), 56-I09 (Example 13 to 13-66), 106-120 (Example 37 to 63-77), 110-120 (Example 37 to 67-77 ).

14 and other sequences as described herein or predictable by amino acid sequence analysis. The MPTF-1 polypeptide variant and / or fragment may have the same or nearly the same structure as one or combination of these MPIF-I regions.

For MPIF-1 variants and / or fragments that are identical or nearly identical to one or more MPIF-1 regions, the structures may be contiguous to each other. In one embodiment, the structures are not adjacent to each other. That is, they are separated by one or more amino acid residues. Preferably, the structure is also arranged in line with the structure of MPIF-I. Preferably, the structure is superimposed on the structure of MPIF-I. In a preferred embodiment, the structure has a structure that is relatively identical to the corresponding structure in MPIF-1 (i.e., the same tertiary structure).

For example, the MPIF-1 variant and / or fragment has the same or nearly the same structure as the N-terminal loop of MPIF-1, the first beta strand, the second beta strand, the third beta strand, and alpha helix. Thus, in a preferred combination, the MPIF-1 variant and / or fragment comprises amino acids 56-63 of SEQ ID NO: 2 (13-20 of Example 37), 70-74 (27-31 of Example 37), 82-87 (39-44 of Example 37), 91-95 (48-52 of Example 37), and 99-109 (56-66 of Example 37).

In another embodiment, the MPIF-1 variant and / or fragment comprises an N-terminal loop, a 3 ₁₀ loop, a first beta strand, a first type III turn, a second beta strand, a type 1 turn, 2 type III turn and MPIF-1 alpha helix. Thus, the MPIF-1 variant and / or fragment may comprise amino acids 56-63 of SEQ ID NO: 2 (13-20 in Example 37), 64-67 (21-24 of Example 37), 70-74 27-31), 75-81 (32-38 of Example 36), 82-87 (39-44 of Example 37), 88-90 (45-47 of Example 36), 91-95 Have the same or nearly the same structure as that of Example 37 (8-52), 96-98 (53-55 of Example 37), and 99-109 (56-66 of Example 37).

In a preferred embodiment, the MPIF-1 variant and / or fragment has the same or nearly the same structure as amino acids 56-109 (13-66 in Example 37) of SEQ ID NO: 2.

In yet another embodiment, the MPIF-I polypeptide comprises or consists of one or more amino acid sequences of MPIF-1. For polypeptides comprising or consisting of two or more regions of amino acid sequence, the regions may be adjacent to each other. In one embodiment, the regions may not be adjacent to each other. That is, they can be separated by one or more amino acid residues. Preferably, the amino acid sequences are aligned with the amino acid sequence of the corresponding region of MPIF-1 and they are separated by the same number of amino acid residues that separate them in MPIF-1.

However, in another embodiment, the MPIF-1 variant and / or fragment comprises an amino acid modification (substitution, deletion and insertion) in one or more of the above regions but does not include any modification in one or more other regions Do not. Polynucleotides encoding such polypeptides are also encompassed by the present invention.

Other preferred polypeptide fragments are MPIF-1 fragments that are biologically active. Biologically active fragments exhibit similar but not necessarily identical activities to the activity of MPIF-1 polypeptides. The biological activity of the fragment may include an improvement in a desired activity or a decrease in an undesirable activity. Polynucleotides encoding such polypeptide fragments are encompassed by the present invention.

However, many polynucleotide sequences, such as the EST sequence, are publicly accessible and available through the sequence database. Some of the sequences relate to SEQ ID NO: 1 or 6 and were publicly available prior to the concept of the present invention. Preferably, the relevant polynucleotides are specifically excluded from the scope of the present invention. Listing all relevant sequences would be cumbersome. Thus, a compound of the general formula ab wherein a is any integer between 1 and 349 of SEQ ID NO: 1, b is any integer between 15 and 363 and both a and b are at the position of the nucleotide residue shown in SEQ ID NO: 1 And b is equal to or greater than a + 14), it is preferred that one or more of the polynucleotides comprising or consisting of the nucleotide sequence is excluded from the present invention.

Amino-terminal and carboxy-terminal deletions of MPIF-1137 amino acid splice variants: As described above, the present invention further provides human MPIF-1 splice variants. The cDNA sequence and the 137 amino acid sequence are shown in Figure 20 A (SEQ ID NOS: 6 and 7, respectively). Using eukaryotic expression systems, the present invention has discovered three N-terminal deletion mutants of the MPIF-1 splice variant. This is His (60) -Asn (137); Met (46) -Asn (137); And Pro (54) -Asn (137). Thus, in a further aspect, an MPIF-1 splice variant N-terminal deletion mutation is provided by the present invention. The mutation comprises or consists of the amino acid sequence shown in Figure 20A (SEQ ID NO: 7) with deletion of the first 45 N-terminal amino acid residue or deletion of the first 59 N-terminal amino acid residue of Figure 20A (SEQ ID NO: 7) . Alternatively, the deletion will comprise the first 59 N-terminal amino acid residue to the first 59 N-terminal amino acid residue of Figure 20A (SEQ ID NO: 7).

Additional N-terminal deletions of the 137 amino acid splice variant polypeptides of the invention shown in SEQ ID NO: 7 include polypeptides comprising or consisting of the amino acid residues: K-2 to N-137 of SEQ ID NO: 7; V-3 to N-137; S-4 to N-137; V-5 to N-137; A-6 to N-137; A-7 to N-137; L-8 to N-137; S-9 to N-137; C-10 to N-137; L-11 to N.137; M-12 to N-137; L-13 to N-137; V-14 to N-137; T-15 to N-137; A-16 to N-137; L-17 to N-137; G-18 to N-137; S-19 to N-137; Q-20 to N-137; A-21 to N-137; R-22 to N-137; V-23 to N-I37; T-24 to N-137; K-25 to N-137; D-26 to N-137; A-27 to N-137; E-28 to N-137; T-29 to N-137; E-30 to N-137; F-31 to N-137; M-32 to N-] 37; M-33 to N-I37; S-34 to N-137; K-35 to N-137; L-36 to N-I37; P-37 to N-137; L-38 to N-137; E-39 to N-137; N-40 to N-137; P-4] to N-137; V-42 to N-137; L-43 to N-137; L-44 to N-137; D-45 to N-137; M-46 to N-137; L-47 to N-137; W-48 to N-137; R-49 to N-137; R-50 to N-137; K-51 to N-137; I-52 to N-137; G-53 to N-137; P-54 to N-137; Q-55 to N-137; M-56 to N-137; T-57 to N-137; L-58 to N-137; S-59 to N-137; H-60 to N-137; A-61 to N-137; A-62 to N-137; G-63 to N-137; F-64 to N-137; H-65 to N-137; A-66 to N-137; T-67 to. N-137; S-68 to N-137; A-69 to N-I37; D-70 to N-] 37; C-71 to N-137; C-72 to N-137; J-73 to N-I37; S-74 to N-I37; Y-75 to N-I37; T-76 to N-137; P-77 to N-137; R-78 to N-137; S-79 to N-137; l-80 to N-137; P-8I to N-137; C-82 to N-137; S-83 to N-137; L-84 to N-137; L-85 to N-137; E-86 to N-137; S-87 to N-137; Y-88 to N-] 37; F-89 to N-137; E-90 to N-137; T-91 to N-137; N-92 to N-137; S-93 to N-I37; E-94 to N-137; C-95 to N-137; S-96 to N-137; K-97 to N-137; P-98 to N-I37; G-99 to N-137; V-100 to N-137; LI0l to N-137; F-I02 to N-137; L-103 to N-I37; T-104 to N-137; K-105 to N-137; K-106 to N-137; G-107 to N-137; R-108 to N-137; R-109 to N-137; F-110 to N-137; C-11l to N-137; A-112 to N-137; N-113 to N-137; P-114 to N-137; S-115 to N-137; D-116 to N-137; K-117 to N-137; Q-118 to N-137; V-119 to N-137; Q-120 to N-137; V-121 to N-137; C-122 to N-137; M-] 23 to N-137; R-124 to N-137; M-125 to N-137; L-126 to N-] 37; K-127 to N-137; L-128 to N-137; D-129 to N-137; T-130 to N-137; R-131 to N-137; Or I-132 to N-137.

Similarly, the C-terminal deletion of the 137 amino acid splice variant mutation shown in SEQ ID NO: 7 is a polypeptide comprising the amino acid sequence of the following residues: M-1 to K-136 of SEQ ID NO: 7; M-1 to R-35; M-1 to T-134; M-1 to K-I33; M-1 to I-132; M-1 to R-131; M-1 to T-130; M-1 to D-129; M-1 to L-128; M-1 to K-127; M-1 to L-126; M-1 to M-125; M-1 to R-124; M-1 to M-123; M-1 to C-122; M-1 to V-12l; M-1 to Q-120; M-I to V-I19; M-1 to Q-I18; M-I to K-117; M-1 to D-116; M-1 to S-I15; M-1 to P-114; M-1 to N-113; M-1 to A-112; M-1 to C-111; M-1 to F-110; M-1 to R-109; M-1 to R-108; M-1 to G-107; M-1 to K-I06; M-1 to K-105; M-1 to T-104; M-1 to L-103; M-1 to F-102; M-1 to I-101; M-I to V-100; M-1 to G-99; M-1 to P-98; M-1 to K-97; M-1 to S-96; M-1 to C-95; M-1 to E-94; M-1 to S-93; M-1 to N-92; M-1 to T-91; M-1 to E-90; M-1 to F-89; M-1 to Y-88; M-1 to S-87; M-1 to E-86; M-1 to L-85; M-1 to L-84; M-1 to S-83; M-1 to C-82; M-1 to P-81; M-1 to I-80; M-1 to S-79; M-1 to R-78; M-1 to P-77; M-1 to T-76; M-1 to Y-75; M-1 to S-74; M-1 to I-73; M-1 to C-72; M-1 to C-71; M-1 to D-70; M-1 to A-69; M-1 to S-68; M-1 to T-67; M-1 to A-66; M-1 to H-65; M-1 to F-64; M-1 to G-63; M-1 to A-62; M-1 to A-61; M-1 to H-60; M-1 to S-59; M-1 to L-58; M-1 to T-57; M-1 to M-56; M-1 to Q-55; M-1 to P-54; M-1 to G-53; M-1 to I-52; M-I to K-51; M-1 to R-50; M-1 to R-49; M-1 to W-48; M-1 to L-47; M-1 to M-46; M-1 to D-45; M-1 to L-44; M-1 to L-43; M-1 to V-42; M-1 to P-41; M-1 to N-40; M-1 to E-39; M-1 to L-38; M-1 to P-37; M-1 to L-36; M-1 to K-35; M-1 to S-34; M-1 to M-33; M-1 to M-32; M-1 to F-31; M-1 to E-30; M-1 to T-29; M-1 to E-28; M-1 to A-27; M-1 to D-26; M-1 to K-25; M-1 to T-24; M-1 to V-23; M-1 to R-22; M-1 to A-21; M-1 to Q-20; M-1 to S-19; M-1 to G-18; M-1 to L-I7; M-1 to A-16; M-1 to T-15; M-1 to V-14; M-1 to L-13; M-1 to M-12; M-1 to L-I1; M-1 to C-10; M-1 to S-9; M-1 to L-8; or M-1 to A-7.

The polypeptide of the present invention is preferably provided in a separated form, and it is preferable that the polypeptide is substantially purified. Recombinantly produced forms of MPIF-2 polypeptides can be nearly purified by the one-step method described in Smith and Johnson, Gene 67: 31-40 (1988).

The polypeptide of the present invention comprises a polypeptide encoded by a deposited cDNA comprising a leader (i.e., a full-length protein), a polypeptide encoded by a deposited cDNA minus leader (i.e., a mature protein) (SEQ ID NO: 2), except for the polypeptide of FIG. 1 (SEQ ID NO: 2), except that the N-terminal methionine residue comprises the polypeptide of SEQ ID NO: 2, , 85%, 90%, 92% or 95% similarity, more preferably at least 96%, 97%, 98%, or 99% similarity. Additional polypeptides of the invention include polypeptides encoded by the deposited cDNA, polypeptides having at least 80%, 85%, 90%, or 95% identity with the polypeptide of Figure 1 (SEQ ID NO: 2), more preferably at least 96 %, 97%, 98%, or 99% identity to a polypeptide having at least 30 amino acids, more preferably at least 50 amino acids.

The default settings for the best-fit program (Wisconsin Sequence Analysis Package, Version 8 for Unix, Generic Computer Group, University Research Park, 575 Science Drive Madison, WI 53711) and similarity determinations by "% similarity" And the similarity score is calculated by comparing the amino acid sequences of the two polypeptides. Best-fit uses the Smith and Waterman-only local homology algorithm (Advances in Applied Mathematics 2: 482-489, 1981) to find fragments with the best homology between two sequences.

Polypeptides having an amino acid sequence that is at least 95% "identical" in the reference amino acid sequence of an MPIF-1 polypeptide are those in which the amino acid sequence of the polypeptide comprises no more than 5 amino acids per 100 amino acids of the reference amino acid sequence of the MPIF-1 polypeptide The same reference sequence as the reference sequence. That is, in order to obtain a polypeptide having 95% or more identical amino acid sequence to the reference amino acid sequence, 5% or less of the amino acid residue in the reference sequence may be deleted or substituted with another amino acid, and the number of amino acids May be inserted into the reference sequence. Such alteration of the reference sequence may occur anywhere between the amino or carboxy terminal position of the reference amino acid sequence or between one such terminal position in one or more adjacent groups in the reference sequence or separately in the residue in the reference sequence.

As a matter of fact, any particular polypeptide may have at least 95%, 96%, 97%, 98%, or 99% identity with the amino acid sequence shown in Figure 1 (SEQ ID NO: 2) or the amino acid sequence encoded by the deposited cDNA clone May be routinely determined using known computer programs such as the Best Fit program (Wisconsin Sequencing Package, Version 8 for Unix, Generic Computer Group, University Research Park, 575 Science Drive Madison, WI 53711). According to the present invention, when a best-fit or any other sequence-sequenced program is used to determine if a particular sequence is 95% identical to the reference sequence, the parameters are set and the percent identity is calculated for the reference amino acid sequence of the total length, Lt; RTI ID = 0.0 > 5% < / RTI > of the total number of amino acid residues.

The polypeptides of the present invention can be used as molecular weight markers on molecular sieve gel filtration columns or on SDS-PAGE gels using methods known to those skilled in the art.

As described in detail below, the polypeptides of the present invention can be used to raise polyclonal and monoclonal antibodies. It is used as an antagonist or agonist capable of enhancing or inhibiting MPIF-1 protein function or in an assay for detecting MPIF-1 protein expression as described below. Furthermore, such polypeptides can be used in yeast 2-hybrid systems to " capture " MPIF-1 protein binding proteins, candidates for antagonists and agonists of the invention. Yeast two-hybrid systems are described in Fields and Song, Nature 340: 245-246 (1989).

MPIF-1 epitope-bearing polypeptide

In another aspect, the invention provides a peptide or polypeptide comprising or consisting of an epitope-bearing portion of a polypeptide of the invention. The epitope of the polypeptide portion is an immunogenic or antigenic epitope of the polypeptide of the invention. An " immunogenic epitope " is defined as the portion of a protein that elicits an antibody response when the whole protein is an immunogenic agent. These immunogenic epitopes are thought to be limited to some of the left side of the molecule. That is, the region of a protein molecule to which an antibody can bind is defined as an " antigenic epitope ". The number of immunogenic epitopes of a protein is generally smaller than the number of antigenic epitopes (see, e.g., Geysen et al., Proc. Natl. Acad Sci. USA 81: 3998-4002 (1983)].

Fragments that act as epitopes can be produced by any conventional means (see, e.g., Houghten, R.A., Proc. Natl. Acad. Sci. USA 83: 5131-5135 (1985), U.S. Patent No. 4,631,211)

In the selection of a peptide or polypeptide-retaining antigen epitope (i. E., Including a region of a protein molecule to which the antibody is capable of binding), relatively short synthetic peptides that mimic a portion of the protein sequence are routinely (Reference, Sutcliffe, JG, Shinnick, TM, Green, N. and Learner, RA, Science 219: 660-666 (1983)).

Peptides capable of inducing protein-reactive sera often appear in the primary sequence of a protein and may be characterized by a set of simple chemical laws. It is not limited to the amino or carboxy terminus of the immunogenic region of the native protein (i. E., The immunogenic epitope). Extremely hydrophobic peptides and their 6 or fewer residues are ineffective for inducible antibodies that bind to the protein of interest. It is usually effective to contain a duggin peptide, especially a proline residue [Sutcliffe et al., Supra, page 661]. For example, 18 out of 20 peptides are designed according to these guidelines, contain 8-39 residues covering 75% of the sequence of the influenza virus hemagglutinin HA1 polypeptide chain, and react with the HA1 protein or native virus Lt; / RTI > In addition, 18/12 from the 12/12 peptide and the labile glycoprotein from MuLV polymerase induce antibodies that precipitate each protein.

The antigenic epitope-bearing peptides and polypeptides of the present invention are used to raise antibodies and specifically bind to the polypeptides of the invention by inducing monoclonal antibodies. Thus, a high proportion of hybridomas obtained by fusion of spleen cells from donors immunized with antigenic epitope-bearing peptides generally secrete antibodies reactive with natural proteins (Sutcliffe et al., Supra, p. 663). Antibodies raised by antigenic epitope-bearing peptides or polypeptides are useful for detecting the protein of interest, and antibodies against the different peptides can be used to track the fate of the various regions of the protein precursor that underwent post-translational processing. Since it has been found that short peptides (e.g., about 9 amino acids) can also bind and present to long peptides in immunoprecipitation assays, peptides and anti-peptide antibodies can be used for a variety of qualitative or quantitative analyzes of the expressed protein, such as competitive assays Wilson et al., Cell 37: 767-778 (1984), page 777]. The anti-peptide antibodies of the present invention are useful for the purification of proteins that are impaired by, for example, adsorption chromatography using known methods.

Preferably, the antigenic epitope-bearing peptides and polypeptides of the present invention designed according to the above guidelines comprise at least seven amino acid sequences contained within the amino acid sequence of the polypeptide of the invention, more preferably at least 9, To about 30 amino acids are most preferred. However, peptides or polypeptides comprising or consisting of a large portion of the amino acid sequence of the polypeptide of the present invention, comprising about 30 to about 50 amino acids or any length, and comprising the entire amino acid sequence of the polypeptide of the invention, Is considered an epitope-bearing peptide or polypeptide of the invention, and is useful for inducing antibodies that react with the protein of interest. Preferably, the amino acid sequence of the epitope-bearing peptide is selected to provide substantial solubility in the aqueous solvent (i. E. The sequence comprises relatively hydrophilic residues and preferably avoids high hydrophobic sequences); Sequences with proline residues are also particularly preferred.

In the present invention, the antigen epitope is preferably at least 4, at least 5, at least 6, at least 7, more preferably at least 8, at least 9, at least 10, at least 15, at least 20, at least 25, and most preferably at least about 15 To about 30 amino acids. Preferred polypeptides comprising or consisting of an immunogenic or antigenic epitope are selected from the group consisting of 10,15,20,25,30,35,40,45,50,55,60,65,70,75,80,85,90, 95 or 100 amino acid residues in length.

Non-limiting examples of antigenic polypeptides or peptides that can be used to generate MPIF-1 specific antibodies include polypeptides comprising or consisting of amino acid residues from about 21 to about 30 in SEQ ID NO: 2; A polypeptide comprising or consisting of about 31 to about 44 amino acid residues in SEQ ID NO: 2; A polypeptide comprising or consisting of about 49 to about 55 amino acid residues in SEQ ID NO: 2; A polypeptide comprising from about 59 to about 67 amino acid residues in SEQ ID NO: 2; A polypeptide comprising amino acid residues from about 72 to about 83 in SEQ ID NO: 2; A polypeptide comprising amino acid residues from about 86 to about 103 in SEQ ID NO: 2; And a polypeptide comprising about 110 to about 120 amino acid residues in SEQ ID NO: 2. As described above, the present inventors determined that the polypeptide fragment was an antigenic region of the MPIF-1 protein.

Additional antigenic polypeptides or peptides that can be used to generate MPIF-1 specific antibodies include the N-terminal and C-terminal deletions described above.

The present invention also provides an epitope fragment of a 137 amino acid splice variant of MPIF-1 (SEQ ID NO: 7). In particular, the invention provides polynucleotides having the following amino acid sequence residues: M-1 to T-l5 of SEQ ID NO: 7; K-2 to A-16; V-3 to L-17; S-4 to G-18; V-5 to S-19; A-6 to Q-20; A-7 to A-21; L-8 to R-22; S-9 to V-23; C-10 to T-24; L-11 to K-25; M-I 2 to D-26; L-13 to A-27; V-14 to E-28; T-15 to T-29; A-16 to E-30; L-17 to F-31; G-18 to M-32; S-19 to M-33; Q-20 to S-34; A-21 to K-35; R-22 to L-36; V-23 to P-37; T-24 to L-38; K-25 to E-39; D-26 to N-40; A-27 to P-41; E-28 to V-42; T-29 to L-43; E-30 to L-44; F-31 to D-45; M-32 to M-46; M-33 to L-47; S-34 to W-48; K-35 to R-49; L-36 to R-50; P-37 to K-51; L-38 to I-52; E-39 to G-53; N-40 to P-54; P-41 to Q-55; V-42 to M-56; L-43 to T-57; L-44 to L-58; D-45 to S-59; M-46 to H-60; L-47 to A-6I; W-48 to A-62; R-49 to G-63; R-50 to F-64; K-51 to H-65; I-52 to A-66; G-53 to T-67; P-54 to S-68; Q-55 to A-69; M-56 to D-70; T-57 to C-7I; L-58 to C-72; S-59 to I-73; H-60 to S-74; A-61 to Y-75; A-62 to T-76; G-63 to P-77; F-64 to R-78; H-65 to S-79; A-66 to I-80; T-67 to P-8I; S-68 to C-82; A-69 to S-83; D-70 to L-84; C-71 to L-85; C.72 to E-86; I-73 to S-87; S-74 to Y-88; Y-75 to F-89; T-76 to E-90; P-77 to T-91; R-78 to N-92; S-79 to S-93; I-80 to E-94; P-81 to C-95; C-82 to S-96; S-83 to K-97; L-84 to P-98; L-85 to G-99; E-86 to V-100; S-87 to I-101; Y-88 to F-102; F-89 to L-103; E-90 to T-104; T-9I to K-105; N-92 to K-106; S-93 to G-107; E-94 to R-108; C-95 to R-109; S-96 to F-110; K-97 to C-111; P-98 to A-112; G-99 to N-113; V.100 to P-11 4; I-10l to S-115; F-102 to D-116; L-103 to K-117; T-104 to Q-118; K-105 to V-119; K-106 to Q-120; G-107 to V-121; R-108 to C-122; R-109 to M-123; F-l10 to R-124; C-11l to M-125; A-112 to L-126; N-113 to K-127; P-114 to L-128; S-115 to D-129; D-116 to T-130; K-117 to R-131; Q-18 to I-132; V-119 to K-133; Q-120 to T-134; V-121 to R-135; C-122 to K-136; or M-123 to N-137.

The epitope-bearing peptides and polypeptides of the present invention may be prepared by any conventional method of preparing peptides or polypeptides comprising recombinant methods using the nucleic acid molecules of the present invention. For example, short epitope-bearing amino acid sequences can be fused to large polypeptides that act as carriers during recombination and purification as well as immunization to produce anti-peptide antibodies. The epitope-bearing peptides can also be synthesized using known methods of chemical synthesis. For example, Houghten reported that 10 to 20 mg of a large number of peptides, such as 248 different 13 residue peptides, produced and identified (by ELISA-type binding studies) a single amino acid variant of a fragment of HA1 polypeptide within 4 weeks A simple method for synthesis has been described [ref. Houghten, R. A. (1985) General method of rapid solid phase synthesis of multiple peptides: specificity of antigen-antibody interaction at the level of individual amino acids. Proc. Natl. Acad. Sci. USA 83: 5131-5135]. This " simultaneous multiple peptide synthesis (SMPS) " method is described in U.S. Patent No. 4,631,211 to Houghten et al (1986). In this way, the individual resins for the solid phase synthesis of the various peptides are contained in a separate solvent-permeable packet, enabling the optimal use of multiple identical repetitive steps involved in the solid-phase method. A complete passive method can simultaneously perform 500-1000 or more syntheses [Houghten et al., Supra, p. 5134].

In the present invention, the preferred nucleic acid fragment comprises a nucleic acid molecule encoding an epitope-bearing portion of the MPIF-1 protein.

In particular, the nucleic acid fragment of MPIF-1 of the present invention comprises a polypeptide comprising or consisting of about 21 to about 30 amino acid residues in SEQ ID NO: 2; A polypeptide comprising about 31 to about 44 amino acid residues in SEQ ID NO: 2; A polypeptide comprising about 49 to about 55 amino acid residues in SEQ ID NO: 2; A polypeptide comprising from about 59 to about 67 amino acid residues in SEQ ID NO: 2; A polypeptide comprising amino acid residues from about 72 to about 83 in SEQ ID NO: 2; A polypeptide comprising amino acid residues from about 86 to about 103 in SEQ ID NO: 2; A nucleic acid molecule encoding a polypeptide comprising an amino acid residue of about 110 to about 120 in SEQ ID NO: 2, or any range or value therefrom.

The present inventors have determined that the above-mentioned polypeptide fragment is an antigenic region of the MPIF-1 protein. A method for determining the epitope-bearing portion of the MPIF-1 protein is described next.

The epitope-bearing polypeptides of the present invention can be used to induce antibodies according to methods known in the art. This includes, but is not limited to, in vivo immunization, in vitro immunization and phage display methods (see, for example, Sutcliffe et al., Supra; Wilson et al., Supra, and Bittle et al., J. Gen. Virol. 66: 2347-2354 (1985)]. If in vivo immunization is used, the animal can be immunized with a free peptide; However, the anti-peptide titer can be elevated by coupling the peptide to a macromolecular carrier such as keyhole limpet hemocyanin (KLH) or the tetanus toxin. For example, a peptide containing cysteine may be coupled to a carrier using a linker such as m-maleimidobenzoyl-N-hydroxysuccinimide ester (MBS), and other peptides may be coupled to a carrier such as glutaraldehyde Coupling can be made to the carrier using a common linker agent. Animals such as rabbits, rats and mice are immunized with free or carrier-coupled peptides by intraperitoneal and / or subcutaneous injection of about 100 ug of peptide or an emulsion containing carrier protein and prune adjuvant. Several booster injections will be needed at intervals of about two weeks to provide useful potency of anti-peptide antibodies that can be detected by ELISA analysis using free surface adsorbed solid surfaces. Reverse transcription of the anti-peptide antibodies in the serum from the immunized animal may be increased by selection of the anti-peptide antibodies, for example, by elution of the antibody selected according to methods known in the art and adsorption of the solid support peptide .

When the immunogenic epitope-bearing peptide of the present invention, that is, the entire protein, is an immunological cause, part of the protein that induces the antibody reaction is identified according to methods known in the art. For example, the document of Geysen et al. Discloses a method for rapid simultaneous synthesis on solid supports of hundreds of peptides of sufficient purity that react in enzyme-linked immunosorption assays. The interaction of the antibody and the synthetic peptide is readily detected without removing them from the support. Peptides that retain an immunogenic epitope of the desired protein in this way can be routinely identified by those skilled in the art. For example, the immunologically important epitope of the coat protein of the foot-and-mouth disease virus is the resolution of the seven amino acids by synthesis of a duplicate set of all 208 possible hexapeptides, including the entire 213 amino acid sequence of the protein It was located by Geysen et al. A complete alternative set of peptides was then synthesized, in which a total of 20 amino acids were alternately substituted at all positions within the epitope, to determine specific amino acids that confer specificity for reaction with antibodies. Thus, peptide mimetics of the epitope-bearing peptides of the present invention can be routinely produced by the above methods. Geysen, U. S. Patent No. 4,708, 771 (1987), describes a method for identifying peptides bearing an immunogenic epitope of a desired protein.

Geysen U.S. Patent No. 5,194,392 (1990) also discloses the use of monomers (amino acids or other compounds) that are topological equivalents of epitopes (i.e., " mimotopes ") that are complementary to a particular paratope A general method for determining or detecting sequences is described. More generally, US Patent 4,433,092 (1989) by Geysen describes a method for determining or detecting the sequence of a monomer that is a topological equivalent of a ligand that is complementary to the ligand binding site of a particular receptor of interest. Similarly, U.S. Pat. No. 5,480,971 to Houghten, RA, et al. (1996) on peralkylated oligopeptide mixtures, discloses linear C _1-7 -alkylperalkylated oligonucleotides and sets and libraries of such peptides, Disclosed are methods of using such oligopeptide sets and libraries to determine the sequence of a peralkylated oligopeptide that preferentially binds to a receptor molecule. Thus, non-peptide analogs of the epitope-bearing peptides of the present invention can be routinely produced by such methods.

The complete disclosure of each of the documents cited in the field of " polypeptides and peptides " is incorporated herein by reference.

According to the evaluation of those skilled in the art, the MPIF-1 polypeptide of the present invention and its epitope-bearing fragment described above can be combined with a part of the constant part of immunoglobulin (IgG) to produce a chimeric polypeptide. Such fusion proteins are easy to purify and exhibit an increase in half-life in vivo. A chimeric protein consisting of the first two domains of a human CD4-polypeptide and the various domains of the heavy or light chain constant portion of a mammal's immunoglobulin [EPA 394,827; Traunecker et al., Nature 331: 84-86 (1988)]. A fusion protein having a disulfide bond dimer structure due to the IgG moiety may be more effective in binding and neutralizing molecules other than the monomeric MPIF-1 protein or protein fragment itself [Foun ~ ulakis et al., J. Biochem 270: 3958-3954 (1995)).

As assessed by those of skill in the art and discussed above, the polypeptides of the invention comprising or consisting of an immunogenic or antigenic epitope may be fused to a heterologous polypeptide sequence. For example, a polypeptide of the present invention may comprise an invariant portion of an immunoglobulin (IgA, IgE, IgG, IgM) or a portion thereof (CH ₁ , CH ₂ , CH ₃ , a complete domain, ) To produce a chimeric polypeptide. Such fusion proteins facilitate purification and increase in vivo half-life. A chimeric protein consisting of the first two domains of a human CD4-polypeptide and the various domains of the immunoglobulin heavy or light chain constant region of a mammal [EPA 0,394,827; Traunecker et al., Nature 331: 84-86 (1988)]. A fusion protein having a disulfide bond dimer structure due to the IgG moiety may be more effective in binding and neutralizing molecules other than the monomer polypeptide or fragment thereof itself (Fountoulakis et al., J. Biochem 270: 3958-3954 1995). The nucleic acid encoding the epitope may be recombined with a gene of interest as an epitope tag to aid in the detection and purification of the expressed polypeptide.

Additional fusion proteins of the invention may be generated through techniques of gene-shuffling, motif-shuffling, exon-shuffling, and / or codon-shuffling (collectively referred to as "DNA shuffling"). DNA shuffling is used to regulate the activity of the polypeptide corresponding to SEQ ID NO: 2, effectively producing antagonists and agonists of the polypeptide (see, for example, U.S. Patent Nos. 5,605,793, 5,811,238, 5,830,721, 5,834,252 and 5,837,458 And Patten, PA, et al., Curr. Opinion Biotechnol. 8: 724-33 (1997); Harayama, S., Trends Biotechnol. 16 (2): 76-82 (1998); Hanson. L. O., et al., J. Mol. 287: 265-76 (1999); And Lorenzo, M. M. and Blasco, R., Biotechniques 24 (2): 308-13 (1998), each of which patents and publications are incorporated herein by reference). In one embodiment, variants of the polynucleotide corresponding to SEQ ID NO: 1 or 6 and corresponding polypeptides can be achieved by DNA shuffling. DNA shuffling involves the assembly of two or more DNA fragments into the desired molecule of the polypeptide corresponding to SEQ ID NO: 1 or 6 by homologous or site-specific recombination. In yet another embodiment, the polynucleotide corresponding to SEQ ID NO: 1 or 6 and the corresponding polypeptide may be modified through any mutagenesis method by error-prone PCR, any nucleotide insertion or other preceding recombination method. In another embodiment, a polypeptide encoded by one or more components, motifs, portions, fragments, domains, fragments, etc., or the like of a coding polynucleotide corresponding to SEQ ID NO: 1 or 6, , A motif, a part, a part, a domain, a fragment, and the like.

Polypeptide Purification and Isolation

MPIF-1 can be prepared by a method including ammonium sulfate or ethanol precipitation, acid extraction, anion or cation exchange resin, phosphocellulose chromatography, hydrophobic interaction chromatography, affinity chromatography, hydroxylapatite chromatography and lectin chromatography And recovered from the recombinant cell culture and purified. The protein refolding step can be used to complete the structure of the mature protein. Finally, high performance liquid chromatography (HPLC) can be used for the final purification step.

The polypeptides of the present invention may be naturally purified products or products of chemical synthesis steps or they may be produced by recombinant techniques from prokaryotic or eukaryotic hosts (e. G., Bacteria, yeast, higher plants, insects And mammalian cells). Depending on the host used in the recombinant production step, the polypeptides of the present invention may be glycosylated or non-glycosylated into carbohydrates of mammals or other eukaryotes. Polypeptides of the invention may comprise an initial methionine amino acid residue.

Also, the polypeptides of the present invention can be chemically synthesized using techniques in the art (see, for example, Creighteon, 1983, Proteins: Structures and Molecular Principles, W.H. Freeman & Co., N. Y, and Hunkapiller et al., Nature 310: 105-111 (1984)]. For example, a polypeptide corresponding to a fragment of MPIF-1 polypeptide can be synthesized by use of a peptide synthesizer. In addition, unconventional amino acids or chemical amino acid analogs may be introduced as substitutions or additions into the MPIF-I polypeptide sequence. Unconventional amino acids include, but are not limited to, the D-isomer of a common amino acid, 2,4-diaminobutyric acid, a-aminoisobutyric acid, 4-aminobutyric acid, Abu, 2- aminobutyric acid, gAbu, eAhx, , Aib, 2-aminoisobutylic acid, 3-aminopropionic acid, ornithine, norleucine, norvaline, hydroxyproline, sarcosine, citrulline, homocitrulline, cysteic acid, t-butylglycine, t- But are not limited to, glycine, cyclohexylamine, b-alanine, fluoro-amino acids, designed amino acids such as b-methyl amino acid, Ca-methyl amino acid, Na-methyl amino acid and common amino acid analogs. In addition, the amino acid may be D (right) or L (left).

The present invention is also directed to a process for the manufacture of a medicament for the treatment or prevention of a disease or disorder which is differentially modified during or after translation (e.g., by glycosylation, acetylation, phosphorylation, amidation, derivatization by known protective / Lt; / RTI > polypeptide). Any of a variety of chemical modifications can be carried out by the known techniques, and for instance specific chemical cleavage by cyanogen bromide, trypsin, chymotrypsin, papain, V8 protease, NaBH _4; Acetylation, formalization, oxidation, reduction; Metabolic synthesis in the presence of tunicamycin, and the like.

Additional post-translational modifications pertaining to the present invention include, for example, N-linked or O-linked carbohydrate chains, N-terminal or C-terminal processing, attachment of chemical moieties of amino acid backbones, N-linked or O- linked carbohydrate chains Addition or deletion of N-terminal methionine residues as a result of chemical modification of prokaryotic host cells and expression of prokaryotic host cells. The polypeptide may be modified with a detectable label, such as an enzymatic, fluorescent, isotopic or affinity label that enables detection and isolation of the protein.

In addition, chemically modified derivatives of the polypeptides of the present invention that can provide additional advantages such as solubility, stability and increased circulation time of the polypeptide or immunogenicity are provided by the present invention (see U.S. Patent No. 4,179,337). The chemical moiety for the derivatization may be selected from water-soluble polymers such as polyethylene glycol, ethylene glycol / propylene glycol copolymer, carboxymethyl cellulose, dextran, polyvinyl alcohol and the like. The polypeptide may be modified at any position in the molecule or at a predetermined position in the molecule and includes one, two, three, or more attached chemical moieties.

The polymer can be of any molecular weight and can be branched or unbranched. The preferred molecular weight in polyethylene glycol is from about 1 kDa to about 100 kDa for ease of handling and manufacture (the term " about " means that some molecules in the production of polyethylene glycol are somewhat less or more than the indicated molecular weight). Other sizes may be used depending on the treatment file desired (e. G., Slow release, bioactive, ease of handling, degree or lack of antigenicity and other known effects of therapeutic proteins or analogs).

The polyethylene glycol molecule (or other chemical moiety) must be attached to the protein in view of its effect on the functional domain or antigenic domain of the protein. There are a number of attachment methods that are readily available to those skilled in the art (e.g., EP 0 401 384, incorporated herein by reference (PEG or GCSF coupling); Malik et al., Exp. Hematol. 20: 10281035 (1992) (reporting pegylation of GMCSF using testeryl chloride). For example, polyethylene glycol can be covalently bound through amino acid residues by a reactor such as free amino or carboxy groups. The reactor is capable of binding activated polyethylene glycol molecules. An amino acid residue having a free amino group will comprise a lysine residue and an N-terminal amino acid residue; The residue having a free carboxyl group includes an aspartic acid residue, a glutamic acid residue, and a C-terminal amino acid residue. The sulfhydryl group can be used as a reactor for attaching polyethylene glycol molecules. Attachment to amino groups such as attachment to the N-terminal or lysine group is preferred for therapeutic purposes.

Proteins chemically modified at the N-terminus are particularly preferred. The use of polyethylene glycol as an example of the present composition can be used to identify variants (by molecular weight, branching, etc.) of the polyethylene glycol molecules, the ratio of the polyethylene glycol molecules to the protein (polypeptide) molecules in the reaction mixture, A method for obtaining an N-terminal pegylated protein can be selected. A method for obtaining an N-terminal pegylated agent (i. E., If necessary separating this moiety from other mono-pegylated moieties) is by purification of the N-terminal pegylated material from a population of pegylated protein molecules. Chemically modified selective proteins in N-terminal modification can be achieved by reductive alkylation using different types of different reactivities of primary amino acids (lysine to N-terminal) that are easy to induce in a particular protein. Under appropriate reaction conditions, substantially selective induction of the protein at the N-terminus is achieved with the polymer containing the carbonyl group.

Antibody

The MPIF-1 protein-specific antibody used in the present invention can be elevated against the native MPIF-1 protein or antigenic polypeptide fragment thereof. It can be presented in animal systems (e. G., Rabbits or mice) with carrier proteins such as albumin, or does not require carriers long enough (greater than about 25 amino acids).

As used herein, the term "antibody" (Ab) or "monoclonal antibody" (Mab) is meant to include unique molecules and antibody fragments (eg, Fab and F (ab ') ₂ fragments) . They can bind specifically to the MPIF-1 protein. Fab and F (ab ') ₂ fragments may lack Fc fragments of apparent intrinsic antibodies more rapidly than circulation and may be less sensitive to non-specific tissue binding of intrinsic antibodies [Wahl et al., J. Nucl. Med. 24: 316-325 (1983)]. Thus, such fragments are preferred.

The polypeptides, fragments or derivatives thereof, or analogs thereof, or cells expressing them may be used as immunogens to generate antibodies thereto. Such an antibody may be a monoclonal antibody or a polyclonal antibody. The present invention also includes chimeric, single chain and humanized antibodies and products of Fab fragments or Fab expression libraries. A variety of methods known in the art can be used to generate such fragments and antibodies.

The present invention relates to a polypeptide sequence comprising or consisting of an epitope of the polypeptide sequence shown in SEQ ID NO: 2 or 7, or a polypeptide sequence encoded by a cDNA contained in a deposited clone. Polynucleotides encoding such epitopes (e. G., Sequences set forth in SEQ ID NO: 1 or 6) are included in the present invention. The nucleotide sequence of the complementary strand of the polynucleotide encoding this epitope is also the same as that of a polynucleotide that hybridizes to the complementary strand under stringent hybridization conditions or low hybridization conditions.

In the present invention, the term " epitope " refers to a polypeptide fragment having an antigenic or immunogenic activity in an animal, particularly a human. Preferred embodiments of the present invention refer to a polynucleotide encoding this fragment and a polypeptide fragment comprising or consisting of an epitope. The region of the protein molecule to which the antibody binds is defined as " antigenic epitope ". In contrast, an " immunogenic epitope " is defined as part of a protein that induces an antibody response (see, e.g., Geyen et al., Proc. Natl. Acad. Sci. USA 81: 3998-4002 (1983)].

Antigenic epitopes are useful for raising antibodies, including monoclonal antibodies that specifically bind to the epitope (see, e.g., Wilson et al., Cell 37: 767-778 (1984); Sutcliffe et al., Science 219: 660-666 (1983)].

Similarly, immunogenic epitopes can be used, for example, to induce antibodies according to methods known in the art (see, for example, Sutcliffe et al., Supra; Wilson et al., Supra; Chow et al., Proc. Natl. Acad. Sci. USA 82: 910-914; And Bittle et al., J. Gen. Virol. 66: 2347-2354 (1985)]. Preferred immunogenic epitopes include secreted proteins. An immunogenic epitope can be presented in animal systems (e. G., Rabbits or mice) with carrier proteins such as albumin, or does not require carriers long enough (greater than about 25 amino acids). However, immunogenic epitopes comprising 8 to 10 amino acids have been shown to be sufficient to raise antibodies capable of binding at least a linear epitope in the modified polypeptide (e.g., Western blotting).

The present invention relates to antibodies and T-cell antigen receptors (TCRs) that specifically bind polypeptides of the invention. The antibodies of the present invention include _{IgG (IgG 1, IgG 2,} IgG 3 and IgG _4), IgG (IgA ₁ and _{IgA 2), IgD, IgE,} IgM , and IgY. As used herein, the term " antibody " (Ab) refers to a whole antibody comprising a single chain whole antibody and its antigen-binding fragment. The antibody is most preferably a human antigen binding antibody fragment of the present invention and is most preferably selected from the group consisting of Fab, Fab 'and F (ab') ₂ , Fd, single chain Fvs (scFv), single chain antibodies, disulfide-linked Fvs (sdFv) _L or V _H domains. Antibodies can be derived from any animal origin, including algae and mammals. Preferably, the antibody is human, murine, rabbit, goat, guinea pig, camel, horse or chicken.

An antigen-binding antibody fragment comprising a single-chain antibody will comprise a variable region, alone or in combination with all or part of the following: a hinge region, a CH ₁ , a CH ₂ and a CH ₃ domain. The invention also encompasses any combination of variable region (s) and hinge region, CH ₁ , CH ₂ and CH ₃ domains. The invention further encompasses monoclonal, polyclonal, chimeric, humanized and human monoclonal and human polyclonal antibodies. Which specifically binds to the polypeptide of the present invention. The present invention includes antibodies that are anti-genotypic for the antibodies of the invention.

The antibodies of the present invention may be monospecific, bispecific, triple specific or multispecific. Multispecific antibodies may be specific for different epitopes of a polypeptide of the invention or may be specific for a heterologous composition (e.g., a solid support material or heterologous polypeptide) as well as the polypeptides of the invention (see, for example, WO 93/17715 ; WO 92/08802, WO 91/00360; WO 92/05793; Tutt et al., J. Immonol. 146: 60-69 (1991); U.S. Patent Nos. 5,573,920, 4,474,892, 5,601,819, 4,714,681, 4,925,648; Kostelny et al., J. Immunol. 148: 1547-1553 (1992)).

An antibody of the invention may be described or defined in terms of the epitope (s) or portion (s) of the polypeptide of the invention that is specifically bound or recognized by the antibody. The epitope (s) or polypeptide portion (s) are defined by the size of the adjacent amino residues, for example by N-terminal or C-terminal positions as described herein, and are listed in tables and figures. Antibodies that specifically bind to any epitope or polypeptide of the invention may be excluded. Thus, the present invention includes antibodies specifically binding to the polypeptides of the invention, and allows the exclusion of the same.

The antibodies of the present invention may be described or defined in terms of cross-reactivity. Any other analog of the polypeptide of the invention, an ortholog, an antibody that does not bind to a homologue. Polypeptides having an identity of 95% or less, 90% or less, 85% or less, 80% or less, 75% or less, 70% or less, 65% or less, 60% or less, 55% or less and 50% or less Non-binding antibodies are encompassed by the present invention. An antibody that binds only to a polypeptide encoded by a polynucleotide that hybridizes with a polynucleotide of the present invention under stringent hybridization conditions (as described herein) is encompassed by the present invention. The antibodies of the present invention may be described or defined in terms of their binding affinity. The preferred binding affinity is ^{5X10 -6 M, 10 -6 M,} 5X10 -7 M, 10 -7 M, 5X10 -8 M, 10 -8 M, 5X10 -9 M, 10 -9 M, 5X1O -10 M, 10 ^{-10 M, 5XI0 -11 M, 10} -11 M, 5X10 -12 M, 10 -12 M, 5X1O -13 M, 10 -13 M, 5X10 -14 M, 10 -14 M, 5X10 -15 M, and and having a decomposition constant or Kd of 10 ^-15 M or less.

The antibodies of the present invention include, but are not limited to, methods known in the art for purifying, detecting and targeting polypeptides of the invention, including in vitro and in vivo diagnostic and therapeutic methods. For example, antibodies are used in immunoassays for quantitative or qualitative determination of polypeptide levels of the invention in biological samples (see, e.g., Harlow et al., Antibodies: a Laboratory Manual, Cold Spring Harbor Laboratory Press, Edition 1988, incorporated herein by reference in its entirety].

The antibodies of the present invention may be used alone or in combination with other compositions. The antibody may further be recombinantly fused with a heterologous polypeptide at the N- or C-terminus or may be chemically conjugated (including covalent and non-covalent conjugation) to a polypeptide or other composition. For example, an antibody of the invention can be conjugated or recombinantly fused with effector molecules such as heterologous polypeptides, drugs or toxins, and molecules useful as markers in detection assays (see, e.g., WO 92/08495; WO 91/14438; WO 89/12624; U.S. Patent No. 5,314,995; And European Patent No. 0 397 387].

The antibodies of the present invention can be prepared by any suitable method known in the art. For example, a polypeptide of the invention or an antigenic fragment thereof may be administered to an animal to induce the production of serum containing polyclonal antibodies. The term " monoclonal antibody " is not limited to antibodies generated through hybridoma techniques. The term " monoclonal antibody " means an antibody derived from a monoclonal and includes eukaryotic, prokaryotic or phage clones. This does not mean how it is created. Monoclonal antibodies can be prepared using a variety of techniques in the art by the use of hybridomas, recombinants and phage display techniques.

Hybridoma techniques can be performed using methods known in the art and in Harlow et al., Antibodies: a Laboratory Manual, Cold Spring Harbor Laboratory Press, 2nd ed., 1988; Hammerling, et al., In: Monoclonal Antibodies and T cell Hybridomas 563681 (Elsevier, NY, 1981) (herein incorporated by reference). Fab and F (ab ') ₂ fragments can be generated by proteolytic cleavage using enzymes such as papain (producing a Fab fragment) or pepsin (producing an F (ab') ₂ fragment).

Alternatively, the antibodies of the present invention can be generated through the application of recombinant DNA and phage display techniques or through synthetic chemistry using methods known in the art. For example, the antibodies of the present invention can be prepared using a variety of phage display methods known in the art. In a phage display method, the functional antibody domain is presented on the surface of a phage particle bearing a polynucleotide sequence encoding them. A phage with the desired binding properties is selected from a repertoire or combination of antibody libraries (e. G., Human or murine) by direct selection with the antigen, typically a solid surface or bead bound or captured antigen. The phage used in this method is a typical fibrous phage comprising fd with Fab recombinantly fused to phage gene III or gene VIII protein and Fv antibody domain stabilized with M13, Fv or disulfide bond. Examples of phage display methods that can be used to prepare antibodies of the present methods are described in Brinkman et al., J. Immunol. Methods 182: 4l-50 (1995); Ames et al., J. Immunol. Methods 184: 177-186 (1995); Kettleborough et al., Eur. J. Immunol. 24: 952-958 (1994); Persic et al., Gene 187: 9-18 (I997); Burton et al., Advances in Immuno1ogP 57: 191-280 (1994); PCT / GB91 / 01134; WO 90/02809; WO 91/10737; WO 92/01047; WO 92/18619; WO 93/11236; WO 95/15982; WO 95/20401; and US Patents 5,698,426, 5,223,409, 5,403,484, 5,580,717, 5,427,908, 5,750,753, 5,821,047, 5,571,698, 5,427,908, 5,516,637, 5,780,225, 5,658,727, and 5,733,743 (herein incorporated by reference).

After screening of the phage, the antibody coding region can be separated from the phage and used to generate whole antibodies, including human antibodies or any other desired antigen-binding fragments, as described in the above-mentioned documents, and can be used in mammalian cells, insect cells, Plant cells, yeast, and bacteria. For example, techniques for recombinantly producing Fab, Fab 'and F (ab') ₂ fragments are described in WO 92/22324; Mullinax et al., Bio Techniques 12 (6): 864-869 (I992); and Sawai et al. , AJR 34: 26-34 (1995); and Better et al., Science 240: 1041-1043 (1988), incorporated herein by reference).

Examples of techniques that can be used to prepare single-chain Fvs and antibodies include those described in U.S. Patent Nos. 4,946,778 and 5,258,498; Huston et al., Methods in Enzymology 203: 46-88 (1991); Shu, L. et al., PNAS 90: 7995-7999 (I993); and Skerra et al., Science 240: 1038-1040 (1988). For some uses involving in vivo antibody use in humans and in vitro detection assays in humans, chimeric, humanized or human antibodies may be preferred. Methods for generating chimeric antibodies are known (see, for example, Morrison, Science 229: 1202 (1985); Oi et al. , ≪ / RTI > Bio Techniques 4: 214 (1986); illies et al., J. Immunol. Methods 125: 191-202 (1989); and US Patent 5,807,715. Antibodies can be humanized using a variety of techniques including CDR-grafting (EP 0 239 400; WO 91/09967; US Patent 5,530,101; and 5,585,089), veneering or resurfacing (EP 0 592 106; EP 0 519 596; Padlan EA, Molecular Roguska. et al., PNAS 91: 969-973 (1994)). , and chain shuffling (US Patent 5,565,332). Human antibodies can be prepared by a variety of known methods, including the phage display methods described above (see, for example, U.S. Patent Nos. 4,444,887, 4,716,111, 5,545,806, and 5,814,318; And WO 98/46645, WO 98/50433, WO 98/24893, WO 98/16654, WO 96/34096, WO 96/33735, and WO 91/10741, incorporated herein by reference).

The invention includes recombinantly fused or chemically conjugated (including both covalent and non-covalent) antibodies to polypeptides of the invention. The antibody may be specific for an antigen other than the polypeptide of the invention. By fusing or conjugating the polypeptide of the present invention to an antibody specific for a particular cell surface receptor, for example, in vitro or in vivo, the antibody can be used to target a polypeptide of the invention to a particular cell type. Antibodies fused or conjugated to the polypeptides of the invention can be used in in vitro immunoassay and purification methods using methods known in the art. Reference example, Harbor et al. WO 93/21232; EP 0 439 095; Naramura e1 al., Lmmuno1. Lett. 39: 91-99 (1994); U.S. Patent 5,474,981; Gillies et al., PNAS 89: 1428-1432 (1992); Fell et al., J. Immunol. 146: 2446-2452 (1991).

The invention also encompasses a composition comprising a polypeptide of the invention fused or conjugated to an antibody domain other than a variable region. For example, a polypeptide of the invention can be fused or conjugated to an antibody Fc region or a portion thereof. The antibody portion fused to a polypeptide of the invention may comprise a hinge region, a CH ₁ domain, and a combination or part of a CH ₃ domain or an entire domain. The polypeptides of the invention may be fused or conjugated to a portion of the antibody for use in immunoassays using known methods or for increased in vivo half-life of the polypeptide. The polypeptide may be fused or conjugated to a portion of the antibody to form a multimer. For example, a portion of the Fc fused to a polypeptide of the invention can form a dimer through a disulfide bond between the Fc moieties. Highly multimeric forms can be produced by fusing polypeptides to a portion of IgA and IgM. Methods for fusing or conjugating the polypeptides of the present invention to a portion of an antibody are known in the art. Reference example. U.S. Patent Nos. 5,336,603, 5,622,929, 5,359,046, 5,349,053, 5,447,851, 5,112,946; EP 0 307 434, EP 0 367 166; WO 96/04388, WO 91/06570; Ashkenazi et al., PNAS88: 10535-10539 (1991); Zheng et al., J. Immunol. 154: 5590-5600 (1995); And Vil et al., PNAS (89: 11337-1134l (1992), incorporated by reference).

The invention also relates to antibodies that act as antagonists or agents of the polypeptides of the invention. For example, the invention includes antibodies that partially or wholly inhibit the interaction of a polypeptide of the invention with a receptor / ligand. Receptor-specific antibodies and ligand-specific antibodies. Receptor-specific antibodies that do not abrogate ligand binding but inhibit receptor activity. Receptor activity (i. E., Signaling) is determined by the techniques described herein or is known in the art. It also includes receptor-specific antibodies that inhibit both ligand binding and receptor activity. Similarly, antibodies that bind to ligands and neutralize antibodies that interfere with the binding of the ligand to the receptor, as well as antibodies which bind to the ligand to interfere with receptor activity but do not prevent the ligand from binding to the receptor. It also includes an antibody that activates the receptor. Such antibodies may act as agonists for all or less of the biological activities affected by the ligand-mediated receptor activity. The antibody may be defined as an antagonist or agonist of biological activity including the specific activity disclosed herein. The antibody agonist may be prepared using methods known in the art. Reference example. WO 96/40281; U.S. Patent 5,811,097; Deng et al., Blood 92 (6): 1981-1988 (1998); Chen, et al., Cancer Res. 58 (16), 3668-3678 (1998); Harrop et al., J. Immunol. 161 (4): 1786-1794 (1998); Zhu et al., Cancer Res. .58 (5): 3209-3214 (1998); Yoon, et al, J. Immunol. 160 (7): 3170-3179 (1998); Prat et al., J. Cel1. Sci. 111 (Pt 2): 237-247 (1998); Pitard et al., J. Immunol. Methods 205 (2): 177-190 (1997); Liautard et al., Cytokine 9 (4): 233-241 (1997); Carlson et al., J. Biol. Chem. 272 (17): 1295- 11301 (1997); Taryman et al., Neuron 14 (4): 755-762 (1995); Muller et al., Structure 6 (9): 1153-1167 (1998); Bartunek et al., Cytokine 8 (1): 14-20 (1996), incorporated herein by reference).

As described above, antibodies to polypeptides of the invention can be used alternatively to produce anti-genotypic antibodies that "mimic" the polypeptides of the invention using instrumental techniques known to those skilled in the art (see, eg, Greenspan & Bona , FASEB J. 7 (5): 437-444 (1989) and Nissinoff, J. Immumol. 147 (8): 2429-2438 (1991)]. For example, antibodies that bind to a ligand to competitively inhibit the binding of the polypeptide of the present invention and / or the multimerization of the polypeptide may be used to generate an anti-genotype that " mimics " polypeptide oligomerization and / or domain binding, To a polypeptide and / or its ligand to neutralize it. Such neutralized anti-genotype or anti-genotype Fab fragments can be used in therapeutic treatment to neutralize polypeptide ligands. For example, the anti-genotypic antibody can be used to block the biological activity of the polypeptide of the present invention and / or bind to its ligand / receptor.

The antibody or its in vivo receptor produced against a polypeptide corresponding to the sequence of the present invention can be obtained by direct injection of the polypeptide into an animal or by administering the polypeptide to an animal, preferably a human. The antibody thus obtained will then bind to the polypeptide itself. Only sequences that encode only a fragment of the polypeptide in this manner can be used to generate antibodies that bind to the entire native polypeptide. Such antibodies can then be used to isolate polypeptides from tissues expressing the polypeptide.

For the production of monoclonal antibodies, any method may be used which provides an antibody produced by continuous cell line culture. For example, the hybridoma technique [Kohler and Milstein, 1975, Nature, 256: 495-497], the trioma technique, the human B-cell hybridoma technique [Kozbor et al., 497], produces human monoclonal antibodies Human B-cell hybridoma technique [Cole et al., 1985, in Monoclonal Antibody and Cancer Therapy, Alan R. Liss, Inc., pp77-96].

Methods for the production of single chain antibodies [US Pat. No. 4,946,778] can be modified to produce single chain antibodies to the immunogenic polypeptide products of the invention.

The antibody of the present invention can be prepared by various methods. For example, a cell expressing MPIF-1 protein or an antigen fragment thereof can be administered to an animal to induce the production of serum, including polyclonal antibodies. As a preferred method, a preparation of MPIF-1 protein is prepared and purified so that there is little natural contaminants. The preparation is then introduced into the animal to produce polyclonal antiserum having high specific activity.

As a most preferred method, the antibody of the present invention is a monoclonal antibody (or its MPIF-1 protein binding fragment). Such monoclonal antibodies can be prepared using hybridoma techniques (Kohler et al., Nature 256: 495 (1975); Kohler et al., Eur. J. Immunol. 6: 511 (1976); Kohler et al., Eur. J. Immunol. 6: 922 (1076); Hammerling et al., In: Monoclonal Antibodies and T-Cell Hybridomas, Elsebier, N. Y., (1981) pp.563-681. In general, this method comprises immunizing an animal (preferably a mouse) with an MPIF-1 protein antigen or more preferably with MPIF-1 protein-expressing cells. Appropriate cells can be recognized by its ability to bind to anti-MPIF-1 protein antibodies. Such cells can be cultured in any suitable tissue cell culture medium; However, Earle's modified earbuds supplemented with 10% fetal bovine serum (inert at about 56 ° C), about 10 μg / l nonessential amino acids, about 1000 U / ml penicillin and about 100 μg / ml streptomycin It is preferred to cultivate cells in Earle's modified ear buds supplemented. The mouse spleen cells are extracted and fused with an appropriate myeloma cell line. Any suitable myeloma cell line may be used in accordance with the present invention; However, it is preferred to use a parental myeloma cell line (SPO2) obtained from the American Type Culture Collection (Rockville, Maryland). After fusion, the resulting hybridoma cells are selectively maintained in HAT medium and cloned by limiting dilution as described in Wands et al., Gastroenterology 80: 225-232 (1981). The hybridoma cells obtained through the above screening are analyzed to identify clones secreting antibodies capable of binding to the MPIF-1 protein antigen.

Alternatively, additional antibodies capable of binding to MPIF-1 protein antigens can be generated in a two-step method by the use of anti-genotype antibodies. This method utilizes the fact that the antibody itself is an antigen, and thus an antibody that binds to the second antibody can be obtained. According to this method, the MPIF-1 protein specific antibody is used to immunize an animal, preferably a mouse. These animal spleen cells are used to generate hybridoma cells, and hybridoma cells are screened to express antibodies that have the ability to bind MPIF-1 protein-specific antibodies that can be blocked by MPIF-1 protein antigens Identify the clone to be generated. Such antibodies may include anti-genotypic antibodies to MPIF-1 protein-specific antibodies and may induce the formation of additional MPIF-1 protein-specific antibodies by immunizing animals.

It will be appreciated that Fab and F (ab ') ₂ and other fragments of the antibodies of the invention may be used in accordance with the methods described herein. These fragments are typically produced by proteolytic cleavage using enzymes such as papain (to produce Fab fragments) or pepsin (to generate F (ab / ₂ ) fragments). Alternatively, MPIF-1 protein-binding fragments can be generated through synthetic chemistry or through the application of recombinant DNA techniques.

It is preferred to use a " humanized " chimeric monoclonal antibody. Such an antibody may be produced using a gene construct derived from a hybridoma cell producing the above-described monoclonal antibody. Methods for generating chimeric antibodies are known. Reference example; Morrison, Science 229: 1202 (1985); Oi et al., Bio Techniques 4: 214 (I986); CabiIIy et al. U, S. Patent No. 4,816,567; Taniguchi et al., EP 171496; Morrison et al., EP 173494; Neuberger et al., WO 8601533; Robinson et al., WO 8702671; Boulianne et al., Nature 312: 643 (1984); Neuberger et al., Nature 314: 268 (1985).

In addition, suitable labels for the MPIF-1 protein-specific antibodies of the invention are provided as follows. Examples of suitable yeasthop labels include malate dehydrogenase, staphylococcus nuclease, delta-5-steroid isomerase, yeast-alcohol dehydrogenase, alpha-glycerol phosphate dehydrogenase, triose phosphate isomerase, peroxidase , Alkaline phosphatase, asparaginase, glucose oxidase, beta-galactosidase, ribonuclease, urease, catalase, glucose-6-phosphate dehydrogenase, glucoamylase and acetylcholinesterase.

An example of a suitable radioactive labeled ^{3 H, 111 ln, 125 I} , 131 I, 32 P, 35 S, 14 C, 51 Cr, 57 To, 58 Co, 59 Fe, 75 Se, 152 Eu, 90 Y, ⁶⁷ Cu, ²¹⁷ Ci, ²¹¹ At, ²¹² Pb, ⁴⁷ Sc, ¹⁰⁹ Pd, and the like. ¹¹¹ In is a preferred isotope when used in in vivo imaging. This is because it solves the problem of dehalogenation of ¹²⁵ I or ¹³¹ I-labeled monoclonal antibodies by the liver. In addition, radioactive nucleotides have very useful gamma emission energies for imaging [Perkins et al., Eur. J Nucl. Med. 10: 296-301 (1985); Carasquillo et al., J. Nucl. Med. 28: 281-287 (1987)].

Examples of suitable non-radioactive isotope labels include ¹⁵⁷ Gd, ⁵⁵ Mn, ¹⁶² Dy, ⁵² Tr, and ⁵⁶ Fe.

Examples of suitable fluorescent labels include the ¹⁵² Eu label, fluorescent label, isothianate label, rhodamine label, phycoerythrin label, phycocyanin label, allophycocyanin label, o-phthaldehyde label, and fluorescent label .

Examples of suitable toxin markers include diphtheria toxin, lysine and cholera toxin.

Examples of chemiluminescent labels include luminescent labels, isoleucine labels, aromatic acridinium labels, imidazole labels, acridinium label labels, oxalate label, luciferin label, leuciferase label, and equolin label .

Examples of nuclear magnetic resonance contrast agents include heavy metal nuclei such as Gd, Mn and iron.

Conventional methods of conjugating the aforementioned labels to antibodies are described in Kennedy et al., Clin. Chim. Acta 70: 1-31 (1976) and Schurs et al., Clin Chim. Acta 81: 1-40 (1977). The coupling techniques mentioned in the Schurs et al. Literature are the glutathaldehyde method, peridate method, dimaleimide method, m-maleimidobenzyl-N-hydroxy-succinimide ester method, Quot;

Chromosome analysis

Further, the nucleic acid molecule of the present invention is useful for chromosome identification. The sequence can be specifically targeted to individual human chromosomes and hybridized to specific positions on individual human chromosomes. Moreover, there is a current need to identify specific sites on chromosomes. In order to mark chromosomal locations, few are currently available as chromosome markers based on substantial sequence data (repetitive polymorphisms). Mapping of DNA to a chromosome according to the present invention is an important first step in relation to a sequence retaining a gene associated with a disease.

In certain preferred embodiments related thereto, the genomic DNA of the MPIF-1 protein gene was cloned using the cDNA disclosed herein. This can be accomplished using a variety of commonly known and commercially available techniques and libraries. The genomic DNA then performs chromosomal mapping in situ using well known techniques for this purpose. Typically, according to the general procedure for chromosomal mapping, some trial and error may be required to identify a genomic probe to provide a good in situ hybridization signal.

Roughly, sequences can be mapped to chromosomes by preparing PCR primers (preferably 15-25 bp) from the cDNA. Computer analysis of the cDNA can be used to quickly select primers that do not span more than one exon in the genomic DNA, which complicates the amplification process. Subsequently, somatic cell hybrids containing individual human chromosomes are PCR-screened using these primers. Only hybrids containing human genes corresponding to the primers will yield amplified fragments.

PCR mapping of somatic cell hybrids is a rapid method of assigning specific DNA to specific chromosomes. In accordance with the present invention using the same oligonucleotide, the sub-orientation can be performed in a similar manner using a panel of some chromosomes or a panel derived from a pool of large genomic clones. Other mapping techniques that can be used to map chromosomes similarly include in situ hybridization, preliminary screening using labeled flow-sorted chromosomes, and pre-selection by hybridization to constitute a chromosome specificity-cDNA library do.

Using fluorescent in situ hybridization (FISH) of cDNA clones for mid-term chromosomal spreads can provide one-step accurate chromosomal location. This technique can be performed using a probe derived from cDNA and as short as 50 or 60 bp. This technique is described in, for example, Verma et al., Human Chromosomes: A Manual of Basic Techniques , Pergamon Press, New York (1988).

Once the sequence is mapped to the correct chromosomal location, the physical location of the sequence on the chromosome may be related to the gene map data. Such data can be found, for example, in V. McKusick, Mendelian Inheritance In Man, available online from the Welch Medical Library at Johns Hopkins University. The relationship between mapped genes and diseases in the same chromosomal region is confirmed through an association (co-inheritance of physically adjacent genes) analysis.

Next, it is necessary to identify differences in the cDNA or genomic sequence between the affected and unaffected individuals. If a mutation is observed in some or all of the affected individuals and no mutation is observed in any normal individuals, the mutation may be the cause of the disease.

Given the current resolution of physical mapping and genetic mapping techniques, cDNAs that are precisely aligned to disease-associated chromosomal regions can be one of 50 to 500 possible causative genes. This assumes the mapping resolution of 1 mb (megabase) and the presence of one gene per 20 kb.

A comparison of affected and unaffected individuals generally involves a chromosomal structural change, such as a deletion or translocation, that can be detected visually from the chromosomal spread or by PCR based on the cDNA sequence as the first step do. Ultimately, the complete sequence of a gene from some individuals is necessary to confirm the presence of the mutation and is necessary to distinguish the mutation from the polymorphism.

The present invention also relates to inhibiting MPIF-1 in vivo using antisense technology. Antisense techniques can be used to regulate gene expression through triple helix formation or antisense DNA or RNA, both based on the binding of polynucleotides to DNA or RNA. For example, the 5 'coding portion of a polynucleotide sequence encoding a polypeptide of the invention is used to construct an antisense RNA oligonucleotide of about 10 to 40 bp in length. By constructing the DNA oligonucleotides to be complementary to the region of the gene involved in transcription (see Triple Helix - Lee et al. , Nucl. Acids Res. , ≪ / RTI > 6: 3073 (1979); Cooney et al., Science , 241: 456 (1988); And Dervan et al., Science , 251: 1360 (1991)], inhibit the transcription and production of MPIF-1. Antisense RNA oligonucleotides hybridize to mRNA in vivo and block the translation of mRNA molecules into MPIF-1 protein (see Antisense - Okano, J. Neurochem. 56: 560 (1991); Oligodeoxynucleotides as Antisense Inhibitors of Gene Expression , CRC Press, Boca Raton, Florida (1988)].

Alternatively, the oligonucleotide can be delivered to the cell by a conventional procedure in which the antisense RNA or DNA can be expressed in vivo to inhibit the production of MPIF-1 in the same manner as described above.

Thus, antisense constructs against MPIF-1 can be used to treat MPIF-I induced or MPIF-I augmented disorders such as atherosclerosis, autoimmune diseases (e.g., multiple sclerosis and insulin dependent diabetes), and chronic inflammatory and infectious Idiopathic shock, histamine-mediated allergic response, prostaglandin-dependent fever, and regeneration deficiency, as well as other chronic inflammatory diseases of the lung, including, but not limited to, inflammatory bowel disease, histamine-mediated allergic reaction, rheumatoid arthritis, silicosis, sarcoidosis, idiopathic pulmonary fibrosis, Anemia and other bone marrow disorders diseases can be treated.

Antagonists, working substances and methods

The present invention also provides a method of screening for compounds to identify antagonists and agonists for the chemokine polypeptides of the invention. Antagonists are compounds that block their function, while the active agent is a compound that possesses or enhances a biological function similar to the polypeptide of the present invention. The chemotaxis can be analyzed by placing the cells colonized by the polypeptides of the present invention on top of a filter with a pore size of about 5 탆 (about 5 탆) sufficient to pass through the cells. The potential working substance solution is placed at the bottom of the chamber with the appropriate conditioning medium in the upper compartment and the concentration gradient of the working substance is measured by counting cells moving into or through the porous membrane over time.

When antagonists are to be measured, the chemokine polypeptides of the present invention are placed at the bottom of the chamber and a potent antagonist is added to determine whether chemotaxis of the cells is hindered.

Alternatively, the mammalian cell or membrane preparation expressing the receptor of the polypeptide is incubated with a labeled, e.g. radioactively labeled, chemokine polypeptide in the presence of the compound. The ability to block this interaction can then be measured. When analyzing the working substance in this manner, the chemokine is absent and measures the ability of the working substance itself to interact with the receptor.

Examples of potential MPIF-1 antagonists include antibodies, and in some cases, oligonucleotides that bind to the polypeptide. Another example of a potential antagonist is the (-) dominant mutant of the polypeptide. The (-) dominant mutant is a polypeptide that binds to the receptor of the curing polypeptide but does not possess biological activity.

In addition, antisense constructs prepared using antisense techniques are potential antagonists. Antisense techniques can be used to control gene expression through triple helix formation or intense DNA or RNA (both methods are based on binding of polynucleotides to DNA or RNA). For example, the 5 'end of the polynucleotide sequence encoding the mature polypeptide of the invention is used to construct an antisense RNA oligonucleotide of about 10 to 40 bp in length. By constructing the DNA oligonucleotides to be complementary to the region involved in transcription (see Triple Helix - Lee et al. , Nucl. Acids Res. , ≪ / RTI > 6: 3073 (1979); Cooney et al., Science , 241: 456 (1988); And Dervan et al., Science , 251: 1360 (1991)] to prevent transcription and production of chemokine polypeptides. Antisense RNA oligonucleotides hybridize in vivo to mRNA and block the translation of mRNA molecules into polypeptides (see Antisense - Okano, J. Neurochem. 56: 560 (1991); Oligodeoxynucleotides as Antisense Inhibitors of Gene Expression , CRC Press, Boca Raton, Florida (1988)]. In addition, the oligonucleotide is transferred to a cell, and antisense RNA or DNA can be expressed in vivo to inhibit the production of a chemokine polypeptide.

Other potential chemokine antagonists are naturally or synthetically modified analogues of the polypeptide that have lost biological function but still recognize the receptor of the polypeptide and effectively block the receptor by binding to the receptor. Examples of peptide derivatives include, but are not limited to, small peptides or peptide-like molecules.

Antagonists can be used to treat MPIF-1 induced or MPIF-I augmented diseases, such as autoimmune diseases and chronic inflammatory and infectious diseases. Examples of autoimmune diseases include multiple sclerosis and insulin-dependent diabetes mellitus.

In addition, antagonistic substances can be used to prevent the mobilization and activation of monocytes, thereby treating infectious diseases including silicosis, sarcoidosis, and idiopathic pulmonary fibrosis. They can also be used to treat idiopathic hyperacid syndrome by inhibiting eosinophil production and migration. In addition, endotoxic shock can also be treated by inhibiting the migration of macrophages and their production of the chemokine polypeptides of the present invention using antagonists.

Antagonists may also be used to treat atherosclerosis by inhibiting monocyte infiltration in the arterial wall.

Antagonists can also be used to treat histamine mediated allergic reactions and immunological disorders, including late allergic reactions, chronic urticaria and atopic dermatitis, by interfering with chemokine mediated mast cell and basophil degranulation and histamine release . IgE mediated allergic reactions, such as allergic asthma, rhinitis and eczema, can also be treated.

Antagonists can be used to treat chronic and acute inflammation by preventing monocyte induction to the wound site. They can also be used to regulate normal cloned macrophages because chronic and acute inflammatory lung diseases are associated with the sequestration of mononuclear cells in the lung.

In addition, antagonists can be used to treat rheumatoid arthritis by inhibiting monocyte invasion into the synovial fluid of the patient ' s joints. Mononuclear cell inflow and activation plays an important role in the pathogenesis of degenerative and inflammatory arthropathy.

Antagonists can interfere with deleterious cascades that are primarily responsible for IL-1 and TNF, preventing the biosynthesis of other inflammatory cytokines. In this way, antagonists can be used to prevent inflammation. Antagonists can also be used to inhibit chemokine-induced prostaglandin-dependent fever.

Antagonists can also be used to treat bone marrow disorders such as aplastic anemia and myelodysplastic syndrome.

In addition, antagonists can be used to treat asthma and allergies by inhibiting eosinophil accumulation in the lungs. In addition, antagonists can be used to treat episodic basement membrane fibrosis, a hallmark of asthmatic lungs.

Working substance

The MPIF-I agonist comprises any small molecule that possesses similar activity to any one or more of the polypeptides as described herein. For example, MPIF-1 activity can be enhanced using MPIF-I agonists. For example, MPIF-1-induced myelogenesis can be augmented in patients undergoing chemotherapy or bone marrow transplantation.

Vaginal Diagnosis and Prognosis

As described below, the particular disease or disorder is selected from the group consisting of MPIF-1 protein and mRNA encoding the MPIF-1 protein, as compared to a corresponding " standard " mammal, Of the total number of employees. In addition, an increased level of MPIF-I protein may be associated with a particular body fluid taken from a mammal having the disease or disorder (e. ≪ RTI ID = 0.0 > g. Serum, plasma, urine, and spinal cord). Thus, the present invention provides a diagnostic method comprising: analyzing the expression level of a gene encoding MPIF-1 in a mammalian cell or body fluids; comparing the gene expression level to a standard MPIF-1 gene expression level Thereby increasing the level of gene expression above the standard expression level as an indicator of a particular disease or disorder.

The present invention is useful as a prognostic indicator when a diagnosis of a disease or disorder has already been made according to conventional methods since patients exhibiting enhanced MPIF-1 gene expression are more likely to be at a lower level than patients expressing the gene This is because they will experience bad clinical outcomes.

As used herein, the phrase " analyzing the expression level of a gene encoding an MPIF-1 protein " can be used to directly (for example, by measuring or estimating an absolute protein level or mRNA level) or indirectly Means to qualitatively or quantitatively measure or estimate the level of MPIF-I or the level of mRNA encoding the MPIF-1 protein in the first biological sample by comparing the MPIF-1 protein level or mRNA level in the biological sample .

Comparing or comparing MPIF-I protein levels or mRNA levels in a first biological sample with standard MPIF-I protein levels or mRNA levels that are standards taken from a second biological sample from an individual not having the disease or disorder . As is understood in the art, once the standard MPIF-1 protein level or mRNA level is known, it can be used repeatedly as a standard for comparison.

As used herein, the term "biological sample" refers to any biological sample obtained from an individual, cell line, tissue culture, or other source containing the MPIF-1 protein. The biological sample may be a mammalian body fluid (e.g., serum, plasma, urine, synovial fluid and spinal cord) containing secreted mature MPIF-I protein and may also be an ovarian, testis, heart, placenta, pancreas, Skin, line, kidney, liver, spleen, lung, bone, bone marrow, eye, peripheral nerve, central nervous system, breast and fetal tissue. Methods for obtaining tissue biopsies and body fluids from mammals are well known in the art. If the biological sample contains mRNA, the breakfast biopsy is the preferred source.

The present invention is useful for detecting diseases in mammals. In particular, the invention is useful for the diagnosis or treatment of various immune system related disorders in mammals, preferably humans. Examples of such diseases include, but are not limited to, erroneous regulation of tumor, cancer and immune cell function such as autoimmunity, arthritis, leukemia, lymphoma, immunosuppression, sepsis, wound healing, acute and chronic infections, cell mediated immunity, humoral immunity, Intestinal disease, bone marrow suppression, and the like. Preferred mammals include monkeys, apes, cats, dogs, cows, pigs, horses, rabbits and humans. A particularly preferred mammal is a human.

The entire cellular RNA may be purified by any suitable technique, for example, Chomczynski and Sacchi, Anal. Biochem. 162: 156-159 (1987)]. [0154] < - > The level of mRNA encoding the MPIF-1 protein can then be analyzed using any suitable method. Examples of these analytical methods include Northern blot analysis, S1 nuclease mapping, polymerase chain reaction (PCR), combination of reverse transcription and polymerase chain reaction (RT-PCR), and reverse transcription and ligase chain reaction (RT- LCR).

Northern blot analysis can be performed as described in Harada et al., Cell 63: 303-312 (1990). Roughly, total RNA is prepared from biological samples as described above. For Northern blot, the RNA is denatured in a suitable buffer (e.g., glyoxal / dimethyl sulfoxide / sodium phosphate buffer), subjected to agarose gel electrophoresis and transferred onto a nitrocellulose filter. After the RNA is bound to the filter by an UV linker, the filter is prehybridized in a solution containing formamide, SSC, Denhardt's solution, denatured salmon sperm, SDS and sodium phosphate buffer. The labeled MPIF-1 cDNA is used as a probe according to any suitable method (for example, ³² P-multiple primed DNA labeling system (Amersham)). After overnight hybridization, the filters are washed and exposed to X-ray film. The cDNA for use as a probe according to the present invention is described in the above paragraph, and the length is preferably 15 bp or more.

S1 mapping can be performed as described in the literature (Fujita et al., Cell 49: 357-367 (1987)). In order to prepare the probe DNA for use in the S1 mapping, the labeled antisense DNA is synthesized using the sense strand of the cDNA as a template. The antisense DNA may be digested with suitable restriction enzymes to generate additional DNA probes of a predetermined length. Such antisense probes are useful for visualizing protected bands corresponding to the target mRNA (i. E. MRNA encoding the MPIF-1 protein). Northern blot analysis can be performed as described above.

The level of mRNA encoding the MPIF-1 protein is preferably analyzed using the RT-PCR method described in Makino et al., Technique 2: 295-301 (1990). According to this method, the radioactivity of the " amplicon " in the polyacrylamide gel band is linearly related to the initial concentration of the target mRNA. Roughly, the method comprises adding total RNA isolated from a biological sample into a reaction mixture containing an RT primer and a suitable buffer. After incubation for primer annealing, the mixture can be supplemented with RT buffer, dNTP, DTT, RNase inhibitor and reverse transcriptase. After the reverse transcription of the RNA by incubation, PCR is performed using the primers labeled for the RT products. Alternatively, the labeled dNTPs may be included in the PCR reaction mixture without labeling the primers. PCR amplification can be performed in a DNA thermal cycler according to conventional techniques. After amplification by manipulating the appropriate number of times, the PCR reaction mixture is electrophoresed on polyacrylamide gel. After drying the gel, the radioactivity of the appropriate band (corresponding to the mRNA encoding the MPIF-1 protein) is quantitated using an image analyzer. RT and PCR reaction components and conditions, reagent and gel concentration and labeling methods are well known in the art. Modifications of RT-PCR will also be apparent to those skilled in the art.

Any set of oligonucleotides that amplify the transcribed target mRNA can be used and can be constructed as described above.

Analysis of MPIF-1 protein levels in biological samples can be accomplished using any known method. A preferred method of analyzing MPIF-I protein levels in a biological sample is an antibody-based method. For example, MPIF-1 protein expression in tissues can be performed using classical immunohistochemical methods. In this case, the specific recognition is provided by the primary antibody (polyclonal or monoclonal), but the secondary detection system uses fluorescence, enzymes or other conjugated secondary antigens. As a result, immunohistochemical staining of tissue for pathological examination can be performed. In addition, tissue can be extracted using urea and neutral detergent to liberate MPIF-1 protein for Western blot or dot / slot analysis (Jalkanen, M. et al . , J. Cell. Biol. 101: 976-985 (1985); Jalkanen, M. et al . , J. Cell. Biol. 105: 3087-3096 (1987)]. In this technique based on the use of a cationic solid phase, the quantification of the MPIF-1 protein can be carried out using a separate MPIF-1 protein as a standard. The technique may also be applied to body fluids. Using these samples, the molar concentration of MPIF-1 protein will help determine the standard value of MPIF-1 protein content for different body fluids, such as serum, plasma, urine, spinal cord, Subsequently, the steady-state value of the amount of MPIF-1 protein is determined using the values obtained from healthy individuals, which can be compared with the amount obtained from the tested individuals.

Other antibody-based methods useful for detecting MPIF-1 gene expression include immunoassays, such as enzyme linked immunosorbent assay (ELISA) and radioimmunoassay (RIA). For example, MPIF-1 protein can be detected and quantified using MPIF-1 protein-specific monoclonal antibody as both an immunoadsorbent and an enzyme-labeled probe. The amount of MPIF-1 protein present in the sample can be calculated with reference to the amount present in the standard preparation, using a linear regression computer algorithm. In other ELISA assays, two distinct specific monoclonal antibodies can be used to detect MPIF-1 protein in body fluids. In this assay, one of the antibodies is used as an immunoadsorbent and the other is used as an enzyme labeled probe.

The technique may be performed as needed in a "one-step" or "two-step" assay. The " one step " assay involves contacting the immobilized antibody with the MPIF-1 protein and contacting the mixture with the labeled antibody without washing. The " two-step " assay involves a washing procedure prior to contacting the mixture with the labeled antibody. It may also be suitably carried out using other conventional methods. In general, it is desirable to allow one component of the analysis system to be fixed on a support, thereby allowing other components of the analysis system to contact the component and to be easily removed from the sample.

Suitable enzyme labels include, for example, those derived from an oxidase group that catalyzes the reaction to produce hydrogen peroxide by reaction with a substrate. Glucose oxidase is particularly preferred because its stability is good and its substrate (glucose) can be easily obtained. The activity of the oxidase tag can be analyzed by measuring the concentration of hydrogen peroxide formed by the enzyme-labeled antibody / substrate reaction. In addition to enzymes, other suitable labeled with a radioactive isotope, such as iodine ⁽¹²⁵ I, ¹²¹ I), carbon ⁽¹⁴ C), sulfur ⁽³⁵ S), tritium ⁽³ H), indium ⁽¹¹² In), and technetium ( ^99m Tc) and fluorescent labels, such as fluorescein and rhodamine, and biotin.

The polypeptides of the present invention and the polynucleotides encoding the polypeptides can be used as research reagents for the purpose of developing therapeutics and diagnostic agents for in vivo purposes related to scientific research, synthesis of DNA and production of DNA vectors, and human diseases. . For example, the immature hematopoietic progenitor cells, such as granulocytes, macrophages, or monocytes, can be inflated by temporarily interfering with the differentiation of immature hematopoietic progenitor cells using MPIF-1. These bone marrow cells can be cultured in vitro.

The fragment of the full-length MPIF-1 gene can be used as a hybridization probe for cDNA library for isolating the above-mentioned full-length gene and isolating another gene having a large sequence similarity with the above gene or having similar biological activity. However, it is preferred that the probe has at least 30 bases, for example, at least 50 bases. In addition, the probe can be used to identify genomic clones or clones containing a complete cDNA sequence including the cDNA clone and the regulatory region, the promoter region, the exon, and the intron corresponding to the full-length transcript. One screening method involves isolating the coding region of the gene using a known DNA sequence that synthesizes an oligonucleotide probe. A library member in which the probe hybridizes can be determined by screening a human cDNA, a genomic DNA, or an mRNA library using a labeled oligonucleotide having a sequence complementary to the gene sequence of the present invention.

The invention also relates to a method of using the MPIF-1 gene as part of a diagnostic assay for detecting a disease or susceptibility to a disease associated with the presence of a mutation in a nucleic acid sequence. These diseases are associated with impaired expression of chemokine polypeptides.

Individuals carrying mutations within the MPIF-1 gene can be detected at the DNA level using a variety of techniques. The nucleic acid for diagnosis can be obtained from a parent cell, for example, blood, urine, saliva, tissue biopsy material and autopsy material. Genomic DNA can be used directly for detection, or amplified using PCR (see Saiki et al ., Nature 324: 163-166 (1986)) prior to analysis. RNA or cDNA can also be used for the same purpose. As an example, MPIF-1 and mutations can be identified and analyzed using PCR primers complementary to the nucleic acid encoding MPIF-1. For example, deletion and insertion can be detected by a change in the size of the amplified product compared to a normal genotype. Point mutations can be identified by hybridizing the amplified DNA to radioactively labeled MPIF-1 RNA or radiolabeled MPIF-1 antisense DNA sequences. RNase A or melting temperature difference can distinguish between a perfectly matched sequence and a non-matching double helix.

Sequence testing based on DNA sequence differences can be performed by detecting alterations in the electrophoretic mobility of the DNA fragments in the gel in the presence or absence of a denaturant. Small sequence deletions and insertions can be visualized by high resolution gel electrophoresis. DNA fragments of different sequences can be distinguished in denaturing formamide gradient gels wherein the mobility of different DNA fragments is retarded in the gel at different positions depending on their particular melting temperature or partial melting temperature (see, e.g., Myers et al. , Science 230: 1242 (1985)).

Sequence changes at specific positions can also be confirmed by nuclease protection assays, such as RNase and S1 protection or chemical degradation (see, for example, Cotton et al., PNAS , USA 85: 4397-4401 (1985 )].

Thus, the detection of a particular DNA sequence may be accomplished by methods such as hybridization, RNase protection, chemical degradation, direct DNA sequencing or restriction enzyme use (e.g., restriction fragment length polymorphism (RFLP)) and Southern blotting of genomic DNA Can be performed.

In addition to conventional gel electrophoresis and DNA sequencing, mutations can also be detected by in situ analysis.

The present invention also relates to diagnostic assays for detecting altered levels of the MPIF-1 protein in various tissues because of overexpression of the protein compared to normal control tissue samples, such as a disease, The presence of sensitivity to < RTI ID = 0.0 > Assays used to detect the level of MPIF-1 protein in a sample derived from a host are well known to those skilled in the art and include radioimmunoassays, competitive binding assays, Western blot assays, ELISA assays and " sandwich & . ELISA assays (Coligan et al., Current Protocols in Immunology 1 (2), Ch. 6 (1991)] involves primarily preparing an antibody, preferably a monoclonal antibody, specific for the MPIF-1 antigen. In addition, a reporter antibody is prepared for the monoclonal antibody. The reporter antibody is attached to a detectable reagent, such as radioactivity, fluorescence, or the horseradish peroxidase enzyme of this embodiment. The sample is separated from the host and incubated in a solid support, for example a polystyrene dish, which binds the protein in the sample. Thus, any free protein binding site on the dish is co-incubated with a non-specific protein such as BSA. The monoclonal antibody is then incubated in the dish while the monoclonal antibody is attached to any MPIF-1 protein attached to the polystyrene dish. All unconjugated monoclonal antibodies are incubated with a buffer Wash it out. A reporter antibody conjugated to horseradish peroxidase is placed into the dish to allow the reporter antibody to bind to any monoclonal antibody bound to MPIF-1. The unattached reporter antibody is then washed away. Next, the peroxidase substrate is added to the dish, the amount of color developed at a predetermined time is measured, and compared with the standard curve, the amount of MPIF-1 protein present in a certain volume of the patient sample is measured.

Competition assays can be used in which an antibody specific for MPIF-1 is attached to a solid support and the labeled MPIF-1 and sample derived from the host are passed over a solid support. The amount of label detected was measured, for example, by liquid scintillation chromatography, which may be related to the amount of protein in the sample.

The "sandwich" assay is similar to the ELISA assay. In the " sandwich " assay, MPIF-1 is passed over a solid support to bind to the antibody attached to the solid support. Then, the second antibody is bound to MPIF-1. A third antibody, labeled and specific for the second antibody, is then passed over the solid support to bind the second antibody, the amount bound being quantifiable.

The present invention provides a method for identifying a receptor for a chemokine polypeptide. Gene encoding the receptor may be obtained by a variety of methods known to those of skill in the art, for example, ligand panning and FACS classification (Coligan et al., Current Protocols in Immun. 1 (2), Ch. 5 (1991)]. It is preferred to use expression cloning, wherein the polyadenylated RNA is prepared from cells reactive with the polypeptide, the cDNA library generated from the RNA is split into pea, and the other cells that are non-reactive with the COS cell or polypeptide are transformed Used to infect. Transfected cells growing on glass slides are exposed to labeled polypeptides. Polypeptides can be labeled by a variety of methods including iodination or insertion of recognition sites for position-specific protein kinases. After fixation and incubation, an automated radioactivity assay is performed on the slide. The positive pool is identified, the subpulle is produced, retransfected using repeated subfuiling, and a re-screening process is performed to yield a single clone encoding the putative receptor substantially.

As an alternative to receptor recognition, labeled polypeptides may bind to an extract product that expresses a cell membrane or receptor molecule. The crosslinked material is analyzed by PAGE analysis and exposed to X-ray film. A labeled complex containing a receptor of the polypeptide is ablated, digested into peptide fragments, and protein microsequence determinations are performed. A cDNA library can be screened to identify a gene encoding a putative receptor by constructing a set of deoxyribonucleic acid oligonucleotide probes using the amino acid sequence obtained by microsequencing.

remedy

The polypeptide of the present invention can be used for various immunoregulatory and inflammatory actions and also for a number of disease states. MPIF-1 belongs to the chemokines, and thus MPIF-1 is a coin factor for leukocytes (for example, monocytes, neutrophils, T lymphocytes, eosinophils, basophils, etc.).

As can be seen by Northern blot analysis, MPIF-1 is significantly expressed in hematopoietic source tissues.

MPIF-1 Therapeutic / Diagnostic Uses

MPIF-1 has been shown to play an important role in the regulation and inflammation of the immune response. As can be seen in Fig. 13, the lipopolysaccharide induces the expression of MPIF-1 from human monocytes. Thus, in response to the presence of endotoxin, MPIF-1 is expressed from monocytes, and thus MPIF-1 may be administered to control the immune response of the host. MPIF-1 can be used as an anti-inflammatory agent.

As shown in Fig. 4, the coinization activity of MPIF-1 on THP-1 cells (A) and PBMC (B) is remarkable. In addition, MPIF-1 induces a significant amount of calcium mobilization in THP-1 cells (FIG. 5), which implies that MPIF-1 possesses a biological effect on monocytes. MPIF-1 also exhibits dose-dependent chemotaxis and calcium mobilization in human monocytes.

The polypeptides of the present invention may also be useful in antitumor therapy because of the evidence that chemokine expressing cells injected into the tumor cause tumor shrinkage and can be used, for example, in the treatment of Kaposi's sarcoma. MPIF-1 induces the secretion of TNF-α, a cell known as a tumor suppressor, which can be used to induce tumor shrinkage. In addition, MPIF-1 can induce monocytes to secrete other tumor and cancer inhibitors, such as IL-6, IL-1 and G-CSF. In addition, MPIF-1 stimulates the invasion and activation of host defense (tumor-killing) cells, such as cytotoxic T cells and macrophages, by their coinizing activity and thus can be used to treat solid tumors.

Bone marrow protection

In addition, the polypeptide can be used to inhibit proliferation and differentiation of hematopoietic cells, and thus the bone marrow stem cells can be protected from chemotherapeutic agents during chemotherapy using the polypeptide. As illustrated in Figures 6 and 7, MPIF-1 inhibits colony formation by low proliferation potential colony-forming cells (LPP-CFC). As illustrated in FIG. 8, M-CIF specifically inhibits M-CSF-stimulated colony formation, whereas MPIF-1 does not. Since MPIF-1 specifically inhibits the growth and / or differentiation of bone marrow cells, this antiproliferative effect allows for the administration of larger amounts of chemotherapeutic agents, thus achieving a more effective chemotherapeutic effect .

The inhibitory effect of MPIF-1 polypeptides on subpopulations of sensitized progenitor cells (e.g., granulocytes, and macrophages / monocytes) can be used therapeutically to inhibit the proliferation of leukemia cells.

In addition, the present inventors have confirmed that MPIF-1 and its mutants (for example, MPIF-1? 23) inhibit proliferation and differentiation of human myeloid precursors and granulocyte precursors in vitro. Similarly, as can be seen from animal studies, for example, MPIF-1.DELTA.23 inhibits the development of hypoproliferative potential colony forming cells (LPP-CFC) and granulocyte / mononuclear cell sensitized progenitor cells both in vivo and in vitro Specific inhibition. These findings imply that the cytotoxic effect of commonly used chemotherapeutic agents has therapeutic utility as a chemoprotective agent capable of protecting early myeloid progenitor cells.

Because MPIF-1 and its variants have the ability to selectively inhibit myeloid progenitor cells, MPIF-1 can be used to treat myeloproliferative disorders such as clinically closely related thrombocytosis (ET ), Erythropoietic (PV) or unexplained myelogenous (AMM). Each disorder is due to acquired mutations in the stem cells that provide growth benefits to the next generation of single hematopoietic stem cells. The pathophysiology of these disorders is distinguished in the presence of overproduction of different cell types. In PV, there is an overproduction of red blood cells, granulocytes and megakaryocytes. In ET, overproduction of platelets and white blood cells naturally exists. In addition, AMM exhibits platelet hypertrophy or leukocytosis in addition to myelofibrosis.

Stabilization of the PV patient can be achieved by removing the red blood cells by a venous incision. However, there is no comparable treatment for increased platelet levels in ET patients. Several bone marrow suppression therapies have been studied to reduce the risk of platelet hypertrophy. Treatment with radioactive, hydroxyurea, alkylating agents (bisulfate and chlorambucil), interferon or anagrelide all show severe side effects. In particular, the use of each bone marrow suppression regimen, except anagrelide, increases the risk of acute leukemia. Anagrelide is a sure cure. However, the adverse reaction to anagrelide must be considered, and its potential chronic toxicity has not been determined. Currently, interferon is considered a second-line therapy, which may include cost, side effects, and the need for parenteral administration. These facts imply that there is a real need for the treatment of the disease.

In vivo studies in mice pretreated with MPIF-1? 23 and treated with 5-FU showed inhibition of platelet progenitor cell proliferation.

The invention also encompasses the use of MPIF-1 and variants thereof in combination with other bone marrow depression therapies and bone marrow depressants.

In Figures 9, 10 and 11, the cell lines of the used cell lines are removed and the resulting cell populations are contacted with M-CIF or MPIF-1. M-CIF reduced the Mac-1 positive population of cells by almost 50%, consistent with the results of FIG. 8 indicating that M-CIF induces inhibition of M-CSF reactive colony forming cells. As can be seen in FIG. 11, MPIF-1 inhibits the ability of sensitized progenitor cells to form colonies in response to IL-3, GM-CSF and M-CSF. Furthermore, as can be seen in Fig. 12, the dose response of MPIF-1 was found to inhibit colony formation. This inhibition is due to blocking of the differentiation signal mediated by these factors or blocking the cytotoxic effect on progenitor cells. In addition, as demonstrated in Examples 9 and 10, MPIF-1 possesses in vitro and in vivo bone marrow protective activity against cytotoxicity of chemotherapeutic agents. Thus, MPIF-1 may be useful as a bone marrow-protecting agent for patients undergoing chemotherapy.

As noted above, major complications due to chemotherapy and radiotherapy are destruction of non-pathological cell types. The present invention provides a method for protecting bone marrow from radiation and chemotherapeutic agents by inhibiting myeloid cell proliferation in an individual. These methods include administering a bone marrow-inhibiting amount of MPIF-1 alone or in combination with an agent selected from the group consisting of macrophage inflammatory protein-1 alpha (MIP-1 alpha), macrophage inflammatory protein- 2 alpha (MIP-2 alpha), platelet factor (MRP-2) selected from the group consisting of IL-4 (PF4), interleukin-8 (IL-8), macrophage coenzyme and activator (MACF) and macrophage inflammatory protein- ≪ / RTI > Therefore, the bone marrow-suppressing composition of the present invention provides a bone marrow-protecting effect, and can be usefully used together with therapies showing harmful effects on bone marrow cells. The reason for this is that the bone marrow inhibiting composition of the present invention can be used for the treatment of cancer by positioning the myeloid cells in a low cycle state, for example, using radioactivity using cell cycle activators (e.g., cytosine arabinoside, hydroxyurea, 5-Fu and Ara- Because it provides protection against cellular damage caused by therapy or chemotherapy. Once the chemotherapeutic agent is removed from the system of the individual, it can be used, for example, as a bone marrow stimulator (e.g., interleukin-11 (IL-11), erythropoietin (EPO), GM-CSF, G-CSF, It is desirable to promote rapid amplification and differentiation of MPPC-1-protected progenitor cells using Trombopoietin (Tpo).

The ability of MPIF-1 to protect bone marrow in vivo in the presence of a chemotherapeutic agent is illustrated in Example 13. As can be seen in Example 13, administration of MPIF-1 to an individual prior to administration of the chemotherapeutic agent accelerates the recovery of platelets in the blood even after multiple rounds of 5-Fu treatment. Also, as can be seen from the experiment described in Example 13, MPIF-1 treatment during multiple 5-Fu treatments restores granulocyte more rapidly. In addition, the results of Example 13 show synergistic effects when MPIF-1 and G-CSF are co-administered.

As described, the inventors have found that MPIF-1 and its variants exhibit potent in vivo inhibition of low proliferation potential colony-forming cells (LPP-CFC) derived from bone marrow. LPP-CFC is a positive hematopoietic progenitor cell producing granulocyte and monocyte lineage. In addition, MPIF-1 reversibly inhibits colony formation by human CD34 ⁺ stem cells derived from granulocyte and monocyte colony forming cells. The protection of these hematopoietic progenitor cells against the cytotoxic effects of 5-fluorouracil (5-Fu), cytosine arabinoside and Taxol (TM), as confirmed by the chemical protection experiments performed by the present inventors, -1? 23.

In the in vivo chemotherapeutic model, this results in faster recovery of both bone marrow progenitor cells and neutrophils and platelet peripheral cell populations, as can be seen from the use of the MPIF-1 variant (? 23). In addition, as can be seen in Examples 10 and 13, administration of MPIF-1 rapidly restores neutropenia and thrombocytopenia in experimental animals treated with 5-Fu. Thus, MPIF-1 and its variants can reduce the incidence of bone marrow dysplasia, granulocytopenia and thrombocytopenia associated with chemotherapeutic agents, thereby reducing infection in patients who perform treatment with such agents.

Accordingly, the present invention relates to a method of protecting myeloid progenitor cells and a method of promoting the recovery of platelets and granulocytes, which comprises a treatment which preferentially kills dividing cells (for example, radiation therapy or cell cycle activator Lt; RTI ID = 0.0 > MPIF-1 < / RTI > MPIF-1 provides in vivo bone marrow protection for therapeutic and therapeutic agents that are administered in sufficient amounts to preferentially kill dividing cells. As used herein, the term "administering MPIF-1" means administering a therapeutically effective amount of MPIF-1, an analog of MPIF-1, or a combination thereof. The administration route of MPIF-1 will be described later.

MPIF-1 may be administered before, during or after treatment to preferentially kill dividing cells. In a preferred embodiment, MPIF-1 is administered prior to performing radiotherapy or before administration of a cytostatic agent, and MPIF-1 inhibits the proliferation of myeloid cells for a sufficient time. The present invention is also used to protect bone marrow cells during multiple courses of treatment that preferentially kill dividing cells. In this case, MPIF-1 may be administered single or multiple doses at different times during treatment or treatment.

As described above, MPIF-1 may be used alone or in combination with one or more bone marrow stimulants. Bone marrow stimulators are used in the art to stimulate the proliferation of the bone marrow cells after bone marrow cells are attenuated in an individual undergoing radiotherapy or treatment with a cell cycle activator (see, e.g., Kannan, V. et al. Et al. , Int. J. Radiat. Oncol. Biol. Phys . 37: 1005-1010 (1997); Engelhard, M. et al., Bone Marrow Transplant 19: 529-537 (1997); Vadhan-Raj, S. et al., Ann Intern Med . 126: 673-681 (1997); Harker, L. et al., Blood 89: 155-165 (1997); Basser, R. et al., Lancet 348: 1279-1281 (1996); Grossman, A. et al., Blood 88: 3363-3370 (1996); Gordon, M. et al., Blood 87: 3615-3624 (1996)]. For example, MPIF-1 may be administered prior to treatment to kill dividing cells, and one or more bone marrow stimulants may be administered during or after such treatment. In this case, MPIF-1 will protect the myeloid cells from the treatment, followed by administration of the bone marrow stimulator (s) will cause expansion of the protected myeloid cell population.

Bone marrow stimulants are generally administered to a patient to be treated with a therapeutically effective amount of a chemotherapeutic agent. The dosage form and mode of administration will depend on a variety of factors, including the subject to be treated, the condition of the cell to be stimulated, the treatment step in chemotherapy, and the bone marrow stimulator (s) used. For example, GM-GSF and G-CSF are therapeutically effective at doses of about 1 μg / kg body weight and 5 to 10 μg / kg body weight, respectively, and can be administered daily by subcutaneous injection For example, Kannan, V. et al. , Int. J. Radiat. Oncol. Biol. Phys. 37: 1005-1010 (1997); Engelhard, M. et al., Bone Marrow Transplant 19: 529-537 (1997); Sniecinski, I. et al., Blood 89: 1521-1528 (1997)]. IL-11 can be administered by subcutaneous infusion at a dose of less than 100 [mu] g / kg body weight daily (Gordon, M. et al., Supra). However, doses of less than 10 [mu] g / kg body weight are also believed to be effective in reducing chemotherapy-induced thrombocytopenia. Tpo can be administered by intravenous injection at doses of 0.3 to 2.5 [mu] g / kg body weight (Vadhan-Raj, S. et al., Ann Intern Med. 126: 673-681 (1997); Harker, L. et al., Blood 89: 155-165 (1997)]. As will be appreciated by those skilled in the art, the optimal dosage formulation and route of administration will depend upon a number of factors, including a variety of factors, as described above. Administration formulations and routes of administration for additional bone marrow stimulants are well known in the art.

Also, the time of administration of the bone marrow stimulator as part of a treatment protocol involving a treatment to preferentially kill dividing cells will depend on various factors as described above for the dosage form and route of administration. There have been many reports on administering bone marrow stimulants to individuals as part of a therapeutic protocol involving radioactive therapy or cell cycle activators. For example, the literature (Vadhan-Raj, S. et al., Supra) describes the use of a single intravenous dose of Tpo three weeks prior to administration of a chemotherapeutic agent. (Papadimitrou, C. et al., Cancer 79: 2391-2395 (1997) and Whitehead, R. et al. , J. Clin. Oncol. 15: 2414-2419 (1997)) describe chemotherapeutic methods involving administering chemotherapeutic agents for several weeks. In each of these cases, G-CSF is administered with the chemotherapeutic agent at multiple points between the first day of treatment and the last day of treatment. Similar uses for both IL-11 and GM-CSF have been reported in Gordon, M. et al., Supra and Michael, M. et al. , Am. J. Clin. Oncol. 20: 259-262 (1997) However, as will be appreciated by those skilled in the art, the optimal time for administering the bone marrow stimulant will depend on the particular bone marrow stimulant used and the condition in which they are administered.

Thus, administration of a bone marrow stimulator to mitigate the cytotoxic effect of a preferential killing of dividing cells on the bone marrow cells is well known in the art. Bone marrow stimulators can be administered by several routes, such as intravenous and subcutaneous injection. The concentration of the bone marrow stimulating agent to be administered varies depending on various factors, but is generally 0.1 to 100 占 퐂 / kg body weight, and may be administered in a single dose or at multiple points during the course of chemotherapy or radiotherapy. However, the bone marrow stimulating agent is generally administered before or after administration of the chemotherapeutic agent, or before or after performing the radioactive treatment. As will be appreciated by those skilled in the art, the condition in which the bone marrow stimulator is used will depend upon the particular bone marrow stimulator and treatment.

As will be appreciated by those skilled in the art, MPIF-1 can generally be used to enhance the efficiency of hematopoietic growth factors as described above. An example of such a hematopoietic growth factor is erythropoietin, which increases the number of all the blood cell types by stimulating the production of IL-3, a multi-line growth factor that stimulates red blood cells and more primitive stem cells. Other factors include stem cell factor; GM-CSF; And hybrid molecules of G-CSF and erythropoietin; IL-3 and SGF; And GM-CSF and G-CSF.

In addition, the myelosuppressive pharmaceutical composition of the present invention is useful for the treatment of leukemia causing hyperproliferative myeloid cell conditions. Accordingly, the present invention provides a method of treating leukemia comprising administering to a mammal together with one or more chemokines selected from the group consisting of MIP-1 alpha, MIP-2 alpha, PF4, IL-8, MCAF and MRP- Or a bone marrow-suppressing amount of MPIF-I alone to a patient with leukemia.

As used herein, the term " inhibiting myeloid cell proliferation " means reducing cell proliferation in the bone marrow cells and / or increasing the bone marrow cell fraction in the low period. As mentioned above, " individual " means a mammal, preferably a human. Pre-incubation of acetonitrile (ACN) with the myelosuppressive composition of the present invention significantly enhances the specific activity of these chemokines for inhibition of myeloid progenitor cells. Thus, prior to administration, see Broxmeyer HE et al., Ann-Hematol. 71 (5): 235-46 (1995) and PCT Publication WO94 / 13321; Quot ;, incorporated herein by reference in its entirety), it is preferred that the bone marrow inhibiting composition of the present invention is pre-incubated with ACN.

The bone marrow inhibiting composition of the present invention can be used in combination with various chemotherapeutic agents. Examples of these chemotherapeutic agents include alkylating agents such as nitrogen mustard, ethyleneimine, methylmelamine, alkylsulfonate, nitroso urea and triazene; Antimetabolites such as folic acid analogs, pyrimidine analogs, especially fluorouracil and cytosine arabinoside, and purine analogs; Natural products such as vinca alkaloids, epi-vinculotoxins, antibiotics, enzymes and biological reaction modifiers; And miscellaneous products such as platinum coordination complexes, anthracenedione, substituted ureas such as hydroxyurea, methylhydrazine derivatives and adrenocorticoid inhibitors.

The chemotherapeutic agent may be administered at known concentrations according to known techniques. The bone marrow depression composition of the present invention may be administered simultaneously with or simultaneously with the chemotherapeutic agent before or after administration of the chemotherapeutic agent.

Certain chemokines such as MIP-1 [beta], MIP-2 [beta] and GRO- [alpha] can inhibit (at least partially block) the bone marrow suppressive effect of the bone marrow inhibiting composition of the present invention. Thus, in a further embodiment, the present invention provides a method of inhibiting bone marrow suppression comprising administering to a mammal an effective amount of a compound of formula I or a pharmaceutically acceptable salt thereof, alone or in combination with one or more of MIP-1 alpha, MIP-2 alpha, PF4, IL- Comprising administering to said mammal an effective amount of an anti-bone marrow inhibitor selected from the group consisting of MIP-1 [beta], MIP-2 [beta] and GRO-alpha prior to exposure with MCAF, 8, MCAF and MRP-2.

Protection of damage induced by cytotoxic agents

In addition, the polypeptides of the present invention can be used to reduce or prevent cytotoxic agent induced damage / injury to cells, tissues and organs.

Damage to normal tissue occurs as a result of exposure to cytotoxic agents. Cytotoxic agents include radiation and chemotherapeutic agents. Radiation may be incidental radiation, environmental radiation, occupational radiation, diagnostic radiation, and therapeutic radiation. In addition, normal tissue injury is a common side effect of cancer therapy, such as radiation therapy, chemotherapy, and the combination of radiotherapy and chemotherapy. Such normal tissue damage is commonly referred to as normal tissue "effect", tissue "toxicity", "morbid state", "complication" and tissue "reaction" (acute, subacute or late).

Accordingly, the present invention provides compositions and methods for treating and preventing normal tissue damage caused by radiation, radiation therapy, chemotherapy and other cytotoxic agents.

Normal tissue toxicity, particularly acute toxicity (ie, occurring within a few days or weeks of treatment), causes pain and causes additional complications such as infection. In addition, acute toxicity assures effective cancer treatment by limiting the dose of the cancer therapeutic. In addition, if the initial toxic effects are asymptomatic (ie, they do not cause morbidity and are not dose-limiting), they may have a late (also known as chronic) effect (ie, months or years after treatment) . Late effects include, for example, sterility, late onset necrosis and fibrosis, and mitotic enhancer induced tumors.

The present invention provides compositions and methods for treating and preventing acute, subacute, and late normal tissue toxicity.

Several pathways have been implicated in cytotoxic agent-induced damage to normal cells and normal tissues induced, for example, by radiation therapy and chemotherapy. Damage can be direct or indirect. Direct damage is caused by the action of cytotoxic agents on cellular components, including damage to cell membranes and other cellular components caused by breakage of the DNA chain, breakage of the DNA chain, and free radicals and reactive oxygen species in the chromosome do.

Polypeptides and polynucleotides of the invention prevent and treat cytotoxic agent-induced damage to cells, tissues and organs by modulating the pathway of one or more of the direct pathways and the indirect pathways described above.

Damage is the loss of potential mitotic cells (known as stem cell models); Vascular disorders that cause hypoxia and other effects; Normal host repair reactions, such as Jun and induction of early gene, such as EGR1, interleukins and induction of proinflammatory cytokines such as TNF, TGFβ, PDGF, induction of inflammatory cytokines such as BFGF and second cytokine cascade (s ); The effects of inflammatory reactions; May be due to interactions between multiple cell types, such as inflammatory cells, stromal cells and fibroblasts.

Polypeptides and polynucleotides of the invention modulate one or more of the causes of the damage described above.

Fibrosis can be induced in one or more ways: Monocytes and macrophages present in the irradiated tissue will induce the production of infectious cytokines, thereby invoking additional macrophages to the inflammatory response; The initial loss of epithelial cells and stromal cells induces inflammation; The irradiation induces the expression of fibrogenic inducible cytokines through the induction of AP-1.

Polypeptides and polynucleotides of the invention modulate the pathway of one or more of the pathways leading to fibrosis.

Cells respond to radiation and other cytotoxic agents in several ways: the formation of ceramides from the membrane sphingomyelin that activates the JNK pathway leading to apoptosis, which is known by the formation of dead bodies; Death induced by other pathways; Mitotic linked deaths identified by the formation of micronuclei (MN); And cytotoxin-induced aging in which cells can not metabolically activate or divide.

Polypeptides and polynucleotides of the invention modulate one or more of these cellular responses to radiation and other cytotoxic agents.

Formulations such as polypeptides of the invention that modulate normal tissue toxicity are referred to as chemical modifiers, toxic protectants, protectants, cytoprotectants, and structurants. These agents are useful for reducing or preventing side effects of cancer treatment and are useful for preventing or treating tissue damage due to radiation exposure.

The term " cytotoxic agent " as used herein means a chemotherapeutic agent (also referred to as antitumor agent) and radiation, wherein the radiation is incidental radiation, occupational radiation, environmental radiation, therapeutic radiation such as fractional radiation therapy, Quot; includes < / RTI > the combined use of chemotherapy and radiation therapy. In addition, forms of irradiation include ionization (gamma ray) irradiation, particle irradiation, low energy transmission (LET), high energy transmission (HET), ultraviolet irradiation, infrared irradiation, visible light and light sensitization. Cytotoxic agents include agents that preferentially kill tumor cells or disrupt the cell cycle of rapidly proliferating cells and also include agents that interfere with or reduce the growth of tumor cells. In addition, chemotherapeutic agents are known as antitumor agents and are known in the art. The term chemotherapy as used in the sentence is meant to include treatment using a single chemotherapeutic agent or a combination of therapeutic agents. In patients requiring treatment, chemotherapy may be combined with surgical or radiotherapy and may be combined with other anti-tumor therapies.

The irradiation can also be performed by ionizing radiation, such as high energy radiation, for example, X-ray or gamma radiation, high energy transfer irradiation, alpha radiation, beta radiation, neutron beam, An accelerating electron beam and an ultraviolet ray. The irradiation also includes photons and fission spectral neutrons.

Polypeptides and polynucleotides of the invention protect normal cells and tissues from the effects of cytotoxic agents such as radiation and chemotherapeutic agents described herein.

Examples of chemotherapeutic agents include vinca alkaloids, epinephrinepotoxin, anthracycline antibiotics, actinomycin D, flicamycin, puromycin, graminidin D, paclitaxel (registered trademark, Bristol Myers Squibb), colchicine, (Or " mAMSA ") can be mentioned. The vinca alkaloids are described in Goodman and Gilman ' s Pharmacological Basis of Therapeutics (7th edition), pp. 1277-1280 1985). Examples of vinca alkaloids include vincristine, vinblastine and vindesine. Epi-grape pilotoxins are described in Goodman and Gilman ' s Pharmacological Basis of Therapeutics (7th ed., Pp 1280-1281 (1985). Examples of epi-grape pilotoxins include ethoposide, etoposide orthoquinone, and tenofoside. Anthracycline antibiotics are described in Goodman & d Gilman ' s The Pharmacological Basis of Therapeutics (7th ed.), pp. 1283-1285 (1985)] Examples of anthracycline antibiotics include daunorubicin, doxorubicin, mitoxantrone and bisanthene. Actinomycin D, also referred to as dactinomycin, is described in Goodman and Gilman's The Pharmacological Basis of Therapeutics (7th edition), pp. 1281-1283 (1985). Flicamycin, also called mitramycin, [see:. Goodman and Gilman's the Pharmacological Basis of therapeutics (7 edition), pp 1287-1288 (1985)] has been described as a chemotherapeutic agent other than the cisplatin (Playa tinol (R), Bristol-Myers Squibb) (Carrageenan), carboplatin (Paraplatin®, Bristol Myers Squibb), mitomycin (mutamycin®, Bristol Myers Squibb), altretamine (hexalene®, US Bioscience, (BCNU) (BiCNU (registered trademark)), cyclophosphamide (cytoxan (registered trademark), Bristol Myers Squibb), rosmutin (CCNU) (CeeNU (registered trademark), Bristol Myers Squibb) Trademark), Bristol Myers Squibb).

Additional therapeutic agents that may be administered with the polypeptides and polynucleotides of the present invention include, but are not limited to, acculinomycin A, aclauricin, acronin, acornisine, adriamycin, aldelucin (interleukin- Aminoglutethimide (cytarndrene), aminoimidazolecarboxamide, amsacrine (m-AMSA; amcinidin), anastrazole (aryimidex), anthracitabine , Anthracycline, anthramycin, asparaginase (Elsfar), azacitidine, azacitidine (ladakamycin), azaguanine, azaserine, azaziridine, 1,1 ', 1 " (2 ', 3'; 3,4) pyrrole (1,2-a) indole-4,7-dione, BCG (terracis), BCNU, BCNU chloroethylnitrosourea, Benzamide, 4- (bis (2-chloroethyl) amino) benzenebutanoic acid, bicalutamide, bischloroethyl, nitrosourea, Carboxymethyl ester, carboplatin, carboplatin (paraplatin), carbohydrate (carbohydrate), carbohydrate (carbohydrate), carbohydrate (BCNU; BiCNU), chlorambucil (Luceran), chloroethylnitrosourea, chlorochomotocin (DCNU), cholomycin A3, cis-retinoic acid, cisplastin ; Platinol), cladribine (2-chlorodeoxyadenosine 2cda; lustatin), coformin, cyclo- leucine, cyclophosphamide, anhydrous cyclophosphamide, chlorambucil, cytarabine, But are not limited to, ribavirin HCl (cytosar-u), 2-deoxy-2 - ((methylnitrosoamino) carbonyl) amino) -D- glucose, dacarbazine, dactinomycin Noruvine HCl (cerubidine), decarbazine, decarbazine (DITC-dome), demecoxin, dexamethasone, dianhydrogalactose , Doxorubicin hydrochloride, eflornithine, estramustine, estramustine phosphate sodium (emcyt), diethoxylthioproxen (etoposide) (Fudr), fludarabine (fludarra), fluoro (etoposide), etoposide (VP16-213), fenretinide, (5-FU), fluoxymasterone (halothestine), flutamide, flutamide (urexin), flux uridine, gallium nitrate (GRANITE) , 2-deoxy-2- (3-methyl-3-nitroso listido) -D-glucopyranose, goserelin (Zolandex), hexestrol, hydroxyurea (hydra), adarbicin Isomycin), iphospazemcitabine, iphosphamide (iflex), mesna holding ipsho Amide (MAID), interferon, interferon alfa, interferon alfa-2a, alpha-2b, alpha-n3, interleukin-2, iobenzuan, ioventuanibenzuan, irinotecan (camptothear), isotretinoin ), Ketoconazole, 4- (bis (2-chloroethyl) amino) -L-phenylalanine, L-serine diazoacetate, lentian, lucophorin, looprolide acetate (LHRH-analog), levamisole ), Romestin (CCNU; (maleic anhydride), cine-NU, mannormustine, maytansine, mechlorethamine, mechlorethamine HCl (nitrogen mustard), medroxyprogesterone acetate (probera, deproporbera), megestrol acetate (Methionine), methanesulfonic acid, ethyl esters, methotrexate (mtx), methionine Methotrexate), methyl-cnu, mimosin, microdizole, mitramycin, mitanthrone, mitobronitol, mitoguazone, mitotol, mitomycin (mitamycin), mitomycin C, N-bis (2-chloroethyl) tetrahydro-2H-1,3,2-oxazaphosphorine (hereinafter referred to as "-DDD; lysodrene), mitogantrone, mitoxantrone HCl (2-chloroethyl) amine, N-methyl-bis (2-chloroethyl) (Doxorubicin), paclitaxel, parkramycin, fasaglutamate (nilandrolone), nilustin, nitracrin, nitrogen mustard, nocodazole, nogalamycin, octreotide (PEG _X- 1), pentostatin (2'-deoxycoformylmine), perfluoromycin, pepticemio, photophoresis, picamycin (mitracin), fisivanil, Puromycin HCl (Materlan), propidium, puromycin, puromycin amino nucleoside, PUVA (prolaren + ultraviolet a ), Pyran copolymers, rapamycin, s-azacytidine, 2,4,6-tris (1-anilidinyl) -s-triazine, taxol, promodicin, sylolimus, Zanthoxyl, zanthoxyl, zanosar), suramin, taxosyl pentacyrate (novadex), taxon, tegafur, tennifoside (VM-26; (Thiophene), thiolone, topotecan, tretinoin (vesanoid), triazicone, tricorderin, triethylene glycol diglycidyl ether, triethyleneglycol diglycidyl ether, (1-aziridinyl) phosphine oxide, tris (1-aziridinyl) phosphine sulfide, triethanolamine, triethyleneterephosphamide, triethylenetriphosphoramide, (Aziridinyl) phosphine sulfide, uracil mustard, vidarabine, vidarabine phosphate, vinblastine, vinblastine sulfate (berbane), vincristine sulfate (2-chloroethyl) -3- (4-methylcyclohexyl) -1-nitroso urea (1-nitrobenzyl) , (8S-cis) -10 - ((3-amino-2,3,6-trideoxy-alpha-L-ribo-hexopyranosyl) ) -7,8,9,10-tetrahydro-6,8,11-trihydroxy-8- (hydroxyacetyl) -1-methoxy-5,12-naphthacenedione, 131-metha- Benzylguanidine (I-131 MIBG), 5- (3,3-dimethyl-1-triazenyl) -1H- imidazole- 4 (1H, 3H) -pyrimidinedione, 2,4,6-tris (1-aziridinyl) -s- thiazine, 2,3,5-tris (1-aziridinyl) N, N-bis (2-chloroethyl) tetrahydro-2H-1,3,2-cyclohexadiene-1,4-dione, 3-iodobenzylguanidine, 5,12-naphthacenedione, 5-azacytidine, 5-fluorouracil, (1aS, 8S, 8aR, 8bS) -6-Amino-8 - (((aminocarbonyl) oxy) methyl) -1,a, 2,8,8a, 8b-hexahydro-8a-methoxy- (2 ', 3': 3,4) pyrrolo (1,2-a) indole-4,7-dione, 6-azauridine, 6-mercaptopurine, .

Preferred therapeutic and compounding agents that may be co-administered with the polynucleotides and polypeptides of the present invention include doxorubicin and docked laundry cells, topotecan, paclitaxel, carboplatin and Taxol, taxol, cisplatin and radiation, 5- FU), 5-FU and radiation, toxotheter, fludarabine, ara C, etoposide, vincristine and vinblastine.

Examples of chemotherapeutic agents also include dock laundry cells (toxotere (R)) and topotecan (hycartin (R)). Additional chemotherapeutic agents and other cytotoxic agents include those provided in the " pharmaceutical compositions " described below.

Additional chemotherapeutic agents and other cytotoxic agents include those described in the " epitopes and antibodies " and other items discussed below and those well known in the art.

The polynucleotides and polypeptides of the present invention can be used for the prevention and / or treatment of radiation damage.

The polynucleotides and polypeptides of the present invention, which are administered prophylactically or therapeutically to protect cells, tissues and organs against low, medium and high dose radiation, protect human or animal populations and populations from damage due to radiation exposure . Such impairments include, but are not limited to, gastrointestinal disorders, weight loss, radiation pain, radiation burns, endocrine disorders, goiters, eye diseases such as dry eye syndrome, inflammatory diseases, mental illnesses, respiratory diseases, genitourinary disorders, As well as other diseases well known in the art. Damage due to exposure to radiation includes damage to cells and tissues as described above, damage of cellular DNA, destruction of cellular functions due to destruction of DNA function, cell necrosis, treatment induced second tumor and other cancer induction And cancer induction.

The proliferation of nuclear weapons and nuclear tests are on the increase in many parts of the world.

In addition, the development of industries such as the use of nuclear power, the treatment of nuclear fuels and the decomposition of nuclear weapons have increased the risk of exposure to radiation. For example, the past four decades have seen four major industrial radiation scales, such as the Kiss Team (USSR) and Wind-Scale (UK) in 1957, Tri Mile Island (USA) in 1979, and Chernobyl (USSR) An accident was reported. Radiation leaks due to those accidents caused extensive exposure of radiation at various concentrations. The accident at the nuclear plant in Chernobyl severely affected residents and livestock in and around the area.

In the early 1950's, cysteamine and related aminoalkyl thiols were found to protect organisms from radiation. Particularly, when such a substance is administered to a mouse before exposure to the X-ray, the substance reduces the lethal effect of the X-ray irradiation. Studies have been conducted to develop better radiation protection agents. For example, aminostatin and other aminoalkyl dihydrogenphosphorothioates (US 3,892,824) have been developed first as protectants for use against X-ray or nuclear radiation, which may be exposed, especially during military conflicts. The most anticipated is amipostin (WR2721 (S-2 (3-aminopropylamino) -ethylproporothioic acid), which is broken down into aminoalkyl thiols in the body and its effect is similar to cysteamine. However, the use of WR2721 has been limited due to poor clinical tolerance [Cairnie, Radiation Res. 94 : 221 (1983); Turrisi et al., In Radioprotetors and Anticarcinogens, Nygaard and Simic, Eds., Academic Press, New York, p. 681-694 (1983); Blumberg, Int. J. Radiation Oncology Biol. Phys. 8 : 561 (1982)].

The present invention provides a method of protecting radiation-induced damage, which can be applied before and / or concurrently with, and / or after radiation administration, since this method is effective at relatively low concentrations of low toxicity, Lt; / RTI > and can also be administered in a single dose as well as in multiple doses.

As described above, the polypeptides and polynucleotides of the present invention protect, alleviate, and treat cells, tissues and organs from damage caused by radiation or other cytotoxic agents, and increase the survival rate of individuals exposed to cytotoxic agents such as radiation. Polypeptides and polynucleotides of the present invention administered before, during and / or after exposure reduce or reduce the degree of radiation damage.

Radiation exposure can be the result of, for example, accidental exposure, intentional exposure, internal exposure, external exposure, occupational exposure, environmental exposure, radon, nuclear contamination, And the like. Such exposure may occur, for example, to workers in the nuclear power industry, military personnel, civilian personnel, emergency personnel, nuclear explosion and nuclear accident survivors, health care workers, patients and space navigators. The polynucleotides and polypeptides of the present invention may also be used to treat or protect against emergency personnel, civilians, or military personnel, or from other sources such as those that may occur to astronauts.

That is, the nucleotides and polypeptides of the present invention may be administered for prophylactic and / or therapeutic purposes in preventing, alleviating or treating sunburn caused by radiation exposure.

The present invention provides a method of protecting or treating an individual against damage, including administering to the individual an effective amount of the polypeptide or polynucleotide of the present invention. Polypeptides and polynucleotides of the present invention can be administered before, during and / or after exposure to radiation as a protective agent against radiation damage. As described above and below, protective agents such as the polypeptides and polynucleotides of the present invention are useful in preventive and therapeutic treatments.

The polypeptides and polynucleotides of the present invention may be used in a variety of applications including, but not limited to, nuclear industry workers, soldiers, storm survivors, medical personnel involved in radiology-related diagnostics and therapies, or individuals susceptible to radiation, such as patients exposed to diagnostic or therapeutic radiation It is useful for preventive and therapeutic treatment. As disclosed herein, the polypeptides and polynucleotides of the present invention are also useful as adjuvants in the radiation treatment of cancer wherein the polypeptides and polynucleotides are capable of selectively destroying cancer tissues by radiation therapy, do.

Thus, the polypeptides and polynucleotides of the present invention can be administered for prophylactic or therapeutic purposes in individuals at risk of radiation exposure. Prevention or alleviation of possible radiation damage in the case of preventive purposes, treatment or alleviation of the damage occurring for therapeutic purposes and slowing down, alleviating or preventing further damage.

Effective amounts should be understood as the amounts of the polypeptides and polynucleotides of the invention sufficient to achieve the desired effect. Such effects may be preventative or therapeutic effects or both. The effective amount depends on various factors such as the type and amount of radiation exposed to the subject, the method of administration, etc., which are known in the art or described later. For example, dosages are as set forth herein and throughout this specification.

The polypeptides of the present invention have been shown to protect the gastrointestinal tract during treatment with chemotherapeutic agents and radiation (see Examples 16 to 18). In addition, the polypeptides of the present invention have been shown to reduce radiation-induced damage in the gastrointestinal tract when administered post-irradiation (see Examples 16-18).

That is, the polypeptide of the present invention can be used to protect epithelial cells. The term " epithelium " is a term encompassing the endothelium and epithelium of the body including the inner membrane of blood vessels and other small intestines. It consists of cells bound by a small amount of binding material. The epithelium is divided into types based on the number of layers, depth, and surface cell morphology. Epithelial cells of the epithelium of the cornea, epithelium of the barrette, epithelium of the ciliate, epithelium of the ciliate, epithelium of the cornea, epithelium of the cube, epithelium of the ciliated epithelium, enamel epithelium, caustic epithelium, epithelium, gingival epithelium, The epithelium of the lens epithelium, mesenchymal epithelium, olfactory epithelium, pustular epithelium, pigment epithelium, protective epithelium, multiple epithelium, vertebral epithelium, respiratory epithelium, hepatic epithelium, rectal tube epithelium, sensory epithelium, monolayer epithelium, squamous epithelium, Epithelium, mature epithelium, mosaic epithelium, transition epithelium, and epithelium of the eye, tongue, gland, oral mucosa, duodenum, ileum, plant, cecum, nasal passages, esophagus, tubules, There are cells.

"Line" is a generic term for cells with special functions that secrete or release substances that are not related to the need for normal metabolism. Examples of lines that may include eukaryotic cells include, but are not limited to, absorption lines, barriers, phallocytes, acid glands, parotid gland, adrenal gland, A biliary tract, a biliary tract, a blood vessel, a blood vessel, and a blood vessel, and a blood vessel line, an arterial line, an arterial line, a tear line, an asiali line, an abian senna line, The main line is the bow line, the upper arm line, the bronchial line, the bruch line, the brunner line, the narrow line, the corpus callosum line, the paranchymal line, the carotid artery line, the abdominal line, the ear wax line, the cervical line, Ancillary lines, cholecal vessels, kobelin lines, coccyx lines, coiled lines, composite lines, spheroids, conjunctival lines, cooper lines, skin lines, cell glands, endocrine glands, duodenal lines, duburn lines, Gastric, endocrine, intraepithelial, esophageal, exocrine, exocrine glands, gastric, gastric, hypogastric, Glacier line, Glacier line, Glaine line, Glacier line, Dune merchant line, Improvement of sludge, Gergen line, Throat line, Haller line, Haul line, Haas line, Pleasure line, Vessel line, Vascular lymphatic line, Hematopoietic line, Hemolymph line, , Trunk, heterozygous gland, hibernating gland, exocrine gland and endocrine gland.

Another example of the line is the carotid artery trunk line, median line, scapular trunk line, epilepsy line, intestinal line, intraepithelial line, dorsal extension line, jugular line, crowus line, laryngoscope, lacrimal line, Lens line, Lens line, Lieber queen line, Snow line, Legend line, Litet line, Lucicus line, lymphatic line, parotid lymph node, narrow line, wired line, submandibular line, Mandu line, Meliss line, Mybal line, partial gland line, Mucous glands, mucous glands, mucus glands, mucous glands, mucinous mucus, duodenal mucus, eustachian tubes, mucinous glands, multicellular glands, uterus, mucosa, Parotid gland, parathyroid gland, stanchion line, parotid gland, parotid gland, thoracic gland, stomach line, stomach line, There are hypocrisy, one line, fire line, pharynx, Philip line, pineal gland and sewer.

Another example of a line is a polygonal line, a multilayer line, a line, a pregnancy line, a legend bone line, a foreskin line, a prostate line, a pubic line, a Yongun line, a dam line, a Husserin line, , An abdominal salivary gland, an external salivary gland, an internal salivary gland, a zander line, a Schiller line, an sebaceous gland, a conjunctival gland, a first gland, The paranasal sinuses, the paranasal sinuses, the paranasal sinuses, the sinuses, the sinuses, the synovial lines, the target lines, the tile lines, the thymus, the thyroid, the parathyroid, the larynx, the tracheal line , Tubular line, supernumerary line, high solid line, tyson line, unicellular line, yodon line, female yodon line, meridian line, uterine line, ovoid line, vine line, Daejeon Choonsoon line, Sound line, foot tire line, Weber line, ball flight line, Zais line, and Jukker line.

Other examples of lines include albumin line, agminate line, auditory line, auxiliary line, urethral line, esophageal line, Yusataki line, sphero line, Galley line, narrow line, harbor line, , Marine-lymphoid line, master line, maxillary line, mery line, eye line, checkerboard line, tracheal line, phleuse line, semi-edge line, injured line, Tereson Line, Tieder Bay Line, Coronal Alveolar Line, Tacoma Line, Outer Sound Line, Wassmann Line, Webper Line and Woolfer Line.

That is, the MPIF-1 of the present invention can be used to protect either cells or cells in such lines.

MPIF-1 is an endothelial cell such as scaly epithelial cells including myocardial cells, myeloid cells and smooth muscle cells, endothelial cells, cuboidal epithelial cells and columnar epithelial cells, neuronal cells such as neurons and neuroglial cells Can be used to protect or alleviate damage induced by cytotoxic agents. MPIF-1 can also be used to protect or alleviate cytotoxic agent-induced damage to muscle tissue, nervous tissue, epithelial tissue, endothelial tissue and connective tissue.

MPIF-1 can also be used to protect or alleviate cytotoxic agent-induced damage to split cells, non-dividing cells, terminal differentiated cells, pluripotent stem cells, antigen-sensitized progenitor cells and non-antigen-expressing stem cells.

That is, MPIF-1 is a neurotrophic factor, such as neurons, including neurons including Schizophrenic neurons, internal neurons, central functional neurons, peripheral functional neurons, and bipolar neurons, Schwann cells, It can be used to treat damage to nervous system cells.

Endocrine and endocrine-related cells can also be treated or protected from cytotoxic agents using MPIF-1. Examples of such cells are: epithelial cells, sewage somatic cells, glial cells, non-granulocyte- Pituitary gland cells including granular callus pigment cells (eosinophils and basophils); Adrenal gland cells including epinephrine-secreting cells, non-epinephrine-secreting cells, water-quality cells, cortical cells (glomerular cells, fiber bundles and reticular cells); Thyroid gland cells including epithelial cells (main lobes and brachial leaves); Parathyroid cells including epithelial cells (chief cells and eosinophilic cells); Pancreatic cells including Langerhans islet cells (alpha, beta and delta cells); Pineal cells including parenchymal cells and glial cells; Thymocytes including brachypterous cells; Testicular cells, including vaginal epithelial cells, epithelial cells, stromal cells (" ladig cell "), mature cells, chimeric cells (primary and secondary), neonatal cells, sperm, Sertoli cells and muscle cells; Ovarian cells including oocytes, horn cells, oocyte cells, granulosa cells, intestinal cells (internal and external), epithelial cells and vesicular cells (primitive cells, vesicular cells, mature cells and occluded cells).

MPIF-1 can be used to treat cytotoxic agent-induced damage of muscle cells such as myofibrils, myofascial fibers and myofascial fibers. MPIF-1 can be used to treat cytotoxic agent-induced damage of skeletal cells such as osteoblasts, bone cells, osteoclasts and their progenitor cells.

Circulatory cells can also be treated or protected from cytotoxic agents by using MPIF-1, which includes cardiac cells (myocardial cells); Erythropoietin, stromal cells, erythrocytes, leukocytes (Hoeiocin, basophils, neutrophils (granular cells) and lymphocytes and mononuclear cells (granulosa cells)), platelets, tissue giant cells Lymphocytes (cytotoxic T cells, helper T cells and suppressor T cells, etc.), gigantic subtypes, monoepithelial cells, myeloid cells, lymphoid cells, lymphoid cells, (Eg, bone marrow aspirate, fragmented bone marrow, and polymorphonuclear granulocyte, etc.), the late phase, And include blood cells and lymph, including reticulocytes, reticulocytes, and bone marrow cells.

Respiratory cells such as capillary endothelial cells and alveolar cells may also be treated with MPIF-1 to reduce or prevent cytotoxic agent induced damage. Neuronal cells such as nephrons, capillary endothelial cells, granulocytes, tubular endothelial cells and podocytes can also be treated or protected. In addition, digestive tract cells such as simple coronary epithelial cells, mucous cells, follicular cells, wall cells, chief cells, enzyme source cells, digestive cells, intestinal chromaffin cells, goblet cells, -1. ≪ / RTI > Auditory cells (parent cells); Horny cells including olfactory receptive cells and tubular epithelial cells; Equilibrium / vestibular organ cells including parent cells and supporting cells; Sensory cells, including pigment cells, epithelial cells, impregnated neurons (rod-shaped and horned), ganglion cells, amacrine cells, bipolar cells and horizontal cells, can also be treated with MPIF-1 to prevent or alleviate cytotoxic damage .

In addition, cells of the mesenchymal stem cells, stromal cells, parent cells / hair follicles, adipocytes, simple epithelial tissues (pharyngeal epithelium, cuboidal epithelium, columnar epithelium, ciliary epithelium and pseudo-layered ciliated tubular epithelium) Cells of glioblastoma, mesenchymal endothelial cells, endothelial cells of the small intestine, endothelial cells of the large intestine, vasculature capillaries (eosinophilic granulosa cells) Endothelial cells of the microvessel structure, endothelial cells of the arterial artery, endothelial cells of the small arteries, endothelial cells of the vein, endothelial cells of the alveolar vessels, and endothelial cells of the bladder were also injected into MPIF-1 Can be processed.

MPIF-1 also prevents and treats cytotoxic damage in the dermis and other loose connective tissue, dense fibrous connective tissue, elastic connective tissue, reticular connective tissue, and connective tissue. Cells of connective tissue that can be protected and treated with MPIF-1 include chondrocytes, adipocytes, periosteum cells, bone cells, ancestral cells, osteoblasts, bone cells, and osteoclasts.

MPIF-1 also protects endothelial cells, hepatocytes, keratinocytes and basal keratinocytes, muscle cells, central nervous system and peripheral nervous system cells, prostate cells and lung cells.

MPIF-1 will also alter the proliferation of epithelial cells in the lung, breast, pancreas, stomach, small intestine, and large intestine. MPIF-1 inhibits the proliferation of epithelial cells, such as sebaceous glands, hair follicles, hepatocytes, type II alveolar cells, mucin-producing goblet cells, and other epithelial cells and their striations in the skin, lung, liver, .

MPIF-1 stimulates hepatocyte proliferation. In other words, MPIF-1 is also used to prevent or reduce acute viral hepatitis and chronic viral hepatitis as well as fulminant or quasi-fulminant hepatic failure due to cancer therapy (e.g., chemotherapy and / or radiation therapy) Or for therapeutic purposes

MPIF-1 can also be used to reduce intrinsic toxicity, a side effect of treatment with cytotoxic agents such as radiation therapy or chemotherapy. MPIF-1 has a cytoprotective effect on small intestinal mucus. MPIF-1 is also used for preventive or therapeutic purposes in order to prevent or alleviate mucositis and to alleviate mucositis (e.g. oral, esophagus, bowel, tubular, kidney and anal ulcers) by chemotherapy or other cytotoxic agents Can be used.

Inflammatory bowel disease such as Crohn's disease and ulcerative colitis is a disease caused by destruction of the mucosal surface of the small intestine or colon, respectively. That is, MPIF-1 can be used to accelerate resurfacing of the mucosal surface to aid faster healing and to prevent or reduce the progression of inflammatory bowel disease. Treatment with MPIF-1 is expected to have a significant effect on the production of mucous membranes throughout the gastrointestinal tract and can be used to protect the mucosa from digestive toxicants or post-operatively. That is, the present invention also provides a method for preventing or treating a mucosal disease or pathological condition, including ulcerative colitis, Crohn's disease, and other diseases in which the mucosa is damaged, including administering an effective amount of MPIF-1. The present invention also provides a method of preventing or treating oral (including swallowing associated with mucosal damage of the pharynx and hypopharynx), esophagus, stomach, intestinal, coronary and renal mucositis irrespective of the drug or mode of use that is the cause of such damage .

MPIF-1 can also be used to prevent and alleviate pulmonary damage by various drugs. MPIF-1 can prevent or treat damage to the alveoli and bronchial epithelium. For example, dyspnea due to smoke inhalation and radiation injury, which can cause necrosis of the bronchial epithelium and alveoli, can be effectively treated with MPIF-1. MPIF-1 can also be used to protect type II lung cells.

MPIF-1 can also be used to reduce intrinsic toxicity, a side effect of radiation therapy, chemotherapy, or other cytotoxic therapies. MPIF-1 has a cytoprotective effect on small intestinal mucus. MPIF-1 may also be used to prevent or alleviate mucositis and to prevent or treat mucositis (e.g., oral, esophagus, intestinal, tubular, renal, and anal ulcers) by chemotherapy, radiation and other cytotoxic agents It can be used for therapeutic purposes. That is, the present invention also provides a method for preventing or treating a mucosal disease or pathological condition, including ulcerative colitis, Crohn's disease, and other diseases in which the mucosa is damaged, including administering an effective amount of MPIF-1. The present invention also provides a method of preventing or treating oral (including swallowing associated with mucosal damage of the pharynx and hypopharynx), esophagus, stomach, intestinal, coronary and renal mucositis irrespective of the drug or mode of use that is the cause of such damage .

MPIF-1 may also be used for the treatment and / or prevention of the following conditions: blisters and burns caused by chemicals, such as ovarian damage due to treatment with a chemotherapeutic agent or treatment with cyclophosphamide, radiation Or cystitis due to chemotherapy, or intestinal damage due to high-dose chemotherapy.

MPIF-1 can be used to prevent or alleviate nephrotoxicity due to chemotherapy, radiation, or other cytotoxic agents.

MPIF-1 can be used clinically to treat or prevent damage to the epidermis, eye tissue, dental tissue, oral cavity and complex sites associated with systemic or topical treatment with radiation therapy and anti-neoplastic agents. MPIF-1 can also be used to treat skin loss.

The present invention also provides a method of protecting an individual from the effects of radiation, chemotherapy or other cytotoxic agents, including administering an effective amount of MPIF-1. The present invention also provides a method for alleviating or preventing tissue damage due to exposure to radiation, chemotherapy or other cytotoxic agents, including administering an effective amount of MPIF-1. An individual may be exposed to ionizing radiation (e.g., radiation) for many reasons, including therapeutic purposes (e.g., treatment of hyperproliferative diseases), release of radioactive isotopes into the environment or invasive or noninvasive medical diagnostic procedures Lt; / RTI > Also, a significant number of individuals are exposed to radioactive radon in the workplace and at home. Expected shortening lifetimes have been calculated using exposures to such an environment over a long period of time [Johnson, W. and Kearfott, K., Health Phys. 73 : 312-319 (1977)]. As disclosed in Examples 17-18, the proteins of the present invention improve the survival rate of animals exposed to radiation. That is, MPIF-1 can increase the survival rate of individuals affected by radiation, protect individuals from sub-lethal doses of radiation, and increase the rate of irradiation in the treatment of diseases such as hyperproliferative disorders.

MPIF-1 can also be used to protect individuals against the dose of radiation, chemotherapeutic agents, or other cytotoxic agents that are not normally tolerated. For such use, or when used as described herein, MPIF-1 may be administered before, after, and / or during treatment with radiation therapy / exposure, chemotherapy or other cytotoxic agents. High dose radiation and chemotherapeutic agents may be particularly useful when treating individuals at advanced disease stages, such as hyperproliferative diseases.

The present invention also relates to the use of an effective amount of MPIF-1 in a subject for the treatment of radiation-induced oral and gastrointestinal injuries, mucositis, intestinal fibrosis, rectalitis, radiation-induced pulmonary fibrosis, radiation- induced pneumonitis, And a method of preventing or treating a symptom such as a gonadal syndrome, radiation induced spinal cord toxicity.

That is, the MPIF-I polynucleotide or polypeptide of the present invention inhibits damage of normal cells, for example, damage to bone marrow stroma, during radiation therapy, chemotherapy, and target radiation therapy for malignant tumors, metastasis and related diseases Such as acute leukemia such as acute lymphocytic leukemia, acute myelogenous leukemia (myeloblastic, zygomorphic, bone morphogenetic, monocytic, and leukemia, etc.) And chronic lymphocytic leukemia such as chronic spinal cord cysts (granulocytic leukemia and chronic lymphocytic leukemia), intrinsic erythropoiesis, lymphomas (for example, Hodgkin's disease and non-Hodgkin's disease) , Multiple myeloma, Waldenstrom's macroglobulinemia, heavy chain disease, and solid tumors, such as sarcomas and carcinomas, for example, Sarcoma sarcoma, sarcoma sarcoma, sarcoma sarcoma, sarcoma sarcoma, sarcoma sarcoma, endometritis sarcoma, lymphatic sarcoma, lymphatic endothelial sarcoma, synovial sarcoma, mesothelioma, Ewing's tumor, leiomyosarcoma, rhabdomyosarcoma, colon cancer Pancreatic cancer, breast cancer, ovarian cancer, prostate cancer, squamous cell carcinoma, basal cell carcinoma, adenocarcinoma, gland carcinoma, gland carcinoma, papillary carcinoma, papillary carcinoma, cystadenocarcinoma, follicular carcinoma, renal cell carcinoma, liver cancer, Wilm's tumor, cervical cancer, testicular tumor, lung carcinoma, small cell lung carcinoma, bladder cancer, epithelioid carcinoma, glioma, astrocytoma, malignant melanoma, squamous cell carcinoma, squamous cell carcinoma But are not limited to, craniopharyngioma, adenocarcinoma, pineal gland, angioblastoma, auditory neoplasm, oligodendroglioma, menangioma, melanoma, neuroblastoma, and retinoblastoma. Such diseases also include metastatic water quality thyroid cancer, degenerative astrocytoma, glioblastoma, follicular lymphoma, colon cancer, heart tumor, pancreatic cancer, melanoma, retinoblastoma, glioblastoma, lung cancer, Osteosarcoma, osteosarcoma, adenoma, breast cancer, prostate cancer, Kaposi's sarcoma, and ovarian cancer. The term " myeloma " Other diseases that can be treated using the polypeptides, polypeptide fragments and variants, agonists and antagonists (including antibodies) and other antibodies of the invention are known in the art and are also disclosed herein.

MPIF-1 may be used alone or in combination with one or more additional drugs that protect against radiation or other drugs. Several cytokines, including IL-1, TNF, IL-6, IL-12, exhibit such protective effects {Neta, R. et al . , J. Exp. Med. 173 : 1177 (1991)}. IL-11 has been implicated in the treatment of small intestinal mucosal cells {Du, XX et al., Blood 83 : 33 (1994)} and radiation induced chest injuries {Redlich, CA et al., The Journal of Immunology 157: 1705-1710 )}. Various growth factors such as fibroblast growth factor and transforming growth factor beta-3 have also been found to perform protection against radiation exposure [Ding, I. et al . , Acta Oncol. 36 : 337-340 (1997); Potten, C. et al . , Br. J. Cancer 75 : 1454-1459 (1977)).

Additional radioprotectants that may be administered with the polypeptides and polynucleotides of the present invention include calcium antagonists (WO 93/02670), polyethylene glycols (US 4,676,979), polyvinyl pyrrolidones (US 4,676,979), polyethylene glycol monoethyl ethers 4,676,979), amides, amines and salts of methoxypolyethylene glycol with chelating agents such as EDTA, DTPA and EGTA (W098 / 47858), manganese and other metallothionein inducing substances (US 5,008,119), WR-2721 5,424,471), WR-1065, other phosphorothioates (US 5,869,338), polyamide thiols (US 5,217,964), SCF / IL-3 / GM-CSF combination or single treatment therapies (US 5,620,685), beta carotene and Dunaalella (Dunaliella) formulation (US 5,948,823), the derivative (US 5,981,512), tea dried and L-Glu-L-Trp ( US 5,770,576), IL-1, tumor necrosis factor, stem cell factor and IL-12 of the alkaloid in Arkite (Neta, Stem Cells 15 (Suppl 2) : 207-10 (1997)), copper chelate (Sorenson, etc., Proc Soc EXP Biol Med 210: ..... 191-204 (1995)), D- factor / GH / IL-1 / TNF combination or single therapy (US 5,843,422), liquid Tiha Amide (CASRNNo. 37239-28-4), amipostin (WR2721), monosodium salt of 2-amino-ethanethiol dihydrogenphosphate (ester) (cystafos, 2-aminoethanethiol (cysteamine), 2,2'-dithiobis (ethylamine) (cystamine), S-2-amino Cu (II) 2 (3,5-DIPS) 4, Cu (II) 2 (3,5-diisopropyl salicylate) , Essential metal element chelates of Fe, Mn and Zn (Sorenson et al . , Proc. Soc. EXP. Biol. Med. 210 : 191-204 (1995)), 2- (allylthio) pyrazine, phosphorus derivatives of alkaloids (Austrian patents 377 988 and 354 644; US 5,981, 512), and the like and combinations thereof.

The polynucleotides and polypeptides of the present invention may also be used in the form of 2- (ethylthio) -10- (3- (4-methyl-1-piperazinyl) propyl) -10H-phenothiazine (m-methylbenzyl) -piperazine (mechlorine, methicillin), and the like, and combinations thereof, in combination with an anti-vomiting agent such as p-chloro-alpha-phenylbenzyl) -4- Polynucleotides and polypeptides of the invention may also be administered with other therapeutic agents and combinations thereof disclosed herein or known in the art.

Hemorrhagic cystitis is a syndrome associated with exposure to drugs, viruses, and toxins as well as certain disease states. And the bleeding of the epithelial lining of the bladder is diffused. Known therapies include intravenous, systemic and nonpharmacologic therapies [West, NJ , Pharmacotherapy 17 : 696-706 (1997)]. Some cytotoxic drugs used clinically have the side effect of causing the inhibition of the normal epithelial proliferation of the bladder and causing the destruction of the ulcer and epithelial lining which are life threatening. For example, cyclophosphamide is a cytotoxic drug that is biologically transformed mainly in the liver and actively alkylates the metabolite by the complex action of the microsomal oxidase system. Such metabolites interfere with the growth of rapidly growing malignant cells. The mechanism of action is thought to be related to the crosslinking of tumor cell DNA (Physician's Desk reference, 1997).

Cyclophosphamide is an example of a cytotoxic drug that causes haemorrhagic cystitis in some patients and can lead to severe complications and, in some cases, death. Fibrosis of the urethra bladder may also progress without or with cystitis. This damage is thought to be caused by cyclophosphamide metabolites excreted in the urine. Hematuria caused by cyclophosphamide occurs for several days but may persist. In severe cases, medication or surgical treatment is needed. For severe haemorrhagic cystitis, nonconsecutive cyclophosphamide therapy should be used. In addition, deterioration of the urethral bladder generally occurs within two years after cyclophosphamide treatment and occurs in patients already with hemorrhagic cystitis [CYTOXAN (cyclophosphamide) package insert]. Cyclophosphamide is harmful to the prostate and the male reproductive system. Cyclophosphamide therapy can be the cause of infertility and may cause some regression of the testicles.

Those skilled in the art will appreciate that an effective amount of MPIF-I polypeptide (including the amount of MPIF-1 polypeptide effective for bone marrow suppression, with or without the use of a bone marrow inhibitor or myelosuppressive inhibitor) for treating an individual in need of MPIF- -1 < / RTI > can be determined empirically according to each situation prescribed. The polypeptide having MPIF-1 activity may be administered in the form of a pharmaceutical composition together with one or more pharmaceutically acceptable carriers.

MPIF-1 can also be used to treat leukemia and abnormal proliferating cells, such as tumor cells, by inducing apoptosis. MPIF-1 induces apoptosis in the proliferation of hematopoietic progenitor cells.

MPIF-1 can be used for the expansion of immature hematopoietic progenitor cells, such as granulocytes, giant cells or mononuclear cells, by temporarily preventing differentiation. Such bone marrow cells can be cultured in vitro. That is, MPIF-1 can be used as an adjuvant of hematopoietic stem cells in vitro for the purpose of bone marrow transplantation and / or gene therapy. Since stem cells are rare and most useful for introducing genes into gene therapy, MPIF can be used to isolate stem cell-rich populations. Stem cells can be enriched by culturing cells in the presence of cytotoxins, such as 5-Fu, which kill rapidly dividing cells, where the stem cells are protected by MPIF-1. Stem cells can be used for transfusion into bone marrow transplant recipients or transfection of genes that are desirable for gene therapy. In addition, MPIF-1 can be injected into an individual to cause stem cells to be released from the bone marrow of an individual into peripheral blood. These stem cells can be isolated for autologous bone marrow transplantation or gene therapy manipulation. After chemotherapy or radiation therapy, the isolated stem cells can be returned to the patient.

In addition, since MPIF-1 is effective not only in giant cells but also in T-lymphocytes, MPIF-1 enhances the ability of antigen-bearing cells to capture viruses, bacteria, or other foreign substances and treat them with lymphocytes reactive to the immune response Exist. MPIF-1 can also regulate the interaction of APC with T-lymphocytes and B-lymphocytes. MPIF-1 provides a costimulatory signal during the presence of the antigen to allow the responding cells to survive, proliferate, differentiate, secrete additional cytokines or soluble mediators, induce apoptosis or other cytotoxic mechanisms It is possible to selectively remove the reacting cells. Since APC has been shown to promote the transfer of HIV to CD4 + T-lymphocytes, MPIF-1 can affect its activity and prevent infection of lymphocytes by HIV or other viruses mediated by APC. It also ensures initial infection of APC, T-lymphocytes or other cell types by HIV, EBV or any other such virus.

In addition, recently, the MIP-1 alpha receptor has been shown to act as a cofactor to promote the introduction of HIV into human mononuclear cells and T-lymphocytes, suggesting that MPIF-1 and its variants affect the introduction of HIV into cells (See Example 1). That is, MPIF-1 can be used as an antiviral agent of viruses and retroviruses which are promoted to be introduced by the MIP-1α receptor.

MPIF-1 may act as an immune enhancing factor by stimulating the intrinsic activity of T-lymphocytes to fight bacterial and viral infections as well as other foreign substances. Such activity is useful for immune responses to neoplasms or benign growth, including solid tumors and leukemia, as well as normal responses to external antigens such as allergy infections.

For these reasons, the present invention is useful for the diagnosis or treatment of various immune system related diseases of mammals, preferably the human body. Such diseases include but are not limited to tumors, cancer and immune cells including autoimmune, arthritis, leukemia, lymphoma, immunosuppressive response, sepsis, wound healing, acute and chronic infections, cell mediated immunity, humoral immunity, inflammatory bowel disease, Functional disorders, and the like.

Thus, MPIF-1 can be used to control wound infiltration of target immune cells to promote wound healing. In a similar aspect, the polypeptides of the invention may enhance host defense against chronic infections, e. G., Mycobacteria, through attack and activation of antibiotic leukocytes.

The polypeptides of the present invention and polynucleotides encoding such polypeptides are research reagents for in vitro research related to scientific research, DNA synthesis and DNA vector production, and as research reagents for the development of therapeutics and diagnostic agents for the treatment of human diseases Can be used. For example, MPIF-1 can be used to inflate immature hematopoietic progenitor cells, such as granulocytes, giant cells or mononuclear cells, by temporarily preventing differentiation. Such bone marrow cells can be cultured in vitro.

Another use of polypeptides is to inhibit T-cell proliferation through inhibition of IL-2 biosynthesis, for example in autoimmune diseases and lymphocytic leukemia.

MPIF-1 can also be used to inhibit epidermal keratinocyte proliferation, which is useful for psoriasis (keratinocyte hyperplasia) because skin Langerhans cells have been shown to produce MIP-1α.

MPIF-1 can be used to prevent scar formation during wound healing through tissue debris cleansing and reinforcement of connective tissue-promoting inflammatory cells and through the control of hypertriglycerid TGF-mediated fibrosis, Increased vascular permeability can be used to treat pulses, thrombocytosis, pulmonary embolism, and myeloproliferative disorders.

Pharmaceutical composition

The MPIF-1 polypeptide pharmaceutical composition comprises an isolated MPIF-1 polypeptide of the invention, particularly an effective amount of MPIF-1 in a mature form effective to increase MPIF-1 activity in an individual. Such compositions should be administered in accordance with the preferred medical treatment regimens, taking into account the clinical conditions of each patient (especially the side effects associated with the MPIF-1 polypeptide alone), the delivery site of the MPIF-1 polypeptide composition, the method of administration, the schedule of administration and other factors known to practitioners Can be formulated and administered in the same manner. An " effective amount " of the MPIF-1 polypeptide for such purpose is determined in view of such considerations.

Polypeptides, agonists and antagonists of the invention may be used in conjunction with suitable pharmaceutical carriers to constitute pharmaceutical compositions. Such compositions contain a therapeutically effective amount of the protein and a pharmaceutically acceptable carrier or excipient. Such carriers include, but are not limited to, saline, buffered saline, dextrose, water, glycerol, ethanol, and combinations thereof. The formulations should be appropriate for the mode of administration.

&Quot; Pharmaceutically acceptable carrier " means any non-toxic solid, semi-solid or liquid carrier, diluent, encapsulating material or formulation auxiliary. The term " parenteral " as used herein refers to modes of administration including intravenous, intramuscular, intraperitoneal, intrasternal, subcutaneous and intraarticular injection and infusion.

In addition, the MPIF-1 polypeptide is preferably administered by a sustained-release system. Suitable examples of sustained release compositions include a semipermeable polymer matrix in the form of a shaped article, for example in the form of a film or microcapsule. Examples of sustained-release matrices include polylactides (US Pat. No. 3,773,919, EP 58,481), copolymers of L-glutamic acid and? -Ethyl-L-glutamate (U. Sidman et al., Biopolymers 22: 547-556 (2-hydroxyethyl methacrylate (R. Langer et al . , J. Biomed. Mater. Res. 15 : 167-277 (1981) and R. Langer, Chem. Tech. 1982), ethylene vinyl acetate (see R. Langer et al., Supra) or poly-D- (-) - 3-hydroxybutyric acid (EP 133,988) The liposomes containing MPIF-1 can be prepared by known methods, such as DE 3,218,121, Epstein et al., Proc. Natl. Acad. Sci. USA 82: 3688-3692 (1985) , EP 52,322, EP 36,676, EP 143,949, EP 142,641, Japanese Patent Application No. 83-118008, USP 4,485,045 and 4,544,545 ( Proc. Natl. Acad. Sci. USA 77: 4030-4034 , And EP 102, 304. Usually, liposomes are prepared by liposome (About 200-800 ANGSTROM) monolayer type with a cholesterol of about 30 mol% or more, and the selected ratios are adjusted for optimal MPIF-1 polypeptide treatment.

In one embodiment, in parenteral administration, the MPIF-1 polypeptide is administered to a recipient in a pharmaceutically acceptable carrier, i. E., At a given concentration and dose, with an injectable dosage form (solution, suspension or emulsion) It is non-toxic and formulated in admixture with other ingredients of the formulation. For example, it is preferred that the formulation does not contain oxidizing agents and other compounds known to be detrimental to the polypeptide.

Typically, the preparation is prepared by uniformly and intimately contacting MPIF-1 with a liquid carrier or finely divided solid carrier or both. Then, if necessary, the product is molded into the desired formulation. The carrier is preferably a parenteral carrier, more preferably a solution which is isotonic with the blood of the recipient. Examples of such carrier excipients include water, saline, Ringer's solution and dextrose solution. Non-aqueous vehicles such as non-petrol and ethyl oleate are also useful in the present invention, as are liposomes.

It is appropriate that the carrier contains a small amount of additives such as a substance improving isotonicity and chemical stability. Such materials are non-toxic to the recipient at the dosages and concentrations employed, such as buffers such as phosphate, citrate, succinate, acetic acid and other organic acids or their salts, antioxidants such as ascorbic acid, Low molecular weight (with less than about 10 residues) polypeptides such as peptides; Proteins such as serum albumin, gelatin or immunoglobulin; Hydrophilic polymers such as polyvinyl pyrrolidone; Amino acids such as glycine glutamic acid, aspartic acid or arginine; Monosaccharides, disaccharides, and other carbohydrates including cellulose or its derivatives, glucose, mannose, or dextrin; Chelating agents such as EDTA; Sugar alcohols such as mannitol or sorbitol; Counter ions such as sodium; And / or non-ionic surfactants such as polysorbates, poloxamers or PEG.

MPIF-1 polypeptides are typically formulated in such excipients at a concentration of about 0.1 mg / ml to 100 mg / ml, preferably 1 mg / ml to 10 mg / ml, at a pH of about 3 to 8. The use of certain of the excipients, carriers or stabilizers described above results in the formation of MPIF-1 polypeptide salts.

When MPIF-1 and / or its variants are administered as a bone marrow protective agent as part of chemotherapy for the treatment of hyperproliferative diseases of the human body, a suitable dosage range for intravenous administration is from 0.01 [mu] g / kg (body weight) to 10 [ (Weight). In addition, MPIF-1 can be administered intravenously at doses of 0.1, 1.0, 10, and 100 占 퐂 / kg (body weight). Extrapolation of animal test data showed that the dose of MPIF-1 suitable for human bone marrow protection was 0.016 μg / kg (body weight).

When MPIF-1 and / or its variants are administered to treat cytotoxic damage to cells, tissues and organs, they can be administered to the human body after exposure to a cytotoxic agent. When used as a prophylactic agent for cytotoxic damage to cells, tissues and organs, MPIF-1 and / or its variants may be administered after exposure of the cytotoxic agent, or both before and after exposure.

In addition, MPIF-1 and / or its variants can be administered once daily for a certain number of days (e.g., 3 days). In addition, when used in chemotherapy, MPIF-1 may be administered to the human body prior to administration of the chemotherapeutic agent (s). For example, MPIF-1 may be administered 2 days, 1 day and on the day of administration of the chemotherapeutic agent (s).

When MPIF-1 and / or its variants are administered to the human body for the treatment of a myeloproliferative disorder, the dose may be the same as when MPIF-1 is used as a bone marrow protectant. When administered to the human body for the treatment of myeloproliferative disorders, MPIF-1 can be administered subcutaneously.

MPIF-1 used for therapeutic administration should be sterilized. Sterilization can be easily accomplished by filtration through sterile filtration membranes (e. G., 0.2 micron membranes). The therapeutic MPIF-1 polypeptide composition is generally placed into a vial having a sterile access port, for example, an intravenous solution bag, or a vial having a pierceable puncture needle.

MPIF-1 polypeptides will typically be stored in unit or multi-dose containers, such as sealed ampoules or vials, as aqueous solutions or as reconstitution thaw preparations. As an example of a thixotropic agent, a 10 ml vial is filled with 5 ml sterile filtered 1% (w / v) aqueous MPIF-1 polypeptide solution and the resulting mixture is frozen. The infusion solution is prepared by hydrating the frozen MPIF-I polypeptide using bacteriostatic water for injection.

The present invention also provides a pharmaceutical pack or kit comprising one or more containers filled with one or more components of the pharmaceutical composition of the present invention. In connection with such container (s), notices stipulated by a government agency regulating the manufacture, use or sale of drugs or biological agents shall include the permission of the government agency for manufacture, use or sale for human consumption. Reflect. In addition, the polypeptides of the present invention may be used in combination with other therapeutic compounds.

The present invention also provides a method of treating and / or preventing a disease or condition (e. G., Any one or more of the diseases or disorders as described herein) by administering an effective amount of a therapeutic agent to the subject. Therapeutic agent means a polynucleotide or polypeptide (including fragments and variants), an agonist or antagonist thereof, and / or an antibody thereto, in combination with a pharmaceutically acceptable carrier (e. G., A sterile carrier).

MPIF-1 can be formulated and administered in the same manner as the preferred medical treatment, taking into account the clinical conditions of each patient (especially the side effects of the MPIF-1 polypeptide alone), delivery site, method of administration, schedule of administration, and other factors known to practitioners Lt; / RTI > The "effective amount" of such a purpose is determined by considering such matters.

Generally, the total amount of MPIF-1 that is pharmaceutically effective when administered parenterally is in the range of about 1 μg / kg (patient weight) / day to 10 mg / kg (patient weight) / day, Depends on treatment details. This dose is more preferably 0.01 mg / kg (patient body weight) / day or more, and in the case of human body, about 0.01 to 1 mg / kg (patient body weight) / day is most preferable considering hormones. In the case of continuous administration, MPIF-1 is usually administered at a dose of about 1 占 퐂 / kg / hour to about 50 占 퐂 / kg / hour by 1 to 4 times of daily injections or continuous subcutaneous injection using a minipump or the like . Vein bag solutions can also be used. The duration of treatment needed to confirm the change and the interval between subsequent treatments to allow the response to occur to appear different depending on the desired effect.

MPIF-1 may be administered orally, rectally, parenterally, intramuscularly, intravaginally, intraperitoneally, topically (by powder, ointment, gel, drip or percutaneous patch), buccal, or oral or nasal spray. &Quot; Pharmaceutically acceptable carrier " means any non-toxic solid, semi-solid or liquid carrier, diluent, encapsulating material or formulation auxiliary. The term " parenteral " as used herein refers to modes of administration including intravenous, intramuscular, intraperitoneal, intrasternal, subcutaneous and intraarticular injection and infusion.

In addition, MPIF-1 is preferably administered by a sustained-release system. Examples of suitable sustained release MPIF-1 are oral, rectal, parenteral, intramuscular, intrapulmonary, intraperitoneal, topical (by powder, ointment, gel, drip or percutaneous patch), buccal, or oral or nasal spray . &Quot; Pharmaceutically acceptable carrier " means any non-toxic solid, semi-solid or liquid carrier, diluent, encapsulating material or formulation auxiliary. The term " parenteral " as used herein refers to modes of administration including intravenous, intramuscular, intraperitoneal, intrasternal, subcutaneous and intraarticular injection and infusion.

In addition, MPIF-1 is preferably administered by a sustained-release system. Suitable examples of sustained release MPIF-1 include polymeric materials (e.g., molded articles, such as semipermeable polymer matrices in the form of films or microcapsules), suitable hydrophobic materials (such as an acceptable emulsion in oil) There is an insoluble derivative (eg insoluble salt).

Examples of sustained-release matrices include polylactides (US Pat. No. 3,773,919, EP 58,481), copolymers of L-glutamic acid and? -Ethyl-L-glutamate (U. Sidman et al., Biopolymers 22 : 547-556 (2-hydroxyethylmethacrylate) (Langer et al . , J. Biomed. Mater. Res. 15 : 167-277 (1981) and Langer, Chem. Tech. 12 : 98-105 (1982) , Ethylene vinyl acetate (see R. Langer et al., Supra) or poly-D - (-) - 3-hydroxybutyric acid (EP 133,988).

In addition, the sustained release MPIF-I composition contains MPIF-1 captured in liposomes [Langer, Science 249 : 1527-1533 (1990); Treat, et al., In Liposome in the Therapy of Infectious Disease and Cancer , Lopez-Berestein and Fidler (eds.), Liss, New York, pp. 317-327 and 353-365 (1989)). Liposomes containing MPIF-1 can be prepared by known methods, e. G. DE 3,218,121, Epstein et al., Proc. Natl. Acad. Sci. USA 82: 3688-3692 (1985), Huang et al., Proc. Natl. Acad. Sci. USA 77: 4030-4034 (1980)], EP 52,322, EP 36,676, EP 143,949, EP 142,641, Japanese Patent Application No. 83-118008, USP 4,485,045 and 4,544,545, and EP 102,304. Usually, liposomes have a small (about 200-800 A) monolayer type with a lipid component of at least about 30 mol% cholesterol, and the selected ratios are adjusted for optimal MPIF-I treatment.

In another embodiment, MPIF-1 is delivered by a pump [Langer, supra; Sefton, CRC Crit. Ref. Biomed. Enf. 14 : 201 (1987); Buchwald et al., Surgery 88: 507 (1980); Saudek et al., N. Engl. J. Med. 321 : 574 (1989)).

Other controlled release systems have been described in the review section of almost the entire article ( Sciece 249: 1527-1533 (1990)).

In one embodiment of parenteral administration, MPIF-1 is generally administered in a unit dose injection form (solution, suspension or emulsion) in a pharmaceutically acceptable carrier (i. E., Compatible with the other ingredients in the composition, And non-toxic to the subject to whom it is administered at a concentration). For example, it is preferred that the agent does not comprise an oxidizing agent and other compounds known to be detrimental to the polypeptide.

In general, the compositions are prepared by uniformly and intimately bringing MPIF-1 into contact with a liquid carrier or a uniformly distributed solid carrier, or both. Then, if necessary, the product is molded into a predetermined preparation. It is preferable that the carrier is a parenteral carrier, and a solution which is a solution of the subject to be administered and an isotonic solution is more preferable. Examples of such carrier vehicles include water, saline, Ringer's solution and dextrose solution. Non-aqueous vehicles and liposomes such as non-volatile oils and ethyl oleate are also useful in the present invention.

The carrier preferably contains small amounts of additives such as isotonicity and chemical stability enhancing substances. Such materials are non-toxic to the subject at the dose and concentration used, and include phosphate, citrate, succinate, acetic acid, other organic acids or salts thereof; Antioxidants such as ascorbic acid; Low molecular weight (less than about 10 residues) polypeptides, such as polyarginine or tripeptides; Proteins such as serum albumin, gelatin or immunoglobulin; Hydrophilic polymers such as polyvinylpyrrolidone; Amino acids such as glycine, glutamic acid, aspartic acid or arginine; Cellulose or its derivatives, monosaccharides including glucose, mannose or dextrin, disaccharides and other carbohydrates; Sugar alcohols such as mannitol or sorbitol; Counter ions such as sodium; And / or anionic surfactants such as polysorbate, poloxamer, or PEG.

MPIF-1 is typically formulated in such vehicles at a concentration of about 0.1 mg / ml to 100 mg / ml, preferably 1-10 mg / ml, at a pH of about 3-8. Use of the particular excipients, carriers or stabilizers described above will result in the formation of polypeptide salts.

MPIF-1 used for therapeutic administration can be sterilized. Sterilization can be easily accomplished by filtration through sterile filtration membranes (e. G., 0.2 micron membranes). MPIF-1 is typically placed into a container with a sterile access port, for example a vial in an intravenous solution bag, or with a cap that can be pierced by a hypodermic needle.

MPIF-1 will normally be stored in unit or multi-dose containers, such as sealed ampoules or vials, as an aqueous solution or as a reconstitution thaw preparation. As an example of a thixotropic agent, a 10 ml vial is filled with 5 ml sterile filtered 1% (w / v) aqueous MPIF-1 polypeptide solution and the resulting mixture is frozen. The infusion solution is prepared by hydrating the frozen MPIF-I polypeptide using bacteriostatic water for injection.

The present invention also provides a pharmaceutical pack or kit comprising one or more containers filled with one or more components of the pharmaceutical composition of the present invention. In connection with such container (s), the notices prescribed by a government agency regulating the manufacture, use or sale of drugs or biological agents shall reflect the permission of the government agency in the manufacture, use or sale for human administration. MPIF-1 may also be used in combination with other therapeutic compounds.

MPFI-1 may be administered alone or in combination with adjuvants. The adjuvants that can be administered with MPFI-1 include, but are not limited to, Alum, Alum and deoxycholate (Immune Ag), MTP-PE (Biocine Corp.), QS21 (Genentech, Inc.), BCG and MPL It is not. In certain embodiments, MPFI-1 may be administered with an alum. In another embodiment, MPFI-1 may be administered with QS-21. Adjuvants that may be administered with MPFI-1 include monophosphoryl lipid immunomodulators Adju Vax 100a, QS-21, QS-18, CRL1005, aluminum salts, MF-59 and virosome adjuvants, It is not. Vaccines that can be administered with MPFI-1 include MMR (measles, mumps, rubella), polio, chicken pox, tetanus / diphtheria, hepatitis A, hepatitis B, Haemophilus influenza B, whooping cough, pneumonia, influenza , Vaccines for the prevention of Lyme disease, rotavirus, cholera, yellow fever, Japanese encephalitis, asthma, rabies, typhoid fever and pertussis. Combinations may be administered incidentally, e. G. In the form of a mixture, separately, concurrently or sequentially. This includes an indication that the combined drug is administered together as a therapeutic mixture, and that the combined drug is administered separately but administered simultaneously, e.g., via separate intravenous administration to the same individual. &Quot; Together " administration includes administration of one of the given first compound or drug followed by the separate administration of the second compound or drug.

MPIF-1 may be administered alone or in combination with other therapeutic agents. Therapeutic agents that can be administered with MPIF-1 include other drugs of the TNF family, cytotoxic agents, chemotherapeutic agents, radiation, radiation sensitizers, targeted radiotherapy, antibiotics, antivirals, steroids and non- steroidal anti- Therapeutic agents, radiation immunosensors, cytokines, and / or growth factors. Combinations may be administered incidentally, e. G. In the form of a mixture, separately, concurrently or sequentially. This includes an indication that the combined drug is administered together as a therapeutic mixture, and that the combined drug is administered separately but administered simultaneously, e.g., via separate intravenous administration to the same individual. &Quot; Together " administration includes administration of one of the given first compound or drug followed by the separate administration of the second compound or drug.

In one embodiment, the compositions of the invention are administered with other TNF classes. TNF, TNF-related or TNF-like molecules can be produced in soluble forms of TNF-alpha, lymphotoxin-alpha (LT-alpha or TNF-beta), LT- , OPGL, FasL, CD27L, CD30L, CD40L, 4-1BBL, DcR3, OX40L, TNF-gamma (International Patent Publication 96/14328), AIM-I (International Patent Publication WO 97/33899) Alpha (International Patent Publication No. WO 98/07880), TR6 (International Patent Publication No. WO 98/30694), OPG and Neutrokine-alpha (International Patent Publication WO 98/18921, OX40 and NGF, DR3 (International Patent Publication WO 97/33904), DR4 (International Patent Publication WO 98/32856), TR5 (International Patent Publication No. WO 96/34095), TR3 TR6 (International Patent Publication No. WO 98/30694), TR7 (International Patent Publication WO 98/41629), TRANK, TR9 (International Patent Publication WO 98/56892), TR10 (International Patent Publication WO 98 / 54202), 312C2 (International Patent Publication No. WO 98/06842) and TR12 and soluble CD154, CD70 and CD153, It is not limited by only.

In another embodiment, MPIF-I is administered in combination with an antiretroviral agent, a nucleoside reverse transcriptase inhibitor, a non-nucleoside reverse transcriptase inhibitor and / or a protease inhibitor. Nucleotide reverse transcriptase inhibitors that may be administered with MPIF-1 include, but are not limited to, RETROVIR TM (Zidovudine / AZT), VIDEX TM (didanosine / ddl), HIVID TM (Zicitabine / ddC) But are not limited to, EPIVIR (TM) (lamivudine / 3TC) and COMBIVIR (zidovudine / lamivudine). Non-nucleotide reverse transcriptase inhibitors that may be administered with MPIF-1 include VIRAMUNE (registered trademark) (nevirapine), RESCRIPTOR (delavirdine), and SUSTIVA (registered trademark) , But are not limited thereto.

Protease inhibitors that may be administered with MPIF-1 include CRIXIVAN TM (indinavir), NORVIR TM (ritonavir), INVIRASE TM (saquinavir), and VIRACEPT TM (Nelfinavir), but are not limited thereto. In certain embodiments, an anti-retroviral agent, a nucleotide reverse transcriptase inhibitor, a nucleotide reverse transcriptase inhibitor and / or a protease inhibitor may optionally be used in combination with MPIF-1 for the treatment of AIDS and / or for the prevention or treatment of HIV infection. have.

In another embodiment, MPIF-1 may be administered with an anti-opportunistic agent. Anti-opportunistic infectious agents that may be administered with MPIF-1 include TRIMETHOPRIM-SULFAMETHOXAZOLE (registered trademark), DAPSONE (registered trademark), PENTAMIDINE (registered trademark), ATOVAQUONE (registered trademark), ISONIAZID (registered trademark), RIFAMPTIN ), PYRAZINAMIDE (registered trademark), ETHAMBUTOL (registered trademark), RIFABUTIN (registered trademark), CLARITHROMYCIN (registered trademark), AZITHROMYCIN (registered trademark), GANCICLOVIR (registered trademark), FOSCARNET (registered trademark), CIDOFOVIR (registered trademark) Such as FLUCONAZOLE (registered trademark), ITRACONAZOLE (registered trademark), KETOCONAZOLE (registered trademark), ACYCLOVIR (registered trademark), FAMCICOLVIR (registered trademark), PYRIMETHAMINE (registered trademark), LEUCOVORIN (registered trademark), NEUPOGEN (registered trademark) Team / G-CSF), and LEUKINE TM (Sarglamos Team / GM-CSF). In one embodiment, MPIF-1 is selected from the group consisting of TRIMETHOPRIM-SULFAMETHOXAZOLE (R), DAPSONE (R), PENTAMIDINE (R)) for prophylactic treatment or prevention of opportunistic infectious pneumocystis carinii pneumonia infection , And / or ATOVAQUONE (registered trademark). In yet another embodiment, MPIF-I is selected from the group consisting of ISONIAZID (R), RIFABUTIN (R), PYRAZINAMIDE (R)) for prophylactic treatment or prevention of opportunistic infectious Mycobacterium avium complex infections, , And / or ETHAMBUTOL (registered trademark). In yet another embodiment, MPIF-I is selected from the group consisting of RIFABUTIN (R), CLARITHROMYCIN (R), and / or AZITHROMYCIN (R) for prophylactic treatment or prevention of opportunistic infections of Mycobacterium tuberculosis (Registered trademark). In another embodiment, MPIF-1 is optionally used in combination with GANCICLOVIR (R), FOSCARNET (R), and / or CIDOFOVIR (R) for prophylactic treatment or prevention of opportunistic infectious cytomegalovirus infections . In yet another embodiment, MPIF-1 is optionally used in combination with FLUCONAZOLE (R), ITRACONAZOLE (R), and / or KETOCONAZOLE (R) for prophylactic treatment or prevention of opportunistic infectious fungal infections. In yet another embodiment, MPIF-1 is optionally used in combination with ACYCLOVIR (R) and / or FAMCICOLVIR (R) for the prophylactic treatment or prevention of opportunistic infectious herpes simplex virus type I and / or type II infections . In yet another embodiment, MPIF-1 is optionally used in combination with PYRIMETHAMINE TM and / or LEUCOVORIN TM for prophylactic treatment or prevention of opportunistic infectious Toxoplasma gondii infection. In yet another embodiment, MPIF-I is optionally used in combination with LEUCOVORIN (R) and / or NEUPOGEN (R) for prophylactic treatment or prevention of opportunistic infectious bacterial infections.

In yet another embodiment, MPIF-1 is administered with an anti-viral agent. Antiviral agents that may be administered with MPIF-1 include, but are not limited to, acyclovir, ribavirin, amantadine, and lemantidine.

In another embodiment, MPIF-1 is administered with an antibiotic. Antibiotics that may be administered with MPIF-1 include but are not limited to amoxicillin, beta-lactamase, aminoglycoside, beta-lactam (glycopeptide), beta-lactamase, clindamycin, chloramphenicol, cephalosporin, ciprofloxacin, But are not limited to, paclitaxel, erythromycin, fluoroquinolone, macrolide, metronidazole, penicillin, quinolone, rifampin, streptomycin, sulfonamide, tetracycline, trimethoprim, trimethoprim-sulphamethoxazole and vancomycin It is not.

Typical non-specific immunosuppressive agents that may be administered with MPIF-I include, but are not limited to, steroids, cyclosporine, cyclosporine analogs, cyclophosphamide methylprednisone, prednisone, azathioprine, FK-506, 15-deoxyspergualin , And other immunosuppressive agents that act by inhibiting the action of responding T cells.

As a specific embodiment, MPIF-1 is co-administered with an immunosuppressive agent. Immunosuppressants that may be administered with MPIF-1 include ORTHOCLONE ^™ (OKT3), SANDIMMUNE ^™ / NEORAL ^™ / SANGDYA ^™ (cyclosporin), PROGRAF ^™ (tacrolimus), CELLCEPT ^™ (mycophenolate), azathioprine, glucocorticosteroid , And RAPAMUNE ^TM (sirolimus), but are not limited thereto. In certain embodiments, the immunosuppressive agent may be used to prevent rejection of tissue grafting or rejection of bone marrow transplantation.

As an additional embodiment, MPIF-I is administered alone or in combination with one or more intravenous immunoglobulin preparations. Intravenous immunoglobulin preparations that may be co-administered with MPIF-1 include but are not limited to GAMMAR ^™ , IVEEGAM ^™ , SANDOGLOBULIN ^™ , GAMMAGARD S / D ^™ , and GAMIMUNE ^™ . In a particular embodiment, MPIF-1 is co-administered with an intravenous immunoglobulin preparation in tissue graft therapy (eg, bone marrow transplant).

As a further embodiment, MPIF-1 is administered alone or in combination with an anti-inflammatory agent. Antiinflammatory agents that can be co-administered with MPIF-1 include, but are not limited to, glucocorticoids and nonsteroidal anti-inflammatory agents, aminoarylcarboxylic acid derivatives, aryl acetic acid derivatives, arylbutyric acid derivatives, arylcarboxylic acids, arylpropionic acid derivatives, Solvates, pyrazolones, salicylic acid derivatives, thiazinecarboxamide, e-acetamidocaproic acid, S-adenosylmethionine, 3-amino-4-hydroxybutyric acid, amicetyline, Corn, diphen pyramid, dithazol, emmopazone, guiazulene, nabumetone, nymerglide, orotane, oxaceprol, paranilin, ferium anthracis, fiochim, prouazone, proxazole, have.

In another embodiment, the MPIF-I composition is administered in combination with a chemotherapeutic agent. Chemotherapeutic agents that may be co-administered with MPIF-1 include, but are not limited to, antibiotic derivatives (eg, Adriamycin ^™ , bleomycin, daunorubicin, and dactinomycin); Antioxidants such as fluorouracil, 5-FU, methotrexate, phloxuridine, interferon alpha-2b, glutamic acid, flicamycin, mercaptopurine, and 6-thioguanine; A cytotoxic agent such as carmustine, BCNU, rosmutin, CCNU, cytosine arabinoside, cyclophosphamide, estramustine, hydroxyurea, proccarbazine, mitomycin, And estradiol, megestrol acetate, methyl testosterone, diethylstilbestrol diphosphate, chlorotrienisene, and testosterone (such as vincristine sulphate); hormones such as medroxyprogesterone, estramustine phosphate sodium, ethinylestradiol, estradiol, Lactone); Nitrogen mustard derivatives (e.g., mefarene, chroman, mechlorethamine (nitrogen mustard) and thiotepa); steroids and combinations (e.g., betamethasone sodium phosphate); And other species (e.g., dicarbazine, asparaginase, mitotane, vincristine sulfate, vinblastine sulfate, and etoposide).

As a specific embodiment, MPIF-1 is administered in combination with any combination of CHOP (cyclophosphamide, doxorubicin, vincristine, and prednisone) or CHOP components. In another embodiment, MPIF-1 is co-administered with Rituximab. In another embodiment, MPIF-I is administered with retushimab and CHOP, or with any combination of retushimab and CHOP components.

In a further embodiment, MPIF-1 is co-administered with a cytokine. Cytokines that may be administered with MPIF-1 include but are not limited to IL2, IL3, IL4, IL5, IL6, IL7, ILlO, IL12, IL13, IL15, anti-CD40, CD40L, IFN- - Alpha and so on. In another embodiment, MPIF-1 may be administered with any interleukin, including but not limited to IL-1 alpha, IL-1 beta, IL-2, IL-3, IL 14, IL-15, IL-16, IL-10, IL-10, IL-12, , IL-17, IL-18, IL-19, IL-20 and IL-21.

As a further embodiment, MPIF-1 is administered in combination with angiogenesis protein. Angiogenesis proteins that can be co-administered with MPIF-1 include, but are not limited to, glioma-derived growth factor (GDGF) as disclosed in European Patent No. EP-399816; Platelet derived growth factor-A (PDGF-A) disclosed in European Patent No. 682110; Platelet derived growth factor-B (PDGF-B) disclosed in European Patent EP-282317; Platelet Growth Factor (PIGF) as disclosed in International Publication No. WO 92/06194; Platelet Growth Factor-2 (PIGF-2) as disclosed by Hauser et al. Growth Factors 4 : 259-268 (1993); Vascular endothelial growth factor (VEGF) as disclosed in International Publication No. WO 90/13649; Vascular endothelial growth factor-A (VEGF-A) disclosed in European Patent EP-506477; Vascular endothelial growth factor-2 (VEGF-2) disclosed in International Publication No. WO 96/39515; Vascular endothelial growth factor (VEGF-3) as disclosed in International Publication No. WO 96/39515; Vascular Endothelial Growth Factor B-186 (VEGF-B186) disclosed in International Publication No. WO 96/26736; Vascular Endothelial Growth Factor-D (VEGF-D) disclosed in WO 98/02543; Vascular endothelial growth factor-D (VEGF-D) disclosed in WO 98/07832; Vascular endothelial growth factor-E (VEGF-E) disclosed in German Patent DE 19639601, and the like. The above-mentioned documents are incorporated herein by reference.

As a further embodiment, MPIF-1 is administered conjointly with a hematopoietic growth factor. Hematopoietic growth factors that may be co-administered with MPIF-1 include, but are not limited to, LERKINE ^™ (SARGRAMOSTIM ^™ ) and NEUPOGEN ^™ (FILGRASTIM ^™ ).

As an additional embodiment, MPIF-1 is co-administered with a fibroblast growth factor. Fibroblast growth factors that may be administered with MPIF-1 include but are not limited to FGF-1, FGF-2, FGF-3, FGF-4, FGF-5, FGF-6, FGF- , FGF-9, FGF-10, FGF-11, FGF-12, FGF-13, FGF-14 and FGF-15.

As a further embodiment, MPIF-1 is co-administered with other therapeutic or prophylactic regimens, such as, for example, radiation therapy.

In a further embodiment, the polynucleotides, polypeptides, agonists and / or antagonists of the present invention may also be co-administered with anti-angiogenic factors. Representative examples of anti-angiogenic factors include anti-invasive factors, retinoic acid and derivatives thereof, paclitaxel, suramin, tissue inhibitors of metalloproteinase-1, tissue inhibitors of metalloproteinase- Minogene activator inhibitor-1, plasminogen activator inhibitor-2, and a lighter variety of " d group " transition metals.

A light "d group" transition metal is meant to include, for example, vanadium, molybdenum, tungsten, titanium, niobium, and tantalum species. Such transition metal species may form transition metal complexes. Suitable complexes of this transition metal species include oxo-transition metal complexes.

Representative examples of vanadium complexes include oxovanadium complexes such as vanadate and vanadyl complexes. Suitable vanadate complexes include metavanadate and orthovanadate complexes such as ammonium metavanadate, sodium metavanadate and sodium orthovanadate. Suitable vanadyl complexes include, for example, vanadyl acetylacetonate and vanadyl sulfate, examples of which include vanadyl sulfate hydrate (e.g., vanadyl sulfate monohydrate and trihydrate).

Typical examples of tungsten and molybdenum complexes include oxo complexes and the like. Suitable oxotungsten complexes include tungstate and tungsten oxide complexes. Suitable tungstate complexes include ammonium tungstate, calcium tungstate, sodium tungstate dihydrate and tungstic acid. Suitable tungsten oxides include tungsten (IV) oxide and tungsten (VI) oxide. Suitable oxomolybdenum complexes include molybdate, molybdenum oxide and molybdenyl complexes. Suitable molybdate complexes include ammonium molybdate and its hydrates, sodium molybdate and its hydrates, and potassium molybdate and its hydrates. Suitable molybdenum oxides include molybdenum (IV) oxide, molybdenum (VI) oxide, and molybdic acid. Suitable molybdenyl complexes include, for example, molybdenum acetylacetonate. Suitable tungsten and molybdenum complexes include, for example, hydroxo derivatives derived from glycerol, tartaric acid, and sugars.

A wide variety of other anti-angiogenic factors may be used in the context of the present invention. Typical examples include platelet factor 4; Sulfated chitin derivatives (manufactured by Queen Crab Shell), (Murata et al . , Cancer Res. 51 : 22-26, 1991); Sulfated polysaccharide peptidoglycan complex (SP-PG) (the function of this compound may be enhanced in the presence of steroids such as estrogen and tamoxifen citrate), staurosporine, matrix metabolism modulators such as proline analogs, Cis hydroxyproline, d, L-3,4-dihydroxyproline, thiaproine, alpha, alpha-dipyridyl, aminopropionitrile fumarate; 4-propyl-5- (4-pyridinyl) -2- (3H) -oxazolone; Methotrexate; Mitosartron; Heparin; Interferon; Chimp-3 (Pavloff et al., J. Biol. Chem., 267 : 17321-17326, 1992); chymostatin (Tomkinson et al ., Biochem J. 286 : 475-480, 1992); (GST); Matsubara & Ziff, J. Clin. Invest. ≪ RTI ID = 0.0 > et < / RTI > 79 : 1440-1446, 1987); Anti-collagenase-serum; alpha-2-antiplasmin (Holmes et al . , J. Biol. Chem. 262 (4) : 1659-1664, 1987); Bisantrene (National Cancer Institute); Lobenzarit disodium (N- (2) -carboxyphenyl-4-chloroanthronyl disodium or "CCA"; Takeuchi et al., Agents Actions 36 : 312-316, 1992); Thalidomide; Anorthostatic steroid; AGM-1470; Carboxyminolimidazole; And metalloproteinase inhibitors (e.g., BB94).

Administration mode. Symptoms resulting from reduced levels of normal or normal levels of MPIF-1 activity in an individual may be treated by administration of the MPIF-1 protein. Therefore, the present invention provides a pharmaceutical composition containing an isolated amount of an isolated MPIF-1 polypeptide of the invention effective to increase MPIF-1 activity level in such an entity, particularly an effective amount of MPIF-1 in its mature form, To a subject in need thereof.

The dosage and dose regimen of MPIF-1 administered to an individual will depend on a number of factors, such as the mode of administration, the nature of the symptoms to be treated, and the judgment of the prescribing physician. The pharmaceutical composition is administered in an amount effective to treat and / or prevent a particular indication. Generally, the polypeptide is administered in an amount of about 10 [mu] g / kg body weight, taking into account the route of administration and syndrome, and in most cases administered in an amount not exceeding about 10 mg / kg body weight on day 1, It is preferred to administer in an amount of about 10 μg.

As a general suggestion, the total pharmacologically effective dose of MPIF-1 polypeptide administered parenterally per dose will depend on the severity of the treatment but is preferably in the range of about 1 [mu] g / kg / day to 10 mg / kg / day, This dose is at least 0.01 mg / kg / day, most preferably about 0.01 to 1 mg / kg / day in humans. If administered sequentially, the MPIF-1 polypeptide is generally administered at a dose of about 1 [mu] g / kg / h to about 50 [mu] g / kg / day by continuous subcutaneous infusion, kg / hour (h). An intravenous bag solution may also be used. For the reactions that take place, the treatment time required to observe the interval and change after treatment varies depending on the desired effect.

The pharmaceutical compositions containing MPIF-1 of the invention can be administered orally, rectally, parenterally, intramuscularly, intravaginally, intraperitoneally, topically (by powder, ointment, patch, or transdermal patch) Or as a spray for oral or nasal administration.

Gene therapy. Chemokine polypeptides and agonist polypeptides or antagonist polypeptides are used in accordance with the present invention to express the polypeptides in vivo, which is often referred to as " gene therapy ".

Thus, for example, a cell of a patient can be a polynucleotide (DNA or RNA) encoding a polypeptide in vitro that is encoded and encoded by the polypeptide, thereby providing the processed cell to the patient for treatment with the polypeptide. This method is well known in the prior art. For example, the cells may be processed according to the prior art using RNA-containing retroviral particles to encode the polypeptide according to the invention.

Similarly, cells may be processed in vivo for expression of the polypeptide in vivo, for example, according to methods known in the art. As is known in the art, producer cells that produce RNA-containing retroviral particles encoding the polypeptides of the present invention can be administered to a patient to express the polypeptide in vivo by processing cells in vivo. Such methods and other methods of administering the polypeptides of the present invention by such methods should be clearly understood by those skilled in the art from the teachings of the present invention. For example, the expression medium for cell processing is not a retrovirus, but an adenovirus that can be used to process cells in vivo, e.g., in combination with a suitable delivery vehicle.

Retroviral plasmid vectors are derived from retroviruses, including, but not limited to, the sarcoma virus of Moloney murine, the leukemia virus of Moloney murine, the spleen necrosis virus, the Rous sarcoma virus and the Harvey sarcoma virus , But it is not limited to this.

In a preferred embodiment, the retroviral expression vector pMV-7 is contiguous with the long terminal repeat (LTR) of the sarcoma virus of Moloney murine, and under the control of the herpes simplex virus (HSV) thymidine kinase (tk) promoter, It contains a resistant gene neo. The unique EcoRI and HindIII sites facilitate the introduction of coding sequences (Kirschmeier, PT et al., DNA 7 : 219-25 (1988)).

A vector comprising, but not limited to, one or more suitable promoters may be a retroviral LTR; SV 40 promoter; Miller et al., Biotechniques , Vol. 9: 980-990 (1989), and the human cytomegalovirus (CMV) promoter, or any other promoter (such as, but not limited to, cellular promoters such as eukaryotic promoters, histones, pol III, and? -Actin promoter). One skilled in the art can clearly see that a suitable promoter can be selected from the teachings taught herein.

Nucleic acid sequences encoding the polypeptides of the present invention include, but are not limited to, viral thymidine kinase promoters such as the herpes simple thymidine kinase promoter; A retroviral LTR, a beta -actin promoter, and a natural promoter that regulates the gene encoding this polypeptide.

A producer cell line is formed by transfecting a packaging cell line with a retroviral plasmid vector. Examples of packaging cells that can be transfected include, but are not limited to, PE501, PA317, and GP + aml2. The vector can be used to transfect the packaging cells using any means well known in the art. Such means include, but are not limited to, electroporation, use of liposomes, and precipitation of CaPO ₄ .

The producer cell line produces an infectious retroviral vector particle containing a nucleic acid sequence encoding the polypeptide. These retroviral vector particles are then used to transduce eukaryotic cells in vitro or in vivo. The transduced eukaryotic cell expresses a nucleic acid sequence encoding the polypeptide. Transduced eukaryotic cells include, but are not limited to, fibroblasts and endothelial cells.

Another category of the invention relates to gene therapy methods used to treat diseases, diseases and conditions. This gene therapy method introduces nucleic acid (DNA, RNA, and antisense DNA or RNA) sequences into an animal to achieve expression of the MPIF-I polypeptide of the present invention. This method requires polynucleotides encoding MPIF-I polypeptides operatively linked to the promoter and any other genetic elements necessary for expression of the polypeptide by the target tissue. Such gene therapy and delivery techniques are known in the prior art, e.g., WO 90/11092 (incorporated herein by reference).

Thus, a cell obtained in a patient can be processed, for example, with a polynucleotide (DNA or RNA) containing a promoter operatively linked to the MPIF-1 polynucleotide in vitro, the processed cell comprising a patient as a polypeptide It makes treatment possible. These methods are well known in the prior art. See, for example, Belldegrun , A., et al. , J. Natl. Cancer Inst. 85: 207-216 (1993); Ferrantini , M , etc., Cancer Research 53: 1107-1112 (1993 ); Ferrantini, M , etc., J. Immunology 153: 4604-4615 (1994 ); Kaido, T, et al. Int. J. Cancer 60 : 221-229 (1995); Ogura, H et al., Cancer Research 50 : 5102-5106 (1990); Santodonato, L et al., Human Gene Therapy 7 : 1-10 (1996); Santodonato, L., et al., Gene Therapy 4 : 1246-1255 (1997); And Zhang, JF et al., Cancer Gene Therapy 3 : 31-38 (1996), incorporated herein by reference. In one embodiment, the processed cells are arterial cells. Arterial cells can be injected directly into the arteries, tissues around the arteries, or reintroduced into the patient via catheter injection.

As will be described in detail below, MPIF-I polynucleotide constructs can be used in any method of delivering injectable materials to animal cells, such as by injecting into the reperfusion space of tissue (heart, muscle, skin, lung, liver, etc.) Lt; / RTI > The MPIF-I polynucleotide construct can be delivered in a pharmaceutically acceptable liquid or aqueous carrier.

In one embodiment, the MPIF-1 polynucleotide is delivered to a naked polynucleotide. The term " naked " polynucleotide, DNA, or RNA herein refers to a nucleic acid sequence that does not contain any carrier-mediated sequences that aid, facilitate, or facilitate entry into cells, including viral sequences, viral particles, liposomal preparations, lipofectin, It says. However, MPIF-I polynucleotides can also be delivered in liposomal preparations, and lipofectin preparations and the like can be prepared according to methods known in the art. Such methods are disclosed, for example, in U.S. Patent Nos. 5,593,972, 5,589,466, and 5,580,869. These documents are incorporated herein by reference.

The MPIF-1 polynucleotide vector construct used for gene therapy does not contain sequences that allow replication and is preferably not integrated into the host genome. Suitable vectors include pWLNEO, pSV2CAT, pOG44, pxT1 and pSG, which are available from Stratagene; PSVK3, pBPV, pMSG and pSVL, which are available from Pharmacia and also available from Invitrogen as pEF1 / V5, pcDNA3.1, and pRc / CMV2. Other suitable vectors are those readily known to those skilled in the art.

Any strong promoter known to those skilled in the art can be used to express the MPIF-1 polynucleotide sequence. Suitable promoters include, but are not limited to, adenovirus promoters such as the adenovirus main late promoter; Or heterologous promoters such as the cytomegalovirus (CMV) promoter; Respiratory syncytial virus (RSV) promoters, inducible promoters such as MMT promoter, metallothionein promoter; A thermal shock promoter; Albumin promoter; ApoAI promoter; A human globin promoter, a viral thymidine kinase promoter such as a herpes simple thymidine kinase promoter, a retroviral LTR; b-actin promoter; And human growth hormone promoters. These promoters may also be natural promoters of MPIF-1.

Unlike other gene therapy techniques, the primary advantage of introducing a naked nucleic acid sequence into a target cell lies in the transitional nature of polynucleotide synthesis in the cell. The introduction of non-replicating DNA sequences into cells has been shown to produce certain polypeptides for periods of up to six months.

MPIF-1 polynucleotide constructs can be used for the treatment of muscle, skin, brain, lung, liver, spleen, bone marrow, thymus, heart, lymph, blood, bone, cartilage, pancreas, kidney, gallbladder, stomach, , The nervous system, the eye, the mammary gland, and connective tissues. The remodeling space of this tissue is an intercellular fluid mucopolysaccharide matrix in the reticular fibers of long-term tissues, elastic fibers of blood vessels or chamber walls, collagen fibers of fibrous tissues, or a matrix of connective tissue that surrounds muscle cells, Lt; / RTI > Similarly, the space is occupied by lymphatic fluid in circulating plasma and lymphatic channels. The reason why it is preferable to be transported to the open space of the muscle tissue will be described later. They can be conveniently delivered by injection into tissues containing these cells. They are preferably delivered and permanently expressed, but progressed without dividing the differentiated cells. However, delivery and expression can also take place in undifferentiated or totally undifferentiated cells such as blood stem cells or dermal fibroblasts. In vivo, muscle cells are competitive in terms of their capacity to degrade and express polynucleotides.

When injecting a naked nucleic acid sequence, the effective dose of DNA or RNA is in the range of about 0.05 mg / kg (body weight) to about 50 mg / kg. The dose is preferably about 0.005 mg / kg to about 20 mg / kg, more preferably about 0.05 mg / kg to about 5 mg / kg. Of course, as will be appreciated by those skilled in the art, this dose will vary with the site of tissue being injected. Suitable and effective dosages of the nucleic acid sequence will be readily determined by those skilled in the art, depending on the symptoms being treated and the route of administration.

The preferred route of administration is parenteral administration which is injected into the reperfusion space of the tissue. However, other parenteral routes may also be used, such as inhalation of aerosol preparations, which are especially carried in lung tissue or bronchial tissue, throat or nasal mucosa. In addition, the naked MPIF-1 DNA construct can be delivered to the artery during angioplasty by a catheter used during the procedure.

These naked polynucleotides include, but are not limited to, direct needle delivery to the delivery site, intravenous injection, topical administration, catheter fusion, or delivery according to methods known in the art, including so-called " do. These delivery methods are well known in the art.

The construct may also be carried by a carrier medium such as a viral sequence, a viral particle, a liposome preparation, a lipofectin, a precipitant, and the like. Such transportation methods are known in the prior art.

In certain embodiments, the MPIF-I polynucleotide construct is complexed in a liposomal formulation. The liposome formulations used in the present invention include cationic (bipolar) formulations, anionic (negative) formulations and neutral formulations. However, cationic liposomes are particularly preferred because charge-intimately charged complexes can form between the cationic liposome and the polyvalent anionic nucleic acid. Cationic liposomes have been shown to modulate intracellular delivery of plasmid DNA (Felgenr et al., Proc. Natl. Acad. Sci. USA 84 : 74137416 (1987), cited by reference); mRNA (Malone et al . , Proc. Natl. Acad. Sci. USA 86 : 60776081 (1989), incorporated herein by reference); Purified transcription factors (Debs et al. , J. Biol. Chem. 265 : 1018910192 (1990), cited for reference).

Cationic liposomes are readily available. For example, N- [12,3-dioleyloxy) -propyl] -N, N, N-triethylammonium (DOTMA) liposomes are particularly preferred and are commercially available from GIBCOBRL under the trade name Lipofectin. (See also Felgener et al., Proc. Natl. Acad Sci. USA 84 : 74137416 (1987), incorporated herein by reference). Other commercially available liposomes include DDAP / DOPE ) And DOTAP / DOPE (Boehringer).

Other cationic liposomes can be prepared from readily available materials using methods known in the art. PCT Publication No. WO 90/11092 (for reference) describes the synthesis of DOTAP (1,2-bis (oleyloxy) -3- (trimethylammonio) propane) liposomes. DOTMA liposome formulations are described in the literature, examples of which are described in P. Felgner et al . , Proc. Natl. Acad. Sci. USA 84 : 74137417, which is incorporated by reference. Liposomes can be prepared from other cationic lipid materials using similar methods.

Similarly, anionic liposomes and neutral liposomes are readily available, for example, from Avanti Polar Lipids (Birmingham, Ala) or can be readily prepared using readily available materials. Such materials include, among others, phosphatidyl, choline, cholesterol, phosphatidylethanolamine, dioleyl phosphatidyl choline (DOPC), diol rayl phosphatidyl glycerol (DOPG), and diol rayl phosphatidyl ethanolamine (DOPE). These materials can also be mixed with the DOTMA and DOTAP starting materials in suitable proportions. Methods for making liposomes using these materials are known in the art.

For example, conventional liposomes can be prepared without the addition or addition of cholesterol using commercially available dioleoylphosphatidylcholine (DOPC), dioleylphosphatidylglycerol (DOPG), and dioleylphosphatidylethanolamine (DOPE) in various combinations can do. Thus, for example, DOPG / DOPC vesicles can be prepared by drying 50 mg of DOPG and DOPC, respectively, in a sonicator vial under a stream of nitrogen gas. The sample is placed under a vacuum pump overnight and hydrated with deionized water the next day. The sample is then sonicated in a capped vial for 2 hours using a Thermal System Model 350 ultrasonic equipped with an inverted cup (bath-type) probe under maximum setting when bath is circulating at 15EC. Alternatively, the negatively charged follicles may be produced in a manner that produces a multilayered platelet without ultrasonication or produces a single lamellar follicle of a size separated by extrusion through a nuclear scar. Other methods known to those skilled in the art can be used.

Liposomes include multilamellar vesicles (MLV), small unilamellar vesicles (SUV), or large unilamellar vesicles (LUV), of which SUVs are preferred. A variety of liposomal nucleic acid complexes are prepared using methods known in the art. See Straubinger et al., Methods of Immonology 101 : 512527 (1983). For example, a MLV containing nucleic acid can be prepared by immersing the phospholipid film on the wall of the glass tube and then hydrating it with a solution of the substance to be encapsulated. Extension sonication for MLV is performed to produce a uniform population of single-plate liposomes to produce SUV. The material to be encapsulated is added to the suspension of preformed MLV and sonicated. If a liposome containing a cationic lipid is used, the dried lipid film is resuspended in an appropriate solution (e.g., sterile water or isotonic buffer solution-10 mM Tris / NaCl), sonicated, and then the preformed liposome is incubated with DNA ≪ / RTI > Liposomes and DNA form highly stable complexes because they bind positively charged liposomes to cationic DNA. SUVs are used with small nucleic acid fragments. LUV is produced by a number of methods known in the art. Commonly used methods include the use of Ca ^+2- EDTA chelation (Papahadjopoulos et al. , Biochem. Biophys. Acta 394 : 483 (1975); Wilson et al. , Cell 17 : USA 76 : 3348 (1979), Fraey et al. , Proc. Natl. Acad. Sci. USA , Biochem. Biophys . Acta 443 : 629 (1976); Ostro et al. , Biochem. Biophys. ; And detergent dialysis (Enoch, H and Strittmatter, P., Proc. Natl. Acad. Sci. USA 76 : 145 (1979)) and reverse phase evaporation (REV) (Fraley et al., J. Biol . Chem. USA, 75 : 145 (1978); SchaefeRidder et al., Science 215 : 166 (1982)), which is incorporated herein by reference).

Generally, the ratio of DNA: liposomes ranges from about 10: 1 to about 1:10, and the ratio is preferably from about 5: 1 to about 1: 5. Particularly preferably the ratio is from about 3: 1 to about 1: 3, most preferably the ratio is about 1: 1.

U.S. Patent No. 5,676,954 (incorporated herein by reference) relates to injecting a hereditary material complexed with a cationic liposome carrier into a rat. U.S. Patent Nos. 4,897,355, 4,946,787, 5,049,386, 5,459,127, 5,589,466, 5,693,622, 5,580,859, 5,703,055 and International Publication No. WO 94/9469 And cationic lipids for use in transfection to animals. U.S. Patent Nos. 5,589,466, 5,693,622, 5,580,859, 5,703,055, and WO 94/9469 (references) provide a method of delivering DNA-cationic lipid complexes to mammals.

In a particular embodiment, the cells are processed in vitro or in vivo using RNA-containing retroviral particles containing sequences encoding MPIF-1. Retroviruses from which the retroviral plasmid vectors are derived include, but are not limited to, Leukemia viruses of Molloni, spleen necrosis virus, Loss sarcoma virus, Graves' sarcoma virus, avian leukemia virus, platelet monkey leukemia virus, human immunodeficiency virus, Myeloproliferative sarcoma virus and mammalian tumor virus.

Transduction of the packaging cell line with the retroviral plasmid vector results in the production of a producer cell line. Examples of packaging cells that can be transfected include but are not limited to PE501, PA317, R-2, R-AM, PA12, T19-14X, VT-19-17-H2, RCRE, 86, GP + envAm12, and DNA cell lines (Miller, Human Gene Therapy 1 : 5-14 (1990), for reference). The vector may be capable of transducing the packaging cells through any means known in the art. Such means include, but are not limited to, electroporation, use of liposomes, CaPO ₄ precipitation, and the like. As an alternative, the retroviral plasmid vector can be encapsulated in liposomes or coupled to lipids and then administered to the host.

The producer cell line produces infectious retroviral vector particles containing a polynucleotide encoding MPIF-1. Such retroviral vector particles can be used to transduce eukaryotic cells in vivo or in vitro. The transduced eukaryotic cells express MPIF-1.

As a specific embodiment, the cells are processed in vitro or in vivo with the MPIF-1 polynucleotide contained in the adenoviral vector. Adenovirus is engineered to encode and express MPIF-1 and at the same time inactivated in terms of its capacity to replicate into the normal lytic virus life cycle. Adenovirus expression alleviates concerns about insertional mutations because viral DNA is made without insertion into the host cell chromosome.

In addition, adenovirus has been used for many years as an enteric live vaccine, resulting in an excellent stability profile (Schwartz, AR et al., Am. Rev. Respir. Dis. 109 : 233-238 (1974)). Finally, adenovirus mediated gene transfer has been demonstrated in a number of cases, including the transfer of alpha 1 antitrypsin and CFTR to the lungs of curtain rats (Rosenfeld, MAR et al., Science 252 : 431-434 (1991); Rosenfeld et al. Cell 68 : 143-155 (1992)). In addition, extensive studies of attempts to produce adenovirus as a causative agent in human cancers have been collectively negative (Green, M. et al., Proc. Natl. Sci. USA 76 : 6606 (1979)).

Suitable adenoviral vectors useful in the present invention are described, for example, in Kozarsky and Wilson, Curr. Opin. Genet. Devel. 3 : 499-503 (1993); Rosenfeld et al., Cell 68 : 143-155 (1992); Engelhard et al . , Human Genet. Ther 4 : 759-769 (1993); Yang et al. , Nature Genet. 7 : 362-369 (1994); Wilson et al ., Nature 365 : 691-692 (1993); And U.S. Patent No. 5,652,224, the disclosures of which are incorporated herein by reference. For example, adenoviral vector Ad2 is useful because it can be propagated in human 293 cells. These cells contain the E1 region of the adenovirus and constitutively express Ela and Elb, which complements the defective adenovirus by providing a product of the deleted gene in the vector. In addition to Ad2, other anenoviruses (e.g., Ad3, Ad5, and Ad7) are usefully employed in the present invention.

Adenoviruses used in the present invention are replication defects. Replication-deficient adenoviruses require the assistance of helper viruses and / or packaging cell lines to form infectious particles. The resulting virus can infect the cells and can express the target polynucleotide operably linked to the promoter, but it can not be replicated in most cells. Replication-deficient adenovirus may be deleted from all or a portion of one or more of the following genes: E1a, E1b, E3, E4, E2a, or L1 to L5.

In certain embodiments, the cells are processed in vitro or in vivo using adeno-associated virus (AAV). AAV is a natural acid deficient virus that requires helper viruses to produce infectious particles (Muzyczka, N., Curr. Topics in Microbiol. Immunol. 158 : 97 (1992)). It is also one of the few viruses that can insert the DNA into unpartitioned cells. A vector containing as little as 300 base pairs of AAV can be packaged and integrated, but the space of exogenous DNA is limited to about 4.5 kb. Methods for producing and using such AAV are known in the art. See, for example, U.S. Patent Nos. 5,139,941, 5,173,414, 5,354,678, 5,436,146, 5,474,935, 5,478,745, and 5,589,377.

For example, suitable AAV vectors for use in the present invention include all sequences required for DNA replication, inducibility, and host-cell integration. MPIF-1 polynucleotide constructs are inserted into AAV vectors using standard cloning techniques, such as Sambrook et al., Molecular Cloning: A Laboratory Manual , Cold Spring Harbor Press (1989). The recombinant AAV vector is then transfected into packaging cells infected with helper virus using any standard technique, including lipofection, electroporation, calcium phosphate precipitation, and the like. Suitable helper viruses include adenovirus, cytomegalovirus, vaccinia virus or herpes virus. Once the packaging cells are transfected and infected, they produce infectious AAV virus particles containing the MPIF-I polynucleotide construct. These viral particles are used to transduce eukaryotic cells in vitro or in vivo. The transduced cells contain the MPIF-I polynucleotide construct inserted into the genome and express MPIF-1.

Other gene therapies are those that operatively associate a heterologous control region with homologous recombination and an endogenous polynucleotide sequence (encoding for MPIF-1) (e. G., U.S. Patent No. 5,641,670, June 24, 1997 WO96 / 29411, filed September 26, 1996, International Publication No. WO 94/12650, filed August 4, 1994, Koller et al . , Proc. Natl. Acad. Sci. USA 86 : 89328935 (1989); Zijlstra et al ., Nature 342 : 435438 (1989)). This method involves the activation of genes that are present in the target cell but are not normally expressed in the cell or are expressed at a level lower than necessary.

Polynucleotide constructs are made using standard techniques known in the art, which contain a promoter and the target sequence is adjacent to the promoter. Suitable promoters are described herein. The target sequence is sufficiently complementary to the endogenous sequence to allow homologous recombination of the promoter-target sequence by the endogenous sequence. The promoter is operably linked to the endogenous sequence through homologous recombination since such a target sequence is sufficiently close to the 5 ' end of MPIF-1, the desired endogenous polynucleotide sequence.

Promoters and target sequences can be amplified using PCR. It is preferable that the amplified promoter contains a restriction enzyme which is distinguished on the 5 ' and 3 ' The 3 'end of the first target sequence contains the same restriction site as the 5' end of the extended promoter and the 5 'end of the second target sequence contains the same restriction site as the 3' end of the extended promoter. The amplified promoter and target sequence are digested and ligated together.

The promoter-target sequence construct is delivered to the cell either as a naked polynucleotide or with the aforementioned transfection-facilitator (liposome, viral sequence, viral particle, whole virus, lipofectin, precipitant, etc.). The P promoter-target sequence may be delivered by any method, for example, by direct needle injection, intravenous injection, topical administration, catheter injection, particle accelerators, and the like. These methods are described in detail below.

The promoter-target sequence construct is degraded by the cell. When homologous recombination between constructs and endogenous sequences occurs, endogenous MPIF-1 sequences are placed under promoter control. The promoter then directs expression of the endogenous MPIF-1 sequence.

Polynucleotides encoding MPIF-1 may be administered with other polynucleotides encoding angiogenic proteins. Angiogenic proteins include, but are not limited to, acid and basic fibroblast growth factors, VEGF-1, VEGF-2, VEGF-3, epithelial growth factor alpha and beta, platelet-derived endothelial growth factor, , Tumor necrosis factor alpha, hepatocyte growth factor, insulin-like growth factor, colony stimulating factor, macrophage colony stimulating factor, granulocyte / macrophage colony stimulating factor and nitrite oxide synthase.

The polynucleotide encoding MPIF-1 contains a secretory signal sequence that facilitates secretion of the protein. Generally, the signal sequence is located at the 5 'end of the coding region or at the coding region of the polynucleotide to be expressed at this end. This signal sequence may be homologous or heterologous to the polynucleotide of interest and the cell to be transfected. In addition, the signal sequence may be chemically synthesized using methods known in the art.

Any mode of administration of the polynucleotide constructs described above can be used as long as the mode expresses a sufficient amount of one or more molecules to provide a therapeutic effect. (E. G., A " gene gun "), a gelfoam sponge reservoir, other commercially available storage material, an osmotic pump (e.g., Alzmani pump), an oral (Tablet or ophthalmic) pharmaceutical preparations of solid suppositories, and sloping or topical administration at the time of surgery. For example, direct injection of naked calcium phosphate precipitated plasmid into the liver and spleen of rats or direct injection of the protein-coated plasmid into the context produces gene expression of the foreign gene in the liver of rats (Kaneda et al., Science 243 : 375 (1989)).

The preferred method of topical administration is by direct infusion. The recombinant molecules of the invention complexed with the carrier medium are administered either directly to the arterial site or topically. It is preferred to topically inject the composition in centimeters, preferably in millimeters, at the arterial site.

As a topical administration method, the polynucleotide construct of the present invention is brought into contact with a surgical wound site or its periphery. For example, the patient may experience surgery and the polynucleotide construct may be coated on the tissue surface in the wound site or the structure may be injected into the tissue site of the wound site.

Therapeutic compositions usefully employed for systemic administration include the recombinant molecules of the invention complexed with the target delivery vehicle of the present invention. Suitable delivery vehicles for use in systemic administration include liposomes containing a ligand for targeting the media to a specific site.

Preferred modes of systemic administration include intravenous injection, aerosol, oral and percutaneous (topical) delivery. Intravenous injection may be carried out using standard methods known in the art. Aerosol delivery can be carried out using standard methods known in the art (Stribling et al., Proc. Natl. Acad. Sci. USA 189 : 11277-11281, which is incorporated by reference). Oral delivery can be performed by complexing the polynucleotide construct of the present invention with a carrier capable of withstanding digestion by the digestive enzymes in the intestine of an animal. Examples of such carriers are plastic capsules or tablets, such as those known in the art. Topical delivery can be performed by mixing the polynucleotide construct of the invention with a lipophilic reagent (e.g., DMSO) that can penetrate into the skin.

Determining the effective amount of the component to be delivered will depend on, for example, the chemical structure and biological activity of the component, the age and weight of the animal, the exact symptoms and severity requiring treatment, and the route of administration. The number of treatments depends on a number of factors, such as the amount of polynucleotide construct administered per dose, as well as the health and medical history of the patient. The exact amount, frequency of dosing, and timing of dosing may be determined by the relevant surgeon or veterinarian.

The therapeutic compositions of the present invention may be administered to any animal, preferably a mammal and a bird. Preferred mammals are humans, dogs, cats, rats, rabbits, sheep, horses, and pigs, of which humans are particularly preferred.

Antisense and ribozymes (antagonists). As a preferred embodiment of the present invention, the antagonist of the present invention may be a nucleic acid corresponding to SEQ ID NO: 1 or 6, or a complementary strand thereof, and / or a nucleic acid corresponding to the nucleotide sequence contained in the deposited clone 75676. As a preferred embodiment, the antisense sequence is internally generated by the organism, or in another embodiment the sequence is administered separately (O'Connor, J. Neurochem 56 : 560 (1991)); Oligodeoxynucleotides as Antisense Inhibitors of Gene Expression, CRC Press, Boca Raton, FL (1988); See oligodeoxynucleotides, CRC Press, Boca Raton, FL (1988) as antisense inhibitors of gene expression. Antisense technology can be used to control gene expression by forming antisense DNA or RNA or triple helix. Antisense technology is described by Okano, J. Neurochem, 56 : 560 (1991); Oligodeoxyneucleotides as Antisense Inhibitors of Gene Expression, CRC Press, Boca Raton, FL (1988). The formation of triple helix is described, for example, by Lee et al., Nucleic Acids Research 6 : 3073 (1979); Cooney et al. Science 241 : 456 (1988); And Dervan et al., Science 251 : 1300 (1991). The method is based on binding polynucleotides to complementary DNA or RNA.

For example, the use of c-myc and c-myb antisense RNA constructs to inhibit the proliferation of non-lymphoid constitutive leukemia cell line HL-60 and other cell lines has been previously disclosed (Wickstrom et al., (1988); Anfossi et al. 1989). These experiments were performed in vitro by incubating the cells and incubating with the ribonucleotides. A similar procedure suitable for in vivo use is disclosed in WO 91/15580. Briefly, a pair of oligonucleotides for a given antisense RNA is prepared as follows: the sequence complementary to the first 15 bases of the open reading frame is contiguous with the EcoRI site on the 5 'end and the 3'Gt; HindIII < / RTI > Next, one pair of oligonucleotides was heated at 90 ° C for 1 minute and then quenched in 2X binding buffer (20 mM Tris HCl pH 7.5, 10 mM MgCl ₂ , 10 mM dithiothreitol (DTT) and 0.2 mM (ATP) Binds to the EcoRI / HindIII site of the viral vector PMV7 (WO 91/15580).

For example, the 5 'coding region of a polynucleotide encoding the mature polypeptide of the invention can be used to design antisense RNA of about 10 to 40 base pairs in length. DNA oligonucleotides are designed to be complementary to the gene locus in the transcription therapy to prevent transcription and receptor production. Antisense RNA oligonucleotides hybridize to mRNA in vivo and block the translation of mRNA molecules into receptor polypeptides.

In one embodiment, the MPIF-1 antisense nucleic acid of the present invention is produced intracellularly by transcription from an exogenous sequence. For example, a vector or a portion thereof is transcribed to generate an antisense nucleic acid (RNA) of the present invention. Such vectors may contain sequences encoding the MPIF-1 antisense nucleic acid. Such a vector may be present as a genetic entity that has been transcribed to produce the desired antisense RNA or may be chromosomal integrated. This vector can be prepared by recombinant DNA technology by standard techniques. Vectors are those known in the art for use in the replication and expression of plasmid, viral or vertebrate cells. Expression of the sequence coding for MPIF-I or a fragment thereof can be by any promoter known in the art of working in vertebrate, preferably human, cells. Such promoters may be inducible or structural. These promoters include, but are not limited to, the promoter region (Bernoist and Chambon, Nature 29 : 304-310 (1981) in the SV 40 early promoter region, the promoter contained in the 3 'terminal repeat of the sarcoma virus (Yamamoto et al., Cell 22: 787-797 (1980), the herpes thymidine promoter (Wagner et al . , Proc. Nall. Acad Sci. USA 78 : 1441-1445 (1981)), the regulatory sequence of the metallothionein gene (Brinster et al ., Nature 296 : 39 -42 (1982)).

The antisense nucleic acid sequence of the present invention is complementary to at least a part of the RNA transcription site of the MPIF-1 gene. Absolute complementarity, though desirable, is not necessary. As used herein, the term " sequence complementary to at least a portion of the RNA " refers to a stable duplex as a sequence having sufficient complementarity to be capable of hybridizing with RNA. In the case of double-stranded MPIF-1 antisense nucleic acid, DNA strands of DNA can be tested or triplex formation can be analyzed. Competence for hybridization depends on the complementarity and the degree of length of the antisense nucleic acid. Generally, the longer the hybridization nucleic acid is, the more the base mismatches with the MPIF-1 RNA. This forms a stable duplex (or in the case of a triplex). The skilled artisan will use standard procedures to determine the melting point of the hybridized complexes by ensuring mismatch tolerance.

Oligonucleotides complementary to the 5 ' end of a message, such as the AUG start codon or a 5 ' compared to it, must work most effectively at the restriction site. However, a sequence complementary to the 3 '-dependent sequence of mRNA is also effective in inhibiting the detoxification of mRNA. Wagner, R., 1994, Nature 372 ; 333-335. Therefore, oligonucleotides complementary to the 5'- or 3'-relative non-toxic non-coding regions of MPIF-1 shown in Figure 20 can be used in an antisense approach to inhibit the detoxification of endogenous MPIF-1 mRNA. Oligonucleotides complementary to the 5 'untranslated region of mRNA include AUG-initiated codon complement. Antisense oligonucleotides complementary to mRNA coding regions are ineffective in inhibiting detoxification, but can be used in accordance with the present invention. Regardless of whether the antisense nucleic acid is designed to hybridize to the 5 'or 3' or coding region of MPIF-1 mRNA, the antisense nucleic acid should be at least 6 nucleotides in length and the oligonucleotide should have a length in the range of 6 to about 50 nucleotides desirable. In a particular embodiment, the oligonucleotide is at least 10 nucleotides, at least 17 nucleotides, at least 25 nucleotides, or at least 50 nucleotides.

The polynucleotide of the present invention may be DNA or RNA, or a chimeric mixture or derivative or variant thereof, a naturally occurring chain or a double-stranded chain. The oligonucleotide can modify the base portion, the sugar portion, or the phosphate framework portion to improve the stability of the molecule, the hybridization, and the like. Oligonucleotides may also be used in conjunction with other suspending groups such as peptides (e.g., targeting host cell receptors in vivo), or agents that facilitate transport across cell membranes (e.g., Letsinger et al., Proc. Natl. Acad, Sci. : 648-652 (1987)); (Eg, Krol et al., BioTechniques 6 : 958-976 (1988)) or insert preparations (eg, Zon, et al. Pharm. Res. 5 : 539-549 (1988)). For this purpose, oligonucleotides can be conjugated to other molecules, such as peptides, cross-linking agents for hybridization targets, transfer agents, disintegration of hybridization targets, and the like.

Antisense oligonucleotides include, but are not limited to, 5-fluorouracil, 5-bromouracil, 5-chlorouracil, 5-iodouracil, hypoxanthine, xanthine, 4- acetylcytosine, 5- (carboxyhydroxymethyl) Carboxymethylaminomethyl uracil, dihydrouracil, beta-D-galactosylquinosine, inosine, N-6-isopentenyl adenine, 1-methyl Guanine, 1-methylinosine, 2,2-dimethylguanine, 2-methyladenine, 2-methylguanine, 3-methylcytosine, 5-methylcytosine, N-6- 5-methoxycarboxymethyluracil, 5-methoxyuracil, 2-methylthio-N-6-isopentenylsuccinyl, 5-methoxyaminomethyl-2-thiouracil, Adenine, uracil-5-oxyacetic acid (v), toxotoxin, pseudouracil, quiozine, 2-thiocytosine, 5-methyl-2-thiouracil, 5-oxyacetic acid (v), 5-methyl-2-thiouracil, 3- (3-amino-3-N2-carboxypropyl) uracil, (acp3) w, and 2,6-diaminopurine, and the like.

Antisense oligonucleotides include, but are not limited to, one or more modified sugar moieties selected from the group including arabinose, 2-fluoroarabinose, xylulose and hexose.

In a preferred embodiment, the antisense polynucleotides include, but are not limited to, phosphorothioate, phosphorodithioate, phosphoramidothioate, phosphoramidate, phosphodiesteride, methylphosphonate, alkylphosphate, At least one modified phosphate skeleton selected from the group including phytosteres and phosacetals or the like.

In another preferred embodiment, the antisense oligonucleotide is an a-anomeric oligonucleotide. The a-anomeric oligonucleotides form specific double-stranded hybrids complementary to RNA, wherein the RNAs are stranded against each other in contrast to the bunits (Gautier et al . , Nacl. Acids Res. 15 ; 6625-6641 (1987)). The oligonucleotide may be a 2'-O-methyl ribonucleotide (Inoue et al . , Nacl. Acids. Res. 15 : 6131-6148 (1987)) or a chimeric RNA- DNA analogue (Inoue et al., FEEBS Lett., 215 : 327-330 1987).

Polynucleotides can be synthesized by automated DNA synthesizers according to standard methods known in the art (e.g., commercially available from Biosearch, Applied Biosystems, etc.). For example, phosphorothioate oligonucleotides can be synthesized by the method by Stein et al. (1988, Nacl. Acids Res. 16 : 3209), methylphosphonate oligonucleotides are prepared using a controlled pore glass polymer support (Sarin et al . , Proc. Natl. Acad Sci. USA 85 : 7448-7451 (1988)).

When an antisense nucleotide complementary to the MPIF-1 ciprotein sequence is used, it is most desirable to complement the transcribed dock region.

Possible antagonists according to the present invention include catalytic RNA or ribozymes (e.g., PCT International Publication No. WO 90/11364, published October 4, 1990; Sarver et al. Science 247 : 1222-1225 (1990) The ribozyme that degrades the mRNA in the recognition sequence can be used to disrupt MPIF-1 mRNA, but it is preferable to use a hammerhead ribozyme. The hammerhead ribozyme forms a complementary base pair with the target mRNA The only requirement is that the target mRNA should have the following sequence of two bases: 5'-UG-3 '. Construction of a hammerhead-shaped ribozyme and Production is well known in the art and is described in more detail in Haseloff and Gerlach, Nature 334 : 585-591 (1988). There are a number of possible hammer-like ribozymes within the nucleotide sequence of MPIF-1 (see, B) And the degradation recognition site is located near the 5 'end of MPIF-1 mRNA; that is, it increases the efficiency of non-functional mRNA transcripts and minimizes intracellular accumulation.

As with the antisense approach, the ribosomes of the invention can be composed of modified oligonucleotides (e.g., improved stability, targeting, etc.) and must be delivered to cells expressing MPIF-1 in vivo. A DNA construct encoding a ribosome can be introduced into a cell in the same manner as described above in connection with an antisense entry encoding DNA. A preferred method of delivery involves using DNA constructs to " encrypt " the ribozyme under the control of a strong constitutive promoter such as a pol III or pol II promoter, wherein the transfected cells produce a sufficient amount of ribozymes It inhibits the detoxification by destroying the endogenous MPIF-1 message. Unlike antisense molecules, ribosomes are catalysts, so lower intracellular concentrations are needed in terms of efficiency.

(Ie, in tumor formation or proliferation) by inhibiting cell proliferation and proliferation of the cell polypeptides of the invention on tumor tissue cells and tissues, that is, by stimulating angiogenesis of the tumor Substance / agonist compounds may be used.

Antagonists / agonists are used to prevent hypervascular diseases and to prevent the growth of endogenous lens cells after extracapsular cataract surgery. Inhibiting the mitotic action of the polypeptides of the invention is beneficial in cases such as restenosis after balloon angioplasty.

Antagonists / agonists may be used to treat the disease.

Therefore, the present invention provides a method of treating a disorder or disease, including but not limited to, the treatment of a disorder or disease caused by overexpression of a polynucleotide of the present invention, referred to in this application, ) An antisense molecule relating to the polynucleotide of the present invention and / or (b) a ribozyme relating to the polynucleotide of the present invention.

Epitopes and antibodies

The present invention encompasses an epitope of a polypeptide having the amino acid sequence of SEQ ID NO: 2 or a polynucleotide sequence contained in ATCC Accession No. 75676 under the above-mentioned low stringency condition or stringent hybridization condition, Or an epitope of a polypeptide sequence encoded by a polynucleotide that hybridizes to a complement of the sequence contained in ATCC Deposit No. 75676.

The invention encompasses a polynucleotide sequence encoding an epitope of the polypeptide sequence of the invention (e. G., The sequence described in SEQ ID NO: 1 or 6) at low stringency conditions or stringent hybridization conditions described above, a poly A polynucleotide sequence of the complementary strand of the nucleotide sequence, and a polynucleotide sequence that hybridizes to the complementary strand.

&Quot; Epitope " relates to a portion of a polypeptide having an antigenic or immunogenic activity in an animal, preferably a mammal, and most preferably a human. In a preferred embodiment, the invention comprises a polypeptide containing an epitope as well as a polynucleotide encoding said polypeptide. As used herein, an " immunogenic epitope " is defined as any protein that elicits an antibody response in an animal, as determined by methods known in the art, for example, methods of producing antibodies (e.g., Geysen et al . , Proc. Natl Acad Sci USA 81 : 3998-4002 (1983)). As used herein, the term " antigenic epitope " refers to a portion of an antibody that immunospecifically binds to an antigen measured by a method known in the art. Immunospecific binding excludes non-specific binding but does not inevitably exclude cross-reacting with other antigens. An antigenic epitope does not have to be present.

Fragments that function as epitopes can be prepared by conventional means (e.g., Houghten, Proc. Natl. Acad. Sci. USA, 82 : 5131-5135 (1985), details of which are described in U.S. Patent No. 4,631,211) .

The antigenic epitope in the present invention is preferably at least 4, at least 5, at least 6, at least 7, more preferably at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, At least 20, at least 25, at least 40, at least 50, and most preferably from about 15 to about 30 amino acids. Preferred polypeptides comprising an immunogenic or antigenic epitope comprise at least 10,15,20,25,30,35,40,45,50,55,60,65,70,75,80,85,90,95 or < RTI ID = 0.0 > It has 100 amino acid residues. An additional non-exclusive preferred antigenic epitope comprises an antigenic epitope as well as a portion thereof, as described herein. Antigenic epitopes are usefully used to generate antibodies, including monoclonal antibodies that specifically bind epitopes. Preferred antigenic epitopes include not only the antigenic epitopes described herein but also combinations of 2, 3, 4, 5, or more of these antigenic epitopes. Antigenic epitopes can be used as target molecules in immunoassays (eg, Wilson et al., Cell 37 : 767-778 (1984); Sutcliffe et al., Science 219 : 660-666 (1983)).

Similarly, immunogenic epitopes can induce antibodies according to methods known in the art (see, for example, Sutcliffe et al., Supra; Wilson et al ., Supra; Chow et al., Proc., Natl. 82 : 910-914; Bittle et al ., J. Gen. Virol 66: 2347-2354 (1985)). Preferred immunogenic epitopes include the immunogenic epitopes herein, as well as combinations of 2, 3, 4, 5, or more of these immunogenic epitopes. A polypeptide consisting of one or more immunogenic epitopes or polypeptides comprising the same can be used to elicit an antibody response in an animal (rabbit or mouse) animal together with a carrier protein, such as albumin, or when the polypeptide is long enough (at least about 25 amino acids) , The polypeptide may be used without a carrier. However, it has been found that immunogenic epitopes containing from 8-10 small numbers of amino acids are sufficient to generate antibodies capable of binding the least number of linear epitopes in the denatured polypeptide (e. G., In Western blotting).

The epitope-producing polypeptides of the present invention are used to induce antibodies according to methods well known in the art. Examples of such techniques include, but are not limited to, in vivo immunization, in vitro immunization, phage display methods (Sutcliffe et al. ; Wilson et al. Supra, and Bittle et al., J. Gen. Virol. 66: 234-2354 (1985). When the in vivo immunoassay is used, the animals can be immunized with the free peptide; however, the reverse of the anti-peptide antibody is the ability to couple the peptide to a macromolecular carrier such as Keyhole limpethemhemicin (KLH) or tetanus toxoid To stimulate additional antigens. For example, peptides containing cysteine residues can be coupled to a carrier using a linker such as maleimido benzoyl-N-hydroxysuccinimide ester (MBS), while other peptides use many common binders such as glutaraldehyde To the carrier. Animals such as rabbits, rats, and mice can be injected intraperitoneally and / or intradermally with about 100 [mu] g of peptide or carrier protein and an adjuvant or other adjuvant known to counteract the immune response, ≪ / RTI > Several additional antigen injections can be injected at intervals of about two weeks to provide useful titer of the detectable anti-peptide antibodies, e. G., By ELISA assays using free peptides adsorbed to the solid surface. Reverse transcription of anti-peptide antibodies in serum from immunized animals can be increased by screening for anti-peptide antibodies (e. G., Adsorption to peptides on a solid support) and by eluting the selected antibodies according to methods known in the art.

Those skilled in the art will be able to fuse the polypeptides of the invention containing an immunogenic or antigenic epitope as described above to other polypeptide sequences. For example, by fusing a polypeptide of the present invention to immunoglobulins (IgA, IgE, IgG, IgM), the fixed domain or a portion _{(CH 1, CH 2, CH} 3, or a portion of the water and their any combination thereof) thereof in the chimeric A polypeptide can be produced. Such fusion proteins facilitate purification and increase half-life in vivo. This is evidenced in the case of chimeric proteins consisting of the first two domains of the human CD4-polypeptide and the various immobilized domains of the heavy or light chain of the mammalian immunoglobulin. EP 394,827; Traunecker et al ., Nature, 331 : 84-86 (1988). Increased delivery of antigen across the endothelial barrier to the immune system is evidenced in the case of an antigen conjugated to an FcRn binding partner such as an IgG or Fc fragment (see, e.g., PCT Publication No. WO 96/22024 and WO 99/04813) . IgG fusion proteins with dimeric structures of disulfide-linkages due to disulfide bonds in the IgG region have also been found to be more efficient in binding and neutralizing other molecules than monomeric polypeptides or fragments thereof. Fountoulakis et al. , J. Biochem. 270 : 3958-3964 (1995). The nucleic acid encoding the epitope may also recombine with an epitope tag (e.g., a hematoglutinin (" HA ") tag or a flag tag) to aid in the detection and purification of the expressed polypeptide. For example, the system by Janknecht et al. Facilitates the purification of nondenaturing fusion proteins expressed in human cell lines (see Janknecht et al., 1991, Proc. Natl. Acad. Sci. USA 88 : 8972-897). In such a system, the gene of interest is subcloned into the vaccine recombinant plasmid and the open reading frame of the gene is fused to an amino-terminal tag consisting of six histidine residues. Such a tag functions as a matrix-binding domain of a fusion protein. Extracts from cells infected with recombinant Vaccinia virus can be eluted using a Ni ⁺² nitroacetic acid-agarose column and a histidine-tagged protein, optionally with an amidazole-containing buffer.

Additional fusion proteins of the invention may be generated through the techniques of gene-shuffling, motif-shuffling, exon-shuffling, and / or codon-shuffling (collectively referred to as " DAN shuffling & . DNA shuffling can be used to tune the activity of the polypeptides of the invention, which can produce polypeptides with altered activity, as well as working substances and antagonists of the polypeptides. U.S. Patent No. 5,605,793; 5,811, 238; 5,830, 721; 5,834,252; And 5,837, 458 and Patten et al., Curr. Opinion Biotechnol. 8 : 724-33 (1997); Harayama, Trends Biotechnol 16 (2) ; 76-82 (1998); Hansson et al. , J. Mol. Biol. 287 : 265-76 (1999); And Lorenzo and Blasco, Biotechniques 24 (2); 3-8-13 (1998) (each of these patents and publications are hereby incorporated by reference). In one embodiment, variants of SEQ ID NOs: 1 or 6 and polypeptides encoded by these polynucleotides can be made by DNA shuffling. In another embodiment, modification of the polynucleotide encoding the polypeptide shown in SEQ ID NO: 2 and modification of the polypeptide encoded by these polynucleotides can be achieved by DNA shuffling. DNA shuffling uses an assembly of two or more DNA segments by homologous recombination or site-specific recombination to produce a modification in the polynucleotide sequence. In another embodiment, polynucleotides or encoded polypeptides of the invention can be modified by random-mutagenesis by error-prone PCR, random nucleotide insertion or other methods prior to recombination. In another embodiment, one or more components, motifs, cross-sections, fragments, domains, fragments, etc. of a polynucleotide encoding a polypeptide of the present invention may comprise at least one component, motif, cross-section, A fragment or the like can be recombined.

Antibody: Another polypeptide of the present invention relates to the polypeptide, polypeptide fragment, or variant of SEQ ID NO: 2 of the present invention, and / or antibody and T-cell antigen receptor (TCR) that immunospecifically binds to an epitope As measured by immunoassays well known in the art for antibody-antigen binding assay. The antibodies of the present invention can be used for the production of antibodies by polyclonal antibodies, monoclonal antibodies, multispecific antibodies, human antibodies, humanized antibodies or chimeric antibodies, single chain antibodies, Fab fragments, F (ab ' But are not limited to, the resulting fragment, an anti-gen (anti-Id) antibody (e.g., an anti-Id antibody to an antibody of the invention) and an epitope-binding fragment of any of the foregoing. The term " antibody ", as used herein, refers to a molecule comprising an immunoglobulin molecule and an immunologically active portion of the immunoglobulin molecule, i. E., An antigen binding site that immunospecifically binds to the antigen. Immunoglobulin molecules of the present invention, any type of (e.g., IgG, IgE, IgM, IgD , IgA and IgY), any class _{(e.g., IgG 1, IgG 2, IgG} 3, IgG 4, IgA 1 and IgA ₂₎ Or subclass of immunoglobulin molecules.

It is most preferred that the antibody is a human antigen-binding antibody fragment of the present invention, and is selected from the group consisting of Fab, Fab 'and F (ab') ₂ , Fd, single chain Fvs (scFv), single chain antibodies, disulfide bonded Fvs (sdFv) V _L, or V _H domains. Antigens, including the single chain antibody-binding antibody fragments can be included with any or all of the sole of the variable region (s), or a hinge region, CH _1, CH ₂ and CH ₃ domains. The invention also hinge region, CH _1, CH ₂ and CH ₃ antigen to include any combination of domains and a variable region (s) includes a binding fragment thereof. The antibodies of the present invention may be derived from animals, including birds and mammals. Preferably, the antibody is a human antibody, a murine (e.g., mouse and rat) antibody, a donkey antibody, a monoclonal antibody, a rabbit antibody, a goat antibody, a guinea pig antibody, a camel antibody, a horse antibody or a chicken antibody. As used herein, a " human " antibody comprises an antibody having the amino acid sequence of a human immunoglobulin, and may be derived from a human immunoglobulin library, or from an animal that is transformed with one or more human immunoglobulins and does not express an endogenous immunoglobulin Isolated antibodies, as described in, for example, U.S. Patent No. 5,939,598 to Kucherlapati et al.

The antibodies of the present invention may be monospecific, bispecific, triple specific or more specific. Multispecific antibodies may be specific for the different epitopes of the polypeptides of the invention or may be specific for both the polypeptides of the invention and heterologous epitopes (e.g., heterologous polypeptides or solid support materials). See, for example, PCT Publication WO 93/17715; WO 92/08802; WO 91/00360; WO 92/05793; Tutt et al., J. Immunol. 147: 60-69 (1991); U.S. Patent No. 4,474,893; 4,714,681; 4,925,648; 5,573,920; 5,601, 819; Kostelny et al., J. Immunol. 148: 1547-1553 (1992).

The antibodies of the present invention may be described or embodied as epitope (s) or part (s) of a polypeptide of the invention in which they recognize or specifically bind. The epitope (s) or polypeptide moiety (s) may be represented as described herein, for example, at the N-terminal and C-terminal positions, the size of the adjacent amino acid residues, or as described in the tables and figures. Antibodies that specifically bind to any epitope or polypeptide of the invention may be excluded. Accordingly, the present invention includes an antibody that specifically binds to the polypeptide of the present invention, and it is also possible to exclude the antibody.

In addition, the antibodies of the present invention can be described or embodied in terms of their cross-reactivity. The invention includes antibodies that do not bind any other analogs, orthologs, or homologs of the polypeptides of the invention. The identity of the polypeptide of the present invention is at least 95%, 90%, 85%, 80%, 75%, 70%, 65%, 60%, 55%, 50% (As calculated using the methods described herein and known in the art). As a specific example, the antibodies of the invention cross-react with human proteins and their corresponding epitope rats, rats and / or rabbit homologues. Less than 95%, less than 85%, less than 80%, less than 75%, less than 70%, less than 65%, less than 60%, less than 55%, less than 50% identity to the polypeptide of the present invention Lt; RTI ID = 0.0 > and / or < / RTI > calculated using the methods described herein). In one embodiment, the cross-reactivity described above includes any single specific antigenic polypeptide or immunogenic polypeptide, or two, three, four, five, or six of the specific antigenic and / or immunogenic polypeptides disclosed herein (S) above. An antibody that binds to a polypeptide encoded by a polynucleotide that hybridizes under stringent hybridization conditions (as described herein) to a polynucleotide of the invention is also encompassed by the present invention. The antibodies of the invention may also be described or embodied in a binding affinity for the polypeptides of the invention. The preferred binding affinity or dissociation constant, that is, Kd is ^{5 x 10 -2 M, 10 -2} M, 5 x 10 -3 M, 10 -3 M, 5 x 10 -4 M, 10 -4 M, 5 x 10 - ^{5 M, 10 -5 M, 5} x 10 -6 M, 10 -6 M, 5 x 10 -7 M, 10 -7 M, 5 x 10 -8 M, 10 -8 M, 5 x 10 -9 M ^{, 10 -9 M, 5 x 10} -10 M, 10 -10 M, 5 x 10 -11 M, 10 -11 M, 5 x 10 -12 M, 10 -12 M, 5 x 10 -13 M, 10 ^-13 M, 5 x 10 ^-14 M, 10 ^-14 M, 5 x 10 ^-15 M, and 10 ^-15 M or less.

The present invention also provides antibodies that competitively inhibit binding of an antibody to an epitope of the invention by any competitive binding assay known in the art, such as immunoassays described herein. In a preferred embodiment, the antibody competitively inhibits binding to epitopes greater than 95%, greater than 90%, greater than 85%, greater than 80%, greater than 75%, greater than 70%, greater than 60%, or greater than 50%.

The present invention also provides an antibody that competitively inhibits the binding of a monoclonal antibody to a polypeptide of the invention, preferably a polypeptide of SEQ ID NO: 2. Competitive inhibition can be measured using any method known in the art, such as competitive binding assays described herein. In a preferred embodiment, the antibody competitively inhibits binding of the monoclonal antibodies of the invention to the polypeptide of SEQ ID NO: 2 at least 90%, at least 80%, at least 70%, at least 60%, at least 50%.

The antibodies of the present invention may act as agonists or antagonists of the polypeptides of the invention. For example, the invention encompasses antibodies that partially or completely destroy receptor / ligand interactions with the polypeptides of the invention. Preferably, an antibody of the invention binds to an antigenic epitope disclosed herein or a portion thereof. The present invention is characterized by both receptor-specific and ligand-specific antibodies. The invention also features a receptor-specific antibody that does not interfere with ligand binding, but interferes with receptor activation. Receptor activation (i. E., Signal transduction) may be described herein, or may be determined by techniques known in the art. For example, receptor activation can be measured by immunoprecipitation followed by Western blot analysis (e.g., as described above) to detect phosphorylation of the receptor or receptor substrate (e.g., tyrosine or serine / threonine). In one embodiment, the antibody is provided to inhibit ligand activity or receptor activity of greater than 95%, 90%, 85%, 80%, 75%, 70%, 60%, 50% do.

The invention also features antibodies recognizing receptor-ligand complexes, as well as receptor-specific antibodies that both interfere with ligand binding and receptor activation, and which do not specifically recognize unbound ligand or unbound ligand. The invention also encompasses neutralizing antibodies that bind to a ligand and prevent binding of the ligand to the receptor, as well as antibodies that inhibit binding of the ligand to the receptor by binding to the ligand to inhibit receptor activation. Antibodies that activate the receptor are also encompassed by the present invention. Such antibodies act as receptor agonists, e. G., By enhancing or activating all or a portion of the biological activity of ligand mediated receptor activation, e.g., by inducing dimerization of the receptor. Antibodies can be described as being agonists, antagonists or inverse agonists of biological activity including the specific biological activity of the peptides of the invention disclosed herein. The above antibody agents can be produced using methods known in the art. See, for example, PCT Publication WO 96/40281; U.S. Patent No. 5,811,097; Deng et al., Blood 92 (6) : 1981-1988 (1998); Chen et al ., Cancer Res. 58 (16) : 3668-3678 (1998); Harrop et al., J. Immunol. 161 (4) 1786-1794 (1998); Zhu et al ., Cancer Res. 58 (15) : 3209-3214 (1998); Yoon et al., J. Immunol. 160 (7) : 3170-3179 (1998); Prat et al ., J. Cell. Sci. 111 ( Pt 2 ) : 237-247 (1998); Pitard et al., J. Immunol. Methods 205 (2) : 177-190 (1997); Liautard et al., Cytokine 9 (4) : 233-241 (1997); Carlson et al ., J. Biol. Chem. 272 (17) : 11295-11301 (1997); Taryman et al., Neuron 14 (4) : 755-762 (1995); Muller et al., Structure 6 (9) : 1153-1167 (1998); See Bartunek et al., Cytokine 8 (1) : 14-20 (1996), all of which are incorporated herein by reference.

The antibodies of the present invention can be used to purify, detect, and target the polypeptides of the invention, including, but not limited to, both in vitro and in vivo diagnostic and therapeutic methods. For example, antibodies are used in immunoassays to qualitatively and quantitatively measure polypeptide levels of the invention in biological samples. See, for example, Harlow et al., Antibodies: A Laboratory Manual , (Cold Spring Harbor Laboratory Press, 2nd ed. 1988), incorporated herein by reference.

As described in more detail below, the antibodies of the present invention may be used alone or in combination with other compositions. The antibody may further be fused recombinantly to the heterologous polypeptide at the N- or C-terminus, or may be chemically conjugated (e. G., Covalent and noncovalent) to the polypeptide or other composition. For example, the antibodies of the invention may be fused or conjugated recombinantly to molecules useful as labels and effector molecules in detection assays, such as heterologous polypeptides, drugs, radionuclides or toxins. See, for example, PCT Publication WO 92/08495; WO 91/14438; WO 89/12624; See U.S. Pat. No. 5,314,995 and EP 396,387.

Antibodies of the invention include derivatives in which the modified, i.e., covalent, bond is modified by covalently attaching any type of molecule to the antibody such that the antibody does not interfere with the generation of an anti-genotypic response. By way of non-limiting example, the antibody derivatives may be modified by, for example, glycosylation, acetylation, pegylation, phosphorylation, amidation, derivatization with known protecting / blocking groups, proteolytic cleavage, binding to cellular ligands or other proteins, Lt; / RTI > Any of a number of chemical modifications may be performed by known techniques including, but not limited to, specific chemical cleavage, acetylation, formylation, and metabolic synthesis of tonicamycin and the like. In addition, derivatives may include one or more non-typical amino acids.

The antibodies of the present invention can be produced by any suitable method known in the art. Polyclonal antibodies to the desired antigen can be produced by a variety of procedures well known in the art. For example, the polypeptides of the present invention can be administered to a variety of host animals, including, but not limited to, rabbits, mice, rats, and the like, to induce the production of serum, including antigen-specific polyclonal antibodies. Various adjuvants may be used to increase the immune response depending on the host species and adjuvants include Freund ' s (complete and incomplete), mineral gels (e.g., aluminum hydroxide), surface active substances (e.g., lysolecithin), pluronic Potentially useful adjuvants such as polyols, polyanions, peptides, oil emulsions, keyhole limpet hemocyanin, dinitrophenol and human adjuvants [e.g., BCG; bacille Calmette-Guerin, and Corynebacterium parvum. < / RTI > Such adjuvants are also well known in the art.

Monoclonal antibodies can be prepared using a variety of techniques known in the art, including hybridoma, recombinant technique and phage display technique, or a combination thereof. For example, monoclonal antibodies are known in the art and are described in, for example, Harlow et al., Antibodies: A Laboratory Manual (Cold Spring Harbor Laboratory Press, 2nd ed., 1988; Hammerling et al., Monoclonal Antibodies and T- Including Hybridoma techniques, including those taught in Cell Hybridomas 563-681 (Elsevier, NY, 1981), which references are incorporated herein by reference. As used herein, the term " monoclonal Quot; mononuclear antibody " is not limited to antibodies produced by hybridoma techniques. The term " monoclonal antibody " refers to any antibody that is derived from a single clone, including any eukaryotic cell clone, prokaryotic cell clone or phage clone. Refers to an antibody, which is not limited by the method of production.

Methods for producing and screening specific antibodies using hybridoma techniques are conventional and are well known in the art and are described in detail in the Examples herein (e.g., Examples 22 and 30). As a non-limiting example, mice can be immunized with the polypeptides of the invention or cells expressing the peptides. When an immune response is detected, for example, an antigen-specific antibody is detected in mouse serum, mouse spleen is recovered and spleen cells are isolated. The spleen cells are then fused to any appropriate myeloma cells (e. G., Cells derived from the cell line SP20 available from the ATCC) by well known techniques. The hybridomas are screened and cloned with limited dilution. The hybridoma clones are then analyzed to find cells that secrete antibodies capable of binding to the polypeptides of the invention in a manner known in the art. Mice carrying a positive hybridoma clone can be immunized to generate multiple liquids, typically containing high levels of antibody.

Accordingly, the present invention provides a method for producing a monoclonal antibody and an antibody produced by the method, which method comprises culturing a hybridoma cell secreting the antibody of the invention, To obtain a hybridoma clone that secretes an antibody capable of binding to the polypeptide of the present invention. The hybridoma produced from the fusant is then harvested to find a hybridoma clone that secretes an antibody capable of binding to the polypeptide of the present invention. Lt; / RTI >

Antibody fragments that recognize a specific epitope can be generated by known techniques. For example, the Fab and F (ab ') ₂ fragments of the present invention can be prepared using enzymes such as papain (to produce a Fab fragment) or pepsin (to produce an F (ab') ₂ fragment) It can be produced by proteolytic cleavage. F (ab ') ₂ fragments contain the variable region, the light chain constant region and the heavy chain of the CH ₁ domain.

For example, antibodies of the invention may be generated using a variety of phage display methods known in the art. In the phage display method, functional antibody domains are displayed on the surface of a phage particle carrying a polynucleotide sequence encoding them. In one embodiment, such phages can be used to display the antigen binding domain expressed in a repertoire or in a combinatorial antibody library (e. G., Human or murine animal). The phage expressing the antigen binding domain that binds to the desired antigen can be screened or identified, for example, using a labeled antigen, or an antigen bound or captured on a solid surface or beid. Phages used in these methods are typically filamentous phage containing the fd and M13 binding domains expressed from a phage having an Fab, Fv or a disulfide stabilized Fv antibody domain fused recombinantly to phage gene III or gene VIII protein . Examples of pegylation methods that can be used to prepare antibodies of the present invention are described by Brinkman et al., J. Immunol. Methods 182: 41-50 (1995); Ames et al., J. Immunol. Methods 184: 177-186 (1995); Kettleborough et al ., Eur. J. Immunol. 24: 952-958 (1994); Persic et al. Gene 187: 9-18 (1997); Burton et al., Advances in Immunology 57: 191-280 (1994); PCT Application No. PCT / GB91 / 01134; PCT Publication WO 90/02809; WO 91/10737; WO 92/01047; WO 92/18619; WO 93/11236; WO 95/15982; WO 95/20401; And U.S. Patent No. 5,698,426; 5,223,409; 5,403,484; 5,580, 717; 5,427,908; 5,750,753; 5,821,047; 5,571, 698; 5,427,908; 5,516, 637; 5,780,225; 5,658,727; 5,733, 743 and 5,969, 108, each of which is incorporated herein by reference.

As described in the above references, the antibody coding regions from the phage can be separated after phage screening to be used to generate whole antibodies, including human antibodies, or any other desired antigen-binding fragments, Animal cells, insect cells, plant cells, yeast, and bacteria. For example, techniques for recombinantly producing Fab, Fab 'and F (ab') ₂ fragments can also be performed using methods known in the art, examples of which include PCT Publication WO 92/22324; Mullinax et al., BioTechniques 112 (6): 864-869 (1992); Sawai et al., AJRI 34: 26-34 (1995), and Better et al., Science 240: 1041-1043 (1988), which references are incorporated herein by reference.

Examples of techniques that can be used to produce monoclonal Fvs and antibodies are described in U.S. Patent Nos. 4,946,778 and 5,258,498; Huston et al., Methods in Enzymology 203: 46-88 (1991); Those described in Shu et al. PNAS 90: 7995-7999 (1993) and Skerra et al. Science 240: 1038-1040 (1988). For some uses, including in vivo uses of antibodies in the human body and in vitro detection assays, it is preferred to use chimeric, humanized or human antibodies. Chimeric antibodies are molecules in which different portions of the antibody are derived from different animal species, such as antibodies having a variable region derived from a murine monoclonal antibody and a human immunoglobulin constant region. Methods for producing chimeric antibodies are known in the art. See, for example, Morrison, Science 229: 1202 (1985), incorporated herein by reference; Oi et al., BioTechniques 4: 214 (1986); Gillies et al., J. Immunol. Methods 125: 191-202 (1989); U.S. Patent No. 5,807,715; 4,816, 567 and 4,816, 397. A humanized antibody is an antibody molecule from a non-human species antibody that binds to a target antigen having at least one complementarity determining region (CDR) derived from a non-human species and at least one structural region derived from a human immunoglobulin molecule. Often, structural residues within the human structural region can be replaced with corresponding residues from the CDR donor antibody to alter, preferably improve, the antigen binding capacity. These structural substitutions can be determined by methods well known in the art, for example, by modeling the interaction of structural residues with CDRs to identify structural residues important for antigen binding and comparing sequences to identify specific structural residues at specific sites (See, e.g., U.S. Pat. No. 5,585,089 to Queen et al., Nature 332: 323 (1988) to Riechmann et al ., The disclosures of which are incorporated herein by reference). For example, CDR-grafting (EP 239, 400; PCT publication WO 91/09967; US 5,225,539; 5,530,101 and 5,585,089), veneering or resurfacing (EP 592,106; EP 519,596; Padlan, Molecular Immunology 28 Studinicka et al., Protein Engineering 7 (6): 805-814 (1994); Roguska et al., PNAS 91: 969-973 (1994)); The antibody may be humanized using a variety of techniques known in the art, including chain reordering (US Patent No. 5,565,332).

Complete human antibodies are particularly desirable for therapeutic treatment of human patients. Human antibodies can be prepared by a variety of methods known in the art, including the phage display methods described above, using antibody libraries derived from human immunoglobulin sequences. This is also described in U.S. Patent Nos. 4,444,887 and 4,716,11; And PCT publications WO 98/46645, WO 98/50433, WO 98/24893, WO 98/16654, WO 96/34096, WO 96/33735 and WO 91/10741, each of which is incorporated herein by reference References are cited for reference.

Human antibodies can also be produced using transgenic mice that are not capable of expressing functional endogenous immunoglobulin, but are capable of expressing human immunoglobulin genes. For example, human heavy and light chain immunoglobulin gene complexes can be introduced into mouse embryonic stem cells via random or homologous recombination. Alternatively, in addition to human heavy and light chain genes, human variable regions, constant regions, and diversity regions can be introduced into mouse embryonic stem cells. The mouse heavy chain and light chain immunoglobulin genes may be disrupted simultaneously or simultaneously with the introduction of a human immunoglobulin locus by homologous recombination. In particular, homozygous deletion of the JH region prevents endogenous antibody production. Modified embryonic stem cells are amplified and microinjected into blastocysts to produce chimeric mice. The chimeric mice then produce homozygous offspring that express human antibodies. Transgenic mice are immunized in the usual manner with selected antigen, e.g., all or part of the polypeptide of the invention. Monoclonal antibodies directed against this antigen can be obtained from immunized transgenic mice using conventional hybridoma techniques. Human immunoglobulin transgenes possessed by transgenic mice are rearranged during B cell differentiation and then undergo class switching and somatic mutation. Thus, using this technique it is possible to produce therapeutically useful IgG, IgA, IgM and IgE antibodies. Examples of references for an overview of techniques for producing such human antibodies are described in Lonberg and Huszar, Int. Rev. Immunol. 13: 65-93 (1995). For a more detailed description of human antibody and human monoclonal antibody production techniques and such antibody production protocols, see, for example, PCT Publication WO 98/24893; WO 92/01047; WO 96/34096; WO 96/33735; European Patent 0 598 877; U.S. Patent No. 5,413,923; 5,625,126; 5,633,425; 5,569,825; 5,661, 016; 5,545,806; 5,814,318; 5,885, 793; 5,916,771 and 5,939,598, the disclosures of which are incorporated herein by reference. In addition, techniques similar to those described above can be used from companies such as Abgenix, Inc. (Freemont, Calif.) And Genpharm (San Jose, CA) to provide human antibodies directed against selected antigens.

Complete human antibodies that recognize a selected epitope can be generated using a technique called " induced screening ". In this method, selected non-human monoclonal antibodies, such as mouse antibodies, are used to induce selection of whole human antibodies that recognize the same epitope (Jespers et al., Bio / technology 12: 899-903 .

In addition, antibodies to the polypeptides of the invention can be used to generate anti-genotypic antibodies that "mimic" the polypeptides of the invention using techniques well known to those skilled in the art (see, eg, Greenspan & Bona, FASEB J 7 (5): 437-444 (1989)] and the method disclosed in Nissinoff [J. Immunol 147 (8) :. 2429-2438 (1991)] reference). For example, by " mimicking " the multimerization and / or binding domain of a polypeptide using an antibody that binds to the polypeptide of the present invention and competitively inhibits the polypeptide multimerization and / or binding of the polypeptide and ligand, Lt; RTI ID = 0.0 > and / or < / RTI > ligands thereof. Neutralization of anti-gen or anti-gen Fab fragments can be used for treatment qjq to neutralize polypeptide ligands. For example, anti-genotypic antibodies can be used to block the biological activity of the polypeptide of the present invention by binding to the polypeptide and / or ligand / receptor thereof.

The term " binding of a polypeptide of the invention to a ligand " means that the ligand polypeptide of the invention binds to a receptor; The receptor polypeptide of the present invention binds to the ligand; The antibody of the present invention binds to an antigen or epitope; The antigen or epitope of the present invention binds to the antibody; The antibody of the present invention binds to an anti-genotype antibody; The anti-genotype antibody of the present invention binds to the ligand; The anti-genotype antibody of the present invention binds to a receptor; But are not limited to, binding of an anti-anti-genotype antibody of the present invention to a ligand, receptor or antibody.

As another example, a polypeptide binding domain and / or an activation domain may be mimicked using an antibody that binds to and competitively activates a polypeptide of the present invention or a ligand thereof and, as a result, binds to the polypeptide and / RTI ID = 0.0 > anti-genotype < / RTI > Activating Fab fragments of anti-genotype antibodies or anti-genotype antibodies can be used in therapy for activating polypeptide ligands. For example, the biological activity of such an anti-genotype antibody can be activated by binding to the polypeptide of the present invention to activate its biological activity and / or bind to the ligand / receptor of the polypeptide of the present invention.

Polynucleotides Encoding Antibodies: The present invention further provides polynucleotides comprising or consisting of nucleotide sequences encoding the antibodies or fragments thereof of the invention. The present invention also encompasses an antibody that specifically binds to a polypeptide of the present invention, preferably a polypeptide of SEQ ID NO: 2, under conditions of stringent hybridization or under stringent hybridization conditions And a polynucleotide that hybridizes to a polynucleotide encoding an antibody that binds to a polypeptide having an amino acid sequence.

The polynucleotide can be obtained by any method known in the art, and the nucleotide sequence of the polynucleotide can be determined by any method known in the art. For example, where the nucleotide sequence of an antibody is known, the polynucleotide encoding the antibody may be assembled from chemically synthesized oligonucleotides (see, for example, Kutmeier et al., BioTechniques 17: 242 (1994) This method involves briefly synthesizing a superimposing oligonucleotide comprising a sequence part encoding an antibody, annealing and ligating the oligonucleotide, and amplifying the ligated oligonucleotide by PCR.

Alternatively, a polynucleotide encoding the antibody can be generated from a nucleic acid from an appropriate source. A clone containing a nucleic acid encoding a specific antibody can not be obtained, but the sequence of the antibody molecule may be PCR amplified using a synthetic primer capable of hybridizing to the 3 ' and 5 ' ends of the antibody coding sequence , Or by using an oligonucleotide probe specific for a particular gene sequence to chemically synthesize the nucleic acid encoding the immunoglobulin, for example, by identifying a cDNA clone from a cDNA library encoding the antibody, or by chemically synthesizing an appropriate source preferably a polyA ⁺ RNA, cDNA library generated from a cDNA library, i. e., any tissue or cell expressing the antibody (e. g., hybridoma cells selected to express the antibody of the invention) ≪ / RTI > The amplified nucleic acid generated by PCR can then be cloned into a cloning vector that can be cloned using any method well known in the art.

Once the amino acid sequence corresponding to the nucleic acid sequence of the antibody is determined, the nucleotide sequence of the antibody can be manipulated (for example, by using a nucleotide sequence manipulation method well known in the art, such as recombinant DNA technique, site- induced mutagenesis, (See, for example, Current Protocols in Molecular Biology , John Wiely, et al., Supra), as described in Sambrook et al., Molecular Cloning, A Laboratory Manual, 2d Ed , Cold Spring Harbor Laboratory, Cold Spring Harbor &Amp; Sons, NY (1998)], antibodies with different amino acid sequences such as amino acid substitutions, deletions and / or insertions can be generated.

In one embodiment, the amino acid sequences of the heavy and / or light chain variable domains are examined to determine the complementarity determining regions (CDRs) in a manner well known in the art, e.g., by comparison with known amino acid sequences of other heavy and light chain variable regions, The sequence and the hypervariable region can be determined. Using conventional recombinant DNA techniques, non-human antibodies can be humanized by inserting one or more of the CDRs into the structural region, such as the human structural region, as described above. (See, for example, Chothia et al ., J. Mol. Biol. 278: 457-479 (1998) for a list of human structural domains) Reference). Preferably, a polynucleotide generated by combining a structural region and a CDR encodes an antibody that specifically binds to the polypeptide of the present invention. Preferably, as described above, one or more amino acid substitutions can be introduced into the structural region, preferably amino acid substitutions improve binding of the antibody to the antigen. In addition, using the above methods, one or more variable region cysteine residues involved in a chain disulfide bond can be amino acid substituted or deleted to produce antibody molecules lacking one or more of the chain disulfide bonds. Other variations on polypeptides are also encompassed by the present invention and are within the skill of the art.

In addition, a technique designed to produce a " chimeric antibody " by splicing a gene derived from a mouse antibody molecule having an appropriate antigen specificity with a gene derived from a human antibody molecule having an appropriate biological activity (Morrison et al ., Proc. .. Natl.Acad Sci 81: 851-855 ( 1984); et al., Neuberger [Nature 312: 604-608 (1984 )]; et Takeda [Nature 314: 452-454 (1985 )]) to be used have. As described above, a chimeric antibody is a molecule derived from a different animal species in which different parts are different. Examples thereof include those having a variable region derived from a murine animal mAb and a human immunoglobulin constant region (e.g., a humanized antibody) .

Alternatively, techniques described for the production of single chain antibodies (U.S. Patent No. 4,946,778; et Bird [Science 242: 423-42 (1988 )]; et al., Huston [Proc Natl Acad Sci USA 85 :.... 5879-5883 (1988) and Ward et al. Nature 334: 544-54 (1989)). The single chain antibody is formed so that the heavy chain fragment and the light chain fragment of the Fv region are linked through an amino acid bridge to form a single chain polypeptide. Techniques for assembling functional Fv fragments in E. coli can also be used (Skerra et al., Science 242: 1038-1041 (1988)].

Method of Producing Antibodies : The antibodies of the present invention can be prepared using any antibody synthesis methods known in the art, particularly chemical synthesis or preferably recombinant expression techniques.

For the recombinant expression of the antibody of the present invention, or a fragment, derivative or analogue thereof (e.g., a heavy or light chain of the antibody of the present invention or a single chain antibody of the present invention), an expression vector comprising a polynucleotide encoding an antibody is constructed There is a need. Upon obtaining a polynucleotide encoding the heavy or light chain of the antibody molecule or antibody of the invention, or a portion thereof (including preferably the heavy or light chain variable domain), the recombinant DNA technique using techniques well known in the art A vector for the production of the molecule can be produced. Thus, a method for producing a protein by expressing a polynucleotide comprising an antibody encoding a nucleotide sequence is described in detail herein. Methods well known in the art can be used to construct expression vectors containing antibody coding sequences and appropriate transcriptional and translational control sequences. Examples of such methods include in vitro recombinant DNA techniques, synthetic techniques, and in vivo gene recombination. Accordingly, the invention provides a replicable anti-vector comprising a nucleotide sequence encoding an antibody molecule of the invention, or heavy or light chain, or heavy or light chain variable region thereof, operably linked to a promoter. Such a vector may comprise a nucleotide sequence encoding a constant region of an antibody molecule (see, for example, PCT Publication No. WO 86/05807; PCT Publication No. WO 89/01036 and U.S. Patent No. 5,122,464) Or as a vector for the expression of a light chain.

The expression vector is transferred to host cells by conventional techniques, and the transfected cells are cultured by conventional techniques to produce the antibody of the present invention. Accordingly, the invention includes a host cell comprising an antibody of the invention, or heavy or light chain thereof, operably linked to a heterologous promoter, or a polynucleotide encoding the single chain antibody of the invention. In a preferred embodiment for the expression of the heavy chain antibody, a vector encoding both the heavy chain and the light chain as described below can be simultaneously expressed in the host cell for expression of the whole immunoglobulin molecule.

A variety of host expression vector systems can be used to express the antibody molecule of the invention. Such a host expression system will not only represent an agent that allows the desired coding sequence to be produced and subsequently purified, but will also allow the antibody molecule of the invention to be expressed in situ when transfected or transfected with a suitable nucleotide coding sequence Lt; / RTI > These include microorganisms such as recombinant bacteriophage DNA, plasmid DNA or bacteria transformed with cosmid DNA expression vectors (eg, E. coli, B. subtilis), including antibody coding sequences; Yeast transfected with a recombinant yeast expression vector containing an antibody coding sequence (e.g., Saccharomyces, Pichia); An insect cell system infected with a recombinant viral expression vector (e.g., baculovirus) containing an antibody coding sequence; A plant cell system transformed with a recombinant plasmid expression vector (e.g., a Ti plasmid) infected with a recombinant virus expression vector (e.g., cauliflower mosaic virus [CaMV], tobacco mosaic virus [TMV] ; Or a recombinant expression construct comprising a promoter derived from a genome of a mammalian cell (e.g., a metallothionein promoter) or a mammalian virus (e.g., adenovirus late promoter; vaccinia virus 7.5K promoter) But are not limited to, mammalian cell systems (e.g., COS, CHO, BHK, 293, 3T3 cells). Preferably recombinant antibody molecules are expressed using eukaryotic cells, particularly in the case of the expression of whole recombinant antibody molecules, especially bacterial cells such as Escherichia coli. For example, using mammalian cells, such as Chinese hamster ovary cells (CHO), with vectors such as the major intermediate early gene promoter elements from human cytomegalovirus is an effective antibody expression system (Foecking et al. Gene 45 : 101 (1986); Cockett et al., Bio / Technology 8: 2 (1990).

In bacterial systems, multiple expression vectors can be advantageously selected depending on the intended use for the antibody molecule being expressed. For example, if it is necessary to produce a large amount of protein for the production of a pharmaceutical composition of an antibody molecule, a vector that induces a high level expression of the fusion protein product that is easily purified may be preferable. Such vectors include the E. coli expression vector pUR278 (Ruther et al., EMBO J. 2: 1), which allows the fusion of the antibody coding sequences into the vector with the lac Z coding region, 1791 (1983)); pIN vectors (Inouye & Inouye, Nucleic Acids Res. 13: 3101-3109 (1985); Van Heeke & Schuster, J. Biol. Chem. 24: 5503-5509 (1989) But is not limited thereto. The pGEX vector can also be used to express external polypeptides as a fusion protein with glutathione S-transferase (GST). In general, such fusion proteins are soluble and can be easily purified from the dissolved cells by adsorbing and binding to the matrix glutathione-agarose beads and then eluting the free glutathione. The pGEX vector is designed to include a thrombin or factor Xa protease cleavage site, allowing the cloned target gene product to separate from the GST site.

In insect systems, Autographa californica nuclear polyhedrosis virus (AcNPV) is used as a vector for expressing an external gene. The virus grows in Spodoptera frugiperda cells. The antibody coding sequence can be cloned individually into non-essential regions of the virus (e.g., the polyhedrin gene) and placed under the control of an AcNPV promoter (e.g., a polyhedrin promoter).

Many viral expression systems can be used in mammalian host cells. When adenovirus is used as an expression vector, the desired antibody coding sequence can be ligated to an adenovirus transcription / translation regulatory complex, such as a late promoter and a 3-part leader sequence. This chimeric gene can then be inserted into the adenovirus genome in vitro or in vivo. Insertion into the non-essential regions of the viral genome (e. G., Regions El or E3) allows the recombinant virus to survive in an infected host and express the antibody molecule (see, e . G. , Logan & Shenk, Proc. Natl. Acd. USA 81: 355-359 (1984)). A specific initiation signal is also required for efficient translation of the inserted antibody coding sequence. Such a sequence includes the ATG start codon and the adjacent sequence. In addition, the initiation codon should be aligned with the reading frame of the target coding sequence for complete translation of the entire insert. These exogenous translational control sequences and origins of the initiation codon can vary and are both natural and synthetic. Expression efficiency can be increased by including appropriate transcription enhancer components, transcription termination sites, etc. (see Bittner et al ., Methods in Enzymol. 153: 51-544 (1987)).

In addition, host cell lines can be selected that modulate the expression of the inserted sequences, or modify and process the gene product in the desired specific manner. Modifications (eg, glycosylation) and processing (eg, cleavage) of protein products can be important for protein function. Different host cells retain a characteristic and specific mechanism for post-translational processing and modification of proteins and gene products. Appropriate cell lines or host systems can be selected to ensure correct transformation and processing of the expressed external protein. To this end, eukaryotic host cells harboring cell organs for proper processing of the primary transcript, glycosylation, and phosphorylation of the gene product can be used. Such mammalian host cells include but are not limited to CHO, VERY, BHK, Hela, COS, MDCK, 293, 3T3, WI38 and in particular breast cancer cell lines (e.g., BT483, Hs578T, HTB2, BT20 and T47D) And Hs578Bst). ≪ / RTI >

Stable expression is desirable for producing recombinant proteins with high yields over a long period of time. For example, a cell line stably expressing an antibody molecule can be processed. Rather than using an expression vector containing a viral origin of replication, it may contain DNA regulated by appropriate expression control elements (e.g., promoters, enhancers, sequences, transcription termination sites, polyadenylation sites, etc.) The host cell can be transformed with DNA that is regulated with a selectable marker that does not. After the introduction of the external DNA, the processed cells are grown in an enriched medium for 1 to 2 days and transferred to a selection medium. Selectable markers in recombinant plasmids have resistance to selection, allowing the cells to stably integrate the plasmid into their chromosome, allowing them to grow to form a center, which can then be cloned and expanded into cell lines. This method can be advantageously used to process cell lines expressing antibody molecules. Such engineered cell lines may be particularly useful for screening and evaluation of compounds that interact directly or indirectly with antibody molecules.

A number of screening systems can be used, such as the herpes simplex virus thymidine kinase (Wigler et al., Cell 11: 223 (1977)), hippocactin-guanine phosphoribosyltransferase (Szybalska & Szybalski, , Hgprt-, or aprt-cells, respectively, by using the gene of the present invention [ Proc. Natl. Acad Sci. USA 48: 202 (1992)] and adenine phosphoribosyltransferase (Lowy et al., Cell 22: In addition, metabolic antagonistic resistance can be used as a screening criterion for the following genes: dhfr confers resistance to methotrexate (Wigler et al ., Natl. .. Acad Sci USA 77: 357 (1980)]; et O'Hare [Proc Natl Acad Sci USA 78 :.... 1527 (1981)]; gpt imparts resistance to mycophenolic acid (Mulligan &Amp; Berg, Proc. Natl. Acad. Sci. USA 78: 2072 (1981)]; neo is an aminoglycoside G-418 Impart a tolerance [Clinical Pharmacy 12: 488-505]; Wu and articles of Wu [Biotherapy 3: 87-95 (1991 )];... Et Tolstoshev [Ann Rev. Pharmacol Toxicol 32: 573-596 ( Rev. Biochem. 62: 191-217 (1993); May, 1993, TIB TECH 11 (1993)); Mulligan, Science 260: 926-932 (1993); and Morgan and Anderson, (5): 155-215); hygro confers resistance to hygromycin (Santerre et al. Gene 30147 (1984).) Screening recombinant clones for methods generally known in the art of recombinant DNA technology ( Current Protocols in Molecular Biology , John Wiley & Sons, NY (1993)]; Ausubel et al .; Kriegler, GeneTransfer and Expression, A Laboratory Manual , Stockton Press, NY (1990); And Dracopoli et al., Current Protocols in Human Genetics, John Wiley & Sons, NY (1994); Colberre-Garapin et al ., J. Mol. Biol. 150: 1 (1981), the disclosures of which are incorporated herein by reference.

The level of expression of the antibody molecule can be increased by vector amplification (for an overview see Bebbington and Hentschel, " The Use of Vectors for Expression of Cloned Gene in Mammalian Cells " in DNA Cloning , Vol. Academic Press, New York, 1987). If the marker in the vector system expressing the antibody is amplifiable, the copy number of the marker gene will be increased if the level of inhibitor present in the host cell culture is increased. Since the amplified region is associated with the antibody gene, the production of the antibody will also be increased (Crouse et al . Mol. Cell. Biol. 3: 257 (1983)).

The host cell may be co-transfected with the two expression vectors of the invention, a first vector encoding the heavy chain derived polypeptide and a second vector encoding the light chain derived polypeptide. The two vectors may contain the same selectable marker that allows the same expression of the heavy and light chain polypeptides. Alternatively, a single vector can be used that encodes and expresses both the heavy and light chain polypeptides. In this case, the light chain should be placed in front of the heavy chain to avoid excess toxic glass heavy chain (Proudfoot, Nature 322: 52 (1986); Kohler, Proc. Natl. Acad. Sci. USA 77: 2197 1980). Encoding sequences for heavy and light chains may include cDNA or genomic DNA.

When the antibody molecule of the present invention is produced by an animal, chemically synthesized, or recombinantly expressed, it can be purified by any method known in the art for purification of immunoglobulin molecules, such as chromatography (e.g., ion exchange chromatography , Affinity chromatography, in particular affinity chromatography and size-fractionated column chromatography by affinity to specific antigens after protein A), centrifugation, differential solubility, or any other standard protein purification technique have. In addition, the antibodies or fragments thereof of the invention can be fused to heterologous polypeptide sequences to facilitate purification by methods described herein or by other methods known in the art.

The invention encompasses a method of recombinantly recombining polypeptides of the invention (or portions thereof, preferably 10, 20, 30, 40, 50, 60, 70, 80, 90 or more than 100 amino acids of the polypeptide) (Including covalent and noncovalent bonds) to produce a fusion protein. Fusion does not necessarily occur directly, but can occur through linker sequences. The antibody may be specific for an antigen other than a polypeptide of the invention (or a portion thereof, preferably 10, 20, 30, 40, 50, 60, 70, 80, 90 or more than 100 amino acids of the polypeptide). For example, antibodies can be used to target polypeptides of the invention to specific cell types by fusing or conjugating the polypeptides of the present invention to antibodies specific for particular cell surface receptors, either in vitro or in vivo. Antibodies fused or conjugated to the polypeptides of the present invention can be used in in vitro immunoassay and purification methods using methods known in the art. For example, Harbor et al., PCT Publication WO 93/21232; EP 439,095; Naramura et al., Immunol. Lett. 39: 91-99 (1994); U.S. Patent No. 5,474,981; Gillies et al. PNAS 89: 1428-1432 (1992); Fell et al., J. Immunol. 146: 2446-2452 (1991), the disclosures of which are incorporated herein by reference.

Conjugates and chelates: The present invention further comprises compositions comprising a polypeptide of the invention fused or conjugated to an antibody domain outside the variable region. For example, a polypeptide of the invention may be fused or conjugated to an antibody Fc region, or a portion thereof. The antibody portion fused to a polypeptide of the invention may comprise a constant region, a hinge region, a CH ₁ domain, a CH ₂ domain and a CH ₃ domain, or any combination of the entire domain or portions thereof. Polypeptides can also be fused or conjugated to the antibody portion to form multimers. For example, an Fc portion fused to a polypeptide of the invention can form a dimer through a disulfide bond between the Fc portions. Polypeptides can be fused to portions of IgA and IgM to produce larger multimeric forms. Methods of fusing or conjugating the polypeptides of the present invention to the antibody portion are known in the art. Examples of references to this include U.S. Patent Nos. 5,336,603; 5,622,929; 5,359,046; 5,349,053; 5,447,852; 5,112,946; EP 307,434; EP 367,166; PCT Publication WO 96/04388; WO 91/06570; Ashkenazi et al ., Proc. Natl. Acad. Sci. USA 88: 10535-10539 (1991); Zheng et al., J. Immunol. 154: 5590-5600 (1995) and Vil et al ., Proc. Natl. Acad. Sci. USA 89: 11337-11341 (1992), the disclosures of which are incorporated herein by reference.

As described above, a polypeptide corresponding to the polypeptide of SEQ ID NO: 2, a polypeptide fragment, or a variant thereof may be fused or conjugated to the antibody portion to increase the in vivo half-life of the polypeptide, or may be determined using methods known in the art It can be used for immunoassay. In addition, the polypeptide corresponding to SEQ ID NO: 2 can be fused or conjugated to the antibody portion to facilitate purification. One reported example describes a chimeric protein consisting of the first two domains of human CD4-polypeptide and the various domains of the heavy or light chain constant regions of mammalian immunoglobulins (EP 394,827; Traunecker et al., Nature 331: 84 The polypeptides of the present invention that have been fused or conjugated to antibodies (by IgG) having a disulfide-linked dimer structure can also be used to bind and neutralize other molecules than secreted monomeric proteins or protein fragments alone (Fountoulakis et al., J. Biochem., 270: 3958-3964 (1995)]. In many cases, the Fc portion of the fusion protein is useful in therapy and diagnosis and thus may exhibit, for example, improved pharmacokinetic properties (EP A 232,262) Alternatively, it may be desirable to express the fusion protein, detect and purify it, and then delete the Fc portion. For example, In drug development, human proteins such as hIL-5 are fused with the Fc portion for high-throughput screening assays to identify antagonists of hIL-5 (See Bennett et al., J. Molecuar Recognition 8: 52-58 (1995); Johanson et al., J. Biol. Chem. 270: 9459-9471 (1995)).

In addition, the antibody of the present invention, or fragments thereof, can be fused to a marker sequence such as a peptide to facilitate purification. In a preferred embodiment, the marker amino acid sequence is a hexa-histidine peptide, such as a tag provided in a pQE vector (QIAGEN, Inc. 9259, 97511 CATHEWS, Eaton Avenue, CA), many of which are commercially available. Gentz et al ., Proc. Natl. Acad. Sci. USA 86: 821-824 (1989), for example, hexa-histidine makes it possible to conveniently purify the fusion protein. Other peptide tags useful for purification include the " HA " tag (Wilson et al., Cell 37: 767 (1984)), which corresponds to an epitope derived from influenza hemagglutinin protein, and the & no.

As described above, antibodies specifically binding to one or more epitopes of MPIF-1 are included in the present invention, and antibodies that specifically bind to any epitope or polypeptide of the present invention may be excluded. Thus, the antibody may be specific for MPIF-1, or may be specific for a polypeptide or epitope other than MPIF-1.

The invention also encompasses antibodies or fragments thereof conjugated to a diagnostic agent or therapeutic agent. Antibodies can be used for diagnostic purposes, e.g., to monitor the development or progression of a tumor, e.g., as part of a clinical testing procedure, for example, to determine the effectiveness of a particular therapy. Antibodies can be bound to a detectable substance to facilitate detection. Examples of detectable materials include various enzymes, subcomponents, fluorescent materials, luminescent materials, bioluminescent materials, radioactive materials, positron emitting metals using various positron emission X-ray tomography, and non-radioactive paramagnetic metal ions. The detectable substance may be conjugated or conjugated to the antibody (or fragment thereof) directly or indirectly using techniques known in the art using an intermediate (e. G., A linker known in the art). For metal ions that can be conjugated to antibodies for use as diagnostic agents in accordance with the present invention, see, for example, U.S. Patent No. 4,741,900. Examples of suitable enzymes include horseradish peroxidase, alkaline phosphatase, beta-galactosidase, or acetylcholinesterase, and examples of suitable solubility complexes include streptavidin / biotin and avidin / biotin, Examples of suitable fluorescent materials include umbelliferone, fluorescein, fluorescein isothiocyanate, rhodamine, dichlorotriazinylamine fluorescein, dysyl chloride or picoerythrin, an example of a luminescent material is luminol, Examples of bioluminescent materials include luciferase, luciferin, and acousin, and examples of suitable radioactive materials include ¹²⁵ I, ¹³¹ I, ¹¹¹ In, or ⁹⁹ Tc.

In addition, the antibody or fragment thereof can be conjugated to therapeutic moieties such as cytotoxic agents, cell growth inhibitors or cytotoxins such as cell death agents, therapeutic agents or radioactive metal ions (e.g. alpha-emitters such as ²¹³ Bi) have. A cytotoxin or cytotoxic agent includes any substance harmful to the cell. Examples include but are not limited to paracetaxol, cytochalasin B, gramicidin D, ethidium bromide, emetine, mitomycin, etoposide, tenofoside, vincristine, vinblastine, colchicine, doxorubicin, daunorubicin, di Hydroxyquinoxaline, glucocorticoids, procaine, tetracaine, lidocaine, propranolol, and furomycin, and analogues or homologues thereof. have. Therapeutic agents include, but are not limited to, metabolic antagonists (e.g., methotrexate, 6-mercaptopurine, 6-thioguanine, cytarabine, 5-fluorouracil dicarbazine), alkylating agents (e. G., Mechlorethamine, (DDP), cis-dichlorodiamine platinum (II) (DDP), melphalan, carmustine (BSNU) and rhomustine (CCNU), cyclotaspamide, Cisplatin), anthracyclines (e.g., daunorubicin [former name: daunomycin] and doxorubicin), antibiotics (such as dactinomycin [formerly actinomycin], bleomycin, mitramycin and anthramycin ), And anti-mitotic agents (e.g., vincristine and vinblastine).

The conjugates of the invention can be used to modify a given biological response and the therapeutic agent or drug moiety should not be considered to be limited to classical chemical therapeutic agents. For example, the drug moiety may be a protein or polypeptide that possesses the desired biological activity. Examples of such proteins include toxins such as abrin, lysine A, Pseudomonas exotoxin, or diphtheria toxin; Alpha, TNF-beta, AIM I (see International Publication WO < RTI ID = 0.0 > (WO), < / RTI > (International Publication WO 97/34911), Fas ligand (Takahashi et al ., Int. Immunol ., 6: 1567-1574 (1994)), VEGI (International Publication WO 99/23105 (&Quot; IL-1 "), interleukin-2 (" IL-2 "), , Interleukin-6 ("IL-6"), granulocyte macrophage colony stimulating factor ("GM-CSF"), granulocyte colony stimulating factor ("GM-CSF") or other growth factors.

Antibodies can also be attached to solid supports, which are particularly useful for immunoassays or for purification of target antigens. Such solid supports include, but are not limited to, glass, cellulose, polyacrylamide, nylon, polystyrene, polyvinyl chloride, or polypropylene.

In addition, MPIF polypeptides, variants, fragments, agonists and antagonists thereof, can be conjugated to diagnostic agents or agents such as those described herein or well known in the art. MPIF conjugates can be used for diagnostic or therapeutic purposes as described herein and well known in the art.

For example, MPIF can be conjugated to radioisotopes and used for diagnosis or therapy.

Techniques for conjugating such therapeutic moieties to antibodies are well known and are described, for example, in Arnon et al., &Quot; Monoclonal Antibodies For Immunotargeting Of Drugs In Cancer Therapy, & quot ; ed. Monoclonal Antibodies And Cancer Therapy , Reisfeld, Alan R. Liss, Inc (1985), pp. 243-56; Hellstrom et al., &Quot; Antibodies For Drug Delivery, " in Controlled Drug Delivery (Second Edition), Robinson et al., Marcel Dekker, Inc. (1987), pp. 633-53; Thorpe, " Antibody Carriers Of Cytotoxic Agents In Cancer Therapy: A Riview ", Monoclonal Antibodies '84: Biological And Clinical Applications, Ed. Pinchera et al. (1985), pp. 475-506; Radiolabeled Antibody In Cancer Therapy ", in Monoclonal Antibodies For Cancer Detection And Therapy , edited by Baldwin et al., Academic Press (1985), pp.303-16; " Analysis, Results, and Future Prospective Of The Therapeutic Use Of Radiolabeled Antibody In Cancer Therapy "; And Thorpe et al., Immunol. Rev. 62 : 119-58 (1982).

Antibodies, proteins, and small molecules comprising the polypeptides of the present invention can be used as targeting and pretargeting molecules. Such molecules of the present invention can be radiolabeled by methods well known to those skilled in the art, including but not limited to antibody for targeted radioimmunotherapy and radiolabeled chelation of the antibody phage library. See, for example, DeNardo et al., Clin. Cancer Res . 5 (10S): 3213s-3218s (1999)); Quadri et al., QJ Nucl. Med . 42: 250-261 (1998). Each of which is incorporated herein by reference.

For chelation, different chemical bonds can be inserted between the antibody and the radiolabeled chelate compound. A radiolabeled monoclonal antibody that is reactive with the target antigen can selectively transmit cytotoxic or diagnostic radioisotopes to malignant cells in vivo. Construction of the pre-targeting molecule can be provided by utilizing the diversity and flexibility of the antibody gene. A variety of sequences of single-chain antibody fragments (i.e., scFvs) that are reactive with the target antigen can be selected from the unimmunized phage antibody library. ScFvs can also be cloned directly from the hybridoma for subsequent manipulations, such as the construction of a phage library that facilitates affinity maturation and modification of specificity. ScFvs affinity for selected specific antigen targets from these sources has demonstrated extensive binding properties. The antibody heavy chain (V (H)) and light chain (V (L)) genes from selected ScFvs can be cloned into a diabody as a cassette. This application is further described in a method for specifically destroying cells by administering the polypeptide of the present invention bound to a toxin or cytotoxic prodrug as described below.

Alternatively, an antibody can be conjugated to a secondary antibody to form an antibody heterozygosity, as described in Segal, U. S. Patent No. 4,676, 980 (incorporated herein by reference).

Antibodies conjugated or untethered to the therapeutic moiety can be used alone or as a therapeutic agent by administration with cytotoxic factor (s) or cytokine (s).

Radiolabelled antibodies, proteins and small molecules are cytotoxic and can be used for targeted radioactive therapy, such as radioactive immunotherapy, or for radioactive immunodetection, such as radioactive immunodiagnosis and radiographic imaging. In order to protect or treat normal tissues, cells, organs, MPIF-1 may be administered before, or during, or after administration of the radiolabeled antibody, protein or small molecule.

As described above, the present invention includes antibodies specifically binding to at least one epitope of MPIF-1, and may exclude antibodies that specifically bind to any epitope or polypeptide of the present invention. Thus, the antibody may be specific for MPIF-1 or specific for an epitope other than MPIF-1. In one embodiment, a radiolabeled antibody, protein or small molecule is administered with the MPIF polypeptide during radiation therapy of cancer or other disease. MPIF polypeptides protect and / or treat normal cells and other normal cells, tissues, organs, such as bone marrow precursors, from pre-, intra-, and post-administration damage of the radiopaque therapeutic.

In another preferred embodiment, the compositions of the present invention are Rituximab (anti -CD20 monoclonal ^{antibody; Rituxan TM), Ibritumomab tiuxetan (} 90 Y , and wherein the null antibody -CD20 mAbs ^{junction; Zevalin TM), Tositumomab (131} I Bexxar ^TM ), Rituximab (Rituxan ^TM ), Trastuzumab (Herceptin ^TM ), OvaRexMoAb (monoclonal antibody to CA 125 of ovarian cancer), Denileukin difitox (diphtheria toxin conjugate; Ontak ), AA98 (a monoclonal antibody that binds to the microvessel system without binding to normal tissue ( Clin. Cancer Res. 5 (suppl): abstract 82 (1999)), Adrecolomab TAG72 glycoprotein), an anti-CEA antibody (conjugated to ¹³¹ I (Behr et al., Amer. Soc. Of Oncol. 35th Annual Meeting, Atlanta, abstract 1025 Anti-VEGF antibody, anti-CXCR4 antibody, CH225 (chimeric anti-EGFR antibody (Mendelsohn et al., Amer. Soc. Of Clin. Oncol. 35th Ann Abstract 1499 (1999)) and ZD 1839 (1999)), CP-358,774 (Karp et al., Amer. Soc. (Both of which are small molecule inhibitors of EGFr), IDEC-Y2B8 (ligated to a molecule that binds ⁹⁰ Y), and the like, as described in Karp et al., J. Clin. Onc. Anti-CD20 antibody, Witzig et al., &Quot; Phase I / II trial of IDEC-Y2B8radioimmunotherapy for tr eatment of relapsed or refractory CD20-positive B-cell non-Hodgkin's lymphoma, "J Clin Oncol 1999 (in press)]; the literature [Blood 1995, such as Bastion; 86:.. 3257-3262] ), 131 I-MN- 14F (ab) ₂ anti-embryonic carcinoma monoclonal antibody (Juweid et al., J. Nucl. Med. 2000 Jan; 41 (1): 93-103; J. Nucl. Med. (Lym-1 is a mouse monoclonal antibody that primarily targets malignant lymphocytes; the chelating agent is bound to ⁶⁷ Cu), ⁶⁷ Cu-2 IT-BAT-Lym- N'N'N''N''-tetraacetic acid (see O'Donnell et al., J. Nucl. Med. 1999 Dec; 40 (12): 2014-20)], direct radioactivity labeling with rhenium-188-labeled anti-NCA antigen antibody BW 250/183 (anti-granulocyte; tris- (2-carboxyethyl) phosphine (TCEP) Seitz et al., Eur. J. Nucl. Med. 1999 Oct; 26 (10): 1265-73)], IDEC-Y2B8 ( ⁹⁰ Y ibritumomab tiuxetan; MX-DTPA chelating radioactive isotope- tiuxetan) and the animals that share binding murine IgG ₁ kappa anti-null antibody -CD20 mAbs (described in Witzig [J. Clin Oncol 1999 Dec; 17 (12..): 3793-803)]), 213 Bi-HuM195 ( wherein -CD33 (Sgouros et al., J. Nucl. Med. 1999 Nov; 40 (11): 1935-46)), ⁶⁷ copper-2-iminothiolane-6- [p- (bromoacetamido) Benzyl] -TETA-Lym-1 ( 90Y-BC-4 (a mouse MAb that recognizes tenascin; a stable MAb conjugate labeled with ⁹⁰ Y is referred to as < RTI ID = 0.0 > D. < / RTI > (ITC-Bz-DTPA) which is a chelate compound of ethylene triamine pentaacetic acid (Riva et al., Clin. Cancer Res. 1999 Oct; 5 ), ¹³¹ I-labeled cG250 and chimeric monoclonal antibody G250 (cG250) (Steffens et al., Clin. Cancer Res. 1999 Oct; 5 (10 Suppl): 3268s-3274s) ), Bispecific anti-embryonic carcinoma antigen / anti-diethylenetriaminopentaacetic acid (DTPA) antibody and ¹³¹ I di-DTPA hapten (Vuillez et al., Clin. Cancer Res. 1999 Oct; 5 Suppl (10 Suppl): 3259s-3267s]) and additional targets for radiation immunotherapy include HLA-DR, CD19, and CD22.

Methods for analyzing normal tissue protection by the compositions of the present invention during radiation immunotherapy are well known in the art and include, for example, studying bone marrow damage by injecting 89-strontium, There is an in vivo model. The following review articles also provide guidance on antibody, radiation therapy, and immunotherapy: "Physical and biological targeting of radiotherapy" Acta Oncol . 1999; 38 Suppl 13: 75-83; &Quot; Radioimmunodiagnosis and therapy " Cancer Treat Rev. 2000 Feb; 26 (1): 3-10; &Quot; Antibodies in the therapy of the colon cancer " Semin Oncol . 1999 Dec; 26 (6) 683-90; "Overview of the clinical development of rituximab: first monoclonal antibody for treatment of lymphoma" Semin Oncol . 1999 Oct; 26 (5Suppl 14): 66-73; &Quot; Radiolabeled antibody therapy of B-cell lymphomas " Semin Oncol . 1999 Oct; 26 (5uppl 14): 58-65; &Quot; Strategies for development of effective radioimmunotherapy for solid tumors " Clin Cancer Res . 1999 Oct; 5 (10 Suppl): 3219s-3223s; "Technical advances in radiotherapy of head and neck tumors" Hematol Oncol Clin North Am. 1999 Aug; 13 (4): 811-23; And " Use of monoclonal antibodies for the diagnosis and treatment of bladder cancer " Hybridoma . 1999 Jun; 18 (3): 219-24.

In another embodiment, the invention provides a composition comprising a polypeptide of the invention (e. G., A composition comprising a heterologous polypeptide, a heterologous nucleic acid, a MPIF polypeptide conjugated with a toxin or a prodrug, or an anti-MPIF antibody) MP < / RTI > expressing B or T cells, monocytes, macrophages and neutrophils. MPIF polypeptides or anti-MPIF antibodies of the invention can be conjugated to heterologous polypeptides, heterologous nucleic acids, toxins or prodrugs via hydrophobic, hydrophilic, ionic and / or covalent bonds.

In one embodiment, the invention provides a method of specifically delivering a composition of the present invention to a cell by administering a polypeptide of the invention (e. G., MPIF polypeptide or anti-MPIF antibody) conjugated to a heterologous polypeptide or nucleic acid. In one example, the invention provides a method of delivering a therapeutic protein to a target cell. In another example, the present invention provides a method of screening for a target nucleic acid (e. G., An antisense or ribozyme) or a two-stranded nucleic acid (e. G., DNA that can be integrated in the cell genome, Providing a way to communicate.

In another embodiment, the invention provides methods for specifically destroying (e.g., destroying tumor cells) cells by administering a polypeptide of the invention (e.g., MPIF polypeptide or anti-MPIF antibody) conjugated to a toxin or cytotoxic prodrug, .

For example, leukemia can be treated by destroying leukemia cells by administering MPIF conjugated to a radioactive isotope.

In one embodiment, the invention provides a method for specifically destroying T or B cell lines (e.g., T or B cell associated leukemia or lymphoma) by administering a MPIF polypeptide conjugated to a toxin or cytotoxic prodrug .

In another embodiment, the invention provides a method of specifically destroying a cell of a monocytic cell line (e.g., monocytic leukemia or lymphoma) by administering an anti-MPIF antibody conjugated to a toxin or cytotoxic prodrug.

In yet another embodiment, the invention provides a method of specifically destroying a cell (e. G., A cell mediated factor of inflammation, e. G., An inflammatory cytokine) by administering a polypeptide of the present invention conjugated to a toxin or cytotoxic prodrug Destruction of < / RTI > Examples of cell mediators of inflammation include T cells, monocytes, dendritic cells, astrocytes, renal glomerular stromal cells, macrophages, neutrophils, and cells involved in transplant rejection (acute or chronic).

In another embodiment, a non-MPIF molecule conjugated with a toxin or cytotoxic prodrug may be co-administered with MPIF-1 to prevent or treat damage of normal cells, tissues or organs, or to prevent or treat bone marrow precursor cells .

By "toxin" is meant a compound that binds to and activates an endogenous cytotoxic effector system, a radioactive isotope, a holotoxin, a modified toxin, a catalytic subunit of a toxin, or other cytotoxic agent, Means any molecule or enzyme that, under certain conditions, causes the death of a cell. Examples of toxins that may be used in accordance with the methods of the present invention include, but are not limited to, radioactive isotopes known in the art, compounds such as antibodies (or their complement fixation containing moieties) that bind to a native or derived endogenous cytotoxic effector system, But are not limited to, kinase, endonuclease, RNAse, alpha toxin, lysine, abrin, Pseudomonas exotoxin A, diphtheria toxin, saponin, momorphine, gelonin, fowweed antiviral protein, alpha saccine and cholera toxin , But is not limited thereto. &Quot; Toxin " may also be a cytotoxic agent, a cell growth inhibitor or a cell death agent, a therapeutic agent or a radioactive metal ion (e.g., an alpha-emitter such as ²¹³ Bi or other radioactive isotopes such as ¹⁰³ Pd, ¹³³ Xe, ¹³¹ Ion, ⁶⁸ Ge, ⁵⁷ Co, ⁶⁵ Zn, ⁸⁵ Sr, ³² P, ³⁵ S, ⁹⁰ Y, ¹⁵³ Sm, ¹⁵³ Gd, ¹⁶⁹ Yb, ⁵¹ Cr, ⁵⁴ Mn, ⁷⁵ Se, ¹¹³ Sn, ⁹⁰ yttrium, ¹¹⁷ Tn, ¹⁸⁶ rhenium, ¹⁶⁶ holmium and ¹⁸⁸ rhenium), luminescent labels (e.g., luminol), fluorescent labels (e.g., fluorescein and rhodamine), and biotin.

Antibodies of the invention may be labeled using techniques known in the art. Non-limiting examples of such techniques include the use of dual-acting adhesives (see, for example, U.S. Patent Nos. 5,756,065, 5,714,631, 5,696,239, 5,652,361, 5,505,931, 5,489,425, 5,435,990, 5,428,139 No. 5,342,604; 5,274,119; 4,994,560; and 5,808,003, each of which is incorporated herein by reference). A cytotoxin or cytotoxic agent includes any substance harmful to the cell. Examples of these include paclitaxel, cytochalasin B, gramicidin D, ethidium bromide, emetine, mitomycin, etoposide, tenofoside, vincristine, vinblastine, colchicine, doxorubicin, daunorubicin, Dihydrothiostosterone, glucocorticoid, procaine, tetracaine, lidocaine, propranolol and furomycin, and analogues or homologues thereof, may be used in combination with one or more compounds selected from the group consisting of dihydroxy anthracenidone, mitoxantrone, mitramycin, actinomycin D, . Therapeutic agents include, but are not limited to, metabolic antagonists (e.g., methotrexate, 6-mercaptopurine, 6-thioguanine, cytarabine, 5-fluorouracil dicarbazine), alkylating agents (e. G., Mechlorethamine, (DDP), cis-dichlorodiamine platinum (II) (DDP), melphalan, carmustine (BSNU) and rhomustine (CCNU), cyclotaspamide, Cisplatin), anthracyclines (e.g., daunorubicin [former name: daunomycin] and doxorubicin), antibiotics (such as dactinomycin [formerly actinomycin], bleomycin, mitramycin and anthramycin ), And anti-mitotic agents (e.g., vincristine and vinblastine).

&Quot; Cytotoxic prodrug " means a non-toxic compound normally present in a cell that is converted to a cytotoxic compound by an enzyme. Non-limiting examples of cytotoxic prodrugs that may be used in accordance with the methods of the present invention include glutamyl derivatives of benzoic acid mustard alkylating agents, phosphate derivatives of etoposide or mitomycin C, phenoxy derivatives of cytosine arabinoside, daunorubicin and doxorubicin And a set amide derivative.

Another preferred embodiment of the present invention includes, but is not limited to, MPIF polypeptides, MPIF polynucleotides and functional antagonists thereof, as described below.

In one embodiment, administration of the MPIF polypeptide (s) or polynucleotide (s) of the invention, or antagonist (s) or agonist (s) thereof (e. G., Anti-MPIF antibody), to treat chronic myelogenous leukemia, Treatment and / or prevention of leukemia, leukemia, histiocytic leukemia, mononuclear leukemia (e.g., acute mononuclear leukemia), leukemia reticulosis, schilling type monocytic leukemia, and / or monocytes and / or monocytes and / , Detected, and / or diagnosed.

Thus, by administering the MPIF polypeptide (s) or polynucleotide (s) of the invention, or agonist (s) or antagonist (s) thereof (e.g., anti-MPIF antibody), the undifferentiated B cell form, (ALL), which may include pre-B cell and B cell forms; Acute myelogenous (myeloid or myeloid) leukemia, which may include undifferentiated myeloid form, differentiated myelogenous form, APL, bone metastasis, monoepithelial, leukemic, and megakaryocytic forms (AML); Which may include B cell morphology, T cell morphology, pre-lymphocyte morphology, triceps syndrome (leukemia stage of subcutaneous T cell lymphoma), hair cell morphology and leukemia of lymphoma (i. E., Leukemia changes observed at the advanced stages of malignant lymphoma) Lymphoid leukemia (CLL); Prevent, detect and / or diagnose leukemia such as chronic myelogenous (myeloid, myeloid or granular) leukemia (CML), for example, a neoplastic clone is red blood cells, megakaryoes, monocytes, T cells or B cells .

In another specific embodiment, administration of the MPIF polypeptide (s) or polynucleotide (s) or agonist (s) or antagonist (s) thereof of the invention may be used to treat, Diagnosis, and / or improvement.

In yet another specific embodiment, administration of the MPIF polypeptide (s) or polynucleotide (s) of the invention, or agonist (s) or antagonist (s) thereof, to treat mononuclear leukopenia, monocytopenia, Prevent, diagnose and / or ameliorate mononucleosis.

In another specific embodiment, administration of the MPIF polypeptide (s) or polynucleotide (s) of the invention, or agonist (s) or antagonist (s) thereof, to treat, prevent, or prevent graft- Diagnose and / or improve.

In another specific embodiment, the MPIF polypeptide (s) or polynucleotide (s) of the invention, or agonist (s) or antagonist (s) thereof, is administered to treat, prevent, diagnose and / or ameliorate anemia.

In yet another specific embodiment, the B cell cytotoxicity is assessed by administering the MPIF polypeptide (s) or polynucleotide (s) of the invention, or agonist (s) or antagonist (s) - treatment, prevention, diagnosis and / or amelioration of Hodgkin's lymphoma, chronic lymphocytic leukemia, progeny, multiple myeloma, Burkitt's lymphoma and EBV-transfected disease.

In another embodiment, symptoms associated with fibrosis and fibrosis using the MPIF polypeptide (s) or polynucleotide (s) of the invention, or agonist (s) or antagonist (s) thereof, , Cystic fibrosis (e.g., pancreatic cystic fibrosis, Clarke-Hadfield syndrome, fibrotic cystic disease of the pancreas, mucocutaneous adhesions and adhesions), myocardial endocardial fibrosis, idiopathic retroperitoneal fibrosis, Treatment of fibrosis, mediastinal fibrosis, nodular subepidermal fibrosis, periocular fibrosis, peri muscle fibrosis, pipestem fibrosis, substitute fibrosis, subarachnoid fibrosis and Symmer's clay pipestem fibrosis, Prevention and / or diagnosis.

In a highly preferred embodiment, the use of MPIF polypeptide (s) or polynucleotide (s), or agonist (s) or antagonist (s) thereof, for the treatment, prevention and / or prophylaxis of mucositis, Diagnose.

Prevention, and / or diagnosis of organ rejection or graft versus host disease (GVHD) and / or related symptoms using the MPIF polypeptides or polynucleotides of the invention, and / or agonists and / can do. The organ rejection is caused by host immune cell destruction of the transplanted tissue through the immune response. Similarly, the immune response is also involved in GVHD, in which case heterologous transplanted immune cells destroy host tissue. Administration of MPIF polypeptides or polynucleotides of the invention and / or agonists and / or antagonists thereof which inhibit the proliferation, differentiation or chemotaxis of immune responses, particularly T-cells, monocytes or other inflammatory mediators, It may be an effective treatment for organ rejection or prevention of GVHD. In addition, when the CCR1 receptor binding to MPIF-1 in the mouse is deleted, allograft survival is prolonged. (Gao et al., "Targeting of the chemokine receptor CCR1 suppresses development of acute and chronic cardiac allograft rejection" J. Clin. Invest. 2000 Jan; 105 (1): 35-44). Thus, for example, blocking the interaction of MPIF-1 or other CCR1 ligands with the CCR1 receptor via MPIF-1 antagonists is useful for preventing acute and chronic graft rejection.

Similarly, the inflammation can be modulated using the MPIF polypeptides or polynucleotides of the invention, and / or agonists and / or antagonists thereof. For example, the MPIF polypeptides or polynucleotides of the invention, and / or agonists and / or antagonists thereof, may inhibit proliferation and differentiation of cells involved in an inflammatory response. These molecules can be used to treat inflammatory conditions including both chronic and acute conditions such as chronic prostatitis, granulomatous prostatitis and malacoplakias, infection-associated inflammation (e.g., septic shock, sepsis or systemic inflammatory response syndrome (SIRS) Inflammatory bowel disease, Crohn's disease or cytokines (eg, TNF or IL-1), inflammatory bowel disease, inflammatory bowel disease, inflammatory bowel disease, Prevent, and / or diagnose symptoms due to over-production of the compound.

In certain embodiments, the anti-MPIF antibodies of the invention are used to treat, prevent, modulate, detect, and / or diagnose inflammation.

In certain embodiments, the anti-MPIF antibodies of the invention are used to treat, prevent, modulate, detect, and / or diagnose an inflammatory disease.

In a preferred embodiment, the composition of the present invention is administered in combination with a CD40 ligand (CD40L), a soluble form of CD40L (e.g. AVREND ^TM ), a biologically active fragment, variant or derivative of CD40L, an anti-CD40L antibody Anti-CD40 antibody) and / or an anti-CD40 antibody (e.g., an operative or antagonistic antibody).

As is recognized in the art, the conjugates and chelates described herein in connection with antibodies are equally applicable to conjugates and chelates of MPIF polypeptides.

Determination of immune phenotype

The antibody of the present invention can be used to determine the immunophenotype of a cell line and a biological sample. The translation product of the gene of the invention may be useful as a cell-specific marker, or more specifically as a cell marker that is differentially expressed at various stages of differentiation and / or maturation of a particular cell type. A cell population that expresses the marker can be screened with a monoclonal antibody directed against a particular epitope, or a combination of epitopes. A variety of techniques using monoclonal antibodies can be used for screening cell populations expressing the marker (s), such as magnet separation using antibody-coated magnetic beads, antibodies attached to solid matrix (e.g., plates) Panning " and flow cytometry (see, for example, U.S. Patent No. 5,985,660; and Morrison et al., Cell 96: 737-49 (1999)).

These techniques may be used to identify specific non-magnetic cells, such as those that may be found in " non-magnetic " cells in transplantation to prevent hematological malignancies (i.e., microfiltration diseases (MRD) in acute leukemia patients) and graft- Cells can be screened. On the other hand, these techniques can screen hematopoietic stem cells and progenitor cells that can undergo proliferation and / or differentiation, such as can be found in human umbilical cord blood.

Analysis of antibody binding

The antibodies of the invention can be assayed for immunospecific binding by any method known in the art. Immunoassays that can be used include, but are not limited to, Western blot, radioimmunoassay, ELISA (enzyme linked immunosorbent assay), "sandwich" immunoassays, immunoprecipitation assays, sedimentation reactions, gel diffusion settling reactions, But are not limited to, competitive and non-competitive analyzes using techniques such as assays, complement-fixation assays, immunoradiometric assays, fluorescence immunoassays, and protein A immunoassays. Such assays are routine and well known in the art (see, for example, Ausubel et al., Eds., 1998, Current Protocols in Molecular Biology, Vol 1, John Wiley & Sons, ). Exemplary immunoassays are briefly outlined below (not intended to be limiting).

Immunoprecipitation protocols are generally carried out in RIPA buffer (1% NP-40 or Triton X-100, 1% sodium deoxycholate) supplemented with protein phosphatase and / or protease inhibitors (e.g. EDTA, PMSF, aprotinin, sodium vanadate) Dissolving the cell group in a cell lysis buffer such as cholate, 0.1% SDS, 0.15 M NaCl, 0.01 M sodium phosphate, pH 7.2, 1% Trasylol), adding the antibody to the cell lysate, (E.g., 1-4 hours), adding protein A and / or protein G sepharose beads to the cell lysate, incubating at 4 ° C for about 1 hour or more, washing the beads in the lysis buffer , And resuspending the beads in SDS / sample buffer. The ability of the antibody to immunoprecipitate a particular antigen can be assessed, for example, by Western blot analysis. Those skilled in the art will be aware of variables that can be modified to increase binding of the antibody to the antigen and reduce background (e. G., Pre-rinse cell lysate with sepharose beads). See, for example, Ausubel et al., Eds., 1998, Current Protocols in Molecular Biology , Vol 1, John Wiley & Sons, Inc., NY (1994) 10.16.1 for further discussion of immunoprecipitation protocols.

Western blot analysis generally involves the preparation of protein samples, electrophoresis of protein samples in polyacrylamide gels (eg, 8% to 20% SDS-PAGE depending on the molecular weight of the antigen), nitrocellulose, PVDF, (E.g., PBS containing 3% BSA or skimmed milk), membrane washing in washing buffer (e.g., PBS-Tween 20), dilution in blocking buffer (Eg, horseradish peroxidase or alkaline phosphatase) or a radioactive molecule (eg, ³² P or ³² kDa), using a primary antibody (the corresponding antibody) ¹²⁵ also connects to I) is considered a secondary antibody (primary antibody diluted in blocking buffer, for example, anti-existence membrane washing, and the antigen in a human antibody) for using the film contact / probing / incubation, wash buffer Detection . Those skilled in the art will know about variables that can be modified to increase the detected signal and reduce background noise. See, for example, Ausubel et al., Eds., Current Protocols in Molecular Biology , Vol 1, John Wiley & Sons, Inc., NY (1994), 10.8.1.

The ELISA can be used for antigen production, addition of the antibody conjugated to a detectable compound such as an enzymatic substrate (e. G., Horseradish peroxidase or alkaline phosphatase) in the well, and incubation of the 96 well microtiter plate with the antigen for a period of time Lt; / RTI > and detection of the presence of the antigen. In an ELISA, it is not necessary to link the antibody to a detectable compound. Alternatively, a second antibody (recognizing the antibody) linked to a detectable compound can be added to the well. Further, instead of coating the well with the antigen, the antibody can be coated on the well. In this case, a second antibody linked to a detectable compound can be added after the antigen has been added to the coated well (i. E., &Quot; sandwich " ELISA). Those skilled in the art will be aware of variables that can be modified to increase the signal being detected and other variations of ELISAs known in the art. See, for example, Ausubel et al., Eds., Current Protocols in Molecular Biology , Vol 1, John Wiley & Sons, Inc., NY (1994), 11.2.1.

The binding affinity of the antibody to the antigen and the separation rate of the antibody-antigen interaction can be determined by competitive binding assay. One example of a competitive binding assay is radioimmunoassay, which involves incubating the labeled antigen with the antibody while increasing the amount of unlabeled antigen and detecting the antibody bound to the labeled antigen. The affinity of the antibody for a particular antigen and the binding cleavage rate can be determined from the data by Scatchard plot analysis. In addition, competition with the second antibody can be measured using radioimmunoassay. In this case, the antigen is incubated with the antibody conjugated to the labeled compound (eg, ³ H or ¹²⁵ I) while increasing the unlabeled second antibody.

Therapeutic Uses - Antibodies

The invention also relates to anti-system therapies comprising administering an antibody of the invention to an animal, preferably a mammal, most preferably a human patient, for the treatment of one or more of the disclosed diseases, disorders or conditions . Therapeutic compounds of the present invention can be used in combination with an antibody of the invention (including fragments, analogs and derivatives thereof as disclosed herein) and an antibody of the invention (including fragments, analogs and derivatives thereof as disclosed herein, and anti- But not limited to, a nucleic acid encoding an antibody, including an antibody. An antibody of the invention may be used to treat, inhibit or ameliorate one or more of the diseases, disorders, or conditions associated with abnormal expression and / or activity of a polypeptide of the invention, including, without limitation, diseases, Can be prevented. The treatment, and / or prevention of diseases, disorders or conditions associated with abnormal expression and / or activity of the polypeptides of the present invention include, but are not limited to, relieving symptoms associated with these diseases, diseases or conditions. The antibodies of the present invention may be provided in pharmaceutical acceptable compositions known in the art or disclosed herein.

To summarize methods by which the antibodies of the present invention can be used therapeutically, the polynucleotides or polypeptides of the invention may be administered either locally or systemically in vivo or by means of complement (CDC) or effector cell (ADCC) And direct cytotoxicity of the antibody, such as, for example, Some of these approaches are described in more detail below. From the teachings disclosed herein, those skilled in the art will know how to use the antibodies of the invention for diagnostic, monitoring, or therapeutic purposes without undue experimentation.

The antibodies of the present invention may be administered in combination with other monoclonal or chimeric antibodies or with other agents such as, for example, lymphokines or hematopoietic growth factors (e.g., IL-2, IL- 3 and < RTI ID = 0.0 > IL-7). ≪ / RTI >

The antibodies of the present invention may be administered alone or in combination with other types of therapies (e.g., radiotherapy, chemotherapy, hormone therapy, immunotherapy, and anti-cancer agents). In general, administration of products of the same species origin or species responsiveness (in the case of antibodies) as the patient is preferred. Thus, in a preferred embodiment, human antibodies, fragment derivatives, analogs or nucleic acids are administered to a human patient for treatment or prevention.

The use of high affinity and / or strong in vivo inhibition and / or neutralizing antibodies, fragments or regions thereof for the polypeptides or polynucleotides of the present invention may be useful for the treatment of diseases associated with polynucleotides or polypeptides (including fragments thereof) Are preferred for both treatment and immune analysis. Such an antibody, fragment, or region will preferably have affinity for the polynucleotide or polypeptide of the invention (including the fragment). The preferred binding affinity dissociation constant, that is, Kd is ^{5 X 10 -2 M, 10 -2} M, 5 X 10 -3 M, 10 -3 M, 5 X 10 -4 M, 10 -4 M, 5 X 10 - ^{5 M, 10 -5 M, 5} X 10 -6 M, 10 -6 M, 5 X 10 -7 M, 10 -7 M, 5 X 10 -8 M, 10 -8 M, 5 X 10 -9 M ^{, 10 -9 M, 5 X 10} -10 M, 10 -10 M, 5 X 10 -11 M, 1 0 -11 M, 5 X 10 -12 M, 10 -12 M, 5 X 10 -13 M, 10 ^-13 M, 5 X 10 ^-14 M, 10 ^-14 M, 5 X 10 ^-15 M, 10 ^-15 M.

Antibody gene therapy

In certain embodiments, a nucleic acid comprising a sequence encoding an antibody or functional derivative thereof is administered to treat, inhibit, or prevent a disease or disorder associated with abnormal expression and / or activity of a polypeptide of the invention by gene therapy. Gene therapy refers to a therapy performed by administering an expressed nucleic acid or an expressible nucleic acid to a subject. In this embodiment of the invention, the nucleic acid produces an encoded protein that mediates the therapeutic effect.

Any method for gene therapy available in the art may be used in accordance with the present invention. An example of such a method is disclosed below.

For a general review of gene therapy, see, for example, Goldspiel et al., Clinical Pharmacy 12 : 488-505 (1993); Wu and Wu, Biotherapy 3 : 87-95 (1991); Tolstoshev, Ann. Rev. Pharmacol. Toxicol. 32 : 573-596 (1993); Mulligan, Science 260 : 926-932 (1993); and Morgan and Anderson, Ann. Rev. Biochem. 62 : 191-217 (1993); May, TIBTECH11 (5) : 155-215 ( 1993). Methods known in the art as available recombinant DNA techniques are described in Ausubel et al. (eds.), Current Protocols in Molecular Biology , John Wiley & Sons, NY (1993); And Kriegler, Gene Transfer and Expression, A Laboratory Manual , Stockton Press, NY (1990).

In a preferred aspect, the compound comprises a nucleic acid sequence encoding an antibody, wherein the nucleic acid sequence is part of an expression vector that expresses the antibody or fragment thereof or a chimeric protein or heavy or light chain in a suitable host. In particular, such nucleic acid sequences retain a promoter operably linked to the antibody coding region, which is inducible or constitutive and, in some cases, tissue-specific. In another specific embodiment, the use of a nucleic acid molecule located next to a region in which an antibody coding sequence and other predetermined sequences facilitate homologous recombination at a given site in the genome may provide for chromosomal expression of the antibody encoding the nucleic acid [Koller and Smithies, Proc. Natl. Acad. Sci. USA 86 : 8932-8935 (1989); Zijlstra et al., Nature 342 : 435-438 (1989)]. In certain embodiments, the expressed antibody molecule is a single chain antibody; Alternatively, the nucleic acid sequence comprises a sequence encoding both the heavy and light chains of the antibody, or fragments thereof.

Nucleic acid delivery to a patient can be indirect if the patient is directly exposed to the nucleic acid or nucleic acid-carrying vector, either directly or indirectly if the cell is first transfected with the nucleic acid in vitro and then transplanted to the patient. Both of these approaches are known as in vivo or in vitro gene therapy, respectively.

In certain embodiments, the nucleic acid sequence is administered directly in vivo, wherein said nucleic acid sequence is expressed to produce an encoded product. This can be accomplished by any of a number of methods known in the art, for example, by constructing a nucleic acid sequence as part of a suitable nucleic acid expression vector and administering it to the cell, such as a defective or attenuated retrovirus or other viral vector (See, for example, U.S. Patent No. 4,980,286), or a method of directly injecting a naked DNA, or a method using a microparticle impact method (for example, a gene gun; biolistic, DuPont) A method of coating with a surface receptor or a transforming agent, a method of encapsulating in a liposome, a microparticle or a microcapsule, a method of binding to a peptide known to be introduced into the nucleus, or a method of binding to a ligand for receptor-mediated intracellular uptake To a subject (see, for example, Wu and Wu, J. Biol., Chem. 262 : 4429-4432 (1987)], which may be used to target cell types that specifically express the receptor. In another embodiment, lysosomal degradation of the nucleic acid can be avoided by forming a nucleic acid-ligand complex in which the ligand comprises a fusogenic viral peptide to destroy the endosome. In another embodiment, nucleic acids can be targeted in vivo for cell-specific uptake and expression by targeting specific receptors (see, for example, PCT Publication Nos. WO 92/06180; WO 92/22635; WO 92 / WO 93/14188, WO 93/20221). Alternatively, with homologous recombination, the nucleic acid can be introduced into the cell and then incorporated into the host cell DNA for expression (Koller and Smithies, Proc. Natl. Acad. Sci. USA 86 : 8932-8935 (1989); Zijlstra et al., Nature 342 : 435-438 (1989)].

In certain embodiments, a viral vector containing a nucleic acid sequence encoding an antibody of the invention is used. For example, retroviral vectors can be used [Miller et al., Meth. Enzymol. 217 : 581-599 (1993)). These retroviral vectors contain the components necessary to accurately package the viral genome and integrate it into the host cell DNA. The nucleic acid sequence encoding the antibody used in the gene therapy is cloned into one or more vectors that are easy to deliver the gene to the patient. A more detailed description of retroviral vectors can be found in Boesen et al., Biotherapy 6 : 291-302 (1994), which describes the use of retroviral vectors to increase the resistance of hematopoietic stem cells to chemotherapy A method of delivering the mdr1 gene to the hepatocyte is described. Other references describing the use of retroviral vectors in gene therapy include Clowes et al., J. Clin. Invest. 93 : 644-651 (1994); Kiem et al., Blood 83 : 1467-1473 (1994); Salmons and Gunzberg, Human Gene Therapy 4 : 129-141 (1993); And Grossman and Wilson, Curr. Opin. in Genetics and Devel. 3 : 110-114 (1993).

Adenovirus is another viral vector that can be used for gene therapy. Adenovirus is a particularly effective mediator for delivering genes to the respiratory epithelium. Adenovirus infects the respiratory epithelium originally and causes symptoms of mild disease. Other targets for adenoviral delivery systems are liver, central nervous system, endothelial cells, and muscle. Adenovirus has the advantage of infecting non-dividing cells. Adenoviral gene therapy has been proposed in Kozarsky and Wilson, Current Opinion in Genetics and Development 3 : 499-503 (1993). Bout et al., Human Gene Therapy 5 : 3-10 (1994) describes the use of adenoviral vectors to deliver genes to the respiratory epithelium of rhesus monkeys. Other examples of adenovirus use in gene therapy are described in Rosenfeld et al., Science 252 : 431-434 (1991); Rosenfeld et al., Cell 68 : 143-155 (1992); Mastrangeli et al., J. Clin. Invest. 91 : 225-234 (1993); PCT Publication No. WO 94/12649; And Wang, et al., Gene Therapy 2: 775-783 (1995). In a preferred embodiment, an adenovirus vector is used.

Adeno-associated virus (AAV) has been proposed for use in gene therapy [Walsh et al., Proc. Soc. Exp. Biol. Med. 204 : 289-300 (1993); U.S. Patent No. 5,436,146].

Other approaches to gene therapy include the delivery of genes to cells in tissue culture by methods such as electroporation, lipofectin, calcium phosphate mediated transfection or viral infection. Typically, the delivery method involves delivering a selection marker to the cell. The cells are then sorted to isolate cells expressing the transferred gene. These cells are then delivered to the patient.

In this embodiment, the nucleic acid is introduced into the cell prior to in vivo administration of the resulting recombinant cell. Such introduction may be accomplished by methods known in the art such as, for example, transfection, electroporation, microinjection, infection with a virus comprising a nucleic acid sequence or using a bacteriophage vector, cell fusion, chromosome- Mediated gene transfer, and spiroplast fusion. Various techniques for introducing foreign genes into cells are known in the art (see, e.g., Loeffler and Behr, Meth. Enzymol. 217 : 599-618 (1993); Cohen et al., Meth. Enzymol. 217 : 618-644 (1993); Cline, Pharmac. Ther. 29 : 69-92m (1985)], which can be used according to the present invention. However, the necessary developmental and physiological functions of recipient cells should not be destroyed. This technique requires that the nucleic acid be stably transferred to the cell so that the cell can express the nucleic acid, and preferably can be inheritably expressed in the progeny of the cell.

The resulting recombinant cells can be delivered to the patient using a variety of methods known in the art. Recombinant blood cells (e.g., hematopoietic stem cells or progenitor cells) are preferably administered intravenously. The amount of the cell to be used depends on the desired effect, the condition of the patient, and the like, and can be determined by those skilled in the art.

Cells in which the nucleic acid can be introduced for gene therapy include any desired cell type of use, including epithelial cells, endothelial cells, keratinocytes, fibroblasts, muscle cells, hepatocytes; Blood cells such as T-lymphocytes, B-lymphocytes, monocytes, macrophages, neutrophils, eosinophils, megakaryocytes, granulocytes; Various hepatocytes or progenitor cells, particularly hematopoietic stem cells or progenitor cells such as those obtained from bone marrow, umbilical cord blood, peripheral blood, fetal liver, and the like, but are not limited thereto.

In a preferred embodiment, the cells used for gene therapy are derived from the patient's autologous tissue.

In embodiments using recombinant cells for gene therapy, the cells or their offspring are introduced into cells to express nucleic acid sequences encoding the antibodies, and the recombinant cells are then administered in vivo for therapeutic effect. In certain embodiments, hepatocytes or progenitor cells are used. Any hepatocytes and / or progenitor cells that can be maintained in vitro and maintained in vitro can be used according to this embodiment of the invention (see, for example, PCT Publication No. WO 94/08598; Stemple and Anderson, Cell 71 : 973-985 (1992); Rheinwald, Meth. Cell Bio. 21A : 229 (1980); And Pittelkow and Scott, Mayo Clinic Proc. 61 : 771 (1986)).

In certain embodiments, the nucleic acid to be introduced for gene therapy can include an inducible promoter operably linked to the coding region to control the expression of the nucleic acid by modulating the presence or absence of a suitable transcription inductor. Demonstration of Therapeutic or Prophylactic Activity.

The compounds or pharmaceutical compositions of the invention are preferably tested in vitro and then tested in vivo for a given therapeutic or prophylactic activity prior to use in humans. For example, in vitro assays to demonstrate the therapeutic or prophylactic utility of a compound or pharmaceutical composition include the effect of a compound on a patient tissue sample or cell line. The effect of a compound or composition on a cell line and / or tissue sample can be determined using techniques known in the art, such as, but not limited to, rosette formation assays and cell lysis assays. In accordance with the present invention, in vitro assays that can be used to determine whether administration of a particular compound is required include, but are not limited to, growing a patient tissue sample in culture, exposing it to a compound or administering it, And in vitro cell culture assays to observe the effects of the compounds.

Therapeutic / Preventive Administration and Composition - Antibodies

The present invention provides a method of treating, inhibiting and preventing by administering to a subject an effective amount of a compound or pharmaceutical composition of the invention, preferably an antibody of the invention. In a preferred aspect, the compound is substantially purified (e.g., the substance that substantially limits its effectiveness or produces undesirable side effects). The subject is preferably an animal, such as, but not limited to, cows, pigs, horses, chickens, cats, dogs, and the like, mammals being preferred, and humans being most preferred.

Dosage formulations and methods of administration that can be used when the compound comprises a nucleic acid or an immunoglobulin are described above. Additional suitable dosage formulations and routes of administration may be selected from those described below.

Various delivery systems are known, and the compounds of the present invention may be administered using these systems. Such delivery systems include, for example, encapsulation in liposomes, microparticles, microcapsules, recombinant cells capable of expressing such compounds, receptor-mediated intracellular uptake [see, e.g., Wu and Wu, J. Biol. Chem. 262 : 4429-4432 (1987)), construction of nucleic acids as part of retrovirus or other vectors, and the like. Methods of introduction include intradermal, intramuscular, intraperitoneal, intravenous, subcutaneous, intranasal, dermal, and oral routes, but are not limited thereto. The compound or composition may be administered by any conventional route, for example, by infusion or bolus injection, by absorption through the epithelial or mucosal sub-layer (e.g., oral mucosa, rectum and intestinal mucosa, etc.) May be administered with other biologically active agents. Systemic or topical administration. It may also be desirable to introduce the compounds or pharmaceutical compositions of the present invention into the central nervous system via any suitable route, for example, intracameral and intrathecal injection. For example, it can be easily in-chamber injected by an intraventricular catheter attached to a reservoir, such as an Ommaya reservoir. For example, pulmonary administration can be achieved using an inhaler or sprayer and a formulation containing an aerosolizing agent.

In certain embodiments, it may be desirable to topically administer the compounds or pharmaceutical compositions of the invention to the area in need of treatment. This can be accomplished, for example, by injection, by catheter means, by suppository means, or by implantation means, in conjunction with local injection during surgery, topical application, such as post-surgical wound dressing, no. The implanting means may be porous, non-porous, or a gelatinous material, such as a membrane, such as a sialastic membrane, or a fiber. Preferably, when administering a protein, including an antibody of the invention, care should be taken to use a substance that does not absorb the protein.

In another embodiment, the compound or composition may be delivered in a medium, particularly a liposome (Langer, Science 249: 1527-1533 (1990); Treat et al., In Liposomes in the Therapy of Infectious Disease and Cancer, Lopez-Berestein and Fidler (eds.), Liss, New York (1989), pp. 353-365; Lopez-Berestein, ibid., Pp. 317-327; See generally the above literature].

In another embodiment, the compound or composition may be delivered to a controlled release system. In one embodiment, a pump can be used [Langer, supra ; Sefton, CRC Crit. Ref. Biomed. Eng. 14 : 201 (1987); Buchwald et al., Surgery 88 : 507 (1980); Saudek et al., N. Engl. J. Med. 321 : 574 (1989)). In other embodiments, polymeric materials can be used ( Medical Applications of Controlled Release, Langer and Wise, eds., CRC Pres., Boca Raton, Florida (1974); Controlled Drug Bioavailability, Drug Product Design and Performance , Smolen and Ball, eds., Wiley, New York (1984); Ranger and Peppas, J., Macromol. Sci. Rev. Macromol. Chem. 23 : 61 (1983); See also Levy et al., Science 228 : 190 (1985); During et al., Ann. Neurol. 25 : 351 (1989); Howard et al., J. Neurosurg. 71 : 105 (1989)). In another embodiment, the controlled release system may require only a portion of the systemic dose by locating the therapeutic target, i.e., in the vicinity of the brain (see, for example, Goodson, Medical Applications of Controlled Release , supra , vol. 2 (1984), pp. 115-138).

Other controlled release systems are discussed in Langer, Science 249 : 1527-1533 (1990).

In certain embodiments in which the compound of the present invention is a nucleic acid encoding a protein, the nucleic acid may be constructed as a part of a suitable nucleic acid expression vector to induce cell death, for example, a method using retroviral vectors (U.S. Patent No. 4,908,286 A method of coating with a lipid or cell-surface receptor or a transfection agent, a method known in the art to be introduced into the nucleus, a method using direct injection, or a method using microparticle bombardment (e.g., gene gun; biolistic, DuPont) Methods for administering by binding to homeobox-like peptides (see, for example, Joliot et al., Proc. Natl. Acad. Sci. USA 88 : 1864-1868 (1991)), the expression of the encoded protein can be promoted by administering the nucleic acid in vivo. Alternatively, the nucleic acid can be introduced into the cell by homologous recombination and incorporated into the host cell DNA for expression.

The present invention also provides pharmaceutical compositions. Such compositions comprise a therapeutically effective amount of the compound and a pharmaceutically acceptable carrier. In certain embodiments, the term " pharmacologically acceptable " means that it has been approved by the federal or state regulatory agency for use in animals and, more specifically, in humans, or as described in the US Pharmacopoeia or other commonly known pharmacopoeias . The term " carrier " means a diluent, adjuvant, excipient or implant that is used in conjunction with the administration of the therapeutic. Such pharmaceutical carriers may be sterile liquids such as water and oils such as petroleum, animal, vegetable or synthetic oils such as peanut oil, soybean oil, mineral oil, sesame oil, and the like. When the pharmaceutical composition is administered intravenously, water is the preferred carrier. Saline solution and dextrose and aqueous glycerol solutions can be used particularly as liquid carriers for injection solutions. Suitable pharmaceutical excipients include starch, glucose, lactose, sucrose, gelatin, malt, rice, flour, chalk, silica gel, sodium stearate, glycerol monostearate, talc, sodium chloride, dried skim milk, glycerol, Ethanol and the like. The composition may, if desired, contain a wetting or emulsifying agent, or a small amount of pH buffering agent. These compositions may be in the form of solutions, suspensions, emulsions, tablets, pills, capsules, powders, sustained release formulations and the like. The composition may be formulated into suppositories using conventional carriers and binders such as triglycerides. Oral formulations may include standard carriers such as mannitol, lactose, starch, magnesium stearate, sodium saccharin, cellulose, magnesium carbonate, and the like. Examples of suitable pharmaceutical carriers are described in Remington ' s Pharmaceutical Sciences, < RTI ID = 0.0 > Martin. Such a composition will comprise a therapeutically effective amount of a compound, preferably a tablet, in combination with an amount of a carrier that provides a dosage form suitable for administration to a patient. The formulation should be appropriate for the mode of administration.

In a preferred embodiment, the composition is formulated according to conventional procedures as a pharmaceutical composition suitable for intravenous administration to humans. Usually, the composition for intravenous administration is a solution in a sterile isotonic aqueous buffer solution. If desired, the composition may also contain local anesthetics and solubilizers, such as lignocaine, which alleviate pain at the injection site. In general, the ingredients are supplied either separately in unit dosage form or mixed therewith, for example as a lyophilized powder or anhydrous concentrate in a sealed container such as a sachette or ampoule indicating the amount of active agent. When the composition is administered by injection, the composition may be injected into an injection bottle containing sterile pharmaceutical grade or salt water. When the composition is administered by injection, the injectable sterile water or salt water ampoule may be provided so that the components can be mixed prior to administration.

The compounds of the present invention may be formulated in a neutral or salt form. Pharmaceutically acceptable salts include those formed using anions such as those derived from hydrochloric acid, phosphoric acid, acetic acid, oxalic acid, tartaric acid and those formed with anions such as sodium, potassium, ammonium, calcium, iron hydroxide, isopropylamine, triethylamine, , Histidine, procaine and the like, and the like.

The amount of a compound of the present invention that is effective for the treatment, inhibition and prevention of a disease or disorder associated with abnormal expression and / or activity of the polypeptide of the present invention can be determined by standard clinical techniques. In addition, in-vitro assays can be used to determine the optimal dosage range. In addition, the exact dosage used in the formulation will depend on the route of administration and the severity of the disease or disorder, and should be determined by the judgment of the practitioner and the circumstances of each patient. Effective doses can be deduced from dose-response curves derived from in vitro tests or animal model test systems.

In the case of antibodies, the dose administered to the patient is typically 0.1-100 mg per kg body weight of the patient. The dose to be administered to the patient is preferably 0.1 to 20 mg, more preferably 0.1 to 10 mg, per 1 kg of the patient's body weight. In general, due to the immune response to foreign polypeptides, the half-life of a human antibody in the human body is longer than the half-life of antibodies derived from other species. Thus, human antibodies can be administered with reduced dosages and times. Further, the dosage and the frequency of administration of the antibody of the present invention can be reduced by, for example, modification with lipidation or the like to improve antibody absorption and tissue penetration (e.g., penetration into the brain).

The present invention also provides a pharmaceutical pack or kit comprising at least one container filled with one or more components of the pharmaceutical composition of the present invention. In some cases, in conjunction with such container (s), a notice directed by a governmental agency regulating the manufacture, use or sale of pharmaceutical or biological products reflecting the approval of the manufacturer, use or sales company for human administration .

Diagnosis and Imaging - Antibodies

Labeled antibodies, and derivatives and analogues thereof that specifically bind to the polypeptide of the present invention can be used for diagnosis to detect, diagnose, or monitor disease and / or disease associated with abnormal expression and / or activity of the polypeptide of the present invention. The present invention relates to a method of modulating expression of a polypeptide of interest in a cell or body fluid of a subject using (a) one or more antibodies specific for that polypeptide, and (b) comparing the level of expression of the gene with the level of expression of the standard, Wherein the decrease or increase in the level of expression of the polypeptide gene as compared to the expression level of the polypeptide is indicative of abnormal expression.

The present invention relates to a method of modulating expression of a polypeptide of interest in a cell or body fluid of a subject using (a) one or more antibodies specific for that polypeptide, and (b) comparing the level of expression of the gene with the level of expression of the standard, Wherein the decrease or increase in the level of expression of the analyzed polypeptide gene is indicative of a particular disease. In the context of cancer, the presence of a relatively large amount of transcripts in an autopsied tissue from an individual may be indicative of the progression of the disease, or may provide a means of detecting disease prior to the appearance of substantial clinical symptoms have. With this type of more definitive diagnosis, healthcare professionals can use preventive measures or aggressive treatment earlier to prevent development or further progression of cancer.

The antibodies of the present invention can be used to analyze protein concentrations in biological samples using classical immunohistological methods known to those skilled in the art (see, for example, Jalkanen, et al., J. Cell Biol. 101: 976-985 (1985); Jalkanen, et al., J. Cell Biol. 105: 3087-3096 (1987)). Other anti-system methods useful for detecting protein gene expression include immunoassays such as enzyme linked immunosorbent assay (ELISA) and radioimmunoassay (RIA). Suitable antibody assay labels are known in the art and include, for example, enzyme labels such as glucose oxidase; Radioisotopes such as iodine ( ¹²⁵ I, ¹²¹ I), carbon ( ¹⁴ C), sulfur ( ³⁵ S), tritium ( ³ H), indium ( ¹¹² In), and technetium ( ⁹⁹ Tc); Luminescent labels such as luminol; And fluorescent labels such as fluorescein and rhodamine and biotin.

One aspect of the invention is the detection and diagnosis of diseases or disorders associated with an abnormal expression of a polypeptide of interest in an animal, preferably a mammal, most preferably a human. In one embodiment, the diagnosis is a) administering (e.g., parenterally, subcutaneously, or intraperitoneally) to a subject an effective amount of a labeled molecule that specifically binds to the polypeptide; b) waiting for a period of time after administration to cause the labeled molecules to preferentially concentrate (and to remove non-binding marker molecules to background levels) at the site in the subject where the polypeptide is expressed; c) determining the background level; And d) detecting a marker molecule in the subject, wherein detection of the marker molecule above background level indicates that the subject has a particular disease or disorder associated with abnormal expression of the polypeptide. Background levels can be determined by a variety of methods, including comparing the amount of the detected marker molecule to a predetermined standard value for a particular system.

Those skilled in the art will appreciate that the size of the subject and the imaging system used will determine the amount of imaging portion needed to produce the diagnostic image. For the radioisotope portion, for human subjects, the amount of radioactive isotope to be injected will generally range from about 5 to 20 milli-Cycles of ⁹⁹ Tc. The labeled antibody or antibody fragment will then preferentially accumulate at the cell site containing the particular protein. In vivo tumor imaging is described in SW Burchiel et al., &Quot; Immunopharmacokinetics of Radiolabeled Antibodies and Their Fragment, " in Tumor Imaging: The Radiochemical Detection of Cancer , SW Burchiel and BARhodes, eds, Masson Publishing Inc. (1982) .

Depending on several variables, including the type of label used and the mode of administration, the time interval after administration required to allow labeled molecules to preferentially concentrate at the site of the subject and to remove non-binding marker molecules to background levels is between 6 and 48 Hour or 6 to 24 hours or 6 to 12 hours. In other embodiments, the time interval after administration is 5 to 20 days or 5 to 10 days.

In one embodiment, monitoring of the disease or disorder is performed, for example, by repeating the method for diagnosing the disease or disorder one month after the initial diagnosis, six months after the initial diagnosis, and one year after the initial diagnosis.

Known methods for in vivo scanning can be used to detect the presence of a labeled molecule in a patient. These methods depend on the type of label used. One skilled in the art will be able to determine the appropriate method for detection of a particular label. Methods and apparatus that may be used in the diagnostic methods of the present invention include, but are not limited to, computer tomography (CT), whole body scanning such as positional emission tomography, magnetic resonance imaging (MRI), and sonography.

In certain embodiments, the molecule is labeled with a radioactive isotope and detected in a patient using a radiation-sensitive surgical instrument (Thurston et al., U.S. Patent No. 5,441,050). In another embodiment, the molecule is labeled with a fluorescent compound and detected in a patient using a fluorescent reactive scanning instrument. In another embodiment, the molecule is labeled with a positron emitting metal and detected in a patient using positron emission-tomography. In another embodiment, the molecule is labeled with a paramagnetic label and detected in the patient using magnetic resonance imaging (MRI).

Antibodies and other kits

The present invention provides kits that can be used in the methods described above. In one embodiment, the kit comprises an antibody, preferably a purified antibody, of the invention in one or more containers. In certain embodiments, a kit of the invention comprises a substantially separate polypeptide comprising an epitope that specifically immunoreacts with an antibody contained in the kit. The kit of the present invention preferably further comprises a regulatory antibody that does not react with the polypeptide. In another specific embodiment, a kit of the invention comprises means for detecting binding of an antibody to the polypeptide (e.g., the antibody can be conjugated to a detectable substrate, such as a fluorescent compound, an enzyme substrate, A radioactive compound or a luminescent compound, or a second antibody recognizing the first antibody may be combined with a detectable substrate).

In another specific embodiment of the present invention, the kit is a diagnostic kit for use in screening serum containing proliferative and / or cancer polynucleotides and antibodies specific for the polypeptide. Such kits may comprise regulatory antibodies that do not react with the polypeptide of interest. Such a kit may comprise a substantially separate polypeptide antigen comprising an epitope that specifically immunoreacts with one or more anti-polypeptide antigen antibodies. Such kits also include means for detecting binding of the antibody to the antigen (e. G., The antibody can be conjugated to a fluorescent compound such as rhodamine or fluorescein, which can be detected by flow cytometry have). In certain embodiments, the kit may comprise recombinantly produced or chemically synthesized polypeptide antigens. The polypeptide antigen of the kit may be attached to a solid support.

In a more particular embodiment, the detection means of the kit described above comprise a solid support to which the polypeptide antigen is attached. Such kits may include non-attached, reporter-labeled anti-human antibodies. In such embodiments, binding of the antibody to the polypeptide antigen can be detected by binding of the reporter-labeled antibody.

In a further embodiment, the invention includes a diagnostic kit for use in screening sera comprising an antigen of a polypeptide of the invention. The diagnostic kit comprises a substantially separate antibody that specifically immunoreacts with a polypeptide or polynucleotide antigen, and means for detecting binding of the polynucleotide or polypeptide antigen to the antibody. In one embodiment, the antibody is attached to a solid support. In certain embodiments, the antibody may be a monoclonal antibody. The detection means of the kit may comprise a second labeled monoclonal antibody. Alternatively or additionally, the detection means may comprise a labeled competitive antigen.

In one diagnostic configuration, the solid phase reagent with the surface-bound antigen obtained by the method of the present invention is reacted with the test serum. After binding the specific antigen antibody to the reagent and removing the unbound serum components by washing, the reagent is reacted with a reporter-labeled anti-human antibody to increase the ratio of the anti-antigen antibody bound on the solid support The reagent is combined with a reporter. The reagent is washed again to remove unbound labeled antibody and the amount of reporter associated with that reagent is determined. Typically, the reporter is an enzyme that is detected by incubating the solid phase in the presence of a suitable fluorimetric, luminescent, or colorimetric substrate (Sigma, St. Lewis, Missouri, USA).

Solid surface reagents in this assay are prepared using well known techniques for attaching proteinaceous materials to solid support materials such as polymeric beads, dip sticks, 96-well plates or filter materials. These attachment methods generally involve the covalent attachment of a protein to a chemical reactor on a solid support such as an active carboxyl, hydroxyl or aldehyde group, usually via a free amine group, or non-specifically adsorbing the protein to a support . Alternatively, streptavidin-coated plates can be used with biotinylated antigen (s).

Thus, the present invention provides a kit or assay system for performing such diagnostic methods. The kit generally comprises a support with surface-bound recombinant antigen and a reporter-labeled anti-human antibody for detecting surface-bound anti-antigen antibodies.

In order to facilitate understanding of the following examples, some frequently mentioned methods and / or terms are described.

The " plasmid " is indicated by a lowercase p before and / or after uppercase letters and / or numbers. As used herein, the starting plasmid is commercially available and may be constructed from plasmids that are either openly available, or available according to published procedures. Also, plasmids equivalent to those disclosed are known in the art and will be apparent to those skilled in the art.

"Decomposition" of DNA is the catalytic cleavage of DNA with a restriction enzyme that acts only in some sequence within the DNA. The various restriction enzymes used herein are commercially available, and reaction conditions, joins and other conditions known to those skilled in the art are used. For analysis, typically 1 μg of plasmid or DNA fragment is used with about 2 units of enzyme in about 20 μl of buffer. To isolate DNA fragments for plasmid engineering, 5 to 50 [mu] g of DNA are typically digested with 20 to 250 units of enzyme under larger volumes. Suitable quantities of buffer and substrate for a particular restriction enzyme are provided by the manufacturer. A culture time of about 1 hour at 37 ° C is generally used, but can be varied according to the supplier's instructions. After digestion, the reaction product is directly electrophoresed on a polyacrylamide gel to isolate the desired fragment.

Size separation of the cleaved fragments is carried out using an 8% polyacrylamide gel as described in Goeddel, D. et al ., Nucleic Acid Res., 8: 4057 (1980).

&Quot; Oligonucleotide " means a single-stranded polydeoxynucleotide or two complementary polydeoxynucleotide strands that can be chemically synthesized. Since 5 'phosphate is not present in such synthetic oligonucleotides, it is not ligated to other oligonucleotides unless ATP and phosphate are added in the presence of kinase.

&Quot; Ligation " refers to the process of forming a phosphodiester bond between two double stranded nucleic acid fragments (Maniatis, T et al., Supra, p146). Without further suggestions, the ligation may be carried out using known buffers and conditions with 10 units of T4 DNA ligase (" ligase ") per 0.5 [mu] g of the approximate molar amount of DNA fragment to be ligated.

Unless otherwise stated, transformation was carried out according to the method described by Graham F. and Van der Eb A. [ Virology , 52: 456-457 (1973)].

Having generally described this invention, it will be appreciated that the invention may be more readily understood by reference to the following examples, which are presented by way of illustration, and are not intended to limit the invention.

Example 1

Bacterial expression and purification of MPIF-1

A DNA sequence coding for MPIF-1, a DNA sequence coding for MPIF-1, a DNA sequence coding for MPIF-1, a DNA sequence coding for MPIF-1, a sequence of ATCC # 75676 < / RTI > Additional nucleotides corresponding to Bam HI and XbaI were added to the 5 'and 3' sequences, respectively. The 5 'oligonucleotide primer has the sequence 5'-TCAGGATCCGTCACAAAAGATGCAGA-3' (SEQ ID NO: 8), followed by a BamHI restriction enzyme site, followed by the 18 'of the MPIF-1 coding sequence starting from the putative terminal amino acid of the processed protein codon Lt; / RTI > nucleotides. The 3 'sequence 5'-CGCTCTAGAGTAAAACGACGGCCAGT-3' (SEQ ID NO: 9) contains a complementary sequence to the XbaI site.

The restriction enzyme site corresponds to the restriction enzyme site on the bacterial expression vector pQE-9 (Qiagen, Inc. Chatsworth, Calif.). pQE-9 is antibiotic resistance (Amp ^r), bacterial origin of replication (ori), IPTG- regulatable promoter effector (P / O), a password, a ribosome binding site (RBS), 6-His tag and restriction enzyme sites do. The pQE-9 is then digested with BamHI and XbaI. The amplified sequence is ligated into pQE-9 and inserted into the frame with a sequence encoding histidine tag and RBS. The ligation mixture is then used to transform E. coli strain M15 / rep4, marketed by Qiagen. M15 / rep4 contains multiple copies of the plasmid pREP4, which expresses the lacI inhibitor and also confer kanamycin resistance (Kan ^r ). By their ability to grow on LB plates, transformants are identified and ampicillin / kanamycin resistant colonies are screened. Plasmid DNA was isolated and confirmed by overnight (O / N) restriction analysis with liquid culture in LB medium supplemented with both Amp (100 / / ml) and Kan (25 / / ml). A large amount of the culture is incubated at a ratio of 1: 100 to 1: 250 using the O / N culture. The cells are grown to an optical density of 600 (OD ⁶⁰⁰ ) between 0.4 and 0.6. IPTG (" isopropyl-BD-thiogalactopyranoside ") is then added to a final concentration of 1 mM. IPTG induces cleavage of P / O by inactivating the lacI inhibitor, thereby enhancing gene expression. Cells are grown for an additional 3 to 4 hours. The cells are then harvested by centrifugation. The cell pellet is solubilized in a chaotropic agent 6 M guanidine HCl. After washing, the solubilized MPIF-1 is purified from the solution by chromatography on a nickel-chelate column under conditions allowing tight binding by a 6-His tag containing protein. See Hochuli, E. et al., J. Chromatography 411: 177-184 (1984). MPIF-1 (95% purity) was eluted from the column in 6 M guanidine HCl pH 5.0 and eluted with 3 M guanidine HCl, 100 mM sodium phosphate, 10 mM glutathione (reduction) and 2 mM glutathione ). After incubation in the solution for 12 hours, the protein is dialyzed against 10 mM sodium phosphate.

Alternatively, the gene is cloned into plasmid pQE70 using the following non-tag primers:

5 'primer: 5'CCC GCA TGC GGG TCA CAA AAG ATG CAG 3' (SEQ ID NO: 10)

SphI

3 'primer: 5'AAA GGA TCC TCA ATT CTT CCT GGT CTT 3' (SEQ ID NO: 11)

BamHI stop

Construction of E. coli optimization MPIF-1

In order to increase the expression level of MPIF-1 in the E. coli expression system, the codon of the gene was optimized for the E. coli codon used. For the synthesis of the optimized region of MPIF-1, a series of 4 oligonucleotides were prepared: mpif-1 oligonumber 1-4 (described below). The PCR reaction was carried out for 7 rounds using these superimposed oligos under the following conditions:

Degeneration 95 degrees 20 seconds

Annealing 58 degrees 20 seconds

Height 72 degrees 60 seconds

After synthesis for 7 rounds, the 5 'primer [ACA T GC ATG C GU GUU ACC A AAA GAC GCU GAA ACC GAU UUC AUG AUG UCC (SEQ ID NO: 12)] for this region and the 3' primer [GCT GCC AA C C TT AGT TTT TAC TTT GGG TGA TAC GGG (SEQ ID NO: 13)] a, was added to the PCR reaction is raised to 6 containing 1 microliter from the initial reaction of the nucleotide. The product was amplified for 30 revolutions using the following conditions.

Degeneration 95 degrees 20 seconds

Annealing 55 degrees 20 seconds

Height 72 degrees 60 seconds

The product produced by the final reaction was digested with SphI and HindIII and cloned into pQE70 digested with SphI and HindIII. Expressing these clones and showing excellent expression levels without these mutations.

mpif Upload number 1:

mpif-1 Up Number 2:

mpif1 Up Number 3:

mpif-1 Upload number 4:

Construction of MPIF-1 deletion mutants

A deletion mutant was constructed from the 5 'terminal of the MPIF-1 gene using the E. coli optimized MPIF-1 construct described above. The primers used for constructing the 5 'deletion are as follows. PCR amplification was performed as described above for the E. coli optimized MPIF-1 construct. The products for Delta 17-A qe6, Delta 23 and Delta 28 were cut using NcoI for the 5 'site and HindIII for the 3' site and cloned into the plasmid pQE60 digested with NcoI and HindIII Respectively. All other products were digested with SphI for the 5 'site and HindIII for the 3' site and cloned into the plasmid pQE70 digested with SphI and HindIII.

The 5 ' primers used were as follows:

Delta 17-A qe6 (pQE60)

5 'Nco I gc gca g cc atg g aa aac ccg gtt ctg ctg gac 3' (SEQ ID NO: 18)

The resulting amino acid sequence of this deletion mutant:

Delta 16-A qe7 (pQE70)

5 ' SphI gc catg gc atg c tg gaa aac ccg gtt ctg ctg gac (SEQ ID NO: 20)

The resulting amino acid sequence of this deletion mutant:

Delta 23 (pQE60)

5 'Nco I gc gca g cc atg g ac cgt ttc cac gct acc tcc (SEQ ID NO: 22)

The resulting amino acid sequence of this deletion mutant:

Delta 24 (pQE70)

5 'SphI gcc gtg gc atg c gtt tcc acg cta cct cc (SEQ ID NO: 24)

The resulting amino acid sequence of this deletion mutant:

Delta 28 (pQE60)

5 'NcoI gcg cag cc atg c cta cct ccg ctg act gct gc (SEQ ID NO: 25)

The resulting amino acid sequence of this deletion mutant:

S70 is mutated into A variant (Ser at position 70 mutates to Ala) (pQE70)

Antisense ttc gaa gta ggc ttc cag cag (SEQ ID NO: 27)

Sense ctg ctg gaa gcc tac ttc gaa (SEQ ID NO: 28)

5 'SphI total length gcc atg gc atg c gtg tta cca aag acg ctg aaa cc (SEQ ID NO: 29)

The resulting amino acid sequence of this deletion mutant:

3 'primer used for all constructs:

3 'HindIII

gcc c aagctt tca gt ttt tac ggg ttt tga tac ggg (SEQ ID NO: 31)

&Quot; mature " MPIF-1 (mutant-1 in Figure 19)

Example 2

Most vectors used for transient expression of the MPIF-1 gene sequence in mammalian cells should have an SV40 replication origin. This allows replication of the vector to achieve a high copy number in cells (e.g., COS cells) expressing the T antigen required for initiation of viral DNA synthesis. Any other mammalian cell line may also be used for this purpose.

Exemplary mammalian expression vectors include a promoter component that mediates the transcription initiation of mRNA, a protein coding sequence, and signals required for transcription termination and signals required for polyadenylation of the transcript. Additional components include an enhancer, a receptor site for RNA splicing and an intervening sequence and a Kozak sequence with the donor site at the side. (LTR) derived from retroviruses such as SV40-derived early promoter and late promoter, HIVI, HTLVI and RSV, and an early promoter of cytomegalovirus (CMV) High efficiency transfer can be achieved. However, cell signals can also be used (e. G., Human acting promoters). Suitable expression vectors for use in the practice of the invention include, for example, vectors such as pSVL and pMSG (Pharmacia, Uppsala, Sweden), pRSVcat (ATCC 37152), pSV2dhfr (ATCC 37146) and pBC12MI (ATCC 67109) do. Usable mammalian host cells include human HeLa, 283, H9 and Jurkart cells, mouse NIH3T3 and C127 cells, Cos 1, Cos 7 and CV 1, African Green Monkey cells, quail QC1-3 cells, L cells and Chinese ham star ovary cells.

Alternatively, the gene may be expressed in a stable cell line containing the gene integrated into the chromosome. It is possible to identify and isolate transfected cells through co-transfection using selectable markers such as dhfr, gpt, neomycin, hygromycin.

Transfected genes can also be amplified to express large quantities of encoded proteins. DHFR (dihydrofolate reductase) is a useful marker for developing cell lines carrying hundreds or even thousands of copies of the gene of interest. Another useful selection marker is the enzyme glutamine synthase (GS). Murphy et al., Biochem J. 227: 277-279 (1991); Bebbington et al., Bio / Technology 10: 169-175 (1992). Using these markers, mammalian cells are grown in a selective medium and cells with the highest resistance are screened. These cell lines contain the amplified gene (s) integrated into the chromosome. For protein production, Chinese hamster ovary (CHO) cells are often used.

Expression vectors pC1 and pC4 are potent transformants of the Rous sarcoma virus [Cullen et al., Molecular and Cellular Biology, 438-447 (1985. 3.)] and fragments of the CMV-enhancer [Boshart et al., Cell 41: 521-530 Lt; / RTI > For example, multiple cloning sites with restriction enzyme cleavage sites BamHI, XbaI and Asp718 facilitate cloning of the gene of interest. In addition to the 3 ' intron, the vector contains the polyadenylation and termination signals of the rat preproinsulin gene.

A. Expression of Recombinant MPIF-1 in COS Cells

Expression of the CMV-MPIF-1 HA plasmid was determined by the following procedures: (1) SV40 cloning start point, (2) ampicillin resistance gene, (3) E. coli cloning start point, (4) CMV promoter- Is derived from the vector pcDNAI / Amp (Invitrogen) containing a polyadenylation site. Expression of the recombinant protein under the CMV promoter can be performed by cloning the DNA fragment encoding the entire MPIF-1 precursor and the HA tag fused within the frame at its 3 'end into the polylinker region of the vector. The HA tag corresponds to an epitope derived from influenza hemagglutinin protein as described above (Wilson, H. et al., Cell 37: 767 (1991)). Through the injection of the HA tag to the target protein, detection of the recombinant protein using an antibody recognizing the HA epitope can be facilitated.

Plasmid construction strategies are as follows:

5 '-GGAAAGCTTATGAAGGTCTCCGTGGCT-3' (SEQ ID NO: 32) encodes the HindIII site (SEQ ID NO: 32), which encodes MPIF-1 by PCR on the original EST cloned using the following two primers: Followed by 18 nucleotides of the MPIF-1 coding sequence starting from the initiation codon; (SEQ ID NO: 33) encodes the last 20 nucleotides (without stop codon) of the complementary sequence, translational stop codon, HA tag and MPIF-1 coding sequence for the XbaI site in the 3 'sequence 5'-CGCTCTAGATCAAGCGTAGTCTGGGACGTCGTATGGGTAATTCTTCCTGGTCTTGATCC-3' . Thus, the PCR product contains the HindIII site, the HA tag fused to the frame followed by the MPIF-1 coding sequence, the translation termination stop codon adjacent to the HA tag, and the XbaI site. The PCR amplified DNA fragment and the vector pcDNAI / Amp are digested with HindIII and XbaI restriction enzymes and ligated. The ligation mixture is transformed into E. coli strain SURE (commercially available from Stratagene Cloning Systems, 11099 North Torrey Pines Road, La Jolla, CA 92037). The transformed cultures are plated on ampicillin medium plates to screen for resistant colonies. Plasmid DAN is isolated from transformants and examined for the presence of the correct fragments by restriction analysis. In the expression of recombinant MPIF-1, COS cells are transfected into expression vectors by the DEAE-DEXTRAN method [J. Sambrook, E. Fritsch, T. Maniatis, Molecular Cloning: A Laboratory Manual, Cold Spring Laboratory Press, (1989)]. Expression of MPIF-1-HA protein is detected by radiolabelling and immunoprecipitation [E. Harlow, D. Lane, Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory Press, (1988)]. Cells are labeled for 8 hours with ³⁵ S-cysteine 2 days post-transfection. The culture medium was then harvested and the cells were lysed with detergent [RIPA buffer (150 mM NaCl, 1% NP-40, 0.1% SDS, 1% NP-40, 0.5% DOC, 50 mM Tris, pH 7.5] (Wilson, I. et al., Id., 37: 767 (1984).) Both cell lysate and culture medium are precipitated with HA-specific monoclonal antibody. The precipitated proteins are analyzed on 15% SDS-PAGE gel do.

B. Cloning and Expression in CHO Cells

The vector pC1 is used for the expression of the MPIF-1 protein. Plasmid pC1 is a derivative of plasmid pSV2-dhfr (ATCC Accession No. 37146). Both plasmids contain the mouse DHFR gene under the control of the SV40 early promoter. The Chinese hamster ovary- or other cells that have dihydrofolate activity transfected with the plasmids can be selected by growing the cells in a selective medium supplemented with the chemotherapeutic agent methotrexate (Alpha minus MEM, Life Technologies). Amplification of intracellular DHFR genes with resistance to methotrexate (MTX) is disclosed in a number of publications. See, for example, Alt, FW, Kellems, R. M., Bertino, J. R., and Schimkc, R.T., 1978, J. Biol. Chem. 253: 1357-1370, Hamlin, J.L. And Ma, C. 1990, Biochem. et Biophys. Acta, 1097: 107-143, Page, M. J. and Sydenham, M.A. 1991, Biotechnology Vol. 9: 64-68. Cells grown at an increased concentration of MTX develop resistance to the drug by overexpression of the target enzyme DHFR as a result of amplification of the DHFR gene. When the second gene is bound to the DHFR gene, it is usually co-amplified and overexpressed. The development of cell lines capable of achieving more than 1,000 copies of the gene is state of the art. Thus, when removing the methotrexate, cell lines contain an amplified gene integrated into the chromosome (s).

For expression of the gene of interest, the plasmid pC1 has been shown to be a potent promoter of the long terminal repeat unit (LTR) of the Rous sarcoma virus (Cullen et al., Molecular and Cellular Biology, 1985. 3. 438-4470) and human cytomegalovirus (Boshart et al., Cell 41: 521-530, 1985) isolated from the enhancer of the immediate early gene. Downstream of the promoter is the following single restriction site cleavage site capable of integrating the genes: the polyadenylation site of the 3 ' intron and rat preproinsulin gene following BamHI. In addition, long terminal repeating units, such as HIV and HTLVI, derived from other high-efficiency promoters such as the human? -Actin promoter, SV40 early or late promoter or other retroviruses can be used for expression. For polyadenylation of mRNA, other signals derived from, for example, human growth hormone or globin may also be used.

In addition, selective markers such as gpt, G418 or hygromycin can be used to screen stable cell lines that carry the gene integrated into the chromosome during co-transfection. Initially, it is advantageous to use, for example, one or more selection markers in G418 and methotrexate.

Plasmid pC1 is digested with restriction enzyme BamHI and then dephosphorylated using calf intestinal phosphate according to procedures known in the art. The vector is then separated on a 1% agarose gel.

A DNA sequence encoding MPIF-1 using a PCR oligonucleotide primer corresponding to the 5 ' and 3 ' Amplify 75676:

The 5 'primer has the following sequence (SEQ ID NO: 34) containing a portion of the MPIF-1 protein coding sequence (SEQ ID NO: 1) of Figure 1 and an underlined BamHI restriction enzyme site:

5 'AAA GGA TCC GCC ACC ATG AAG GTC TCC GTG GTC 3'

BAMHI KOZAK

As described below, the 5 'end of the amplified fragment encoding human MPIF-1 inserted into an expression vector provides an effective signal peptide. Kozak, M., J. Mol. Biol. 196: 947-950 (1987), an efficient signal for translation initiation in eukaryotic cells is appropriately located in the vector portion of the construct.

The 3 'primer has the following sequence (SEQ ID NO: 35) including the nucleotide portion complementary to the MPIF-1 coding sequence (SEQ ID NO: 1) described in Figure 1 and the Asp718 restriction site, including the stop codon:

5 'AAA GGA TCC TCA ATT CTT CCA GGT CTT 3'

BamHI stop

The amplified fragments were separated on a 1% agarose gel as described above and then digested with endonucleases BamHI and Asp718 and then purified again on 1% agarose gel.

The isolated fragments and the dephosphorylated vector are then ligated with T4 DNA ligase. E. coli HB101 cells are then transfected and bacteria containing plasmid pC1 inserted in the correct orientation using the restriction enzyme BamHI are identified. The sequence of the inserted gene is confirmed by DNA sequencing.

Transfection of CHO-DHFR- cells

Chinese hamster ovary cells bearing active DHFR enzymes are used for transfection. Using the lipofecting method, 5 μg of the expression plasmid C1 is cotransfected with 0.5 μg of the plasmid pSVneo [Felgner et al., Supra]. The plasmid pSV2-neo contains the gene neo from Tn5, which encodes an enzyme that provides resistance to the dominant selection marker, G418 and other antibiotic classes. The cells are seeded in alpha minus MEM supplemented with 1 mg / ml G418. Two days later, the cells are trypsinized, seeded in hybridoma cloning plates (Greiner, Germany) and cultured for 10-14 days. After this period, single clones are trypsinized and then seeded into 6-well Petri dishes using different concentrations of methotrexate (25 nM, 50 nM, 100 nM, 200 nM, 400 nM). The clones grown in the highest concentration of methotrexate are then transferred to a new 6-well plate containing a higher concentration of methotrexate (500 nM, 1 [mu] M, 2 [mu] M, 5 [mu] M). The same procedure is repeated until the clones grow at a concentration of 100 [mu] M.

Western blot analysis and SDS-PAGE are used to analyze the expression of a given gene product.

Example 3

Expression pattern of MPIF-1 in human tissues

Northern blot analysis was performed to examine the expression levels of MPIF-1 in human tissues. Total cellular RNA samples were separated by the RNAzol ^TM B system (Biotecx Laboratories, Inc. 6023 South Loop East, Houston, TX 77033). Approximately 10 [mu] g of total RNA isolated from each specific human tissue was isolated from 1% agarose gel and blotted on a nylon filter (Sambrook, Fritsch, and Maniatis, Molecular Cloning, Cold Spring Harbor Press, . The labeling reaction is carried out using 50 ng of the DNA fragment according to the Stratagene Prime-It kit. The labeled DNA is purified with a Select-G-50 column (5 Prime-3 Prime, Inc. 5603 Arapahoe Road, Boulder, CO 80303). The filter is then hydriled with the radioactively labeled full-length MPIF-1 gene at 1,000,000 cpm / ml in 0.5 M NaPO ₄ , pH 7.4 and 7% SDS overnight at 65 ° C. After washing twice with 0.5 x SSC, 0.1% SDS at room temperature and twice at 60 캜, the filter is exposed overnight at -70 캜 using an intensifying screen.

Example 4

Expression and purification of chemokine MPIF-1 using a baculovirus expression system

SF9 cells are infected with recombinant baculovirus designed to express MPIF-1 cDNA. The cells were infected at MOI of 2 and incubated at 28 DEG C for 72-96 hours. Cell debris from infected cultures was also removed by low-speed centrifugation. Protease inhibitor cocktail was added to the supernatant at a final concentration of 20 ug / ml Pefabloc SC, 1 ug / ml leupeptin, 1 ug / ml E-64 and 1 mM EDTA. The concentration of MPIF-1 in the supernatant was monitored by dropwise addition of 20-30 상 of the supernatant to a 15% SDS-PAGE gel. MPIF-1 was detected as a visible 9 Kd band corresponding to an expression level of several mg per liter. MPIF-1 was further purified through a three-step purification procedure: heparin-binding affinity chromatography. The supernatant of the baculovirus culture was mixed with 1/3 volume of buffer containing 100 mM HEPES / MES / NaOAc pH 6 and filtered through a 0.22 쨉 m membrane. The sample was then applied to a heparin binding column (HE1 Poros 20, Bi-Perceptive System Inc.). pH 6, MPIF-1 was eluted with about 300 mM NaCl in a linear concentration gradient of 50 to 500 mM NaCl in 50 mM HEPES / MES / NaOAc; Cation exchange chromatography. MPIF-1 enriched from heparin chromatography was diluted 5-fold with buffer containing 50 mM HEPES / MES / NaOAc (pH 6). The resulting mixture was then applied to a cation exchange column (S / M Poros 20, Bio-Perceptive System Inc.). pH 6, MPIF-1 was eluted with about 250 mM NaCl in a linear concentration gradient of 25 to 300 mM NaCl in 50 mM HEPES / MES / NaOAc; Size exclusion chromatography. After cation exchange chromatography, MPIF-1 was further purified by applying it to a size exclusion column (HW50, TOSO HAAS, 1.4 x 45 cm). MPIF-1 was fractionated at a location close to the 13.7 Kd molecular weight standard (RNase A), which corresponds to the dimeric form of the protein.

After the three-step purification procedure described above, the resulting MPIF-1 was assessed to be at least 90% pure as determined from Coomassie blue staining of SDS-PAGE gel (FIG. 3).

In addition, purified MPIF-1 was tested against endotoxin / LPS contaminants. Following LAL analysis (Bio Whittaker), the LPS content was less than 0.1 ng / ml.

Example 5

Effect of baculovirus-expressing M-CIF and MPIF-1 on M-CSF and SCF-stimulated colony formation of newly isolated bone marrow cells

Low density populations of mouse bone marrow cells were incubated in a 37 ° C-treated tissue culture dish for 1 hour to remove mononuclear cells, macrophages, and tracheal cells attached to plastic surfaces. A group of non-adherent cells were then plated (10,000 cells / dish) in the agar-containing growth medium, with or without the factors as shown in Fig. The cultures were incubated (88% N ₂ , 5% CO ₂ and 7% O ₂ ) for 10 days at 37 ° C and scored colonies under an inverted microscope. The data are expressed as the average number of colonies, which was obtained from three runs of analysis.

Example 6

Bone marrow cells Lin ^- Effects of MPIF-1 and M-CIF on LI-3 and SCF stimulation proliferation and differentiation of populations

A population of mouse bone marrow cells enriched in primitive hematopoietic progenitors was obtained through a negative selection procedure in which monoclonal antibodies (anti-cdllb, CD4, CD8, CD45R and Gr-1 antigen ) Panel and magnetic beads were used to remove dendritic cells from most lines. (50 ng / ml) in the growth medium supplemented with IL-3 (5 ng / ml) and blastocyte factor (SCF) (100 ng / ml) Depleted cells) were plated (5 x 10 ⁴ cells / ml). After incubation for 7 days in a humidified thermostat (5% CO ₂ , 7% O ₂ and 88% N ₂ environment) at 37 ° C, cells were harvested and analyzed for HPP-CFC and immature progenitor cells. In addition, cells were analyzed for expression of specific different antigens by FACScan. Colony data are expressed as the mean number of colonies (+/- SD), which was obtained from analysis performed in 6 dishes for each group of cells (Figure 9).

Example 7

In response to IL-3, M-CSF and GM-CSF, MPIF-1 inhibits colony formation.

Mouse bone marrow cells were flushed from both the thigh and tibia, separated by Ficoll density gradient, and mononuclear cells were removed by plastic attachment. Cells generated in MEM-based medium supplemented with IL-3 (5 ng / ml), GM-CSF (5 ng / ml), M-CSF (10 ng / ml) and G- Groups were incubated overnight. In the agar-based colony formation assay, in the presence of IL-3 (5 ng / ml), GM-CSF (5 ng / ml) or M-CSF (5 ng / The cells were plated in 1,000 cells / dish. The data presented colony formation as a percentage of the number of colonies formed by the specific agent alone. Both experiments are represented by data presented as the mean of two dishes using error bars representing the standard deviation for each experiment (Figure 11).

Example 8

Expression through gene therapy

Fibroblasts are obtained from the subject by skin biopsy. The tissue obtained is placed in a tissue-culture medium and separated into small pieces. A small piece of the tissue is placed on the wetted surface of the tissue culture flask. At this time, about ten pieces are placed in each flask. The flask is rotated up and down, allowed to adhere, and left at room temperature overnight. After allowing to stand at room temperature for 24 hours, the flask was inverted and a piece of tissue residue was fixed to the bottom of the flask and a fresh medium such as Ham's F12 medium was added with 10% FBS, penicillin and streptomycin . It is then incubated at 37 占 폚 for about one week. At this time, add a new medium and continue to exchange every few days. After an additional 2 weeks of culture, a fibroblast monolayer appears. The monolayer was trypsinized and weighed into a large flask.

The long terminal repeating unit of the Molloinius sarcoma virus is the pMV-7 located at the side [Kirschmeier, P.T. , DNA 7: 219-25 (1988)] is digested with EcoRI and HindIII and then treated with bovine phosphatase. Linear vectors are fractionated on agarose gels and purified using glass beads.

The cDNA encoding the polypeptide of the present invention is amplified using a PCR primer corresponding to each of the 5 'and 3' terminal sequences. A 5 'primer containing the EcoRI site and a 3' primer containing the HindIII site are used. Equal amounts of Moloney murine sarcoma virus linear backbone and EcoRI and HindIII fragments are added together in the presence of T4 DNA ligase. The resulting mixture is maintained under conditions suitable for ligation of the two fragments. The ligation mixture is used to transform bacterial HB101 and then plated on agar-containing kanamycin to ensure that the gene is properly inserted into the vector.

Amphotropic pA317 or GP + aml2 packaging cells were grown in a tissue culture in Dulbecco's Modified Eagles Medium (DMEM) with 10% calf serum (CS) at a density of fusion . An MSV vector containing the gene is then added to the medium and the cells are transduced with the vector. The filled cells now produce infectious viral particles containing the gene (the filled cells are now referred to as producer cells).

New medium is added to the transduced producer cells, and then the medium is harvested on a plate of 10 cm fusion producer cells. The used medium containing the infectious viral particles is filtered through a Millipore filter to remove the separated producer cells and the fibroblasts are infected with this medium. The medium is removed from the sub-fusion plate of the fibroblasts and quickly replaced with medium from the producer cells. Remove the paper and replace with a new medium. If the viral titer is high, all visible fibroblasts will be infected and no screening is required. If the titer is very low, it is necessary to use retroviral vectors with selectable markers such as neo or his .

The engineered fibroblasts are then grown either singly or to confluence on cytodex 3 microcarrier beads and then injected into the host. The fibroblasts now produce protein products.

Example 9

In vitro myeloprotection

As described above, MPIF-1 is a potent inhibitor of myeloid progenitor cells, hypoproliferating potential colony-forming cells (LPP-CFCs) that produce the granulocyte and monocyte lineage. To demonstrate that MPIF-1 protects LPP-CFC from cytotoxicity of cell cycle chemotherapeutic agents, the line-cell of the lineage-depleted population (Lin-cell) was isolated from mouse bone marrow and multiple cytotoxic Lt; / RTI &gt; in the presence of Cyne. After 48 hours, a set of each 5-Fu culture was obtained and incubation continued for an additional 24 hours, at which time a number of live LPP-CFCs were determined by a clonogenic assay . As shown in Figure 21A, while ~ 40% of the LPP-CFC was protected from 5-Fu-induced cytotoxicity in the presence of MPIF-1, the presence of an irrelevant protein and the absence of MPIF- A little protection (<5%). High proliferating potential colony-forming cells (HPP-CFC) were not protected by MPIF-1 under the same culture conditions, demonstrating that the MPIF-1 protection effect is specific.

A similar experiment was performed using the chemotherapeutic agent Ara-C instead of 5-Fu. As shown in FIG. 15B, LPP-CFCs were highly protected by both wild-type MPIF-1 and mutant MPIF-1 (i.e., variant-1, see description of this variant, see Example 11 below). Thus, MPIF-1 can protect LPP-CFC from cytotoxicity induced by both chemotherapy 5-Fu and Ara-C.

Example 10

In vivo bone marrow protection

The results of the in vitro bone marrow protection suggest that MPIF-1 can protect critical cell types within the bone marrow during the duration of the chemotherapeutic drug, by cytotoxic drugs, a serious side effect observed in chemotherapeutic cancer patients Suggesting that the induced bone marrow toxicity may be improved. To demonstrate in vivo bone marrow protection, two types of experiments were performed in mice. In one experiment, MPIF-1 1.0 mg / Kg was intravenously injected (IP) for 3 days at intervals of 24 hours to a group of mice (Group-4), and on day 3, 5-Fu 150 mg / Kg was injected again (IP). Animals treated with saline (Group 1), MPIF-1 alone (Group-2), or animals injected with 5-Fu alone (Group-3) were used as controls. Then, the number of white blood cells (WBC) in the peripheral blood was measured by sacrificing four animals from each group on days 3, 6 and 10 after administration of 5-Fu. As shown in Fig. 16, only the MPIF-1 injection had a slight effect on the number of WBCs. As expected, 5-Fu treatment significantly reduced circulating WBC counts on day 6 after 5-Fu treatment. Animals treated with MPIF-1 before 5-Fu administration showed approximately 2-fold higher numbers of WBCs in the blood than animals treated with 5-Fu alone, which is significant. Thus, treatment of mice with MPIF-1 before 5-Fu treatment may promote recovery of neutropenia.

Hematopoietic stem cells and multipotent progenitor cells in the bone marrow are responsible for restoring all hematopoietic lines following chemotherapy. In normal individuals, these cells are divided at a lower frequency and thus survive a single dose of the chemotherapeutic agent. However, if a second dose of the chemotherapeutic agent is administered within 3 days after the first dose, the critical precursor cell types within the bone marrow are rapidly cleaved during this period, and these cells are killed.

To demonstrate that MPIF-1 can protect these cell types in the bone marrow, the following experiments were performed. Experiments were carried out using three groups of mice (6 per group) treated as follows: Group 1 injected with saline on days 1, 2 and 3; Group-2 injected with 5-Fu on days 0 and 3; Groups injected with 5-Fu on days 0 and 3, and MPIF-1 on days 1, 2 and 3 -3. See FIG. On days 6 and 9, bone marrow was harvested and HPP-CFC and LPP-CFC frequencies were measured according to the proliferative cell assay method known in the art. The results demonstrate that administration of MPIF-1 prior to the second dose of 5-Fu rapidly restores HPP-CFC and LPP-CFC frequencies by 9 days compared to animals treated with 5-Fu only.

Example 11

Studies using MPIF-1 variants

Multiple MPIF-1 variants truncated at the N-terminal were identified and characterized. The amino terminal sequences of these variants measured by Edman degradation are shown in FIG. For example, variant-2, variant-3, variant-7 and variant-8 spontaneously developed during the purification of the mature form of MPIF-1, and this preparation is called formulation K0871. Similarly, variant-2, variant-3, variant-4 and variant-5 were found in batches of other purified MPIF-1 formulations (formulation HG00300-B7). Since these mutants could not be purified from each other, the following experiment was carried out using Formulation K0871 and HG00300-B7. Mutant-6, identical to variant-3 for the amino terminal sequence except for N-terminal methionine, was generated by in vitro mutagenesis. The same variant-1 as the wild type except for the N-terminal methionine was also generated by the mutagenesis method. In addition, a cDNA clone encoding another splicing form of MPIF-1 (variant-9) and 137 amino acids (Fig. 20A) was found (see Fig. 19). By comparing the amino acid sequence of variant-9 with the amino acid sequence of MPIF-1, it was found that 18 amino acids were inserted between residues 45 and 46 of the MPIF-1 sequence and that arginine of MPIF-1 was lost (Fig. 20B). The biological activities of these MPIF-1 variant proteins are summarized below.

Intracellular calcium distribution. In the previous examples, MPIF-1 protein was shown to channel calcium across mononuclear cells. Wild-type and mutant MPIF-1 proteins were tested for their ability to induce intracellular calcium distribution in human monocytes using human MIP-1 alpha as a positive control. Experiments were performed as follows: human monocytes were separated by elutriation and incubated for 30 min at 37 ° C with 2.5 mM indol-1 / acetoxymethyl ester and 1 mM CaCl ₂ , 2 mM MgSO ₄ , 5 1 x 10 &lt; ⁶ &gt; cells in 1 ml of HBSS containing 10 mM HEPES (mM) glucose and 10 mM HEPES (pH 7.4) were incubated and added dropwise with indomethacin / acetoxymethyl ester. Then, the cells washed with HBSS and resuspended in the same buffer with ⁵ x 10 5 cells / ㎖, were stimulated with various concentrations of the proteins indicated at 37 ℃. Fluorescence-induced fluorescence in response to changes in intracellular calcium ((Ca ⁺⁺ ) i) on a Hatchi F-2000 fluorescence spectrometer by monitoring the excitation of Indo-1 at 330 nm and emission at 405 and 485 nm Signal was measured. The results are shown in Fig.

The results show that variant-6 is not distinguishable from wild-type and variant-1 is about twice as active as wild-type, whereas formulation K0871, HG00300-B7 and variant-9 are 10 times more active than wild type Prove that. See FIG. Since MIP-1 alpha and MPIF-1 are 45% identical to the primary amino acid sequence, they were intended to determine whether they interact with the same receptor. To investigate this possibility, the ability of MPIF-1 to de-sensitize MIP-1α-induced calcium circulation was studied. Figures 22A and 22B show that MIP-1 alpha and MPIF-1 wild-type proteins can de-sensitize calcium circulating capacity in mononuclear cells, while MCP-4 (other beta-chemokines) do not.

In a similar experiment, formulations K0871, HG00300-B7, and variant-1, variant-6 and variant-9 were able to block MIP-1α induced calcium circulation. This experiment was performed as follows: In the experiment as disclosed in Fig. 21, calcium channel response of human mononuclear cells to 10 ng / ml of induced protein was measured as described above. For de-sensitization studies, monocytes were first exposed to one factor, and when the response to the first treatment returned to baseline, the second factor was added to the same cells. (-) signal when there is no response to the second factor and a (+) signal indicating the stimulus response to the first factor (see FIG. 23).

Thus, MPIF-1 and its variant variants share a component of cell surface receptors for MIP-1 alpha and appear to interact with it. The recent demonstration that the MIP-1? Receptor functions as a cofactor to facilitate the introduction of HIV into human monocytes and T-lymphocytes raises the interesting possibility that MPIF-1 and its variants may interfere with the process of HIV introduction into the cells .

Main harmony. The chemotaxis of human peripheral blood mononuclear cell (PBMC) fractions (consisting primarily of lymphocytes and mononuclear cells) was measured in response to varying concentrations of MPIF-1 and its variants in 96-well neuroprotective chemotactic chambers. The experiments were performed as follows: Cells were washed three times with HBSS (BSS / BSA) containing 0.1% BSA and resuspended at 2 x 10 ⁶ / ml for labeling. Calcine-AM (molecular probe) was added to a final concentration of 1 mM and the cells were incubated at 37 占 for 30 minutes. After such incubation, the cells were washed three times in HBSS / BSA. The labeled cells were then resuspended at 8 x 10 ⁶ / ml and 25 ml (2 x 10 ⁵ cells) of this suspension were dispensed above the chamber angle of a 96 well chemotactic plate. Various concentrations of chemotactic agents were dispensed into the chamber bottom of each well. The top and bottom of the chamber were separated by a polycarbonate filter (3-5 mm pore size; without PVP; NeuroProbe, Inc.). Cells were transferred for 45-90 minutes and then quantified using a Cytofluor 11 fluorescence plate reader (PerSeptive Biosystems) in the number of transferred cells (all attached to the bottom of the chamber and to the bottom surface of the filter) Respectively. Values represent the concentration at which the peak activity induced at a magnification relative to the background indicated in parentheses is observed.

The results shown in FIG. 24 indicate that variant-1 and variant-6 are indistinguishable from wild-type, whereas formulations K0871 and HG00300-B7 are more potent inducers of chemotaxis than wild type.

Effect on LPP-CFC Colony Formation. To determine the effect of MPIF-1 variants on colony formation by LPP-CFC, a limited number of mouse bone marrow cells were cultured in soft agar containing medium supplemented with multiple cytokines with or without varying concentrations of MPIF-1 variant . IL-1 alpha (10 ng / ml) and M-CSF (5 ng / ml) were added to the following recombinant mouse cytokines IL-3 (5 ng / Ml), a low density population of mouse bone marrow cells was plated (1,500 cells / 3.5 cm diameter dish) in agar containing various concentrations of indicated media with or without indicated MPIF-1. Plates were then incubated in a tissue culture thermostat for 14 days, scoring LPP-CFC colonies under an inverted microscope. The data presented in Figure 25 was obtained from several different experiments that were analyzed twice under each condition.

The results show that the effective concentration required for 50% of the maximal inhibition in the case of preparations K0871 and HG00300-B7 was 20- to 100-fold lower than that of the wild type and 2 to 10 times lower than wild type in the case of variant -6 (See FIG. 25). Thus, deletion of the N-terminal amino acid of the MPIF-1 protein results in an increase in the efficacy of the molecule.

Example 12

In vivo, MPIF-1 induced blastocyst distribution

To demonstrate that MPIF-1 stimulates circulation of the parent cells in vivo, the following experiment was performed. Six mice were used for each treatment group (C57B black 6 / J, female, about 6 weeks old). The mice were injected (I.P.) with saline (vehicle control) or MPIF-1 (5 [mu] g / mouse). After 30 minutes, the mice were bled and analyzed for WBC with a Coulter counter. Blood collected from all six animals in each group was then collected and analyzed for Gr.1 + cells and CD34.Sca-1 + double positive cells using FACScan. WBC counts are expressed as mean ± SD, and FACScan data are expressed as% of total cells. Since the CD34.Sca-1 + double positive cells are thought to represent the anticipated properties of the hematopoietic stem cells, the results shown in FIG. 26 demonstrate that MPIF-1 can be used as a cell transfer agent.

Example 13

MPIF-1 treatment during 5-Fu treatment allows faster recovery of platelets and granulocytes .

Two of the major complications caused by chemotherapy are neutropenia (reduced blood neutrophil count) and thrombocytopenia (reduced platelet count). Granulocyte-colony stimulating factor (G-CSF) is currently being used clinically to alleviate neutropenia. G-CSF is known to stimulate colony formation by colony forming units-granulocyte (CFU-G) in vitro and to stimulate granulocyte generation in animal models. Thrompopoietin (Tpo) is used in clinical trials to alleviate thrombocytopenia. Tpo is known to stimulate colony formation by colony forming unit-megakaryocytes (CFU-Meg) in vitro and to stimulate platelet production in experimentally induced thrombocytopenia in animal models. One of the major limitations of G-CSF in clinical practice is that it is not effective in alleviating neutropenia in patients receiving multiple cycles of chemotherapy. This is likely due to depletion of CFU-G in target cells, bone marrow, to which G-CSF acts. Tpo will also suffer from the same fate as indicated by early clinical trial results. Any drug that can prevent the depletion of Tpo target cells and G-CSF during chemotherapy will be clinically of great value. The data presented below suggest that MPIF-1 may meet these clinical needs.

In the previous examples, MPIF-1 was shown to inhibit colony formation by bipotential granulocyte / mononuclear bone marrow precursor cells in vitro. In particular, Examples 9 and 10 provide data demonstrating that MPIF-1 protects virulent bone marrow progenitor cells from in vitro and in vivo 5-Fu induced cytotoxicity. These pluripotent progenitor cells are expected to generate more dendritic precursor cells of all myeloid lineages, including CFU-G and CFU-Meg. The following experiment was conducted to demonstrate that MPIF-1 treatment during 5-Fu treatment in 2 or 3 cycles can restore platelets and granulocytes more rapidly.

MATERIALS AND METHODS: C57BL6 female mice (7-10 weeks old) with an average body weight of 19.4 g (± 1.1 SD, n = 150) were used. All mice were provided with standard diet and cancer / light cycle residence and temperature throughout the experiment. The MPIF-1 preparation (HG00304-E6) was prepared in E. coli, which has a truncated form of MPIF-1 deficient in the 23 N-terminal amino acid of the mature protein (i.e., Of MPIF-1 variant-3). Clinical grade G-CSF (Neupogen ^R ) was purchased from Shady Grove Pharmacy, Rockville, MD 20850 (Neupogen ^R is manufactured by Amgen Inc., Amgen Center, Thousand Oaks, CA 91320). 5-Fluorouracil (5-Fu) was purchased from Sigma Chemicals and dissolved in hot water immediately before use to prepare it. The MPIF-1 solution was prepared by diluting with normal saline. Similarly, G-CSF was diluted in a buffer consisting of 10 mM sodium acetate, 5% (wt / v) mannitol, 0.004% (v / v) Tween 80, (pH 4.0). A rat monoclonal antibody conjugated with a suitable fluorescent dye for mouse CD41a, Gra.1 and Mac.1 antigens was purchased from Pharmingen.

Five groups of mice (30 mice per group) were treated as follows:

Group 1 received IP injection of 0.1 ml of a saline solution on days -2, -1, 0, 6, 7, and 8, and this was used as a normal control.

Group 2 received IP injection of 0.2 ml (100 mg / kg body weight) of 5-Fu solution on days 0 and 8.

Group 3 was injected with 5-Fu as in Group 2 and IP injection of 0.1 ml (1.0 mg / kg body weight) of MPIF-1 solution on days -2, -1, 0, 6, 7 and 8.

Group 4 was injected with 5-Fu as in Group 2 and IP injection of 0.1 ml (0.5 mg / kg body weight) of G-CSF solution on days 1, 2, 3, 9, 10 and 11.

Group 5 was injected with 5-Fu as in group 2 , MPIF-1 as in group 3 , and G-CSF as in group 4 .

Six animals from each group were then analyzed on the indicated day and platelet and granulocyte recovery was monitored for peripheral blood and bone marrow concentration. It should be noted that the mice analyzed on days 6 and 8 after the first 5-Fu treatment were not treated second with MPIF-1 5-Fu.

Peripheral blood was collected from the retroorbital sinus in EDTA-coated tubes and immediately analyzed by FACS Vantage to determine the number of platelets (CD41a positive events) and granulocytes (Gra.1 and Mac.1 double positive cells) Respectively. It should be noted that the method of analysis and species of animals used are not possible to complete the counting. Instead, granulocytes were expressed as a percentage of total leukocyte cells, and platelets were evaluated as CD41a positive events per 15 seconds on a sorter. The mice were then sacrificed and bone marrow cells were obtained by standard methods. Bone marrow cells were also subjected to FACS analysis to monitor the percentage of Gra.1 and Mac.1 double positive cells in bone marrow cells. Since the steps by which these antigens begin to be expressed in the granulocyte lineage are not precisely known, Gra.1 and Mac.1 double positive cells in the bone marrow are expected to be heterogeneous with respect to their developmental and maturation potential stages.

In addition, the number of progenitor cells in proliferating cells was measured by analyzing bone marrow using in vitro proliferative cell analysis method. In summary, hyperproliferative potential colony forming cells (HPP-CFC) and low proliferating potential colony forming cells (LPP-CFC) analysis were performed in two layers of agar culture system. In a 3.5 cm diameter dish, cells were treated with 20% FBS, 0.5% Difco agar, 7.5 ng / ml mIL-3, 75 ng / ml mSCF, 7.5 ng / ml hM- CSF and 15 ng / ml mIL- The bottom layer was prepared with 1 ml of MEM supplemented. Then 0.5 ml of a murine bone marrow cell suspension containing 2,000 cells / dish in MEM supplemented with 20% FBS and 0.3% agar was superimposed on the layer. The upper agar was allowed to solidify at room temperature for about 15 minutes. The plates were then incubated for 14 days in tissue culture incubators (37 ° C, 88% N ₂ , 5% CO ₂ and 7% O ₂ ) and scored colonies on an inverted microscope. The total number of colonies in this experiment was recorded.

FACS data were obtained by analyzing the substances obtained from 3 animals of each group at each time point, whereas proliferative cell analysis was performed using cells obtained from 6 animals of each group at each time point. Finally, the data points for the dates of the experiments are expressed as values obtained from saline-injected normal mice (Group 1).

Results: To monitor platelet recovery in peripheral blood, the steady state concentration of CD41a-positive cells was measured by FACS vantage. As shown in Figure 27, the group treated with MPIF-1 before treatment with 5-Fu (Group 3) showed faster and more potent platelet recovery than that observed with 5-Fu + saline treated group (Group 2) Respectively. As expected, the kinetics of platelet recovery in mice treated with G-CSF (Group 4) was indistinguishable from that observed in 5-Fu + saline treated mice. In addition, the group treated with 5-Fu treated G-CSF and MPIF-1 (Group 5) showed a higher overall platelet concentration compared to that observed in MPIF-1 treated group (Group 3) There was no effect. Thus, platelet recovery in peripheral blood was rapid in mice pretreated with MPIF prior to 5-Fu treatment.

Granulocyte recovery in peripheral blood was monitored by quantifying steady state concentrations of Gra.1 and Mac.1 double positive cells in the blood. As shown in Fig. 28, steady-state concentrations of Gra.1 and Mac.1 double-positive cells in the blood rapidly decreased after 6 days of the first and second 5-Fu treatments in 5-Fu treated mice. Pre-processing of MPIF-1 provided two advantages; The degree of neutropenia (degree of depletion of Gra.1 and Mac.1 double-positive cells) was much lower and the rate of recovery was much faster compared to that observed in 5-Fu and saline treated mice (Group 2) Respectively. As expected, the recovery rate of Gra.1 and Mac.1 double positive cells in the blood was rapid in the group treated with G-CSF after 5-Fu treatment (Group 4). However, the degree of neutropenia recovery in G-CSF treated mice was much less than that observed in MPIF-1 treated group (Group 3) at 8 days. MPIF-1 and G-CSF dosing effects on granulocyte depletion and recovery (Group 5) were significantly higher than those observed in mice treated with MPIF-1 alone or G-CSF alone in these mice, Gra.1 and Mac. 1 cells were very large in that the steady-state concentrations of double-positive cells were very high. Thus, as shown in Figure 28, MPIF-1 and G-CSF appear to exert additional effects when they are administered together.

As described above, recovery at the bone marrow concentration was monitored by FACS vantage method and proliferative cell analysis. The results obtained by FACS are shown in Fig. As expected, the concentrations of Gra.1 and Mac.1 double positive population of 5-Fu treated bone marrow (Group 2) cells rapidly dropped from 6 to 14 days and returned to normal levels on day 16. The effect of 5-Fu mediated depletion of these Gra.1 and Mac.1 dendritic cells was completely abolished when the mice were treated with MPIF-1 before 5-Fu (Group 3). Surprisingly, G-CSF (Group 4) was able to prevent the depletion of Gra.1 and Mac.1 double positive cells in response to the first 5-Fu dose, but not in the second dose. This seems to be due to the effectiveness of G-CSF target cells and the timing of administration of G-CSF. The recovery after 8 days of the first 5-Fu treatment was much higher than that observed in mice treated with MPIF-1 alone or G-CSF alone, but similar responses were observed in mice treated with MPIF-1 and G-CSF 5).

The data obtained in the proliferation assay is shown in Fig. The frequency of the remaining progenitor cells in the bone marrow was reduced in response to 5-Fu for 14 days, and recovered slightly on the 16th day. The decrease in the frequency of these progenitor cells disappeared in mice treated with MPIF-1 before 5-Fu treatment. On the other hand, even when mice were treated with G-CSF, they were not effective in maintaining the frequency of precursor cells found in normal or MPIF-1-treated bone marrow. The effect of G-CSF and MPIF-1 on myeloid progenitor cells seems to be complex.

Summary of preliminary clinical pharmacology tables

The following tables (Tables 2, 3 and 4) summarize the in vitro and in vivo first and second pharmacological studies.

Abbreviations in the table for the batches mentioned in Tables 2, 3 and 4. The MPIF-1 batch is designed by a multicomponent code that represents the type of organism in which the protein is expressed and the expressed product (e.g., growth, full length or variant). The letter after the hyphen at the end of the design represents the organism in which the protein is expressed or the vector used for expression (for example, B = baculovirus, C = CHO cell, E = E. coli ). The last three letters preceding the hyphen represent the type or modification of the expressed protein (e.g., 300 = full length MPIF-1, 301 = MPIF-1 Δ17 variant, 302 = methionine added to the amine terminus of the mature amino acid sequence Mature MPIF-1 containing the residue, 304 = MPIF-1 [Delta] 23 variant and 311 = full length MPIF-1). Thus, the layout design represents the form of the expressed MPIF-1 protein, whether the protein is secreted from the host cell, and, if secreted, the form of the secreted protein. For example, HG00300-B5 indicates that baculovirus vectors were used to express full-length MPIF-1 protein. Also, because such systems are used to express the MPIF-1 protein, the isolated form of this protein is mature MPIF-1.

One exception to the nomenclature mentioned above corresponds to the case of batch HG00300-B7. This arrangement contains a mixture of four different MPIF-1 polypeptides. The inventors believe that these polypeptides were generated as a result of proteolytic cleavage of MPIF-1 occurring during the purification process. The MPIF-1 variant present in batch HG00300-B7 is discussed in Example 11.

First Pharmacology - In vitro Experimental Design Cell type MPIF-1? 23 dose Chemical agent result Effect of MPIF-1 or MPIF-1? 23 on Colony Formation Using Mouse Bone Marrow HPP-CFCLPP-CFC 0.01-100 ng / mL NA Both MPIF-1 and MPIF-1Δ23 resulted in a dose-dependent decrease in the frequency of LPP-CFCs. MPIF-1Δ23 was significantly more effective than MPIF-1 at all test concentrations. But did not significantly affect the frequency of -CFC. Effects of MPIF-1Δ23 on proliferation of human hematopoietic progenitor cells CD34 ⁺ , human cord blood 1-100 ng / mL NA Treatment with MPIF-1Δ23 resulted in 20% -40% inhibition of cell viability. The results suggest that MPIF-1Δ23 is a myeloid pro-inflammatory factor. Measurement of specific progenitor cell types targeted by MPIF-1? 23 CD34 ⁺ , human 50 ng / mL NA The formation of BFU-E, CFU-G, CFU-M, and CFU-Meg was not inhibited. The results showed that MPIF-1Δ23 inhibited the formation of CFU-GM and CFU- Is defined as an inhibitor of human granulocyte / mononuclear cell precursor cells. Characterization of MPIF-1Δ23 Inhibition on Mouse Bone Marrow Mouse marrow 50 ng / mL NA MPIF-1Δ23 reduced the frequency of myelinated CFU-GM colonies to 30% of the control. The frequency of LPP-CFC colonies was reduced to 25% of the control. MPIF-1Δ23 expresses CFU-E, BFU-E And the formation of HPP-CFC colonies. Performance of MPIF-1Δ23 to protect the systemic deficit of bone marrow cells from cytotoxic action of 5-FU Mouse marrow NA 5-FU MPIF-1Δ23 protected 40% -50% of LPP-CFC from cytotoxicity induced by 5-FU. MPIF-1Δ23 did not protect HPP-CFC.

First pharmacology - In vivo Experimental Design Bell Placement of MPIF-1 MPIF-1 dose, schedule, route Chemical agent Dose, schedule, route End result In vivo effects of MPIF-1 or MPIF-1Δ23 on the frequency of HPP-CFC and LPP-CFC in peripheral blood and bone marrow mouse HG00300-B5HG00304-E2 0.5 mg / kg, twice daily at 8 hour intervals for 2 days, i.p. NA NA MPIF-1Δ23 significantly reduced the frequency of LPP-CFC in the bone marrow. The effect of MPIF-1Δ23 on the frequency of LPP-CFC in the blood was variable. MPIF-1Δ23 did not affect the frequency of HPP-CFC I did. Measurement of optimal MPIF-1Δ23 dosing schedule to protect against 5-FU mouse HG00304-E2 1 mg / kg, varying between -3 days and 0 day, i.p. 5-FU 150 mg / kg, 0 day, i.p. MPIF-1Δ23 administered on days -2, -1, and 0 was most effective in protecting bone marrow from cytotoxic effects of 5-FU. Dose dependence on bone marrow recovery after 5-FU Measurement of MPIF-1? 23 mouse HG00304-E6 0.01-10 mg / kg, -2 days, -1 days, 0 days, i.p. 5-FU 150 mg / kg, i.p. Dose-dependent reactions were observed at day 4, with the best recovery occurring at the lowest test dose (0.01 mg / kg). Dose response was not observed at all on day 6. - dependent curves were obtained on day 8 and optimal activity was observed at 0.1 mg / kg. Performance of MPIF-1Δ23 to protect the in vivo myeloid lineage from cytotoxic therapy mouse HG00304-E6 1 mg / kg, -2 day, -1 day, 0 day, i.p. 5-FU 150 mg / kg, 0 day, i.p. Colony formation from the bone marrow of mice treated with MPIF-1? 23 returned to normal on day 7 after treatment with 5-FU. Bone marrow colony formation from mice treated with only 5-FU Did not show recovery. Measurement of the protective action of MPIF-1? 23 from multiple cycles of chemotherapy mouse HG00304-E6 1 mg / kg, -2 days, -1 days, 0 days, 6 days, 7 days, 8 days 5-FU 100 mg / kg, 0 day and 8 day, i.p. MPIF-1? 23 protected the progenitor cells after two cycles of 5-FU The most dramatic protection appeared after the second cycle of 5-FU The chemical protective action of MPIF-1? ^{+ &} Lt; / RTI ^& gt ^; cells in the peripheral region.

First pharmacology - In vivo (continuous)

Experimental Design Bell Placement of MPIF-1 MPIF-1 dose, schedule, route Chemical agent Dose, schedule, route End result Measurement of MPIF-1Δ23 activity, which can accelerate recovery of bone marrow colonies, neutrophils and platelets after multiple cycles of chemotherapy Measurements of MPIF-1Δ23 activity in combination with G-CSF mouse HG00304-E6 1 mg / kg, -2 days, -1 days, 0 days, 6 days, 7 days, 8 days, i.p. 5-FUG-CSF 100 mg / kg, 0 and 8 days, i.p.0.5 mg / kg, 1 day, 2 days, 3 days, 9 days, 10 days and 11 days The severity of neutropenia as measured by deletion of Gr-1 and Mac-1 diploid cells was significantly less and was significantly lower in mice treated with MPIF-1 and 5-FU compared to mice treated with 5-FU alone The recovery rate was even faster. Treatment with G-CSF after 5-FU resulted in a rapid recovery of the double-positive cells in the blood. The degree of recovery in G-CSF treated mice was significantly less than that observed in MP-F-1 treated mice on day 8. Mice treated with MPIF-1 and G-CSF were significantly more resistant to MPIF-1 or G- CSF compared to those treated exclusively with CSF. Colony formation from the bone marrow of mice treated with 5-FU was significantly reduced. MPIF-1 treatment before 5-FU terminated the action of 5-FU on colony formation. Platelet recovery was faster and stronger in mice treated with MPIF-1 and 5-FU compared to those treated with 5-FU alone. The addition of G-CSF no longer affected.

2nd Pharmacology - In vitro Experimental Design Cell type Placement of MPIF-1 MPIF-1 Dose Range result Measurement of calcium migration by MPIF-1 or MPIF-1? 23 T cells, B cells, mononuclear cells, neutrophils, basophils, dendritic cells, NK cells, THP-1 cells HG00300-B7HG00302-E2HG00302-E3HG00304-E2HG00304-E3HG00304-E6HG003O4-E7HG00301-C1HG00311-C1 1-1000 ng / mL The detectable response was observed from monocytes and dendritic cells at 100 ng / mL. The mononuclear cell line THP-1 reacted with MPIF-1? 23 at a maximum effect of 100 ng / mL. Measurement of the chemotactic activity of MPIF-1Δ23 T cells, monocytes, neutrophils, lymphocytes, eosinophils, basophils, MK cells, platelets HG00300-B5HG00300-B7HG00302-E1HG00302-E2HG00303-E1HG00304-E2HG00304-E6HG00304-E7 0.1-1000 ng / mL MPIF-1Δ23 stimulated chemotactic responses to inactive T cells with maximal response at 10 ng / mL MPIF-1Δ23 showed chemotactic activity for newly isolated monocytes with maximal effect at 100 ng / mL Chemotactic response was observed in neutrophils. There was no response in other cells tested. Effects of MPIF-1 or MPIF-1? 23 on Monocytes Monocyte HG00300-B7HG00302-E1HG00302-E2HG00302-E3HG00304-E3HG00304-E6HG00301-C1HG00311-C1 0.5-1000 ng / mL MPIF-1? 23 induced a low but variable release of ribosomal N-acetyl-? -D-glucosidase from the newly isolated mononuclear cells. MPIF-1? 23 was found to inhibit the lysosomal enzyme elastase, glucuronidase, Did not affect the release of peroxidase MPIF-1? 23 induced monocytes and did not secrete IL-1?, TNF-a, IL-10 or IL-12? MPIF-1? Did not affect oxidative rupture or cytotoxic activity. Effect of MPIF-1 or MPIF-1Δ23 on histamine release Basophils, person HG00300-B5HG00300-B7HG00302-E2HG00304-E6 1-1000 ng / mL MPIF-1 and MPIF-1? 23 did not induce histamine release from basophils. Effect of MPIF-1 or MPIF-1? 23 on NK cell mediated lethality NK cells, human HG00302-E1HG00300-B7 1-100 ng / mL MPIF-1 and MPIF-1? 23 did not affect IL-stimulated NK cell mediated lethality of K562 cells.

Second pharmacology - in vitro (continuous)

Experimental Design Cell type Placement of MPIF-1 MPIF-1 Dose Range result Effects of MPIF-1 or MPIF-1Δ23 on Platelet Aggregation Platelet, person HG00302-E1HG00300-B7 0.1-100 ng / mL MPIF-1? 23 did not induce or regulate platelet aggregation. The effect of MPIF-1? 23 on the growth of untransfected human cells Fibroblasts, astrocytes, smooth muscle cells, epithelial cells, veins and microvascular endothelial cells, bone marrow, B cells, T cells, mononuclear cells, neutrophils, keratinocytes HG00300-B7HG00300-B5HG00302-E1 0.1-1000 ng / mL MPIF-1 [Delta] 23 did not induce, enhance or inhibit the cell proliferation enumerated for study. The effect of MPIF-1 or MPIF-1? 23 on the release of IL-6 and prostaglandin Human primary endothelial cells, pulmonary fibroblasts, and aortic smooth muscle cells HG00300-B5HG00300-B7HG00302-E1HG00300-E2HG00304-E2HG00301-C1 0.1-1000 ng / mL MPIF-1 and MPIF-1? 23 did not affect the release of IL-6 or proglastin. Effects of MPIF-1? 23 on capillary formation The primary microvascular endothelial cells HG00304-E2 0.1-100 ng / mL MPIF-1? 23 did not induce the formation of capillaries in vitro. The effect of MPIF-1 on the performance of tumor cells that can be invaded through the fused monolayer of endothelial cells Primary endothelial cells HG00300-B7 0.1-10 ng / mL ㆍ No action Effects of MPIF-1 or MPIF-1? 23 on adhesion of peripheral blood mononuclear cells or granulocytes to IL-1 activated endothelium Primary endothelial cells HG00300-B5HG00300-B5HG00304-E6HG00304-E7HG00301-C1 0.1-100 ng / mL ㆍ No action

Example 14

Preparation, recovery and purification of MPIF-1? 23 using pHE4-5 expression vector

MPIF-1 is a novel human β-chemokine. The mature form of MPIF-1 is secreted as a 99 amino acid peptide with a molecular weight of 11.2 kDa. In addition, 76 amino acids of length truncated form (MPIF-1? 23) were also identified during the early expression study of MPIF-1. In the baculovirus expression system, MPIF-1 [Delta] 23 was sequenced and subcloned. Biological measurements show that the truncated form is more active than the full-length counterpart.

Cloning and expression

The MPIF-1? 23 gene originally isolated from the aortic endothelial complementary deoxyribonucleic acid library was subcloned into the expression vector pHE4 in the single restriction enzyme cleavage sites NdeI and Asp 718 (Fig. 31), and the K12-derived E. coli strain SG 13009 Maryland, Bethesda, Nathesia Instruments Institute of Health, Susan Gottsman). Additional strains of E. coli that can serve as hosts suitable for protein expression using pHE4 include strains DH5a and W3110 (ATCC Accession No. 27325). The pHE4 vector contains a strong synthetic promoter with two lac operators. Expression from these promoters is regulated by the presence of a lac repressor and is induced using isopropyl beta -D-thiogalactopyranoside (IPTG) or lactose. The plasmid also contains an effective ribosome binding site and a synthetic transcription terminator downstream of the MPIF-1? 23 gene. The vector also contains a clone of the pUC plasmid and a neomycin phosphotransferase gene that forms kanamycin in the transformed bacteria.

Manufacturing method

Overview of the fermentation process. The fermentation process for MPIF-1? 23 is summarized in the following listed steps and is illustrated in FIG.

Master Seed Bank. The major cell bank (MCB) of E. coli transformed with the plasmid expressing MPIF-1? 23 was prepared under the current Good Manufacturing Practices. This bank was prepared in a medium containing glycerol as a cryopreservation agent and frozen at -80 占 폚. After preparation, the MCB was tested to confirm absence of contamination by phage or other microorganisms.

First Inoculation Step. The first inoculation step culture is prepared in a stationary shake flask containing the inoculum preparation medium. The shake flask is inoculated with 1: 2000 dilution using the thawed seed storage stock and placed in a shaker maintained at 225 rpm and 37 ° C for 12 hours.

Create step. Production phase cultures are prepared in a 100 liter Fed-Batch fermentor with DO ₂ , pH, temperature and nutrient source control. This production medium (37 ° C) is inoculated with the first inoculation step culture to provide an optical density (OD) of 0.20 units per millimeter at 600 nm. Protein expression is induced by the addition of IPTG (final concentration 20 mM) when the culture reaches 10 + units per millimeter or OD of -2 units at 600 nm. Cells are collected 4 hours after induction.

Cell collection step. Bacteria are recovered by centrifugation at 18,000 g using a continuous flow centrifuge. The formed cell paste is stored at -80 ° C.

Recovery of MPIF-1? 23

The recovery of MPIF-1? 23 is summarized in FIG.

Cell lysis. The E. coli cell paste is thawed and resuspended in 10 volumes of resuspension buffer. Subsequently, the cells are passaged through a homogenizer (2 times) at 7000 psi and then separated.

Cleaning the inclusion body. NaCl was added to the cell lysate to a final concentration of 0.5 M, and then concentrated twice by tangential flow filtration using a 0.45 μm membrane. The remaining dialysis retentate was dialyzed against 3 volumes of wash-2 buffer (100 mM Tris-HCl, 500 mM NaCl and 25 mM EDTA-Na ₂ ) and then washed with 1 volume of wash-1 buffer -HCl, 25 mM EDTA-Na ₂ ). The dialyzed artifact is diluted 2-fold using wash-1 buffer, and the insoluble fraction is collected by continuous centrifugation. Alternatively, the inclusion bodies can be cleaned by centrifugation.

Solubilization of inclusion bodies. The resulting pellet obtained by performing centrifugation was dissolved in the same volume of solubilization buffer (100 mM Tris-HCl, 1.75 M guanidine-HCl and 25 mM EDTA-Na₂). The suspension is initially stirred at room temperature for 2 to 4 hours and then at 2 to 10 ° C for 12 to 18 hours.

Cultivation (refold) . The suspension is centrifuged and the supernatant is collected and then mixed with 9 volumes of growth buffer (100 mM sodium acetate, 125 mM NaCl and 2 mM EDTA-Na ₂ ). The diluted material is held for about 2 hours (2 DEG C to 10 DEG C) to settle the precipitate. This material can be filtered, treated immediately, or stored for 72 hours or less.

refine

HS-50 cation exchange chromatography. Purification of MPIF-1? 23 is summarized in FIG. The filtrate is loaded onto a POROS HS-50 column equilibrated with 50 mM NaCl, 150 mM NaCl, pH 5.8-6.2. Proteins are eluted stepwise using NaCI (300 mM to 1500 mM). Fractions were eluted with 500 mM NaCl, collected and then diluted 2-fold with water for injection.

HQ-50 / CM-20 Anion / cation exchange chromatography. The collected fractions obtained by performing HS-50 column chromatography are loaded on a serial column set (CM-20 column followed by HS-50 column) equilibrated with CM-1 buffer. MPIF-1? 23 is eluted from the CM-20 column using NaCl (100 mM to 900 mM). The eluted fractions were analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis (SDS-PAGE) and reverse phase high performance liquid chromatography (HPLC) and fractions containing MPIF-1? 23 were collected and then subjected to ultrafiltration Concentrate by passage through additional HS-50 column.

Size Exclusion Chromatography. The CM-20 eluate is loaded onto Sephacryl-100 HR equilibrated with S-100 buffer. Fractions were collected and analyzed by SDS-PAGE and reverse phase HPLC. Fractions containing MPIF-1.DELTA.23 are collected, sterile filtered using a 0.2 μm filter, and stored at 2 ° C. to 10 ° C.

Specification for bulk materials

The following specifications, listed in Table 5 below, are established for bulk phase MPIF-1? 23.

Test and test specifications for the release of bulk MPIF-1Δ23 Specification Explanation Exterior Transparent colorless solution pH 5.8 ± 0.2 Protein concentration by BCA 1-5 mg / mL Purity ^* Reversed phase HPLC size exclusion HPLCSDS-PAGE (Coomassie blue staining) Reduction conditions Non-reducing conditions ≥90% ≥90% ≥90% ≥90% Residual DMA ≤100 pg / mg protein Immunomodulatory Muller cells (ammoebocyte) ≤10 EU / mg protein Biological tests (assessed by Ca ⁺ shift assay) Report results ^* The purity of the MPIF-1Δ23 formulation is compared to standard values, and its specifications are currently in use.

Specification for the drug product

The final drug product meets all the specifications listed for bulk materials in Table 5 and is also tested for sterility (21CRF610.12).

Example 15

Intracellular Ca in monocytes ⁺² MPIF-1? 23 mediated inhibition of colony formation correlated with the performance of MPIF-1 capable of translocating

MPIF-1? 23 inhibits LPP-CFC colony formation in in vitro soft agar assay and induces intracellular calcium mobilization in monocytes, including THP-1 cells (myelomonocytic cell line). Both measurement assays are used to evaluate the biological activity of MPIF-1Δ23 in tablet studies and safety studies. In LPP-CFC assay, freshly isolated rat bone marrow cells were treated with multiple cytokines (5 ng / mL IL-3, 50 ng / mL SCF, 5 ng / mL M-CSF and 10 ng / mL IL- Lt; / RTI &gt; in a soft agar. The culture is inoculated for 14 days, after which the colonies are scored using an inverted microscope.

Calcium migration testing uses newly isolated human monocytes or THP-1 cells loaded with Fura-2 (0.2 nM per milliliter). When cells are stimulated with MPIF-1? 23, Ca ²⁺ migration is assessed with a fluorimeter. The Ca ²⁺ shift assay provides a rapid indicator of the activity of the MPIF-1Δ23 preparation (Table 6).

Intracellular Ca in monocytes ²⁺ MPIF-1? 23 mediated inhibition of colony formation correlated with the performance of MPIF-1 capable of translocating MPIF-1Δ23 structure / arrangement / condition Ca ²⁺ transfer (ng / mL) ^* LPP-CFC inhibition (ng / mL) ^† MPIF-1 / HG00300-B5 1000 20-40 MPIF-1Δ23 / HG003004-E2, stored for 3 months at 4 ° C 100 5-10 MPIF-1Δ23 / HG00304, stored for one week 100 5-10 MPIF-1Δ23 / HG00304, stored for 4 weeks 100 5-10 MPIF-1Δ23 / HG00302-E2, stored at 4 ° C for 3 months 1000 > 100 MPIF-1? 23 / HG00304-E3, the first peak in the CM column 100 5-10 MPIF-1? 23 / HG00304-E4, the second peak in the CM column 100 5-10 MPIF-1? 23 / HG00304-E3, the third peak in the CM column > 1000 > 1000 ^* Minimum concentration required to transfer calcium from human monocytes and / or THF-1 cells ^† Concentration that produces 50% inhibition of LPP-CFC colony formation compared to the control

Formulation and Storage

Bulk phase MPIF-1? 23 is prepared by preservation treatment, and the liquid preparation is a sterile disposable product. The protein is buffered with a buffer of 50 mM sodium acetate, 125 mM NaCl, pH 5.8, filled into a Wheaton Type 1 glass vial and stored at 2-8 [deg.] C.

stability

Stability studies were performed using protein concentrations of 1.0 mg / mL buffered with sodium acetate at pH 5, 6 and 7 at -80 ° C, 2 ° C-8 ° C, 20 ° C-25 ° C, and 2 ° C-8 ° C .MPIF-1 [Delta] 23 was found to be stable for at least 6 months when stored at 2 [deg.] C to 8 [deg.] C in a solution of 10 mM sodium acetate, 125 mM NaCl at pH 5-7. For current ongoing studies, samples were analyzed for appearance, protein concentration, purity [SDS-PAGE (reducing and nonreducing conditions); Reverse phase HPLC and size exclusion HPLC] and activity (Ca ²⁺ mobile bioassay) meet the specifications outlined above.

A stability study for the MPIF-1 [Delta] 23 batch (HG-00304-E10) used in the preliminary clinical toxicology study was disclosed. The MPIF-1Δ23 batch used in this study was formulated at a protein concentration of 4.0 mg / mL in 50 mM NaOAc, 125 mM NaCl, pH 5.9. The storage conditions are -80 ° C, 2 ° C-8 ° C, 25 ° C and 37 ° C at 60% relative humidity and 45 ° C at 75% relative humidity. The stability study period is 12 months at 25 ° C or lower, 6 months at 37 ° C, and 1 month at 45 ° C. Stability is determined by appearance, pH, purity [SDS-PAGE (reducing and non-reducing conditions); Reversed phase HPLC; And size exclusion HPLC], and activity (Ca ²⁺ mobile bioassay). Endotoxin testing and bioburden testing prior to sterilization are performed at selected time points.

Example 16

MPIF-1 to protect gastrointestinal tract from Taxol-induced cytotoxicity

(Subcutaneous) administration of 0.3 mg / kg MPIF-1? 23, which was performed simultaneously with intraperitoneal administration of 10.5 mg / kg of Taxol on Day 0, Day 1 and Day 2, protected the rat from weight loss associated with intestinal toxicity. Rats treated with 0.3 mg / kg MPIF-1? 23 maintained their body weight on day 0 and day 5, and actually weighed after 9 days. Rats treated with 0.1 mg / kg MPIF-1.DELTA.23, or those without MPIF-1.DELTA.23, lost approximately 45 g of body weight between day 0 and day 5.

Paclitaxol (Taxol: registered trademark) and MPIF-1? 23 were administered on day 0, day 1 and day 2 as described above. The individual weights of the litters of all groups were approximately 185 g on day -2 (ie, two days prior to the first administration of paclitaxol and MPIF-1). After 5 days, the individuals in the two test groups (those receiving paclitaxol and those not receiving MPIF-1Δ23 or receiving 0.1 mg / kg MPIF-1Δ23) weighed approximately 155 g. In contrast, individuals receiving 0.3 mg / kg MPIF-1Δ23 weighed approximately 175 g. Control individuals (those with no paclitaxel or MPIF-1Δ23) were 195 g after 5 days of body weight. The ability of MPIF-1Δ23 to protect animals from the acute toxicity of Paclitaxol demonstrates the ability of cytotoxic drugs to protect many types of cells, including hematopoietic stem cells as well as gastrointestinal cells.

Example 17

Sublethal model of gastrointestinal protection

C57B1 / 6 female mice (12 weeks old) were exposed to a total dose of 9 Gy ¹³⁷ cesium acetic acid (dose rate: 25.54 cGy / min) delivered at the same dose of 4.5 Gy at 4 hour intervals on day 0. MPIF-1 (1 mg / kg / BID ip) was administered to a group of mice called "MPIF-1 (before irradiation)" for 4 consecutive days (-2 to +1 day). Groups referred to as " MPIF-1 (post-irradiation) " were administered MPIF-1 (1 mg / kg / BID ip) for 7 consecutive days starting on the first day after the first day of irradiation. In the control group, only diluent (HBSS + 0.1% normal mouse serum) was administered. All mice received acid water for 7 days before irradiation. Three days before irradiation, amoxicillin was added to acidic water (0.5 mg / mL) and continued daily until day 7 after irradiation. Mice were observed for survival, condition and weight change. Data are expressed as percent change in body weight of each group based on the individual body weight on the indicated date relative to body weight percent measured at the start of the experiment (see Figures 37-38). The ability of MPIF-1 to protect animals from the acute toxic effects of radiation demonstrates the ability to protect many types of cells, such as gastrointestinal cells, from cytotoxic drugs, as well as hematopoietic stem cells.

Example 18

Lethality model of camouflage protection

C57B1 / 6 female mice (12 weeks of age) were exposed to a total of 11 Gy ¹³⁷ cesium acute dose (dose rate: 25.54 cGy / min) delivered at two identical doses of 5.5 Gy at 4 hour intervals on day 0. MPIF-1 (1 mg / kg / BID ip) was administered to a group of mice called "MPIF-1 (before irradiation)" for 4 consecutive days (-2 to +1 day). The group called " MPIF-1 (post-irradiation) " was administered MPIF-1 (1 mg / kg / BID ip) for 7 consecutive days starting on the first day after the first day of irradiation. In the control group, only diluent (HBSS + 0.1% normal mouse serum) was administered. All mice received acid water for 7 days before irradiation. Three days before irradiation, amoxicillin was added to acidic water (0.5 mg / mL) and continued daily until day 7 after irradiation. Mice were observed for survival, condition and weight change. Data are expressed as percent change in body weight of each group based on the individual body weight on the indicated date relative to body weight percent measured on the day of the experiment (see Figures 39-40).

All of the controls (without MPIF-1) were killed on day 20 post-irradiation. In contrast, 25% of the MPIF-1 (post-irradiation) group and 57% of the MPIF-1 (pre-irradiation) group survived to 38 days after the irradiation. The ability of MPIF-1 to protect animals from lethal radiation demonstrates the ability to protect many types of cells, such as cells in the gastrointestinal tract, from cytotoxic drugs.

Example 19

How to Measure Cell Protection

One way to measure the protective performance of MPIF-1 relative to a particular cytotoxic drug is to assess the dose-reduction factor (DRF) or dose-modifiying factor (DMF) [Weiss, Environ. Health Perspectives, 105,1473-1478 (1997); Brown et al . , Pharmacol. Ther. 39, 157-168 (1998); Yuhas et al. , Int. J. Radiat. Biol. 15, 233-237 (1973); Weiss et al., Radiation and the Intestinal Tract, ed., Boc., Raton, FL, CRC Press, pp. 183-199 (1995)].

For example, mice are irradiated with a series of doses with and without MPIF-1. The protection of the gastrointestinal tract by MPIF-1 is measured by DMF for 6-7 days survival after irradiation of the entire body with a relatively high dose. DMF for 30-day survival measures MPIF-1 protection of the hematopoietic system.

Example 20

Construction of N-terminal and / or C-terminal deletion mutants

The following general approach can be used to clone N-terminal or C-terminal MPIF-1 deletion mutants. Generally, two oligonucleotide primers of about 15-25 nucleotides are derived from the desired 5 ' and 3 ' positions of the polynucleotide of SEQ ID NO: 1 or 6. The 5 'and 3' positions of the primers are determined on the basis of the desired MPIF-1 polynucleotide fragment. If necessary, an initiation codon and a termination codon are added to the 5 'primer and the 3' primer, respectively, to express the MPIF-1 polypeptide fragment encoded by the polynucleotide fragment. Preferred MPIF-1 polynucleotide fragments are those encoding the N-terminal and C-terminal deletion mutants disclosed above in the " MPIF-I polypeptide "

In addition, additional nucleotides containing restriction sites that facilitate the process of cloning MPIF-I polynucleotide fragments within the desired vector may also be added to the 5 'and 3' primer sequences. MPIF-1 polynucleotide fragments are amplified from genomic DNA or deposited cDMA clones using appropriate PCR oligonucleotide primers and conditions discussed herein or known in the art. MPIF-1 polypeptide fragments encoded by the MPIF-1 polynucleotide fragments of the invention can be expressed and purified in the same general manner as full-length polypeptides, but the difference between the chemical and physical properties between the particular fragment and the full- Therefore, routine modifications may be necessary.

Polynucleotides encoding the MPIF-1 polypeptide fragments R-46 to N-120 are amplified and cloned as follows, exemplifying the invention but not by way of limitation: The N-terminal portion of the polypeptide fragment, termed R-46, Primer containing a consecutive start codon at the restriction enzyme site in a frame containing the polynucleotide sequence encoding it. A complementary 3 ' primer containing a consecutive stop codon at the restriction site in a frame containing the polynucleotide sequence encoding the C-terminus of the MPIF-1 polypeptide fragment terminating with N-120 is generated.

The amplified polynucleotide fragment and the expression vector are digested with a restriction enzyme recognizing a site in the primer. The degraded polynucleotides are then ligated together. The MPIF-1 polynucleotide fragment is inserted into a restricted expression vector, preferably by placing the MPIF-1 polypeptide fragment encoding the region downstream of the promoter. This ligation mixture is transfected into competent E. coli cells using standard procedures and performing as described in the examples herein. Plasmid DNA is isolated from resistant colonies and identification of cloned DMA is confirmed by restriction analysis, PCR and DNA sequencing.

Example 21

Protein fusion of MPIF-1

The MPIF-1 polypeptide is preferably fused to another protein. Such fusion proteins can be used in a variety of applications. For example, fusing an MPIF-1 polypeptide to a His-tag, an HA-tag, a protein A, an IgG domain and a maltose binding protein facilitates purification (see Example 5; EP A 394,827; And Truncker et al ., Nature 331 : 84-86 (1988)]. Similarly, fusion to IgG-1, IgG-3 and albumin increases in vivo half-life. A nuclear localization signal fused to an MPIF-1 polypeptide can target the protein to specific bacterial differentiation, whereas covalent heterodimers or homodimers can increase or decrease the activity of the fusion protein. In addition, the fusion protein may form a chimeric molecule having at least one fusion moiety. Finally, the fusion protein can increase the solubility and / or stability of the fused protein compared to the unfused protein. All types of fusion proteins described above can be prepared by modifying a protocol summarizing the fusion of a polypeptide to an IgG molecule or the protocol described in Example 2.

Briefly, the human Fc portion of an IgG molecule can be PCR amplified using primers spanning the 5 'and 3' ends of the sequences described below. In addition, these primers should have a convenient restriction site that facilitates cloning into an expression vector, preferably a mammalian expression vector.

For example, when using pC4 (accession no. 209646), the human Fc portion can be ligated into the BamHI cloning site. Note that the 3 'BamHI site should be destroyed. The vector containing the human Fc portion is then again restricted to BamHI and the vector is linearized, then the MPIF-1 polynucleotide isolated by the PCR protocol described in Example 1 is ligated into such a BamHI site. It should be noted that the polynucleotide should be cloned without a stop codon. Otherwise, no fusion protein is produced.

When naturally occurring signal sequences are used to generate isolated proteins, pC4 does not require a second signal peptide. Alternatively, if a naturally occurring signal sequence is not used, the vector may be modified to include a heterologous signal sequence (see WO 96/34891).

Human IgG Fc region (SEQ ID NO: 42):

In addition, one or more components, motifs, regions, fragments, domains, fragments, etc. of MPIF-1 can be recombined with one or more components, motifs, regions, fragments, domains, fragments, etc. of the variant molecule. In a preferred embodiment, the variant molecule is a member of the kemokin family. In another preferred embodiment, such heterologous molecules include, for example, platelet derived growth factor (PDGF), insulin growth factor (IGFI), transforming growth factor (TGF) , Fibroblast growth factor (FGF), TGF-beta, bone morphogenetic protein (BMP) -2, BMP-4, BMP-5, BMP-6, BMP-7, activin A and B, (Dpp), 60A, OP-2, dorsalin, GDF, nodal, MIS, inhidin-alpha, TGF-beta1, TGF- Beta2, TGF-beta3, TGF-beta5, and glial-induced neurotrophic factor (GDNF).

Example 22

Generation of antibodies

Hybridoma technique

The antibodies of the present invention can be prepared by a variety of methods (see Current Protocols, Chapter 2). As an example of such a method, cells expressing MPIF-1 are administered to an animal to include the production of serum containing a polyclonal antibody. In a preferred method, a preparation of MPIF-1 protein is prepared and purified to render it free of natural contaminants. The formulation is then injected into the animal to produce a larger specific active polyclonal antiserum.

In a most preferred method, the antibody of the invention is a monoclonal antibody (or a protein binding fragment thereof). Such monoclonal antibodies can be prepared using hybridoma techniques {Kohler et al ., Nature 256 : 495 (1975); Kohler et al . , Eur. J. Immunol. 6 : 511 (1976); Kohler et al . , Eur. J. Immunol. 6 : 292 (1976); Hammerling et al., Monoclonal Antibodies and T-Cell Hybridomas , Elsevier, NY, pp. 563-681 (1981). Generally, such procedures involve the immunization of an animal (preferably a mouse) with an MPIF-1 polypeptide, or more preferably with a secreted MPIF-1 polypeptide expressing cell. Such cells may be cultured in any suitable tissue culture medium, but they may be cultured in a medium containing 10% fetal bovine serum (inactivated at about 56 캜), about 10 g / l of nonessential amino acids, about 1,000 U / ml of penicillin, It is preferable to cultivate the cells in an Earle-modified Eagle's medium supplemented with 100 ug / ml of streptomycin.

Splenocytes from such mice are extracted and fused to an appropriate myeloma cell line. Although any suitable myeloma cell line can be used in accordance with the present invention, it is preferred to use a maternal myeloma cell line (SP2O) available from the ATCC. After fusion, the resulting hybridoma cells are selectively maintained in HAT medium and cloned by restriction of dilution as described in Wands et al., Gastroenterology 80 : 225-232 (1981). The hybridoma cells obtained through such secretion are then analyzed to identify clones secreting antibodies capable of binding MPIF-1 polypeptides.

Alternatively, additional antibodies capable of binding to the MPIF-1 polypeptide can be generated in a two-step process using an anti-genotype antibody. Such a method makes it possible to use the fact that the antibody itself is an antigen, and thus it is possible to obtain an antibody that binds to the second antibody. According to this method, a protein-specific antibody is used to immunize an animal, preferably a mouse. Then, an antibody capable of blocking the ability of MPIF-1 to bind to MPIF-1 protein-specific antibody by screening hybridoma cells and producing hybridoma cells using such animal spleen cells Lt; / RTI &gt; Such antibodies may include anti-genotype antibodies to MPIF-1 protein specific antibodies and may be used to immunize animals to induce the formation of further MPIF-1 protein specific antibodies.

Fab and F (ab &apos;) ₂ and other fragments of the antibodies of the invention can be used in accordance with the methods described herein. Typically, such fragments are produced by proteolytic cleavage using enzymes such as papain (Fab fragment generation) or pepsin (F (ab ') ₂ fragment production). Alternatively, secreted MPIF-1 protein binding fragments can be generated by the application of recombinant DNA technology or by synthetic chemistry.

For in vivo use of antibodies to humans, it may be desirable to use " humanized " chimeric monoclonal antibodies. Such antibodies can be generated using genetic constructs derived from hybridoma cells that produce the aforementioned monoclonal antibodies. Methods for generating chimeric antibodies are known in the art {Morrison, Science 229 : 1202 (1985); Oi et al., BioTechniques 4 : 214 (1986); Cabilly et al., U.S. Patent No. 4,816,567; Taniguchi et al., EP 171496; Morrison et al., EP 173494; Neuberger et al., WO 86/01533; Robinson et al., WO 87/02671; Boulianne et al ., Nature 312 : 643 (1984); Neuberger et al ., Nature 314 : 268 (1985)).

Isolation of antibody fragments derived for MPIF-1 from a library of scFvs

The native V gene isolated from human PBL is constructed as a large library of antibody fragments containing reactivity to MPIF-1 with or without donor exposure (see U.S. Patent No. 5,885,793, which is incorporated herein by reference in its entirety) }.

The structure of the library. The library of scFvs is constructed from the RNA of human PBL as described in WO 92/01047. To rescue the phage expressing the antibody fragment, 50 ml of 2xTY (2xTY-AMP-GLU) containing 1% glucose and 100 ug / ml of ampicillin was inoculated using approximately 10 ⁹ Escherichia coli with phagemid, While being grown to an OD of 0.8. 50 ml of 2xTY-AMP-GLU was inoculated with 5 ml of this culture, 2 x 10 ⁸ TU of a delta gene 3 helper (M13 delta gene III, see WO 92/01047) was added, and the culture was shaken , Incubated at 37 ° C for 45 minutes, and then incubated at 37 ° C for 45 minutes with shaking. The culture is centrifuged at 4,000 rpm for 10 minutes and the pellet is resuspended in 2 liters of 2xTY containing 100 ug / ml ampicillin and 50 ug / ml kanamycin and grown overnight. The phage is prepared as described in WO 92/01047.

The M13 delta gene III is prepared as follows: The M13 delta gene III helper phage does not encode the gene III protein and thus the phage (mid) representing the antibody fragment has a greater desire to bind the antigen. Infectious M13 delta gene III particles are prepared by growing helper phage in cells with pUC19 derivatives supplying the wild-type gene III protein in the phage formative state. The culture is incubated at 37 ° C for 1 hour without shaking, and then incubated at 37 ° C for 1 hour with shaking. Cells were spun down (IEC-Centra-8, 4,000 revs / min for 10 minutes), resuspended in 300 ml 2xTY broth (2xTY-AMP-KAN) containing 100 ug ampicillin / ml and 25 ug kanamycin / Resuspended, grown overnight at 37 ° C with shaking. Sikimyeo purified from the culture medium by the phage particles in the two times of PEG precipitation, and concentrated (Sambrook et al., 1990), resuspended in 2 ㎖ PBS-suspended and, 0.45 um filter; approximately 10 ¹³ transduction units by passing the (Minisart NML Sartorius) / Ml &lt; / RTI &gt; (ampicillin resistant clone).

Panning the library. An immunotube (Nunc) is coated overnight in PBS with 4 ml of the 100 ug / ml or 10 ug / ml polypeptides of the present invention. The tubes are blocked with 2% Marvel-PBS for 2 hours at 37 [deg.] C and then washed 3 times in PBS. Approximately 10 ¹³ TU of the phage is applied to the tube, incubated at room temperature for 30 minutes while turning over the turntable, and left for 1.5 hours. The tubes are washed 10 times with PBS 0.1% Tween-20 and 10 times with PBS. 1 ml of 100 mM triethylamine is added and the solution is eluted by spinning over the turntable for 15 minutes and then the solution is immediately neutralized with 0.5 ml of 1.0 M Tris-HCl (pH 7.4). The phage eluted with the bacteria for 30 minutes at 37 ° C using a phage are then incubated with 10 ml of intermediate log Escherichia coli TG1. The E. coli is then placed on a TYE plate containing 1% glucose and 100 ug / ml ampicillin. The resulting bacterial library is then harvested by delta gene 3 helper phage as described above to produce a phage for subsequent rounds of selection. This process is then repeated for a total of four rounds of affinity purification. For the third and fourth rounds, the tube wash is increased 20 times with PBS, 0.1% Tween-20, and 20 times with PBS.

Characterization of binders. Escherichia coli HB 2151 is infected using leached phage from the third and fourth rounds of selection and soluble scFv is generated from a single colony for analysis (Marks et al., 1991). ELISA is performed with a microtiter plate coated with 10 pg / ml of the polypeptide of the present invention in 50 mM bicarbonate at pH 9.6. Clones positive for ELISA are characterized by PCR fingerprinting (see for example WO 92/01047) followed by sequencing.

Example 23

How to handle MPIF-1 level reduction

The invention is directed to a method of treatment comprising administering to a subject in need thereof an increased level of a polypeptide of the invention in a body, comprising a therapeutically effective amount of an agonist of the invention (including a polypeptide of the invention). It is also recognized that conditions caused by a reduction in the normal or normal expression level of MPIF-I in an individual can be treated by administering MPIF-1, preferably in a secreted form. Thus, the invention also provides a method of treatment comprising administering to a subject a therapeutic agent comprising an amount of MPIF-1 to increase the level of MPIF-1 activity in a subject in need of a level increase of the MPIF-1 polypeptide .

For example, a patient with an increased level of MPIF-1 polypeptide is provided with a daily dosage of 0.1 to 100 ug / kg of polypeptide for 6 consecutive days. The polypeptide is preferably secreted. The precise details of the dosage regimen based on administration and formulation are provided in the " pharmaceutical composition " section herein.

Example 24

Increased levels of MPIF-1 treatment methods

The invention also relates to a method of treatment comprising administering to a subject in need thereof a therapeutically effective amount of an antagonist of the invention (including a polypeptide and an antibody of the invention) to a subject in need of a reduction in the polypeptide level of the invention.

In one example, antisense techniques are used to inhibit the production of MPIF-1. This technique is an example of a method of level reduction of MPIF-1 polypeptide, preferably secretory form, due to a variety of pathologies.

For example, patients diagnosed with an elevated level of MPIF-1 are given intravenous antisense polynucleotides at 0.5, 1.0, 1.5, 2.0, and 3.0 mg / kg for 21 days. If this treatment is well tolerated, this treatment is repeated after 7 days of rest. The preparation of the antisense polynucleotide is provided in the " pharmaceutical composition " section herein.

Example 25

Therapeutic methods using gene therapy - In vitro

One method of gene therapy involves transplanting fibroblasts capable of expressing MPIF-1 into patients. In general, fibroblasts are obtained from a subject by skin biopsy. The resulting tissue is placed in a tissue culture medium and separated into small pieces. A small piece of tissue is placed on the wet surface of the tissue culture flask and approximately 10 pieces are placed in each flask. The flask is sealed and allowed to stand at room temperature overnight, turning it up and down. After 24 hours at room temperature, the flask is inverted and the tissue pieces are fixed to the bottom of the flask and fresh medium (e.g., Ham F12 medium containing 10% FBS, penicillin and streptomycin) is added. The flask is then incubated at 37 [deg.] C for approximately one week. At this time, fresh medium is added, and it is changed every several days. In addition, after two weeks in culture, a monolayer of fibroblasts develops. The faults are trypsinized and transferred to a larger flask.

PMV-7 {Kirschmeier, PT et al., DNA 7 : 219-25 (1988)} in which the long terminal repeat of Moloney murine sarcoma virus is flanked is digested with EcoRI and HindIII and then treated with calf intestinal phosphatase . The linear vector is fractionated on an agarose gel and purified using glass beads.

The cDNA encoding MPIF-1 can be amplified using PCR primers corresponding to the 5 ' and 3 ' end sequences, respectively, as described in Example 1. The 5 'primer preferably contains an EcoRI site and the 3' primer preferably comprises a HindIII site. Equal amounts of Moloney murine sarcoma virus linear scaffold and amplified EcoRI and HindIII fragments are added together in the presence of T4 DNA ligase. The resulting mixture is held under appropriate conditions for ligation of the two fragments. The ligation mixture is then used to transform the bacterial HB101 and then placed in an agar containing kanamycin for the purpose of verifying that the vector contains MPIF-1 properly inserted.

Amphotropic pA317 or GP + am 12 packaging cells are grown in tissue culture at a concentration of adhesion in Dulbecco's modified Eagle's medium (DMEM) containing 10% bovine serum (CS), penicillin and streptomycin. MSV vectors containing the MPIF-1 gene are then added to the medium, and the packaging cells are transduced with vectors. Now, the packaging cells produce infectious viral particles containing MPIF-1 (now the packaging cells are referred to as producer cells).

Fresh medium is added to the transduced producer cells, and then the medium is collected from 10 cm plates of the adhesion producer cells. The spent media containing infectious viral particles are filtered through a Millipore filter to remove desorbed producer cells and then infected with fibroblasts using this medium. The medium is removed from the adherent plate of the fibroblasts and replaced quickly with medium from the producer cells. This medium is removed and replaced with fresh medium. When the viral titer is high, virtually all fibroblasts are infected and do not require secretion. If the titer is very low, a retroviral vector with a selectable marker such as neo or his should be used. When fibroblasts are efficiently infected, fibroblasts are analyzed to determine whether MPIF-1 protein is produced.

The engineered fibroblasts are then grown alone or in adherent concentration on cytodex 3 microcarrier beads and then transplanted into the host.

Example 26

Gene therapy using endogenous MPIF-1 gene

Other methods of gene therapy in accordance with the present invention are described, for example, in U.S. Patent No. 5,641,670 (June 24, 1997); International Application Publication WO 96/29411 (September 26, 1996); International Application Publication WO 94/12650 (Aug. 4, 1994); Koller et al . , Proc. Natl. Acad. Sci. USA 86 : 8932-8935 (1989); And Zijlstra et al., Nature 342 : 435-438 (1989), which is herein incorporated by reference in its entirety). This method involves the activation of a gene that is present in the target cell, but is not expressed in the cell, or is expressed at a lower level than the target value.

A polynucleotide construct containing a targeting sequence and a promoter homologous to the 5 ' unencrypted sequence of endogenous MPIF-1 in which the promoter is flanked is prepared. The targeting sequence is sufficiently close to the 5 'end of MPIF-1 that the promoter is operatively linked to the endogenous sequence upon homologous recombination. Promoters and targeting sequences can be amplified using PCR. It is preferred that the amplified promoter contains a separate restriction enzyme site on the 5 'and 3' ends. The 3'end of the first targeting sequence contains the same restriction enzyme site as the 5'end of the amplified promoter and the 5'end of the second target sequence contains the same restriction enzyme site as the 3'end of the amplified promoter desirable.

The amplified promoter and amplified targeting sequence are digested with appropriate restriction enzymes and then treated with small intestine phosphatase. The degraded promoter and the degraded targeting sequence are added together in the presence of T4 DNA ligase. The resulting mixture is maintained under appropriate conditions for ligating the two fragments. The constituents are size fractionated on an agarose gel and then purified by phenol extraction and ethanol precipitation.

In this example, the polynucleotide construct is administered as a naked polynucleotide by electrophoresis. However, polynucleotide constructs may also be administered in conjunction with transfection agents such as liposomes, viral sequences, viral particles, precipitants, and the like. Such delivery methods are well known in the art.

Once the cells are transfected, homologous recombination is performed to operatively link the promoter to the MPIF-1 sequence. This leads to the expression of MPIF-1 in the cells. Expression can be detected by immunological staining or any other method known in the art.

Fibroblasts are obtained from patients by skin biopsy. The resulting tissue is placed in DMEM + 10% fetal bovine serum. Exponentially growing or early normal fibroblasts are trypsinized and cleaned from the plastic surface with nutrient media. The cell suspension is removed for counting, and the remaining cells are centrifuged. Sucking out the supernatant, and then the pellet resuspended in 5 ㎖ electrophoresis buffer (20 mM HEPES pH 7.3, 137 mM NaCl, 5 mM KCl, 0.7 mM Na 2 HPO 4, 6 mM dextrose), the. The cells are re-centrifuged, the supernatant is drawn off, and the cells are resuspended in electrophoresis buffer containing 1 mg / ml acetylated serum albumin. The final cell suspension contains approximately 3 X ¹⁰⁶ cells / ml. Electrophoresis is performed immediately after resumption.

Plasmid DNA is prepared according to standard techniques. For example, to construct a plasmid for targeting to the left of MPIF-1, the plasmid pUC18 (MBI Perimeter, Amherst, New York, USA) is digested with HindIII. The CMV promoter is amplified by PCR to the XbaI site on the 5 'end and the BamHI site on the 3' end. Two MPIF-1 unencrypted sequences are amplified by PCR; one MPIF-1 unencrypted sequence (MPIF-1 fragment 1) is amplified from the HindIII site at the 5 'end and Xba at the 3' 1 unencrypted sequence (MPIF-1 fragment 2) is amplified from the BamHI site at the 5 'end and the HindIII site at the 3' end. The CMV promoter and MPIF-1 fragments 1 and 2 are digested with appropriate enzymes (CMV promoter-XbaI and BamHI; MPIF-1 fragment 1-XbaI; MPIF-1 fragment 2-BamHI) and ligated together. The resulting ligation product is digested with HindIII and ligated with HindIII digested pUC18 plasmid.

Plasmid DNA is added to a sterile cuvette (BioRad) equipped with a 0.4 cm electrode gap. Generally, the final DNA concentration is at least 120 [mu] g / ml. Then, (containing approximately 1.5 X 10 ⁶ cells) 0.5 ㎖ of the cell suspension was added to the cuvette, and slowly mixed with the cell suspension and DNA solution. Perform electrophoresis with a GenePulser device (BioRad). Capacitance and voltage are set to 960 μF and 250 and 300 V, respectively. As the voltage increases, the cell viability decreases, but the percentage of surviving cells that stably incorporate the introduced DNA into the genome increases dramatically. Given this parameter, a pulse time of approximately 14 to 20 mSec will be observed.

The electrophoresed cells are maintained at room temperature for approximately 5 minutes and then the contents of the cuvette are slowly removed with a sterile transfer pipette. Cells are directly added to 10 ml of pre-warmed nutrient medium (DMEM containing 15% bovine serum) in 10 cm dishes and incubated at 37 ° C. The next day, the medium is aspirated, replaced with 10 ml of fresh medium, and incubated for a further 16-24 hours.

The engineered fibroblasts are then grown either alone or on a cytodex 3 microcarrier bead at an adhesion concentration and then injected into the host. Now, fibroblasts produce protein products. The fibroblasts can then be introduced into the patient as described above.

Example 27

Therapeutic methods using gene therapy - in vivo

Another aspect of the invention is the use of in vivo gene therapy to treat disorders, diseases and conditions. Gene therapy involves the introduction of pure nucleic acid (DNA, RNA and antisense DNA or RNA) MPIF-1 sequences into an animal to increase or decrease the expression of MPIF-1 polypeptide. The MPIF-1 polynucleotide may be operatively linked to a promoter or other gene component necessary for the expression of the MPIF-1 polypeptide by the target tissue. Such gene therapy and delivery techniques and methods are well known in the art {see, for example, WO 90/11092, WO 98/11779, which is incorporated herein by reference; U.S. Patent Nos. 5,693,622, 5,705,151, 5,580,859; Tabata H. et al . , Cardiovasc. Res. 35 (3) : 470-479 (1997), Chao J. et al . , Pharmacol. Res. 35 (6) : 517-522 (1997), Wolff JA, Newuromuscul. Disord. 7 (5) : 314-318 (1997), Schwartz B. et al . , Gene Ther. 3 (5) : 405-411 (1996), Tsurumi Y. et al., Circulation 94 (12) : 3281-3290 (1996)).

The MPIF-1 polynucleotide construct can be delivered by any method of delivering an injectable material to an animal cell, such as an injection into the interstitial space of a tissue (heart, muscle, skin, lung, liver, bowel, etc.). The MPIF-1 polynucleotide construct can be delivered in a pharmaceutically acceptable liquid or aqueous carrier.

The term " naked " polynucleotide, DNA or RNA is intended to encompass any nucleic acid molecule, such as a viral sequence, viral particle, liposomal preparation, lipofectin or precipitant, which serves to aid, facilitate or facilitate entry into the cell Means a sequence lacking a delivery vehicle. However, MPIF-1 polynucleotides can be prepared by liposomal preparations (see, for example, Felgner PL et al . , Ann. NY Acad. Sci. 772 : 126-139 (1995) and Abdallah B. et al . , Biol. Cell 85 (1) : 1-7 (1995).

The MPIF-1 polynucleotide vector construct used in the gene therapy is preferably a construct that is integrated into the host genome or does not contain a sequence that permits replication. Any strong promoter known to those skilled in the art can be used to induce expression of the DNA. Unlike other gene therapy techniques, one major advantage of introducing a pure nucleic acid into a target cell is the transient nature of intracellular polynucleotide synthesis. Studies have shown that non-replicating DNA sequences can be introduced into cells to provide the production of a given polypeptide for a period of less than six months.

MPIF-1 polynucleotide constructs can be used for the treatment of muscle, skin, brain, lung, liver, spleen, bone marrow, thymus, heart, lymph, blood, bone, cartilage, pancreas, kidney, bladder, stomach, , The nervous system, the eye, the hippocampus, and connective tissue. The interstitial space of the tissue includes the same matrix with intercellular fluid, a mucopolysaccharide matrix along the reticular fibers of the organ tissue, elastic fibers in the blood vessels or dissolving walls, connective tissue covering the collagen fibers or muscle cells of the fibrous tissue, . Similarly, it is the space occupied by the lymphatic fluid of the plasma and lymphatic channels of the circulatory system. Delivery of muscle tissue to the interstitial space is desirable for reasons discussed below. This can be easily delivered by injection into a tissue containing these cells. Although it is desirable that they be expressed and transduced to differentiated transient non-dividing cells, the transfer and expression may be made in undifferentiated or less completely differentiated cells, such as blood stem cells or dermal fibroblasts. In vivo muscle cells are particularly competent in their ability to absorb and express polynucleotides.

For pure MPIF-I polynucleotide injections, an effective dose of DNA or RNA ranges from about 0.05 g / kg body weight to about 50 mg / kg body weight. The dosage is preferably about 0.005 mg / kg to about 20 mg / kg, more preferably about 0.05 mg / kg to about 5 mg / kg. Of course, as one of ordinary skill in the art understands, this dosage will vary with the tissue site of the injection. Suitable and effective dosages of the nucleic acid sequences are nodular in the art and depend on the condition and route of administration being treated. The preferred route of administration is by parenteral route injection into the interstitial space. However, other parenteral routes may also be used, such as inhalation of aerosol preparations, particularly for delivery to the lungs or bronchial tissues, throat or nasal mucosa. In addition, pure MPIF-1 polynucleotide constructs can be delivered to the arteries during catheter-assisted angioplasty for use in this procedure.

The dose response effect of intramuscular injected MPIF-1 polynucleotide in vivo is determined as follows. Appropriate MPIF-1 template DNA for the generation of mRNA encoding the MPIF-1 polypeptide is prepared according to the standard recombinant DNA method. The template DNA, which may be circular or linear, is used as DNA complexed with pure DNA or liposomes. Then, various amounts of template DNA are injected into the quadriceps of the mouse's femur.

Five to six week old female and male Balb / C mice are anesthetized by intraperitoneal injection with 0.3 ml of 2.5% avitin. A 1.5 cm incision is made in the anterior femur and the quadriceps muscle is visible. MPIF-1 template DNA is injected from the cut end of the muscle into the knee at a depth of approximately 0.5 cm and a depth of approximately 0.2 cm for 1 minute through a 27 gauge needle with a 0.1 ml carrier in a 1 cc syringe. Place the suture line at the injection site for further localization and seal the skin with a stainless steel clip.

After an appropriate incubation time (e.g., 7 days), the quadriceps muscles are resected to produce muscle extracts. And stained for MPIF-1 protein histochemically at the 15th 15 um cross section of each quadriceps muscle. The time course for MPIF-1 protein expression is done in a similar manner except that the quadriceps femoris from different mice is harvested at different times. The durability of intramuscular MPIF-1 DNA after injection can be determined by Southern blot analysis after preparation of total cytoplasmic DNA and HIRT supernatant from injected control mice. Using the experimental results of the mouse, appropriate dosages and other therapeutic parameters can be estimated in humans and other animals using MPIF-1 pure DNA.

Example 28

MPIF-1 transgenic animal

MPIF-1 polypeptides can also be expressed in transgenic animals. Animals of any species, such as mice, rats, rabbits, hamsters, guinea pigs, pigs, micro-pigs, goats, sheep, cattle and non-human primates such as baboons, monkeys and chimpanzees can be used to make transgenic animals But are not limited to these. In certain embodiments, techniques described herein or other techniques known in the art are used to express polypeptides of the invention in humans as part of a gene therapy method.

Any technique known in the art can be used to introduce an introduced gene (i. E., A polynucleotide of the invention) into an animal to produce an initial line of transgenic animals. These techniques include: pronuclear microinjection (Paterson et al, Appl Microbiol Biotechnol 40: 691-698 (1994); Carver et al, Biotechnology ( NY) 11:..... 1263-1270 (1993); Wright et al,. Biotechnology (New York) 9 : 830-834 (1991); and Hoppe et al ., U.S. Pat. No. 4,873,191 (1989)); (Van der Putten et al ., Proc. Natl. Acad. Sci. , USA 82 : 6148-6152 (1985)), retrovirus-mediated gene transplantation in blastocysts or embryos; Gene targeting with embryonic stem cells (Thompson et al., Cell 56 : 313-321 (1989)); Electroporation of cells or embryos (Lo, Mol Cell. Biol. 3 : 1803-1814 (1983)); Introduction of polynucleotides of the present invention using a gene gun (reference example: Ulmer et al., Science 259 : 1745 (1993)); Introduction of nucleic acid structure into embryo pluripotent stem cells and hepatocyte transplantation by embryo transfer; And sperm-mediated gene transplantation (Lavitrano et al., Cell 57 : 717-723 (1989)). These techniques are described in Gordon " Transgenic Animals &quot;, Intl. Rev. Cytol. 115 : 171-229 (1989).

Any technique known in the art can be used to produce transgene clones containing the polynucleotides of the invention, examples of which include nuclei from cultured embryos, fetal or adult immobilized cells (Campell et al., Nature 380 : 64-66 (1996); Wilmut et al., Nature 385 : 810-813 (1997)).

The present invention provides an animal having a transgene in a cell other than a transgenic animal having an introduced gene in all cells as well as not all cells, that is, a mosaic animal or a chimeric animal. The transgene can be inserted as a single transgene or as a multiple copy, such as in a concatamer, for example in head-to-head connection or head-to-tail connection. In addition, the inserted gene may be selectively introduced into a particular cell type to activate it, for example, as described in Lasko et al. (Lasko et al., Proc. Natl. Acad. Sci. USA 89 : 6232-6236 . The regulatory sequences necessary for these cell type specific activation will depend on the particular cell type of interest and will be apparent to those skilled in the art. Gene target substitution is preferred when the polynucleotide transgene is intended to be inserted into the chromosomal location of the endogenous gene.

In summary, when using these techniques, a vector containing a constant nucleotide sequence homology to an endogenous gene is designed for the purpose of disrupting its function by being inserted into the nucleotide sequence of the endogenous gene through homologous recombination with the chromosomal sequence. In addition, the transgene may be selectively introduced into a specific cell type to activate the endogenous gene only within the cell type, for example, Gu et al. (Gu et al., Science 265 : 10106 (1994) have. The regulatory sequences necessary for these cell type specific inactivation will depend on the particular cell type of interest and will be apparent to those skilled in the art. The contents of each document cited in this paragraph are incorporated herein by reference in their entirety.

Similarly, DNA encoding the full-length MPIF-1 protein can also be inserted into the vector using the following primers for tissue-specific expression.

In addition to expressing the polypeptides of the invention in a ubiquitous manner or in a tissue-specific manner in transgenic animals, the expression of the polypeptide may be regulated by a variety of different methods (e.g., genetically or chemically regulated expression) It will be routine to those skilled in the art to make structures.

Once the transgenic animal is generated, the expression of the recombinant gene can be investigated using standard techniques. Initial screening can be performed by Southern blot analysis or PCR techniques to analyze the tissue of the animal to see if transgene insertion has occurred. In addition, the level of expression of the transgene mRNA in the tissues of transgenic animals can be determined by Northern blot analysis of tissue samples obtained from animals, in situ hybridization and reverse transcriptase-PCR (rt-PCR) Techniques, but are not limited to these techniques. Tissue samples expressing the transgene can also be assessed immunocytochemically or immunohistochemically using antibodies specific for the transgene product.

Once the initial animal is created, it can be mated, inbred, crossed or crossed to produce a population of particular animals. Examples of these crossing methods include a method of crossing an initial animal with one or more insertion sites to establish a separate line, a method of generating a complex transgene that expresses the transgene at a higher level due to the effect of the additional expression of each transgene A method of inbreeding a heterozygous transgenic animal producing a homozygote of an animal at a given insertion site in order to increase expression and eliminate the need for animal screening by DNA analysis, Methods of crossing isolated homozygous lines that produce hybrid heterozygous or homozygous lines, and methods of crossing to place transgene in a separate environment suitable for the experimental model of interest.

The transgenic animals of the present invention are useful for explaining the biological function of MPIF-1 polypeptides, for studying conditions and / or diseases associated with abnormal MPIF-1 expression, and for screening compounds that are effective in ameliorating these conditions and / or diseases But are not limited to, use to characterize an animal model system.

Example 29

MPIF-1 Knock-Out Animals

Endogenous MPIF-1 gene expression can also be reduced by inactivating or knocking out the MPIF-1 gene and / or its promoter using targeted homologous recombination. (Reference Example: Smithies et al, Nature 317: 230-234 (1985); Thomas & Capecchi, Cell 51:. 503-512 (1987); Thompson et al, Cell 5:. 313-321 (1989); each of which Functional polynucleotide that is a mutant of the present invention in contact with DNA that is homologous to an endogenous polynucleotide sequence (the coding or regulatory region of the gene) (or a non-functional polynucleotide DNA sequence) can be used in the presence or absence of a selectable marker and / or a negatively selectable marker. In yet another embodiment, techniques known in the art are used to knock out cells that contain, but do not express, the gene of interest. Insertion of DNA constructs through targeted homologous recombination results in inactivation of the targeted gene. These approaches are particularly suited for academic research and agricultural applications that can be used to produce animal offspring with targeted genes for which transformation to embryonic stem cells is inert (see Thomas & Capecchi 1987 and Thompson 1989, supra). This approach, however, can be changed to a mechanical procedure for use in humans if the recombinant DNA construct is directly administered in vivo or targeted at the site of interest, using suitable viral vectors as would be apparent to those skilled in the art.

In yet another embodiment of the invention, cells genetically engineered to express the polypeptide of the invention, or alternatively, genetically engineered (e. G., Knock-out) so as not to express the polypeptide of the invention, Lt; / RTI &gt; These cells may be obtained from a patient (i. E., An animal including a human being) or an MHC-compatible provider and include but are not limited to fibroblasts, bone marrow cells, blood cells (e.g., lymphocytes), adipocytes, muscle cells, endothelial cells, . Such cells may be used to introduce the coding sequence of the polypeptide of the present invention into the cell, or alternatively, genetically in the laboratory using recombinant DNA techniques to disrupt the coding sequence and / or endogenous regulatory sequences associated with the polypeptide of the invention The plasmid, the cosmid, the YAC, the exposed DNA, the electroporation, the liposome, and the like are transfected into the recombinant technique, and the transfection is carried out using a transfection technique (viral vector, preferably using a vector that inserts the transgene into the cell genome) Including but not limited to the use of The coding sequence of the polypeptide of the present invention may be located under the control of a strong structure or inducible promoter or promoter / enhancer to achieve expression, preferably secretion, of the MPIF-1 polypeptide. The engineered cells expressing and preferably secreted the polypeptides of the present invention may be introduced into the entire body of a patient, for example, circulating or intraperitoneally.

Alternatively, the cells may be blended into the substrate and implanted into the body, for example, genetically engineered fibroblasts may be implanted as part of a skin graft, and genetically engineered endothelial cells may be part of a lymph node or vascular graft (See, for example, Anderson et al ., U.S. Patent No. 5,399,349 and Mulligan & Wilson, U.S. Patent No. 5,460,959, each of which is incorporated herein by reference)

Where the administered cells are non-viscous or non-MHC compatible cells, they may be introduced by known methods to inhibit the expression of a host immune response to the introduced cells. For example, the cell may be transfected with an adjacent extracellular environment, while the introduced cells may be introduced into a capsule-like form such that the introduced cells are not recognized by the host immune system.

The knock-out animals of the present invention describe the biological function of MPIF-I polypeptides, study the conditions and / or diseases associated with the expression of abnormal MPIF-1, and screen compounds that are effective in ameliorating these conditions and / or diseases But are not limited to, uses featuring a useful animal model system.

Example 30

Production of antibodies

Hybridoma technique

The antibodies of the present invention can be prepared by a variety of methods (see Current Protocols, Chapter 2). As one example of these methods, the cell-expressing polypeptides of the present invention are administered to an animal to induce the production of serum containing polyclonal antibodies. In a preferred method, the polypeptide (s) preparation of the present invention is purified so as to be substantially free of natural contaminants after being produced. These products are then introduced into animals to produce polyclonal antiserum with greater specific activity.

Monoclonal antibodies specific for the polypeptides of the invention are prepared using hybridoma techniques (Kohler et al., Nature 256 : 495 (1975); Kohler et al., Eur. J. Immunol. 6 : 511 1976); Kohler et al., Eur. J. Immunol. 6 : 292 (1976); Hammerling et al ., In Monoclonal Antibodies and T-Cell Hybridomas , Elsevier, New York (1981), pp. 563-681). Generally, the animal (preferably a mouse) is immunized with the polypeptide (s) of the invention or more preferably with secreted polypeptide expressing cells. These polypeptide-expressing cells are cultured in any appropriate tissue culture medium and are supplemented with 10% fetal bovine serum (inactivated at about 56 [deg.] C) and incubated with about 10 g / l of an essential amino acid, about 1,000 U / ml penicillin, It is preferable to cultivate in Earle's modified Earle medium supplemented with about 100 占 퐂 / ml of mycin.

Splenocytes from these mice are extracted and fused with an appropriate myeloma cell line. Any suitable myeloma cell line may be used in accordance with the present invention, but it is preferred to use an AM2 cell line (SP2O) available from the ATCC. After fusion, the resulting hybridoma cells are optionally maintained in HAT medium and then cloned by limiting dilution as described by Wands et al. ( Gastroenterology 80 : 225-232 (1981)). The hybridoma cells obtained through these selections are then analyzed to identify clones secreting antibodies capable of binding to the polypeptides of the present invention.

In addition, additional antibodies capable of binding to the polypeptides of the present invention can be produced in a two-step process using an anti-genotype antibody. These methods utilize the fact that the antibody itself becomes an antigen, and thus it is possible to obtain an antibody that binds to the second antibody. According to this method, a protein specific antibody is used to immunize an animal, preferably a mouse. The splenocytes of these animals are used to produce hybridoma cells, and the hybridoma cells are screened to identify clones producing the antibody. The ability of the antibody to bind to a protein-specific antibody is determined by the ability of the polypeptide of the invention (S). These antibodies comprise antibodies of the anti-genotype to the protein-specific antibody and are used to immunize the animal to induce the formation of additional protein-specific antibodies.

In the case of in vivo use of the antibody in humans, the antibody is " humanized &quot;. Such antibodies can be generated using genetic constructs derived from hybridoma cells that produce the aforementioned monoclonal antibodies. Methods for producing chimeric and humanized antibodies are known in the art and are discussed herein (Morrison, Science 229 : 1202 (1985); Oi et al., BioTechniques 4 : 214 (1986); Cabilly Morrison et al. , European Patent 173494; Neuberger et al ., WO 8601533; Robinson et al. , WO 8702671; Boulianne et al., Nature , et al ., U.S. Patent No. 4,816,567; Taniguchi et al. , European Patent 171496; 312 : 643 (1984); Neuberger et al., Nature 314 : 268 (1985)).

Isolation of antibody fragments specific for the polypeptide from the library of scFvs

The naturally-occurring V-gene isolated from human PBL is constructed as an antibody fragment library containing the reactivity to the polypeptide of the present invention in which the provider may or may not be exposed (see, U.S. Patent No. 5,885,793, Incorporated herein by reference).

Formation of libraries . The library of scFvs is constructed from the RNA of human PBL as described in PCT Publication WO 92/01047. To construct a phage representing the antibody fragment, about 10 ⁹ E. coli with phagemid was used to inoculate 50 ml of 2 x TY containing 1% glucose and 100 g / ml ampicillin (2 x TY-AMP-GLU) gkrh and incubate with shaking until the OD reaches 0.8. 5 ml of this medium was used to inoculate 50 ml of 2xTY-AMP-GLU, 2x108 TU of the delta gene 3 helper (M13 delta gene III, see PCT Publication WO 92/01047) was added and the medium was incubated at 37 ° C for 45 The cells were incubated at 37 ° C for 45 minutes after incubation without shaking. The medium was centrifuged at 4000 rpm for 10 minutes and the pellet resuspended in 2 L TY containing 100 μg / ml ampicillin and 50 μg / ml kanamycin and incubated overnight. The phage were prepared as disclosed in PCT Publication No. WO 92/01047.

M13 delta gene III was prepared as follows. The M13 delta gene Ⅲ helper phage does not encode the gene Ⅲ protein, and thus the phage (mid) representing the antibody fragment has greater binding properties with the antigen. The infectious M13 delta gene III particle is made by cultivating helper phage in cells with pUC19 derivatives complementing wild type gene III protein during phage development. The medium is incubated at 37 ° C for 1 hour without shaking and then shaken at 37 ° C for 1 hour. Cells were spun down (IEC-Centra, 8,400 rpm, 10 min) and resuspended in 300 ml 2xTY culture containing 100 / / ml ampicillin and 25 / / ml kanamycin (2xTY-AMP-KAN) Incubate overnight and shake at 37 ° C. Purified from the culture medium by the phage particles in 2 PEG- precipitation times and concentrated and (Sambrook et al., 1990), resuspended in 2ml PBS, passed through a 0.45㎛ filter (Minisart NML; Sartorius), final concentration of about 10 ¹³ Transcription units / ml (ampicillin-resistant clones) were obtained.

Panning the library . The immunotubes (Nunc) are coated overnight in PBS with 100 μg / ml of the polypeptide of the invention or 4 ml of 10 μg / ml. The tubes are blocked with 2% Marvel-PBS for 2 hours at 37 ° C and then washed 3 times with PBS. Approximately 10 ¹³ TU of phage were placed in a tube and incubated for approximately 30 minutes at room temperature with shaking from the turntable on the turntable or from left to right, and then allowed to stand for 1.5 hours. The tubes were washed 10 times with PBS 0.1% Tween-20 and 10 times with PBS. The solution is immediately neutralized with 0.5 ml of 1.0 M Tris-HCl, pH 7.4, and then 1 ml of triethylamine is added and the phage is dissolved by spinning for 15 minutes under turntables or above. The phage were then used to infect 10 ml of mid-log E. coli TG1 by incubating the phages separated and dissolved with bacteria at 37 ° C for 30 minutes. The E. coli is then plated on TYE plates containing 1% glucose and 100 [mu] g / ml ampicillin. And the resulting bacterial library was formed with delta gene 3 helper phage as described above to produce phage for subsequent iterations of selection. These procedures are then repeated for four times in all three affinity purification steps, three and four times with PBS, 20 times with 0.1% Tween-20 and 20 times with PBS, respectively.

Characteristics of binders . The isolated phages were used to infect E. coli HB 2151 and the soluble scFv was produced from a single colony for analysis (Marks et al., 1991). ELISA is performed with a microtiter plate coated with 10 pg / ml of the polypeptide of the present invention in 50 mM bicarbonate at pH 9.6. Clones positive in ELISA are further characterized by PCR fingerprinting (reference example: PCT Publication WO 92/01047) and subsequent sequencing. These ELISA positive clones can also be used to inhibit or competitively inhibit techniques known in the art, such as epitope mapping, binding affinity, receptor signal transduction, antibody / antigen binding and competitive operative or antagonistic activity More and more by technology such as ability.

Example 31

Deadly irradiation model

A schematic diagram of the experimental method of Example 18 is shown in Fig. As shown in Fig. 42, MPIF-1 (? 23) improves the viability of mice that are fatal. MPIF activity, which enhances survival, varies with experimental conditions.

The study described in this example tested the activity of MPIF-1 (? 23). However, those skilled in the art will appreciate that, to test the activity of full-length MPIF-1, or fragments thereof, agonists and / or MPIF-1 antagonists as well as polynucleotides (eg, through gene therapy) The research could easily be modified.

Example 32

Bone marrow protection from radiation in vivo

An experiment showing that MPIF-1 increases mouse viability following a fatal challenge (see Example 18) suggests that such chemokines act as radioprotectants. To further investigate this activity, changes at the bone marrow stroma level were observed in lethal dose-exposed mice and it was determined whether MPIF-1 enhanced recovery of these striations after irradiation.

A schematic diagram of the experimental method is shown in Fig. This is a variation of the method of Example 17. The MPIF-1 used was MPIF-1 (? 23) group HG00304-E11. The negative control group (IRR) received vehicle (HBSS + 0.1% normal mouse serum intraperitoneal injection) from day -3 to day +3 except 24 hours before and after irradiation. For comparison in bone marrow analysis, the control (normal) was not examined at all.

Analysis of bone marrow cells . Mice of each group were sacrificed at 4, 14, 21 and 38 days to obtain bone marrow (BM) samples. Colony formation assays in soft agar were used to determine the number of bone marrow stroma from bone marrow (colony forming units (CFU) -c and CFU-granulocyte macrophage macrophages (CFU-GEMM)). Reference example: Grzegorzewski et al., Blood 183 : 377 (1994); And Metcalf, The Hematopoietic Colony Stimulating Factors, Amsterdam, Elsevier (1984) p.27. Cells were incubated with recombinant human erythropoietin (rhEpo) (8 U / ml) and recombinant mouse interleukin-3 (rmIL-3) (100 U / ml) using a single layer agar based assay system . (CFU-M), granulocyte (CFU-G), erythrocyte (CFU-E, and eruptive unit (CFU-G), and granulocyte BFU) -E) or granulocyte / mononuclear cell (CFU-GM) progenitor cells were designated as CFU-c. CFU-GEMM analysis provides information on the recovery of pluripotent progenitor cells in the bone marrow, while CFU-c analysis provides information on the recovery of more cardiovascular disease in each bone marrow lineage. As shown in Figures 44 and 45, MPIF-1 administration resulted in a significant increase in the levels of mature and immature bone marrow stroma.

The study described in this example tests the activity of MPIF-1 (? 23). However, those skilled in the art will appreciate that the studies exemplified to test the activity of full-length MPIF-1 or its fragments, agonists and / or MPIF-1 antagonists as well as polynucleotides (e.g., by gene therapy) It can be easily modified.

Example 33

Physical, chemical and pharmacological properties of MPIF

Formulations, pharmacological classes and administration. The bone marrow stromal inhibitory factor is a recombinant human protein beta-chemokine, truncated (8.85 kDa). MPIF was expressed in E. coli and homogeneously purified.

MPIF is provided as a sterile, colorless solution intended for injection. MPIF was formulated as a sterile solution containing various concentrations of MPIF (2-8 mg / mL) buffered to pH 6.0 + -0.2 with sodium acetate and sodium chloride. The solution is filled into disposable glass vials. Retention of activity on organisms appeared at 2 ° C to 8 ° C for more than one year. MPIF will be administered intravenously.

The MPIF protein consists of 77 amino acids (△ 23 plus the Met residue at the N-terminus) and has a molecular weight of 8.85 kDa.

General description of manufacturing details. MPIF is expressed in Escherichia coli and purified therefrom. MPIF proteins are often found in protein structures called inclusion bodies. The natural unavailability of these inclusion bodies allows for a purification process that removes components that contaminate E. coli prior to protein solubilization.

Chromatography and filtration methods are used to isolate and purify MPIF proteins. This separation and purification step is investigated as follows.

An analytical table of intermediate products eluted from the chromatography column is continuously irradiated by ultraviolet absorbance at 280 nm.

- The purity and content of MPIF protein in the different purification steps are determined by analytical RP-HPLC.

Analytical methods are used to measure the concentration, purity and identity of MPIF, as well as to detect possible impurities such as DNA, endotoxins and microbiological biohazards.

Quality control. The MPIF drug product is a clear, colorless solution. A single band is observed on sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) under reducing and non-reducing conditions after coumarin blue staining. A single peak is separated in size exclusion high performance liquid chromatography (SE-HPLC). A large peak (&gt; 90%) is observed by reversed phase high performance liquid chromatography (RP-HPLC) with two small peaks. Both of the two small peaks have the same N-terminal sequence (Edman decomposition) with the same molecular weight and large peaks of MPIF. Peptid mapping by trypsin digestion from various amounts of MPIF consistently represents the same number of peptides in a more specialized treatment. The molecular weight of MPIF as measured by a mass spectrometer is 8.85 kDa.

Two biological assays are used to measure the activity of MPIF. Calcium mobilization assays in THP-1 human bone marrow tumor cells measure immediate results of MPIF binding to the known receptor, CCR1. Chemical protection analysis provides a means of measuring the activity of a compound that protects the bone marrow stroma of mice from cytotoxic effects of 5-FU.

The study described in this example tests the activity of MPIF-1 (? 23). However, those skilled in the art will appreciate that the studies exemplified to test the activity of full-length MPIF-1 or its fragments, agonists and / or MPIF-1 antagonists as well as polynucleotides (e.g., by gene therapy) It can be easily modified.

Example 34

Nonclinical study

Introduction and summary . Myelosuppression is one of the most common dose limiting toxicities of cytotoxic chemotherapy. This inhibition results from loss of bone marrow stroma in the bone marrow, and in severe cases increases the risk of bleeding and infection complications. The bone marrow proinflammatory factor (MPIF) is being developed as a chemical protective agent. MPIF is characterized as a human beta-chemokine. Chemokines (chemotactic cytokines) are soluble proteins secreted by various cell types in response to wound, infection or activation of the immune system. These proteins are structurally related by sequence homology, and are functionally related by their activity to chemotactic responses among leukocyte populations.

The biological effects of MPIF on bone marrow progenitor cells distinguish this chemokine from all known chemokines and cytokines (Premack, BA and Schall, U., Nature Med. 2 (11): 1174-1178 (1996); Rollins, BJ , Blood 90 (3): 909-928 (1997)). Nonclinical studies indicate that this novel chemokine protects these cells from cytotoxic effects of chemotherapeutic agents by limiting proliferation and / or differentiation of progenitor cells (Patel et al., J. Exp. Med. 185 : 1163 (1997)). MPIF represents a potent inhibitory potential of slow-growing latent colony-forming cells (LPP-CFCs) in the laboratory. LPP-CFC is an organism capable of producing granulocytes and monocytes. MPIF also reversibly inhibits colony formation by granulocyte and monocyte colony forming cells induced in rat hepatocytes. Chemical protection experiments in the laboratory indicate that MPIF protects these hematopoietic lineages from the cytotoxic effects of antimetabolites (5-FU, ARA-C), antitumor antibiotics, topoisomerase I inhibitors and taxanes.

MPIF protects bone marrow progenitor cells in vivo when administered immediately before each cycle of chemotherapy. In addition, the treatment of MPIF resulted in a faster recovery in both bone marrow progenitor cells and the surrounding cell populations of neutrophils and platelets than the control in the in vivo chemotherapy model. Taken together with the results from laboratory studies, these results demonstrate the clinical applicability of MPIF as a protector of hematopoietic progenitor cells from chemotherapy-induced bone marrow suppression.

If the striatum pool is at its maximum, initiation of MPIF-mediated therapy at the beginning of chemotherapy may result in preservation of the striatum pool at the end of the cycle of chemotherapy. Once clinically confirmed, the results indicate that granulocyte colony-stimulating factor (G-CSF) or granulocyte and mononuclear colony stimulating factor (GM-CSF), which have been unable to prevent progressive loss of progenitor cell preservation following repeated courses of chemotherapy It represents an advantage beyond what can be achieved with the same growth factor. In addition, MPIF intervention after multiple cycles of chemotherapy may be useful in helping to recover from neutropenia and thrombocytopenia, as well as in patients with reduced prostatic cell preservation symptoms.

Phase I studies where placebo was limited and dose escalated stepwise were performed in healthy volunteers (n = 25) who received repeated doses (n = 6). Administration was initiated at a level that was calculated to be 2 logs below the optimal amount of protection and was scaled up stepwise to 100 g / kg of body weight at or below the maximum dose to produce adverse effects that could not be detected in preclinical toxicology studies. Healthy volunteers were treated daily at 0.1, 1, 10, 30 and 100 μg / kg for 6 days. Six patients, five MPIF patients, and one placebo recipient entered each dose level. There were no serious adverse events thought to be associated with the administration of MPIF. In general, the reported side effects are light and temporary, mainly mild headaches, muscle pain and fatigue. Only one serious side effect (possibly TIA) of unknown relevance to the study drug occurred in a 71-year-old woman aged 52 hours after the last injection. The patient's symptoms were soon to be cured after admission. Immune responses such as antibody development were not observed. Analysis of flow cytometry data showed a transient, dose-dependent reduction in CD14-positive mononuclear cells during MPIF administration. However, no reduction in the number of isolated monocytes or neutrophils was observed. No evidence of major changes in blood circulation or inhibition of hematopoiesis was observed in these healthy volunteers with normal hematopoiesis. The pharmacodynamic study revealed that the area below the initial time curve (AUC) of the maximum concentration (C _MAX ) and its concentration without any cumulative drug trail on day 6 after treatment was associated with dose (Example 34).

Pharmacological Classification and Principles . The recombinant human protein, MPIF, is a truncated version of the human β-chemokine, myeloid lineage inhibitor-1 (MPIF-1). One promising indication for MPIF in patients undergoing bone marrow suppression chemotherapy is the protection of bone marrow progenitor cells.

Systemic therapy with cytotoxic drugs is the basis for the most effective treatment of sporadic cancers. In addition, adjuvant chemotherapy is often used after treatment of local conditions by surgery or radiotherapy.

Since bone marrow suppression is one of the most common of the serious toxicities associated with chemotherapy, there is considerable clinical need for the benefits of adjunctive therapy in patients receiving bone marrow suppression therapy. Nonclinical studies indicate that MPIF has considerable potential as a protective agent against hematopoietic progenitor cells from cytotoxic effects of chemotherapeutic agents.

Hematopoietic stem cells and pluripotent progenitor cells play a role in restoring all hematopoietic systems. In normal individuals, these cells do not break up very often and are relatively resistant to a single course of chemotherapy. However, if multiple cycles of chemotherapy occur consecutively, bone marrow progenitor cells respond to increased proliferation and become much more susceptible to toxic effects from the dosing and processing of subsequent chemotherapeutic agents. Many of the processes of cytotoxic chemotherapy result in progressive loss of myeloid progenitor cells, which slows recovery and exacerbates neutrophils and platelets to the lowest amount.

The clinical value of these protectants is to reduce the incidence and / or duration of chemotherapy-induced cytopenias, and potentially reduce the likelihood of infection and bleeding following a high dose or successive cycles of chemotherapy. In addition, MPIF provides an adjunct standard for dosing intensity, timely administration of potent chemotherapy.

Currently available therapies . Colony stimulating factors (CSFs) have been used to maintain standard and increased dosages of chemotherapy for nearly a decade. Nonclinical evidence suggesting that an incremental dose of chemotherapy in stages can substantially increase the destruction of tumor cells and promote survival in selected malignant tumors is worthwhile, but this concept is not clinically validated . CSFs that stimulate the production of granulocytes, macrophages and platelets are used to shorten the duration of neutropenia or thrombocytopenia after chemotherapy. On the other hand, MPIF protects bone marrow progenitor cells by temporarily limiting their proliferation before or during exposure to chemotherapy. Different aspects of this effect prevent the progressive loss of progenitor cell preservation that occurs with repeated administration of chemotherapy, even in conjunction with CSF. Therefore, the use of MPIF makes it possible to increase the dosage strength of the chemotherapy.

Nonclinical studies . MPIF acts as a chemoprotective agent against the myeloid progenitor cells. Compared with other chemoprotective agents that have shown efficacy in laboratory experiments and / or animal models, MPIF inhibits CD34 ⁺ progenitor cell proliferation and inhibits proliferation of granulocytes and monocytes (CFU-GM), a colony forming unit (CFU lt; / RTI &gt; cells) and inhibit colony production by both cells. Some human MPIF scaffolds exhibit similar inhibitory effects on CFU-GM and CFU-GEMM colony formation. Therefore, MPIF inhibits pluripotent striations, ability to strike and order striatum.

In addition, MPIF acts as a potent and reversible inhibitor of low proliferating-colony forming cells (LPP-CFC) in mice, which ultimately contain many marrow myeloid lineages that develop into peripheral mononuclear and granulocytes. In addition, MPIF inhibits the more primitive, highly proliferating colony-forming cells (HPP-CFCs) that exhibit various properties of pluripotent hematopoietic stem cells.

Tissue expression of MPIF . The mRNA expression pattern of MPIF is complex with messages detected in lung, liver, bone marrow, activated mononuclear cells, and bone marrow mononuclear HL-60 and THP-1 cell lines. More recently, isolated transcripts have been identified in the pancreas, heart, skeletal muscle, and a smaller range of bone marrow.

Nonclinical pharmacology. The results of more than 270 experiments in the laboratory indicate that MPIF used for screening anticancer agents did not show any detectable effect in any of the tumor cell lines of the National Cancer Institute tumor record or any of the other 52 tumor cell lines. In addition, more than 75 cell-based assays have been developed to study MPIF activity. Of the normal cell types, the biological activity of MPIF is restricted to specific cell and hematopoietic progenitor cell ranges in the surrounding immune system (Table 7). Specifically, MPIF has been found to achieve the following.

- inducing chemotactic response in stationary T cells and monocytes

- induction of Ca ²⁺ mobilization in monocyte, monocyte-derived dendritic cells and eosinophils

- inhibition of multipotential and afferent progenitor colony formation in rats at concentrations ranging from 0.1 to 100 ng / mL

- inhibition of human CD34 ⁺ cell proliferation at concentrations ranging from 0.1 to 100 ng / mL

MPIF screening analysis of the laboratory room 75-80 Cell-based screening system Endoderm-derived cells Mesenchymal stem cell Ectoderm-derived cells Species quantity Hepatocyte lung epithelium Epithelial cells Vascular epithelial cell osteoclast / osteoblast cartilage cell skin (fibroblast) smooth muscle (aorta) muscle cell immune system cell ^* hematopoietic stem cell ^* Neuronal cell cortex neuron GABA neuron Schwann cell epithelial cell Proliferation cell destruction differentiation transition NCI cancer screening

^* Jim found a biological activity.

The chemotactic activity of MPIF

MPIF is a potent chemoattractant for stalled T lymphocytes with a pronounced maximal response at 10 ng / mL. MPIF did not stimulate chemotaxis in anti-CD3 activated T cells over a wide range (0.1-1000 ng / mL). The newly isolated mononuclear cells showed a chemotactic response to MPIF. The maximal migration was observed at 100 ng / mL, which is 10 times higher than that required to induce a similar response in stationary T cells. A weak chemotactic response occurred in neutrophils. MPIF did not induce chemotaxis in B lymphocytes, eosinophils, basophils, NK cells and platelets.

Effect of MPIF on human blood cells . MPIF has the following experimental effect in human blood cells.

Monocyte and monocyte derived macrophages . The MPIF effect on lysosomal enzyme secretion was measured in newly isolated mononuclear cells. A small but variable secretion of N-acetyl- beta -D-glucosidase was observed over the range of 0.5-500 ng / mL. Although the secretion was detectable, the secretion was less significant than that induced by MIP-1?, MIP-1 ?, RANTES or MCP-1. In another study, MPIF (1 to 100 ng / mL) had no effect on the secretion of lysosomal enzyme elastase, glucuronidase or myeloperoxidase. MPIF (0.1-100 ng / mL) did not induce monocytes to secrete IL-1β, tumor necrosis factor-α (TNF-α), IL-10 or IL-2. In addition, no effect on oxidative detonation or cytotoxic activity was found in activated macrophages.

Gloss . The basophils purified from peripheral blood were incubated with MPIF (1 to 1000 ng / mL) for 10 minutes and the resulting supernatant was analyzed for histamine release. MPIF did not induce histamine release.

Natural killer cells . Purified PBMCs were used as a means to measure the effect of MPIF on natural killer-mediating cells killing K562 cells. MPIF (1 to 100 ng / mL) had no effect on IL-2-stimulated K562 cell killing.

Platelets. MPIF at a concentration of 1 to 100 ng / mL failed to induce or regulate platelet activation or aggregation.

MPIF is a human CD34 ⁺ Suppress the proliferation of striatum.

To measure the effect of MPIF on human hematopoietic progenitor cell proliferation, CD34 ⁺ cells were isolated from umbilical cord blood, resuspended at 5 x 10 ⁴ cells / mL, and cultured in the presence of IL-3 and SCF Lt; / RTI &gt; for 4 days. The resultant bone marrow stroma was washed and conditioned under four conditions, that is, seeding conditions, conditions in which MPIF was added to the medium, addition of a cytokine mixture of IL-3, GM-CSF and erythropoietin (EPO) Under the conditions that the cytokine mixture and MPIF were added to the medium. After six days, the number of viable cells in each medium was measured. As shown in Fig. 46, the bone marrow stroma can not survive when cultured under the conditions of feeding or in the condition that MPIF is added to the medium. On the other hand, the cytokine mixture dramatically increased the survival rate of the striatum. Addition of MPIF (1-1000 ng / mL) resulted in considerable inhibition of cell proliferation (20-40%). (These results are representative of three independent experiments. Values are mean absorbance ± SD .

Inhibition of human CFU-GM by MPIF

In order to investigate whether the inhibitory effect of MPIF is targeting a specific lineage, CD34 ⁺ -induced striatal cells were cultured in BFU-E, CFU-G, CFU-M, CFU-GM, CFU-Meg and CFU-Mix colonies (Methocult TM semi-solid medium containing IL-3, GM-CSF, SCF, EPO and TPO). Comparison of the number and phenotype of colonies occurring in the presence of MPIF at 14 days after culture or the number and phenotype of colonies resulting from the presence of the control cytokine MIP-1α or monocyte chemoattractant protein-4 (MCP-4) did.

The results of two representative experiments indicate that MPIF inhibits the formation of both CFU-GM and CFU-Mix (50-64%). MIP-1α and MCP-4 were not effective on colony formation in any striatal group. These results confirm the inhibitory effect of MPIF on bone marrow stromal development and define MPIF as an inhibitor of human granulocyte and mononuclear cell progenitor cells. (Table 8)

Effect of MPIF on the formation of human CD34 ⁺ striatum Colony frequency (number of colonies per 1000 cells) Culture conditions BFU-E CFU-G CFU-M CFU-GM CFU-Meg CFU-Mix Experiment No. 1 badge 13 ± 2 15 ± 2 14 ± 3 16 ± 3 11 ± 3 11 ± 2 MPIF-1 19 ± 2 18 ± 5 12 ± 2 8 ± 2 12 ± 2 5 ± 1 MIP-1? 17 ± 3 19 ± 5 14 ± 2 14 ± 4 12 ± 3 12 ± 2 Experiment No. 2 badge 14 ± 3 13 ± 3 13 ± 1 12 ± 3 13 ± 1 11 ± 2 MPIF-1 14 ± 2 11 ± 1 12 ± 2 5 ± 1 12 ± 2 4 ± 1 MPIF-1α 12 ± 2 12 ± 3 13 ± 2 13 ± 2 14 ± 2 12 ± 1 MCP-4 12 ± 1 14 ± 2 12 ± 1 12 ± 2 11 ± 1 12 ± 2

Chemical protection in the laboratory

(5 ng / mL), SCF (50 ng / mL) MCSF (5 ng / mL) and IL-1 alpha (10 ng / mL) in the presence or absence of MPIF. After 60-70 hours, the medium was treated with chemotherapeutic agent and the culture then continued for 3-24 hours, at which point the number of viable LPP-CFCs was measured by standard clonality assay. As shown in Figure 47, the cytotoxic effects of cell cycle-specific drugs 5-FU, Ara-C, paclitaxel and daunorubicin were significantly reduced by MPIF. Conversely, MPIF was unable to protect the striatum from the alkalizing agents melphalan and thiotepa. These results support the role of MPIF as an inhibitor of pluripotent myeloid progenitor cell proliferation and provide insight into the range of chemotherapeutic agents against which chemokines can be effective.

In another experiment, the protective effect of MPIF on 5-FU-induced toxicity was measured using MPIF at a concentration of 0.1-100 ng / ml, and two methods of MPIF production in large quantities were performed using Lot # 11 and Lot # 19 Respectively. The data show a dose response curve showing percent colony protection from administration of MPIF. The data show that 0.1 ng / ml of MPIF gives about 30% chemical protection and 1,000 ng / ml of MPIF gives more than 80% chemical protection. (Data not shown.)

Summary of in vivo studies

As shown in Figure 48, in vivo analysis of MPIF was performed in mice first with an adjuvant study on compound safety in rabbits and rats. The results of these studies are as follows.

- MPIF is a powerful bone marrow protectant for hematopoietic progenitor cells. The result of this protection is a faster recovery of the bone marrow stroma and surrounding cells after chemotherapy compared to the control.

- MPIF can be administered daily for periods of up to 14 days without significant toxicity.

-MPIF has no effect on cardiovascular.

-MPIF is not exothermic.

-MPIF is quickly removed from circulation.

In vivo bone marrow stromal protection

Experiments were conducted to investigate whether MPIF protects bone marrow stroma in vivo against the effects of chemotherapeutic agents. Mice were injected with MPIF (1.0 mg / kg intraperitoneal) at 24 hour intervals every day for 3 days and then injected once with 5-FU (150 mg / kg intraperitoneal) at 3 days. Mice were killed at various times after 5-FU treatment and the number of bone marrow colonies was determined by standard HPP-CFC or LPP-CFC analysis. Controls include mice receiving saline and 5-FU alone. As shown in Figure 49, the number of bone marrow colonies detected in MPIF-treated mice returned to normal levels within 7 days after administration of 5-FU. In contrast, bone marrow colony formation from mice treated with 5-FU alone did not show recovery at this time. These results indicate that pretreatment of MPIF prior to chemotherapy allows for a faster recovery of colony forming cells, which may be due to the ability of MPIF to protect bone marrow stroma.

The results of MPIF-mediated striatal protection were evident at the periphery where the number of total white blood cell (WBC) cells increased immediately after colonization was found in the bone marrow. The data presented in Figure 49B summarizes eight unique experiments. The displayed values are expressed as the standard error of the mean WBC value plus or minus averages. The differences observed at day 6 and day 8 were relative to p-values less than 0.001 and 0.0001.

Chemical protection effect of MPIF in several cycles of treatment

Although the results thus far support the role of MPIF as a protective agent for bone marrow stroma, protection throughout the various phases of treatment is the use of clinically relevant MPIF. The results of the experiment in which the chemical protective effect of MPIF is measured during three cycles of treatment are shown in FIG. Analysis of bone marrow colony formation using normal mouse, bone marrow obtained from mice treated with 5-FU (in 100 mg / kg ip) alone or mice treated with 5-FU and MPIF (1.0 mg / kg in situ) (Grzegorzewski, KJ, et al., Blood (abstr: suppl) (disclosure permitted) throughout the three cycles of treatment.

MPIF dose range and dosing schedule

The nonclinical dose response of MPIF ranging from 0.01 to 10 mg / kg varied. This diverse response was the reason for choosing a 3-log range of dosages for clinical trials.

The different non-clinical dosing regimens tested are shown in FIG. The reason for choosing to administer to humans at -2, -1, and 0 days relative to chemotherapy administration was based on the observation that this treatment regimen provided the most consistent and reproducible protection among the many plans tested in mice. In addition, given the relatively short serum MPIF half-life and long cell cycle time of hepatocyte stroma in vivo (McNicec et al., Int. J. Cell Cloning 8 : 146-160 (1990); Bertoncello et al. , Exp. Hematol. 19 : 174-178 (1991)), it is theoretically advantageous to administer multiple doses of MPIF to a patient to maximize protection by maximizing progenitor cell exposure.

When tested both in vivo and in vitro, the results indicate that MPIF is biologically unusual. The activity of this protein, which functions as a powerful bone marrow protector, will be used as a chemical protective agent and will protect the early bone marrow from the effects of commonly used chemotherapeutic agents. The clinical value of these agents is evidenced by their ability to reduce the incidence and severity of cytopenia induced by chemotherapy and thereby reduce the likelihood of infection and bleeding.

Nonclinical toxicology

Three nonclinical toxicological studies were conducted: the 7 day dose range study, the 14 day subchronic study and the 25 day subchronic study, under the direction of Good Laboratory Practice. Doses below 20 mg / kg did not cause significant toxicity. A summary of the multiple dose study (Figure 52) and a summary of the clinical study for non-clinical studies (Figure 53) are presented. Other effects of autonomic nervous system or cardiovascular events were observed at doses of 10 mg / kg or less.

Nonclinical absorption, distribution, metabolism and excretion

The pharmacokinetic study of MPIF was performed in BALB / c female mice receiving one intravenous or subcutaneous MPIF pill at a dose of 20 mg / kg. Three mice were killed from each treatment group and bled at each time point, and the concentration of MPIF was measured by enzyme-linked immunosorbent assay. As shown in Figure 54, intravenous administration of MPIF is rapidly and rapidly removed to a low but detectable level that lasts 24 hours after injection. Subcutaneous injections exhibit similar characteristics with the major difference that the highest serum levels are delayed by 30 minutes. After this period, the elimination becomes indistinguishable from that observed in intravenously treated mice. The elimination of MPIF is expected for small proteins as a rule.

The second pharmacokinetic study of MPIF was performed in mice receiving one large intravenous pill of 20 mg / kg. MPIF was rapidly removed from the serum. MPIF could be detected at 0.1% of the dose at 8 hours after administration.

The study described in this example tested the activity of MPIF-1 (? 23) in connection with its use during chemotherapy. Many of these methods are equally applicable to studies of the use of MPIF-1 in radiotherapy. In addition, those skilled in the art will be able to illustrate the activity of full-length MPIF-1, or fragments thereof, agonists and / or MPIF-1 antagonists as well as polynucleotides (eg, through gene therapy) The researches were easily transformed.

Example 35

Clinical and clinical studies

Previous human experiments

Clinical study 00304-CRX-HV-01. Phase I studies were conducted to investigate safety, pharmacokinetic (PK) and pharmacodynamic effects in healthy volunteers (Louie et al., Blood 90 : 1569A (abst; suppl) (1997)). 30 subjects (14M, 16F, median age 46, range 21-73 years) were randomly selected for blinded, placebo-controlled, continuous dose escalation (0.1, 1, 10, 30, 100 [mu] g / kg), received 6 doses of MPIF or placebo administered for about 6 consecutive days over a period of about 1 minute. Each dose group consisted of 6 subjects randomly extracted at a 5: 1 active vs. placebo ratio. Side effects (AE) were assessed over 4 weeks. After two weeks, the initial stability data was reviewed and it was decided whether to start the next group of experiments. Blood samples were obtained for flow cytometry to evaluate hematology, chemistry, PK, antigenic and peripheral blood cell populations.

This was a Phase I stability study in healthy volunteers, so confirming efficacy was not the primary goal. However, improvements in the following laboratory parameters have been evaluated.

- Number of white blood cells (WBC)

- Separated neutrophil count (ANC)

- Number of red blood cells (RBC)

- platelet count

- Peripheral blood cell composition by flow cytometry

In the number of isolated neutrophils, no difference was observed between the active treatment group and the placebo treated group when measured by a change in the number or percentage of isolated. These results can be compared with preclinical studies in normal animals. Likewise, no significant change from baseline was observed for leukocytes, red blood cells or platelets.

Flow cytometry was continuously monitored to further describe the effect of MPIF in peripheral leukocyte subgroups. The percentage of peripheral blood mononuclear cells (granulocytes, mononuclear cells, T and B lymphocytes) as a function of dose and number of days studied is graphically shown in FIG.

No significant changes from baseline at any time during the study were observed in granulocytes, T lymphocytes or B lymphocytes. The lack of effect on granulocyte counts in healthy volunteers was consistent with the lack of effect observed in healthy animals in preclinical studies.

A temporary dose-dependent reduction in peripheral mononuclear cell ratio producing CD14 was observed during 6 days of administration of MPIF in the group receiving 10-100 占 퐂 / kg / day (days).

Analysis of the relative changes from baseline was performed to assess the significance of mononuclear CD14 inhibition during treatment. Percentage changes relative to the trapped CD14 + action were calculated. Baseline values were considered as controls for each patient. The baseline level can be compared between treatment groups, with the lowest p value = 0.18 (comparison between treatment group and placebo) in paired comparisons.

The overall analysis of the change model was significant at all points in time. For the high dose groups 10, 30 and 100 μg / Kg (p = 0.0001), the change from the baseline was significantly different from zero. There was also a significant difference between the placebo and these three treatment groups. There was a significant difference between the groups of 100 [mu] g / Kg and 30 [mu] g / Kg and 10 [mu] g / Kg to express the dose response.

To further investigate the effects of study drugs on mononuclear cells, isolated monocyte counts (AMCs) were calculated. This was done in the same way as calculating the number of neutrophils isolated using the following equation.

AMC = number of WBCs ×% mononuclear cells (from the number indicating the difference) × 1000

The results are shown in FIG. When monocytes were quantified by morphological means, the expected decrease in AMC was not observed during MPIF treatment. In fact, a slight increase during the treatment period was found to occur at MPIF doses above 1 μg / Kg, which returned to baseline levels within 2-3 days of the final dose.

The simplest interpretation of mononuclear cell data is that MPIF causes a dose-dependent change in monocyte chemotaxis leading to the entry of non-CD14 producing mononuclear cells. This will calculate the appropriate increase and decrease in the number of isolated mononuclear cells in the CD14 producing mononuclear cells observed by the flow cytometer. By these methods, there is clear evidence of transient and reversible biological activity.

Safety . What is expected of MPIF is the protection of myeloid lineage cells in patients undergoing myelosuppressive chemotherapy. In clinical trials of these drugs with limited dose ranges and limited placebo, the most frequently reported AEs are involved in their infectious and hemorrhagic complications associated with bone marrow suppression and the accompanying chemotherapy administration. In preclinical and clinical studies, the effects of MPIF were transient, reversible, and confined to myeloid cells. Although it is predicted that MPIF will reduce the frequency, severity and duration of chemotherapy-induced cytopenias, unexpected delayed action may aggravate cytopenias.

In the healthy volunteer Study I at Phase I, 00304-CRX-HV-01, 18 subjects (14 being active and 4 being placebo) experienced 32 side effects (25 active and 7 placebo) .

Adverse events are coded by researchers as "related to study drug administration", "not associated with study drug administration", or "do not know relationship to study drug administration". To standardize the record of side effects on causality, these adverse events encoded by the investigator as "I do not know the relationship with study drug administration" were considered by the sponsor with a possible cause and were therefore recorded as "related AE".

According to these criteria, 11 out of 25 MPIF treated subjects experienced one or more side effects that are thought to be associated with MPIF treatment. Three of the five subjects receiving placebo experienced one or more side effects that were thought to be associated with drug administration. The occurrence of side effects (relative 44% versus 60%) between the active-treated subject and the placebo-treated subject could be compared.

Twenty-nine of the 32 recorded side effects had NCI CTC Grade 1 or 2 severity. Two were unknown. The most frequently reported adverse events were headache (5 subjects, 4 active / 1 placebo), sleepiness (3 subjects, 2 active / 1 placebo), leukopenia (3 subjects, 2 active / 1 placebo). A summary of all AEs recorded during Phase I studies is shown in Table 9.

One adverse reaction was reported to be serious and dangerous. A 72-year-old white female elderly experienced dizziness, which was manifested by dizziness, nausea and vomiting, and showed instability at approximately 56 hours after the final administration of MPIF (30 μg / Kg). The subjects were admitted overnight during observation and irradiation. Laboratory, head CT and carotid ultrasound tests were not disclosed. The subject had similar experiences in the past. Symptoms quickly recovered and the subject was discharged from the hospital the day after the study was completed without any sequelae. The researchers wrote that they "do not know" the relationship between these adverse events and drug administration studies.

Summary of inverse of body system, COSTART and processed intensity Body system (COSTART) Placebo process N% 0.1 / / KgN% 1.0 mu g / KgN% 10 [mu] g / KgN% 30 / / KgN% 100 [mu] g / KgN% All active Body total abdominal pain incompetence back pain headache injection site edema Injection pain 2 401 200 00 01 200 00 0 0 00 00 00 00 00 00 0 4 800 01 201 204 800 00 0 0 00 00 00 00 00 00 0 0 00 00 00 00 00 00 0 1 200 00 00 01 201 201 20 5 200 01 41 44 161 41 4 Digestive diarrhea 1 200 01 20 0 00 00 0 1 201 200 0 0 00 00 0 0 00 00 0 0 00 00 0 1 41 40 0 Blood / lymphatic anemia hematopoietic hematopoietic leukopenia leukopenia 1 200 00 00 01 20 2 400 01 200 01 20 1 201 200 00 01 20 0 00 00 00 00 0 0 00 00 00 00 0 1 200 00 01 200 0 4 161 41 41 42 8 Musculoskeletal muscle pain 0 00 0 0 00 0 1 201 20 0 00 0 0 00 0 0 00 0 1 41 4 Nervous system dizziness Facial nerve paralysis Surface vasodilation Dizziness 1 201 200 01 201 200 0 0 00 00 00 00 00 0 2 400 00 02 401 200 0 0 00 00 00 00 00 0 2 401 200 00 00 01 20 1 200 01 200 00 00 0 5 201 41 42 81 41 4 Respiratory system sore throat 0 00 0 0 00 0 0 00 0 0 00 0 1 201 20 0 00 0 1 41 4 Skin pruritus 0 00 0 1 201 20 0 00 0 0 00 0 0 00 0 0 00 0 1 41 4 Urogenital system Uterine hemorrhagic vaginitis 0 00 00 0 0 00 00 0 0 00 00 0 1 201 200 0 0 00 00 0 1 200 01 20 2 81 41 4

Parameters of water dynamics. The pharmacokinetic parameters were measured in the 00304-CRX-HV-01 study. Plasma samples were obtained at baseline and following the initial administration at 5, 10, 15, 30 minutes, 1, 2, 4, 24 hours. Other samples were obtained before and after the last dose when drug accumulation was measured.

This data is summarized in FIG. Circulating blood levels are relatively easily measured in the 0.1 and 1.0 μg / kg groups for less than 1 hour and 30 minutes. Higher doses resulted in detectable levels for up to 24 hours after injection. AUC and C _MAX were dose-proportional. Among the subjects who received the highest dose (30 and 100 占 퐂 / kg), T _1/2 was about 0.5 hours. At 6 days, plasma levels before final drug administration did not show signs of drug accumulation.

These studies indicate that MPIF-1 causes side effects associated with G-CSF such as skeletal pain, alopecia, diarrhea, neutropenic fever, mucositis, heat, fatigue, anorexia and the like.

The study described in this example tested the activity of MPIF-1 (? 23). However, those skilled in the art will appreciate that, to test the activity of full-length MPIF-1, or fragments thereof, agonists and / or MPIF-1 antagonists as well as polynucleotides (eg, through gene therapy) The research could easily be modified.

Example 36

Summary of data and guidance for researchers

Summary of preclinical data . In vivo studies indicate that MPIF is a potent protectant for hematopoietic progenitor cells. Inhibition of rat pluripotent and order scaffold colonies was observed at concentrations ranging from 0.1 to 100 ng / mL. Experiments in the laboratory have shown that MPIF confers cytoprotection against the effects of antimetabolites, anticancer antibiotics, topoisomerase inhibitors, and Tapsene. In vivo studies, the outcome of these protections is a more rapid recovery of bone marrow stroma and surrounding cell populations after cytotoxic chemotherapy.

MPIF can be administered daily (by intravenous injection) for up to 14 days without significant toxicity. No adverse effects were observed in animals under an equivalent dose of 1.67 mg / kg in humans. No adverse effects were observed in toxicology studies repeatedly dosed at doses of 20 mg / kg or less for 28 days in animals.

In the experiment, more than 270 experiments showed no growth promoting effect on MPIF tumor cell lines.

Summary of clinical data . MPIF was safely administered to healthy volunteers receiving repeated doses (n = 25) at a Phase I placebo control, dose escalation study (dose range: 0.1-100 μg / kg) and was well accepted.

No serious adverse events that were considered to be associated with MPIF administration were observed in healthy volunteers.

A transient, dose-dependent reduction in CD14 expression was observed in peripheral blood mononuclear cells. The number of isolated monocytes and neutrophils was not affected by other calculations.

MPIF could be detected up to 24 hours after treatment.

MPIF was not immune when administered to healthy volunteers.

Possible risks and side effects . The effects of MPIF were temporary, reversible in clinical and clinical studies, and limited to myeloid cells. Although MPIF is expected to reduce the frequency, severity, and duration of chemotherapy-induced cytopenias, unexpected delayed action may aggravate cytopenias.

Temporary adverse events with comparable frequency and severity compared to placebo were observed in early clinical studies of healthy volunteers receiving intravenous administration of several times MPIF of MPIF. No acute response thought to be associated with MPIF administration was observed. MPIF was non-immunogenic in healthy volunteers.

Although there are few side effects associated with MPIF administration, the introduction of exogenous proteins can potentially result in immunologically mediated local or systemic responses. The subject may experience acute allergic reactions (such as fever, urticaria, hypertension or reactions such as hypotension), or other reactions such as vasculitis or sera. Kidney dysfunction, hepatic dysfunction, immunosuppression, coagulopathy and neuropathy are also rare, but can not be ruled out.

The subject should be closely examined at the time of administration and after administration for any symptoms of silver acute inverse. Emergency supplies (epinephrine, corticosteroids, hypertension, and cardiac defibrillators) should be readily available when acute reactions occur.

taboo. The safety of the product for use during pregnancy or for use with breastfeeding did not appear in clinical trials.

caution

Information for patients. MPIF should be used under the direction of a clinical trial doctor or researcher who is familiar with this product when using any of the investigational products.

The safety of the product for use during pregnancy or for use with breastfeeding did not appear in clinical trials. No laboratory animal studies were conducted to investigate the safety of embryonic or fetal development, migration process and development around birth and postnatal development. A woman who misses the menstrual cycle should be presumed pregnant until other things are proved. Therefore, essential surveys should only be carried out during pregnancy only when the possible benefits exceed the risks to the mother and the fetus.

It is not known whether MPIF is distributed in breast milk. Breastfeeding should be discontinued if study medication is found to be necessary.

Laboratory tests. Peripheral blood volume should be thoroughly examined in patients treated with MPIF. Laboratory tests should be performed according to the accompanying clinical test method.

Product Interaction . MPIF is not involved in causing any serious interactions with other drugs.

Carcinogenesis, mutation, impaired fertility. Animal studies to investigate carcinogenicity and mutagenicity were not performed with MPIF. Studies in the laboratory indicate that MPIF does not detect any effect on tumor cell lines.

Product abuse and dependence. The product abuse and dependence potential of MPIF is not investigated in the nonclinical or mortality population but is considered negligible.

Overdose. There is no clinical experience of overdosing MPIF. No toxicity was observed in animals given doses of 20 mg / kg or less continuously for 14 days. This is equivalent to a dose of 1.67 mg / kg in humans.

Dosage and administration . The dose and dosage regimen for MPIF was not investigated. Early studies evaluated that 0.1 to 100 μg / kg of MPIF administered by intravenous injection for less than 6 days was safe at daily doses. A 2A study to measure biological activity and preliminary efficacy will yield a dose range of 1-100 μg / kg. Sequential dosages and administration regimens will be determined from the results of these initial efficacy studies.

Supply and storage . MPIF is supplied as a sterile liquid formulation. These products should be stored between 2 ° C and 8 ° C in an area that is only available to authorized persons. For patient administration, the diluted drug was stable at room temperature for 12 hours or less without degradation of the product.

The study described in this example tested the activity of MPIF-1 (? 23). However, one of ordinary skill in the art will readily be able to illustrate the activity of full-length MPIF-1, or fragments thereof, agonists and / or MPIF-1 antagonists as well as polynucleotides (eg, through gene therapy) The research could be modified.

Example 37

Solution Structure and Dynamics of MDIF-1

summary

The myeloid precursor inhibitor factor-1 (MPIF-1) is a prodrug inhibitor and monocyte activator. The solution structure of MPIF-1 was determined by nuclear magnetic resonance (NMR) spectroscopy. Unlike many CC chemokines, the MPIF-1 structure is a monomer. This structure is very well known except for the terminal residues and adopts the characteristic 3-helix and the upper kelmine folding of a-helix. In addition to the four cysteines that characterize most of the chemokines, MPIF-1 has two additional cysteines that form a sulphated bond. Chain chain dynamics reveal that the functionally important N-terminal residue, the N-terminal loop residue, and the residue adjacent to the sulfhydryl bond exhibit considerable power compared to the protein core. MPIF-1 is a monomer with reduced activity and under various solution conditions, but is processed from the entire 99 amino acid protein at the N-terminus and the latter is also functional. Thus, MPIF-1 is unique in that it is a longer full-length protein among all other chemokine associates. These studies are consistent with the idea that monomers are sufficient to activate the 7-TM receptor on leukocytes.

introduction

Chemokines (chemotactic cytokines) mediate a variety of biological processes including white blood cell trafficking, hematopoiesis, and angiogenesis and play a fundamental role in host defense against infection (Baggiolini, M et al., Annu. Rev. Immunol. 15: 675-705 (1997); Rollins, BJ, Blood 90: 909-928 (1997); Luster, AD, N. Engl. J. Med. 338: 436-445 (1998)). Approximately 40 chemokines have been identified so far; All of which are 70 to 100 amino acids in length and are characterized by four conserved cysteines. The chemokines are roughly divided into CC and CXC families based on whether the first two cysteines are neighbors (CC) or separated by amino acids (CXC). CXC chemokines can be divided into two subgroups, 'ELR' and 'non-ERL'. All ELR CXC chemokines activate neutrophils, while non-ERC CXC chemokines activate other subset of lymphocytes. CC chemokine activates monocytes, macrophages, eosinophils, basophils, and T-cells but does not activate neutrophils. In addition, a single member of the C family with only two cysteines and a single member of the CX ₃ C family were also identified.

A member of the CC family, myeloid precursor inhibitor-1 (MPIF-1) (known as CK beta 8) was initially identified in a large-scale sequencing effort and was structurally expressed in liver, lung pancreatic and bone marrow (Peter, VP, et al , J. Exp. Med. 185: 1163-1172 (1997)). In addition to inhibiting the formation of bone marrow cell colonies that occur in the granulocyte and monocyte lineage, this is also chemotactic for monocytes and eosinophils (Peter, VP, et al., J. Exp. Med. 185: 1163-1172 ); Young, B.-S., et al., Blood 91: 3118-3126 (1998)). Due to alternating splicing, two forms of the protein are formed, CK beta 8 and CK beta 8 -1, which are 99 amino acids in length and 116 amino acids in length, respectively (Young, B.-S., et al., Blood 91: Interestingly, the truncated forms (? 24-99) of 99 amino acid proteins (hereinafter referred to as MPIF-1) were observed to be substantially more active (Nardelli, B., et al. The cross-desensitization experiment in mononuclear cells and eosinophils showed that MPIF-1 (SEQ ID NO: CCR1 receptor, but incomplete desensitization in both cases suggests that additional receptor (s) may be involved (Nardelli, B., et al., J. Immunol. 162: 435-444 MPIF-1 causes rapid administration-dependent release of [ ³ H] -arachidonic acid from monocytes dependent on extracellular calcium and inhibits phospholipase A ₂ (PLA ₂ ) inhibitors In addition, PLA ₂ activation appears to be required for the formation of filamentous actin in mononuclear cells.

Several CXC and CC chemokine structures have been identified by NMR spectroscopy and X-ray crystallography (Clore GM, et al., Biochemistry 29: 1689-1696 (1990); Fairbrother, WJ, et al., J Malkowski, MG, et al., J. Biol. Chem. (1986) (1994); Skelton, NJ, et al., &Lt; RTI ID = 0.0 &gt;(1996); Kim, KS, et al., FEBS Lett. 395: 277-282 (1996)), &lt; RTI ID = 0.0 &gt;1996); Crump, MP, et al., J. Biol. Chem. 273: 22471-22479 (1998); Sticht, H., et al., Biochemistry 38: 5995-6002 (1999)). It was additionally discovered that the majority of the early characterized chemokines are dimers and CXC and CC chemokines are dimerized using different regions of the protein. In CXC chemokines, the first beta helix constitutes the dimer interface and in the CC chemokine the N terminal moiety constitutes the dimer interface. These observations and the observation that CXC chemokine activated only neutrophils and CC chemokines activated other white blood cells led to the belief that dimer formation is essential for leukocyte 7-TM receptor binding.

Features and subsequent discoveries of chemokines such as SDF-1 (CXC chemokine), MCP-3, eotaxin, HCC-2 and I-309 (CC chemokines) suggest that these chemokines are principally monomers (1998); Sticht, H &lt; / RTI &gt; et al., &Lt; RTI ID = 0.0 &gt; Crump, MP, et al., EMBO J. 16: 6996-7007 (1997)). Solution studies have shown that associations are sensitive to ionic strength, buffer conditions and pH (Mayo, KH, and Chen, M.-J., Biochemistry 28: 9469-9478 (1989); Yang, Y., et al. J. Biol. Chem 269: 20110-20118 (1994); Lowman, HB, et al., Protein Sci 6: 598-608 (1997)). (2000); Paavola, CD, et al., &Lt; RTI ID = 0.0 & Chem., Trapping of monomeric states (Rajarathnam, K., et al., Science 264: 90-92 (1994); Rajarathnam et al., J. Biol. Chem., 273: 33157-33165 , K., et al., J. Biol. Chem. 272: 1725-1729 (1997) showed that monomers are sufficient to bind and activate 7-TM receptors on leukocytes. Suggesting a process that is capable of binding to the proteoglycans and forming a concentration gradient and a process essential for certain leukocyte traffic killing (Hoogewerf, AJ, et al., Biochemistry 36: 13570-13578 (1997) ; Koopmann, W., and Krangel, MS, J. Biol. Chem. 272: 10103-10109 (1997)).

In this study, the solution structure and main chain dynamics of MPIF-1 can be characterized by NMR spectroscopy. In addition, the association tendency of MPIF-1 and full-length MPIF-1 whole protein were studied under various solution conditions by sedimentation ultracentrifuge measurement. The implication and association characteristics of structure and dynamics are discussed in terms of their function. The data in this study form the structural basis of treatment due to mutational studies and the structural assistance of immune-related diseases.

Experimental process

Protein expression and purification. The MPIF-1 gene sequence was chemically synthesized using optimized codons for expression in E. coli. The gene was then subcloned into an expression vector pHE4 containing a strong synthetic promoter with two lac actives, an effective ribosome binding site and a synthetic transcriptional terminator downstream of the inserted gene. The expression plasmids were transformed with E. coli. K12 derived from strain SG13009. After induction with IPTG, MPIF-1 was produced as an insoluble protein and extracted and refolded in 1.75 M guanidine hydrochloride in the presence of 5 mM cysteine. The expressed protein has additional Met (initial codon) at the N-terminus and is considered the first residue of MPIF-1 for simplicity. The protein was passed through a continuous exchange of strong cation (porous HS-50), anion (porous HQ-50) and cation (porous CM-20) exchange columns and finally size exclusion (Sephacryl S-100) . Full-length 99 amino acid matured MPIF-1 was cloned, expressed and purified as described for the MPIF-1 protein.

Sedimentation equilibrium. Analytical ultracentrifugation experiments were performed on a Beckman model XL-A ultracentrifuge at 20 ° C at rotor speeds of 23,000, 28,000 and 40,000 rpm. Experiments were conducted at two different starting concentrations of different buffer and ionic strengths to study their effect on dimerization (Table 10). Absorbance was measured at 280 nm and data were collected with a mean value of five consecutive radial scans using a 0.003 cm step size. The data were fitted according to the following equation.

At this time, the δ _{^{(r 2 -r 0 2),}} H = (1-υρ) (ω / 2RT), C r and C ₀ is the concentration of each of the radii r and r _0, M is the molecular weight of the monomer , υ is the partial specific volume, ρ is the solvent density, ω is the rotor angular velocity, K _a is the association constant of the monomer-dimer equilibrium, and E is the baseline offset. The partial incompleteness was calculated from the weighted average value of each amino acid partial specific volume. The data was fitted to the equation by nonlinear least squares method using Microcal Origin 4.1 software available from Beckman for XL-4. The features of the fitting were χ ² , the sum of the squared errors and error verification for the system deviation. Data were fit for a single species or for a monomer-dimer model. The theoretical molecular weights of MPIF-1 and MPIF-1 (1-99) are 8854.6 and 11367.6, respectively, and the data can be fitted for monomers for both proteins under all solution conditions (Table 10).

MR spectroscopy. All spectra were collected at 35 ° C with a VariantUnity Plus 600 or INOVA 500-MHz spectrometer equipped with field gradient accessories. The protein concentration was 2 mM in 20 mM sodium acetate, 1 mM sodium azide (pH 5.2 at 90% H ₂ O / 10% ² H ₂ O (v / v) or 99.99% ² H ₂ O). Chemical shifts were referenced to DSS using the method of Wishart et al. (Wishart, DS, et al., J. Biomol. NMR 6: 135-140 (1995)). The distribution of electron potential in the main chain NH, N, C _[alpha] and C [ _beta] molecules was based on the HNCACB and CBCA (CO) NH experiments (Muhandiram, DR, and Kay, LE, J. Mag. -216 (1994). chemical shift of the side chain atoms is ¹⁵ N- editing a total correlation spectroscopy ^{(15 N-edited total correlation spectroscopy} (TOCSY)) (Zhang, O., et al., J.Biol.NMR 4 : 845-858 (1994)) and HCCH-TOCSY (Kay, LE, et al., J. Magn. Reson B 101: 333 (1993)). High resolution 2 Dimension ¹ H- ¹ H Nuclear Overhauser enhancement spectroscopy (NOESY), TOCSY and DQF-COZY experiments were used to distribute the aromatic protons. The proton spacing was determined using ¹⁵ N-edited NOESY (mixing time 50 ms and 150 ms) and ¹⁵ N / ¹³ C- The NOE cross-peak intensities were determined to be 2.8, 3.5, and 4.0, respectively (Pascal, S, M., et al., J. Magn. Res. 103: 197-201 , &Lt; / RTI &gt; and 5.0 Angstroms, respectively. The upper limit of the non-stereospecifically distributed methyl and methylene protons was appropriately modified as the center average, and additionally 0.5 Å was added to the upper region to correct for higher intensities of distances including methyl protons (Kuboniwa, H., et al., Nat. Struct. Biol. 2: 768 (1995)), the stereospecific distribution of the β protons was determined by the ³ J The coupling constants (Grzesiek, S., et al., J. Am. Chem. Soc. 117: 5312-5315 (1995)) were obtained at the relative intensities of NOE from CαH to CβH and NH 3 proton in the NOESY spectrum. The stereospecific distribution of leu 25 and 66 methyl protons was made on the basis of the relative intensities of the NOEs of CαH to CH ₃ protons after setting the χ1 angle.

Hydrogen bond restriction. Potential candidates for hydrogen bonding were initially identified based on observing the slow exchange of amide protons from a series of 2D ¹ H- ¹⁵ N HSQC spectra recorded within 24 hours of protein degradation at ² H ₂ O. [ For each hydrogen bond, two distance limits were used (r _NH-O , 1.8-2.3 A and r _NO , 2.4-3.3 A). Hydrogen bond restriction was used only after the initial set of structures was determined. Only amide protons satisfying distance and angular constraints using hydrogen bond acceptors were used for structure determination.

Data processing and structure determination. All NMR spectra were processed using the program's nmrPipe suite (Delaglio, F., et al., J. Biomol. NMR 6: 277-293 (1995)). The structure was determined by a mixed distance geometry-dynamics simulated annealing method using the program XPLOR (Brunger, ATXPLOR Version 3.1 Manual, Yale University, New Haven, CT (1993)). A total of 713 NOE distance limitations (320 residue, 178 sequence, 84 intermediate range and 132 long range NOE) were used. In addition, 82 dimers (53 Φ and 29 χ ₁ ) and 36 hydrogen bonding limits (from 18 hydrogen bonds) were used for final structure determination. The initial structure was fabricated with NOE restriction only and then the structure was determined, including the dihedral and hydrogen bonding limitations. The simulated annealing decision was performed using a set of standard lumped parameters and a topology file in XPLOR version 3.1. A total of 50 structures were fabricated and 30 best structures were selected based on the lowest energy and good stereochemistry.

kinetics. ¹⁵ NT ₁ , T ₂ and [ ¹ H] ¹⁵ N NOE experiments were recorded at 35 ° C. with ¹⁵ N MPIF-1 labeled uniformly using the gradient form of the pulse sequence (Farrow, NA, et al., Biochemistry 33: 5984-6003 (1994)). All spectra were obtained using 544 (t ₂ ) and 128 (t ₁ ) real points, and 3 s recycle delay was used for the T ₂ experiment and 1.2 s was used for the T ₁ experiment. The [ ¹ H] - ¹⁵ N NOE was measured by recording the HSQC spectrum under proton saturation and non-extinguishing conditions. The spectrum of the NOE member was recorded using a delay of 5 s and the NOE presence spectrum was recorded with a 2s delay and 3s of proton saturation recorded to give the same 5s delay between the shortest periods. The spectra were processed using the NMR Pipe and the first two dimensions ¹⁵ NT ₁ and the T ₂ spectra were hand dispensed using the program PIPP. Subsequent spectra were automatically extracted using the program CAPP. The values of T ₁ and T ₂ were obtained by nonlinear least squares fitting of the crossover peaks to the 2-parameter exponential decay. The uncertainty of the values of T ₁ and T ₂ was obtained as the standard deviation of the fitting. The NOE value was obtained from the ratio of the peak intensity recorded in the proton saturation state or the non saturation state. The uncertainty of the NOE value was estimated from the baseline of the spectrum defined by Farrow et al., 1994 (Farrow, NA, et al., Biochemistry 33: 5984-6003 (1994)).

result

Sedimentation equilibrium. The ability of chemokines to associate depends on solution conditions such as pH and ionic strength. The association of MPIF-1 and full-length MPIF-1 were studied using ultracentrifugation at different pH and ionic strengths. A summary of the results is shown in Table 10. The data show that both proteins are monomers and do not tend to associate under exponential conditions. The monomer status of MPIF-1 is consistent with the NMR structure and is also derived from the correlation time determination in ¹⁵ N dynamics.

Structural statistics of MPIF-1. Statistics of the 30 final simulated annealing (SA) structures are shown in Table 11, and overheads of the individual structures on average structure are shown in Figure 58A. The structure of the protein is defined in detail except terminal residues 1-10 and 67-77. The characteristics of the resulting structure are described in the program PROCHECK (Laskowski, RA, et al., J. Appl. Crystallogr. 26: 283-291 (1993)) and VADAR .ualberta.ca) were used to test various criteria such as stereochemistry, hydrogen bonding, occupied areas of the Ramachandran plot, Van der Waals contacts, buried charged residues, multiple buried residues and packing defects. All 30 structures met the above criteria, which is expected to have a high resolution structure.

Structural statistics for 30 determined MPIF-1 structures and atomic r.m.s. Difference Energy (kcal.mol ^-1 ) NOE ^a 3.21 + - 0.48 Dihedral ^a 0.01 ± 0.01 Combination 4.31 ± 0.09 Van der Walls 1.54 0.30 Deviation from Ideal Geometry ^b Bond (A) 0.0019 ± 0.0001 Angle (°) 0.536 ± 0.001 Improwers (°) 0.305 0.001 Atomic rms difference (Å) ^c The main chain atom (11-66) 0.57 + 0.08 The Ruler (11-66) 1.09 ± 0.08

^The values for ^a NOE and twist angle were calculated from the square well potential using a force cosantant of 50 kcal.co, ^-1 Å ² and 200 kcal mol ^-1 rad ^-2 , respectively.

^b The values of the bonds, angles and improper indicate deviations from ideal values based on complete stereochemistry.

^c rms difference of 30 final structures overlaid on average structure

All structure and energy minimum mean structures showed good conalent geometry (Table 11) and minimum NMR costraint violation. The 30 SA structure has a NOE violation greater than 0.2 A and the diphedral angle violation is greater than 2 C. The rms distribution for residues 11-66 between all 30 structures and average structures is 0.57 A for the backbone atoms, And 1.09 A for the heavy atom (Fig. 59A.B). The accuracy of the twist angle is analyzed by the order parameter S (Hyberts, S. G., et al., Protein Sci. 1: 736-751 (1992)). The order parameter of the? And? Twist angles for structure residues 11-66 is > 0.95, indicating that the structure backbone is well defined (Fig. 59C, D). The order parameter for xi is shown in Figure 59E. Figure 60F shows the solvent access area, lower values imply that the side chains are buried and that bulk solvents are inaccessible. These residues tend to be hydrophobic and are highly structured as evidenced by high order parameters, generally χ1> 0.9. One exception to the large hydrophobic material exhibiting a low S is Ile-13. This is an exposed solvent and is conserved in several CXC and CC chemokines, and structure-function studies have identified a key role for this residue in receptor binding. It is observed that all twist angles of the 30 structures are included in the advantageous area of the Ramachandran plot. 72% of the residues are included in the core portion (the most advantageous region), 23% are in the permissible region and 1% is large.

Solution structure of MPIF-1. The structure of MPIF-1 employs a conventional chemokine fold and consists of a stretch loop in three beta helices after the N-terminus and alpha helices in the C-terminus (Figure 58B) (Figure 58B). The first 10 residues preceding the CC motif represent only or no sequence NOE and lack a predetermined structure since they have low order parameters for phi and psi. Followed by an N-terminal loop containing a series of turns (residues 13-20) leading to 3 ₁₀ helix (residues 21-24). The first β helix (residues 27-31) is connected to the second β helix (residues 39-44) by a type III turn, which is connected to the third β helix (residues 48-52) by the I-type. The third helix is directed to helix (residues 56-66) via the III-turn. Residues 67-77 are highly unstructured in agreement with long range NOE, small intermediate NOE and low order parameters. The hydroxyl protons of Thr-31 and Thr-44, respectively located at the ends of the first and second helix, play a structural role because they are hydrogen bonded to the backbone carbonyl across the helix. Of the six cysteines, four cysteines have all CC chemokine properties: Cys-11 forms a disulfide bond with Cys-35, which is part of the turn connection of the first and second β helices, and Cys- It forms a disulfide bond with Cys-51 in the helix. MPIF-1 had two additional cystines (Cys-22 at 3 ₁₀ helix, Cys-62 at alpha -helix). A database of cysteine chemical shifts was made and C _? Shifts were observed to be uniquely different in the free state and in the disulfide bonded form (unknown result). The shifts of Cys-22 and Cys-62 indicate that they are involved in disulfide bond formation. The structure is also evident from NMR properties such as cysteine close to the base to form disulfide bonds and the small coupling constant, the exchange of slow amide protons, and the NOE pattern of Cys-62, which is characteristic of helix residues. Cys-12-Cyw-51 disulfide bonds are adapted to left-handed twist in the main structure while the other two disulfide bonds are unstructured. The core portion of the structure has multiple long range hydrophobic contacts (Ile-20, Leu-25, Tyr-28, Phe-29, Val-40, Phe-42, Phe-50, Ala- 52, Val-59, MEt-63 and Leu-66). In addition to disulfide bonds, the long range NOEs between Thr-31, Gly-30, Tyr-15, Cys-11, Cys-12 and Cys-51 may be essential for their function, - orient to the terminal residue.

kinetics. ¹⁵ NT ₁ , T _2, and NOE annealing data can be obtained at 60 out of 73 predicted intramolecular potentials (Fig. 60A-C). The data can not be used for four prolines because of chemical shift overlapping or because the signal is too weak to be quantified with a reliable intensity and the remaining residues Met-1, Asp-2, His-5, Ala- 8, Ile-13, Ser-19, Ser-33, Glu-34, Ser-36, Lys-46, Leu-66 and Lys-67. ¹⁵ NT ₁ , T ₂ and NOE annealing data were analyzed to describe the internal dynamics of MPIF-1 using the model independence form of Lipari-Szabo (Lipari, G., and Szabo, A., J. Am. Chem Lipari, G., and Szabo, A., J. Am. Chem. Soc. 104: 4559-4570 (1982b)). Extracting data of each residue is S ² -τ _c (model _{^{1), S 2 -τ c -τ}} e ( model _{^{2), S 2 -τ c -τ}} ex ( Model 3), S ² -τ _c --τ _e- R _cx (Model 4) and a 2x magnitude model (Model 5) that allows the internal motion to occur at two different time scales (Clore, G., et al., J. Am. Chem. Soc. 112: 4989-4991 (1990)). By fitting the characteristics of the fittings, we adopted a suitable model for each residue. By minimizing the experiment, the optimal τ _c for each residue criterion was initially determined and the T ₁ , T ₂ and NOE values were determined using the isotropic spectral density function. Under the condition that the internal motion is slower than 100 ps and T ₂ is not significantly shortened due to the structural exchange, the T ₁ / T ₂ ratio (Figure 60D) is considered independent of the order parameter or internal motion and the overall correlation time τ _c Of the market. These residues were identified as having an N [ ¹ H] NOE value of <0.65, whereas the T ₁ / T ₂ ratio is outside of ₁ SD from the mean. On this basis, 26 residues were excluded and τ _c was calculated to be 4.6 ± 0.2 based on the remaining 34 residues.

The generalized order parameter (S ² ), chemical exchange (R _ex ), and local correlation time (τ _c ) as a function of amino acid sequence are shown in Figures 60E-G. The generalized order parameter provides a measure of the magnitude of the inner motion, where S ² = 1 means that the given NH-coupled vector is stiff, whereas S ² = 0 means that the motion is unrestricted. Residues 67-77 at the C-terminus and residues 1-10 preceding the first cysteine at the N-terminus exhibit a low order parameter (S ² < 0.7). Both ends are poorly represented in the NMR structure and kinetic data confirms that these residues are inherently mobile and that structural defects are not due to lack of experimental limitations. Except for the terminal, the average S ² value for residues 11-66 is 0.84 ± 0.06. Other residues representing low order parameters are Arg-18 and Ile-20 (0.7), which are part of the N-terminal loop. The annealing data for the residues Tyr-15, Cys-22, Thr-44, Cys-51, Ala-52 and Asn-77 require exchange terms (Models 3 and 4) Terminal residues 69-73 required a double size model. Residues Arg-3, Phe-4 and Thr-74 can not be fitted for any model. All other residues can not be fitted to the simple model.

discussion

MPIF-1 is a monomer in solution. ¹⁵ Determination of NMR structure from N dynamics and calculation of rotational correlation time indicates that MPIF-1 is present as a monomer under experimental conditions (50 mM acetate, pH 5.2). Ultracentrifugation studies under various solution conditions also indicate that MPIF-1 is present as a monomer (Table 10).

Structure and solution studies provide some intuition on the association properties in chemokines, but the molecular characteristics attributed to the specificity of the interaction are still difficult to grasp. CC chemokines show a greater difference in associative ability and can therefore exist in very high order polymers to monomers. MIP-1 alpha (Czaplewski, LG, et al., J. Biol. Chem. 274: 16077-16084 (1999)), RANTES (Skelton, NJ, et al., Biochemistry 34: 5329-5342 (HCC-2), which is very well associated at neutral pH (> 100 kDa), whereas at low pH it is separated into dimers at low pH, whereas Ibeta (Lodi, PJ, et al., Science 263: 1762-1767 And MCP-3 are monomers (Paolini, JF, et al., J. Immunol. 153: 2704-2717 (1994); Sticht, H., et al., Biochemistry 38: 5995-6002 KS, et al., FEBS Lett. 395: 277-282 (1996)).

On the other hand, CXC chemokines produce dimers and tetramers only because they are more restricted in association behavior (Clark-Lewis, I., et al., J. Leukocyte Biol. 57: 703-711 (1995)) . On the basis of the structure, some residues that were considered important for dimerization were ineffective or almost ineffective, while conversely mutations of residues away from the dimer interface produced monomers (Czaplewsku, LG et al., J. Biol. Chem. , Paolola, CD, et al., J. Biol. Chem. 273: 33157-33165 (1998), 274: 16077-16084 (1999); Laurence, JS, et al., Biochemistry 39: 3401-3409 ). It is clear that the various stabilizing forces by the moieties remote from the first sequence serve to promote association of dimers and tetramers.

However, there is data demonstrating that for both CXC and CC chemokines, the monomer is sufficient to bind and activate 7-TM on leukocytes. It has already been shown that monomers are as active as natural proteins in in vitro functional assays by rendering them non-dimerizable by trapping three neutrophil-activated chemokines (IL-8, NAP-2 and MGSA) in the monomer state (Rjarathnam, K. Similarly, RANTES, MCP-1, MIP-1, MCP-1, and MCP- 1α and MIP-1β revealed that the monomer is a receptor binding class (Czaplewsku, LG et al., J. Biol. Chem., 274: 16077-16084 (1999); Laurence, JS, et al. Biochemistry 39: 3401-3409 (2000); Paavola, CD, et al., J. Biol. Chem. 273: 33157-33165 (1998)).

Thus, the question of what exactly is the role of the dimer and higher order oligomers, if any, arises. Recent studies suggest that there is such a role in the establishment of binding and concentration gradients for proteoglycans, and in processes essential for traffic-leukocyte trafficking (Hoogewerf.A.J., et al., Biochemistry 36: 13570-13578 (1997)); Koopmann, W., and Krangel, M. S., J. Biol. Chem. 272: 10103-10109 (1997)). Important residues for proteoglycan binding tend to be basic residues such as arginine, lysine, and histidine in both CXC and CC chemokines, and tend to be located away from regions that are aggregated and are critical for binding to the 7-TM receptor. In this study, MPIF-1 showed no tendency to form dimers in solution. It has been investigated whether MPIF-1 and the other monomers Kemokin I-309 and HCC-2 are dimerized in the presence of cell surface proteoglycans.

An interesting feature of chemokines is the presence of multiple species that have been differentially processed at both the N-terminus and the C-terminus. Some chemokines (MPIF-1) are secreted as large precursors that have been proteolytically processed at the N-terminus or C-terminus to produce functional proteins. NAP-2, β-thromboglobulin (βTG), and connective tissue-activating protein-III (CATP-III) are N-terminal products of the plate culture base protein (PBP) While the others are considered as inert precursors. All four types of dimers and tetramers (Yang, Y., et al., J. Biol. Chem. 269: 20110-20118 (1994); NAP-2 and CTAP differentially processed at the C- -III was isolated from cell cultures (Ehlert, JE, et al., J. Immunol. 161: 4975-4982 (1998)).

The N-terminal region of the functional chemokine is composed of about 10 amino acids preceding the first conserved cysteine, and a mutation study revealed an essential role for receptor binding and activation of these residues. Truncated CC chemokines exhibit very different and unusual properties (Clark-Lewis, I., et al., J. Leukocyte Biol. 57: 703-711 (1995)). For example, deletion of the first 4 residues in MCP-1 results in loss of activity, whereas deletion of the first 5 residues results in almost complete recovery of activity. Additional deletions result in impairment of function but not with the ability to bind receptors and act as antagonists (Gong, J.-H., and Clark-Lewis, I., J.Exp.Med.181: 631 -640 (1995)). Continuous deletion at the N-terminus of IL-8 (CXC chemokine) results in increased activity, impaired activity and subsequent conversion of the antagonist (Moser, B., et al., J. Biol. Chem. 268: 7125 -7128 (1993)). It was observed that the truncation of the N-terminal residue preceding the first cysteine in CC chemokines favored monomer formation, suggesting that the length of the N-terminal residue is responsible for dimerization.

Alternating splicing of the MPIF-1 gene produces 99 amino acids and 116 amino acid bipartite proteins, respectively (referred to as CKβ8 and CKβ8-1, respectively) (Young, B.-S., et al., Blood 91 : 3118-3126 (1998)). Because differences in these lengths are present in the N-terminal region (32 amino acids and 49 amino acids, respectively, preceding the first cysteine), observations are exceptional between CC chemokines. Two forms of the protein have been found to have myeloid precursor inhibition and similar activity in monocyte chemotaxis. Expression of 99 amino acid proteins in baculovirus produces three forms of protein, full length and two truncated forms. This truncated form was found to be more potent in both monocyte chemotactic and myeloid precursor inhibition assays. MPIF-1 and full-length MPIF-1 data under various solution conditions (Table 10) indicate that there is no correlation between their monomer and their ability to form a dimer with the N-terminal residue length. Because leukocyte reinforcement and activity is regulated spatially and transiently, the effect of mutants of MPIF-1 (similar to other chemokines) acts in vivo. One aspect of leukocyte activation is the release of peptidases and proteases that act on the chemokines, thus regulating their activity and function.

Detailed description of structure and comparison with other chemokines. MPIF-1 adopts the usual chemokine folding of three? -Helix and top? -Helix. The core portion of the structure is well defined and represents a high order parameter for the lowest rmsd, phi, phi and chi for the backbone and core, and most slowly changing amides exhibit H- Lt; / RTI &gt; bonds. Several CC chemokine structures have been elucidated including the structures of MPIF-1 [beta], RANTES, MCP-I, MCP-3, Eotaxin and HCC-I (Lodi, PJ, et al., Science 263: 1762-1767 (1994); Kim, KS, et al., FEBS Lett. 395 (1994); Skelton, NJ, et al., Biochemistry 34: 5329.5342 (1995); Handel, TM, and Domaille, PJ, Biochemistry 35: 6569-6584 : 277-282 (1996); Crump, MP, et al., J Biol. Chem. 273: 22471-22479 (1998); Sticht, H., et al., Biochemistry 38: 5995-6002 (1999)). The secondary and tertiary structural elements of MPIF-1 are similar to those observed in other CC chemokines, although sequence identity between MPIF-1 and other CC chemokines varies between ~ 25 and 60% (Figure 62) (Fig. 61). The main chain overlap of the structured regions of MPIF-1 and other CC chemokines (residues 11-66) represents an rmsd of 1.5 to 2.0 A. The lowest rmsd is observed for the structural region (helices and helix) and the higher rmsd is observed at the N-terminal residue, the N-terminal loop and 30 s turn. Large sequence differences are observed for these regions of the protein, which are relatively less defined regions and are both mobile and functionally important.

15, Ile-20 (N-terminal loop), Leu-25, Tyr-28 (Cys-11, Cys-12, Cys-35, Cys- Val-40, Ile-4l, Phe-42, Thr-44 (second beta strand), Phe-50, Ala- 52 (third beta strand), Val- 59, Met-63 and Leu-66 (a-helices) (ordered according to the MPIF-1 sequence) are conserved or conservatively substituted (FIG. Most are bulky hydrophobic materials located in helix or helix and adopt the same side chain structure in different structures and constitute structural scaffolds. The-31 and Thr-44 are completely buried polar residues and the structure reveals that the hydroxyl proton plays a structural role by hydrogen bonding to the main chain carbonyl across the helix.

The structure reveals that the Cys12-Cys51 disulfide bond adopts almost the left-handed helical structure. ¹⁵ N dynamics data indicate that some cysteine and residues around all three disulfide bonds are structurally exchanged, indicating that these regions of the protein are mobile and perform structural exchange. Cys-11-Cys-35 disulfide bonds exhibit the greatest segmental motion, which, for example, has been shown to result in severe impairment of function without structural alteration due to mild confusion for disulfide bonds in IL-8 (Rajarathnam, K , et al., J. Biochemistry 38: 7653-7658 (1997)). The kinetics in the N-terminal region, disulfide defects and 30 s turn suggest that it plays an important role in specific binding and activation (Clark-Lewis, I., et al., J. Leukocyte Biol. 57: 703-711 1995). MPIF-1 has the highest sequence homology to HCC2, and the latter also has six cysteines, which bind to CCR1 and are stem cell proliferation inhibitors. One difference between MPIF-1, HCC-2 and other CC chemokines is the Trp residue at position 58 in all CC chemokines except MPIF-1 (containing Gln) and HCC-2 (containing Gly). MPIF-1 and HCC-2 structures indicate that the steric bulk of the indole side chain will be present by the disulfide bond method.

In addition to the conserved hydrophobic material, some charged residues are also conserved, and Arg-18 at the N-terminal loop and Lys-45 and Arg-48 at 40 turn are exposed to the solvent and bound to the negatively charged proteoglycan (Chakravarty, L., et al., J. Biol. Chem. 273: 29641-29647 (1998); Koopmann, W., et al., J. Immunol.163: 2120-2127 (1999)). However, the relative importance of the position of the proteoglycan-binding domain and the charged residues involved in binding varies from chemokine to chemokine. (Kuschert, GSV, et al., Biochemistry 37: 11193 (1998)), PF-4 (Mayo, KH, et al., Biochem> J.312: 357 The first β-helical residues in SDF-1 (Amara, A (1984)) and MCP-1 (Chakravarty, L., 279: 23916-25 (1999)) and residues of 40 s turn and MIP-1 alpha (Koopman, W. and Kvangel, MS, J. Biol. Chem. 272: 10103-10109 (1997)) and MIP-1? (Koopmann, W., et al., J. Immunol.163: 2120-2127 (1999)) were related to proteoglycan binding. MPIF-1 is a very basic protein and has additional base residues in the 40 s loop (Lys-46) and a-helix (Lys-57, Arg-64 and Lys-67), indicating that it binds the proteoglycan more tightly 63).

Structure - Function

Activation of monocytes: MPIF-1 has been shown to bind CCR1 with high affinity and being a potent activator of monocytes (Nardelli, B., et al., J. Immunol. 162: 435-444 (1999); Berkhout, TA, et al., Biochem. Pharmacol. 59: 591-596 (2000)). MCP-3, RANTES, MIP-1 alpha and HCC-2 also bind and activate CCR1. Mutagenicity studies have shown that the N-terminal residue preceding the first cysteine and the residue of the N-terminal loop (between the second cysteine and the preceding 3 ₁₀ helix) play an important role in receptor binding to CXC and CC chemokines ( G., J.-H., and Clark-Lewis, I., J Exp, Med., 181: 631-711 (1995), J. Leukocyte Biol. Pakianathan, DR, et al., Biochemistry. 36: 9642-9648 (1997)). Terminal loop moiety of the chemokine ligand is believed to interact with the acceptor N-terminal moiety that constitutes the initial docking site and this interaction is a ligand for interacting with a receptor moiety that evokes stereostructure modification and action response (Clump-Lewis, I., et al., J. Leukocyte Biol. 57: 703-7007 (1997) 711 (1995)).

In the MPIF-1 structure, the N-terminal loop residues are well defined (S> 0.95 for Φ, Ψ) and adopt a unique stereostructure. On the other hand, the ¹⁵ N kinetics data indicates that this protein domain is relatively easy to move, exhibiting fluctuations below nanoseconds and exhibiting steric configuration changes at slower millisecond time magnitudes. Similar observations have been made by Erotica (Crump, MP, et al., Protein Sci. 8: 2041-2054 (1999); Ye, J., et al., J. Biomol. NMR 15: 115-124 (1999) , vMIPII, the viral monomer CCMOKIN (LiWang, AC et al., Biochemistry 38: 442-453 (1999)) and fractalkine, which binds to multiple CXC and CC chemokine receptors, the monomer CX ₃ C chemokine Mizoue, LS, et al., Biochemistry 38: 1402-1414 (1999)). The N-terminal loop domain is adjacent to and close to all three disulfide bonds. Sequence analysis also conserves residues (numbered according to MPIF-1) corresponding to Ile-13, Tyr-15, Arg-18 and Ile-20 and is similarly substituted with other CC chemokines, The induction results in a decrease in binding to each of these receptors. Ile-13 (the first residue after the second cysteine) is solvent-exposed to all CXC and CC chemokines and will most likely play a direct role in receptor activation. In RANTES, N-terminal loop residues are important, which are observed to be receptor specific: Arg-17 is required for binding to CCR1, Phe-12 is required for binding to CCR3, and Phe-12 and Ile-15 Is required for binding to CCR5 (Pakianathan, DR et al., Biochemistry 36: 9642-9648 (1997)). In most structures, Tyr-15 and Ile-20 are buried and packed for other hydrogenated residues that adopt a similar steric configuration and represent a structural role. These observations indicate that MPIF-1, Ile-13 and Arg-18 play a functional role and are directly involved in receptor binding, while Tyr-15 and Ile-20 act as part of the structural scaffold.

NMR structure and kinetic data of MPIF-1 indicate that the N-terminal residue preceding the first cysteine is unstructured. Similar observations have been made in all monomer NMR and dimeric CXC structures, indicating that the mobility of these residues is essential for optimal interaction with the receptor. All CXC chemokines that activate neutrophils have a characteristic 'ELR' sequence preceding the first cysteine essential for binding and activation (Clark-Lewis, I., et al., J. Leukocyte Biol. 57: 703-711 (1995)). The signature sequence is lacking in CC chemokine and sequencing does not provide any insight into receptor specificity. In addition, CC chemokines represent complex ligand-receptor profiles. Most CC chemokines that activate monocytes, macrophages, eosinophils and T cells bind to many receptors and most receptors bind to multiple chemokines. On the other hand, examples of ligand binding (LARC and CCR6) binding to only one receptor are known. The N-terminal residues of MPIF-1 have little or no similarity to other chemokines that bind to CCR1 (HCC-2, MCP-3, RANTES and MIP-1 alpha). HCC-2 exhibits very high total sequence homology to MPIF-1 (~ 60%) but not at the N-terminus. Recent structural-functional studies in RANTES that bind to several receptors in addition to CCR1 revealed receptor-specific responses when mutating specific N-terminal residues: Pro-2, Asp-6 and Thr-7 bind to CCR1 Pro-2 and Tyr-3 are essential for binding to CCR3, and Tyr-3 and Asp-6 are essential for binding to CCR5. None of the N-terminal residues are identical between MPIF-1 and RANTES and only one residue is conservatively substituted (Tyr-3 at RANTES and Phe-4 at MPIF-1). Tyr-3 has been shown to play an essential role in binding to CCR3 in RANTES and remains to be tested whether MPIF-1 can bind to CCR3. Interestingly, it has recently been shown that the length of the N-terminal residue, rather than the nature of the side chain, is important for binding MCP-1 to CCR2 (Jarnagin, et al., Biochemistry 38: 16167-16177 (1999)).

Inhibition of proliferation of progenitor cells. All forms of MPIF-1 were shown to inhibit progenitor cell proliferation, and truncated forms were observed to be more effective in inhibition than whole proteins. Surprisingly, CC and CXC chemokines such as MIP-α, IL-8, GRO-β, PF-4, IP-10 and MCP-1 inhibit the proliferation of progenitor cells, while GRO- α, NAP- Lt; / RTI &gt; and &lt; RTI ID = 0.0 &gt; RANTES &lt; / RTI &gt; The chemokine profile indicates that the ability to regulate proliferative cell proliferation is not related to the activation of their kinemokinergic receptors. In addition, the monomeric form of MPIF-1 alpha is more significant than the form of the aggregated native protein (Czaplewski, LG, et al., J. Biol. Chem. 274: 16077-16084 Molecular-based studies of MPIF-I function are clinically relevant to patients with a variety of diseases, such as those who perform chemotherapy.

It will be apparent that the invention may be practiced otherwise than as specifically described in the foregoing Detailed Description and Examples.

Numerous modifications and variations of the present invention are possible in light of the above teachings and are intended to be within the scope of the appended claims.

All patents, patent applications, and references cited herein are incorporated by reference. U.S. Patent Application No. 08 / 941,020, filed September 30, 1997, is incorporated herein by reference in its entirety.

Certificate of Microorganism Deposit (PCT Rule 13bis)

A. The following certificate is about the microorganism mentioned on the second line of the specification page 39 B. Proof of Deposits Additional deposits are identified on the attached sheet. Name of the Depositary: American Bacterium Culture Collection Address (including postal code and country) of depositary institution: Virginia, USA 20110-2209 Manassas University Seat Bollevard 10801 Date of deposit: September 30, 1997 Accession number: ATCC 209311 C. Additional proof (blank if not necessary) This information will continue on the attached sheet. DNA plasmid pHE4-5 / MPIF? 23 D. Designation of the country requiring proof (if this proof is not for all designated States) E. Provide a separate certificate (blank if not necessary) The certificates presented below will then be submitted to the International Bureau (specify the general type of certificate, eg "Trust Number of Deposit") For office International Office This paper has been repaired with the international application This paper has been repaired by the international collection office. Authorized Authorized

Certificate of Microorganism Deposit (PCT Rule 13bis)

A. The following certificate relates to the microorganism mentioned on line 11 on page 5 of the specification B. Proof of Deposits Additional deposits are identified on the attached sheet. Name of the Depositary: American Bacterium Culture Collection Address (including postal code and country) of depositary institution: Virginia, USA 20110-2209 Manassas University Seat Bollevard 10801 Date of deposit: February 29, 1994 Accession number: ATCC 75676 C. Additional proof (blank if not necessary) This information will continue on the attached sheet. DNA plasmid 99248 D. Designation of the country requiring proof (if this proof is not for all designated States) E. Provide a separate certificate (blank if not necessary) The certificates presented below will then be submitted to the International Bureau (specify the general type of certificate, eg "Trust Number of Deposit") For office International Office This paper has been repaired with the international application This paper has been repaired by the international collection office. Authorized Authorized

Claims (22)

A method of treating or preventing cytotoxicogenic cell damage comprising administering to an individual an effective amount of a polypeptide of SEQ ID NO: 2 or an active variant or fragment thereof, wherein said cell is (a) a bone marrow stem cell, a hematopoietic cell And connective tissue cells, excluding pluripotent progenitor cells; (b) epithelial cells; (c) muscle cells; And (d) neural cells. 2. The method of claim 1, wherein the connective tissue cells are selected from the group consisting of skeletal cells, osteoblasts, osteoclasts, osteocytes, chondrocytes, adipocytes, periosteum cells, bone cells, progenitor cells, blood cells, erythrocytes, leukocytes, eosinophils, Lymphocytes, macrophages, lymphocytes, monocytes, platelets, tissue macrophages, organ specific phagocytes, B-lymphocytes, T-lymphocytes, macrophages, monocytes, myeloids, lymphocytes, Wherein the antigen is selected from the group consisting of progenitor cells, progenitor cells, progenitor cells, progenitor cells, progenitor cells, progenitor cells, progenitor cells, progenitor cells, progenitor cells, progenitor cells, 2. The method of claim 1, wherein said subject is experiencing treatment to kill dividing cells. 4. The method of claim 3, wherein said treatment is selected from chemotherapy, radiation therapy or target radiation therapy. The method of claim 1, wherein said subject is experiencing occupational or fatal exposure to a cytotoxic agent. 5. The method of claim 4, wherein said polypeptide is administered prior to said treatment. 5. The method of claim 4, wherein said polypeptide is administered during said treatment. 5. The method of claim 4, wherein the polypeptide is administered after the treatment. 6. The method of claim 5, wherein said polypeptide is administered prior to said exposure. 6. The method of claim 5, wherein said polypeptide is administered during said exposure. 6. The method of claim 5, wherein said polypeptide is administered after said exposure. 5. The method of claim 4, wherein said treatment is target radiation therapy. 13. The method of claim 12, wherein the target radiotherapy is radiation immunotherapy. 3. The method of claim 1, wherein the subject is experiencing intentional exposure to a cytotoxic agent. 15. The method of claim 14, wherein the cytotoxic agent is radiation. 16. The method of claim 15, wherein said polypeptide is administered prior to said exposure. 16. The method of claim 15, wherein said polypeptide is administered during said exposure. 16. The method of claim 15, wherein said polypeptide is administered after said exposure. 16. The method of claim 15, wherein the radiation is caused by nuclear explosion. 20. The method of claim 19, wherein said subject is in need of treating or preventing a radiation disease or radiological burn. 2. A method of inhibiting the proliferation of leukemia cells comprising administering an effective amount of a polypeptide of SEQ ID NO: 2 or an active variant or fragment thereof, wherein the polypeptide, variant or fragment is conjugated with a therapeutic moiety. 22. The method of claim 21 wherein the therapeutic is a radioactive isotope.