WO1993020107A1 - Recombinant active forms of ecef, proteins that associate therewith, and uses thereof - Google Patents

Abstract

Novel shortened forms of ECEF are described, proteins that associate with ECEF are identified and methods of using such compositions for the inhibition of ECEF activity are presented.

Description

TITLE OF THE INVENTION

RECOMBINANT ACTIVE FORMS OF ECEF, PROTEINS THAT ASSOCIATE THEREWITH, AND USES THEREOF

This invention was made with government support; the government has certain rights in this invention.

Field of the Invention

The invention is in the field of recombinant DNA, and specifically, to shortened recombinant forms of Eosinophil Cytoioxicity-Enhancing Factor (ECEF), proteins that associate with such forms, a unique cultured cell line for the production of ECEF and uses thereof.

Background of the Invention

Physical and functional characteristics of the human eosinophils are altered in association with allergy, asthma, certain parasitic diseases, certain chronic inflammatory diseases, and certain experimental cancer immunotherapies. These changes include reduction in density, hypersegmentation of the nucleus (occasional), enhanced oxidative metabolism and release of toxic oxygen metabolites, enhanced arachidonic acid metabolism via the 5-lipoxygenase pathway, and enhanced cytotoxic capabilities. Recent work demonstrates that the defined cytokines TNF, GM-CFS, IL-3, IL-5 and HILDA/IL-10 can induce most of these physical and functional changes in normal blood eosinophils in vitro (TNF: Silberstein et al. , Proc. Nail. Acad. Sci. USA 83:1055 (1986); GM-CSF: Silberstein et al , J. Immunol. 137:3290 (1986), Lopez et al. , J. Clin. Invest. 78:1220 (1986) and Owen et al. , J. Exp. Med. 166:129 (1987); IL- 3: Lopez et al. , Proc. Natl. Acad. Sci. USA 54:2761 (1987) and Rothenberg et al. , J. Clip. Invest Si: 1986 (1988); IL-5: (Lopez et al , J. Exp. Med. 167:219 (1988) and Rothenberg et al, J. Immunol. 143:2311 (1989) and HILDA/IL-10 (Moreau et al, J. Immunol 738:3844 (1987)). Thus, these cytokines have a potential role in the patholophysiology of eosinophilia.

In the conditioned medium of LPS-stimulated monocytes or PMA/LPS-stimulated U937 cells, the dominant cytokine species (with respect to units of activity in the assay of eosinophil cytotoxic function) has a low isoeletric point (pi - 3.7 - 4.9) and is resistant to boiling temperatures (Veith et al J. Exp. Med. 757: 1828 (1983); Lenzi et al, Cell. Immunol. 94:333 (1985); Silberstein et al. J. Immunol 138:979 (1987); and Silberstein et al J. Immunol 143:979 (1989)). This cytokine, which was designed ECEF (for Eosinophil Cytotoxicity-Enhancing Factor)), was purified and identified as a 10 kDa polypeptide with a pi of 3.7. It was possible to determine 17 of the first 20 amino acids, and at the time the work was done, the Gembank and PIR databases contained no similar polypeptide sequences (Silberstein et al , J. Immunol 143:979 (1989)).

In the cell, ECEF appears to exist in association with other proteins. One of the components of the ECEF macromolecular complex has been identified as TNF (Silberstein et al, Proc. Natl Acad. Sci. USA 83:1055 (1986)).

The complete structure of 10-kDa ECEF is not known, however, partial amino acid sequence analysis shows that its N-terminal is identical to that of a recently described 13.5-kDa cytokine (Rimsky et al. , J.

Immunol 735:3304 (1986); Wakasugi et l , Proc. Natl Acad. Sci. USA 84-.S04 (1987); Tagaya et al, J. Immunol 140:2614 (1988); Wollman et al., J. Biol Chem. 263: 15506 (1988); and Tagaya et al, EMBO J. 8:757 (1989)). The 13.5-kDa substance functions in a manner similar to IL-1, in that it promotes proliferation of murine thymocytes and fibroblasts (Rimsky et al., J. Immunol. 735:3304 (1986); Wa asugi et al , Proc. Natl Acad. Sci. USA 54:804 (1987)) and induces expression of IL-2R in a T cell line (Tagaya et al, J. Immunol. 140:2614 (1988); Tagaya et al, EMBO J. 8:757 (1989)). The 13.5 kDa cytokine also contains a conserved dithiol reductase moiety that is identical to that of the enzyme thioredoxin

(Wollman et al, J. Biol Chem. 253: 15506 (1988); Tagaya et al , EMBO J. 8:757 (1989)). Because of this property and its functions, the 13.5 kDa molecule has become known as "3B6-derived IL-1" (Rimsky et al , J. Immunol 735:3304 (1986); Wakasugi et l , Proc. Natl. Acad. Sci. USA 84:S04 (1987)), "human thioredoxin" (Wollman et al, J. Biol. Chem. 253:15506 (1988)), or "adult T cell leukemia-derived factor" (Tagaya et al, J. Immunol 140:2614 (1988); Tagaya et al, EMBO J. 8:757 (1989)) and is designated hereafter as TRX.

A 14 kDa molecule was isolated by a French group on the basis of its ability to support the growth of an EBV-transformed B-lymphocyte line in the manner of IL-1 (Rimsky et al. J. Immunol. 735:3304 (1986)). Subsequently, a cDNA encoding this molecule was cloned, expressed, and also designated "thioredoxin" (Wollman et al , J. Biol Chem. 253: 15506 (1988)). In the same time frame, a Japanese group was studying a 14 kDa substance designated ADF from an HTLV-1 transformed lymphocyte cell line. This substance induced the expression of IL-2 receptors in a large granular lymphocyte cell line from an ATL patient, also in the manner of IL-1. This group isolated a cDNA clone encoding this substance and found that it was nearly identical to the clone of the French group (differing only in two nucleotide bases and consequently two amino acids) (Tagaya et al. J. Immunol. 140:26X4 (1988) and Tagaya et al. EMBO J. 8:757 (1989)). In April of 1991 , a lab in Israel reported the isolation of a similar cDNA in a different context. This group studied gene products that mediated the growth inhibitory properties of IFN-7 in HeLa cells, using a "knockout" method (cDNA in the reverse orientation in an expression vector that generated anti-sense RNA). A clone was isolated that permitted rapid cell growth, presumably by knocking out a growth inhibitor. Sequence analysis showed that the insert was identical to the cloned sequence from the Japanese group. Thus, this gene product appears to have an intracellular role as a growth inhibitor in certain cells (Deiss, L.P. et al., Science 252:117 (1991)).

The other independent finding related to this molecule was reported by a laboratory in England. The investigators were studying the presence of reduced sulfhydryl groups on the surface of phagocytic cells. They found that a single 11 kDa protein species on the surface of THP-1 cells was susceptible to rapid alkylation with radiolabeled iodoactamide. When this substance was purified and its sequence was determined, it was found to have the same NH₂-terminal as ECEF/thioredoxin/ADF (Martin, H. et al., Biochem. Biophys. Res. Comm. 775:123 (1991)). Its size and its χrøsition on the cell surface suggest that it may be equivalent to the 10 kDa ECEF species, rather than the 14 kDa species.

Cloning allowed the determination of the amino acid sequence. In the amino acid sequence predicted by the thioredoxin/ADF cDNAs, the region from amino acids 30-34, encoding amino acid sequence "WCGPC," is homologous to the catalytic site of the enzyme thioredoxin, conserved in all animal and plant species that have been studied (Wollman et al., J. Biol Chem. 253:15506 (1988)) and Tagaya et al , EMBO J. 8:757 (1989)). The work of Holmgren (Holmgren, A., /. Biol. Chem. 254.-9627 (1979) and Holmgren, A., Ann. Rev. Biochem. 54:237 (1985)) with E. coli and spinach thioredoxin shows that the paired cysteines can accept reducing equivalents from a number of sources and transfer them efficiently to cleave disulfide bonds in other proteins. Though the remaining parts of the gene have no relationship to thioredoxin from non-mammalian species or other known proteins, the purified recombinant "thioredoxin" did have classical thioredoxin activity (reduction of insulin in the presence of suboptimal DTT concentrations). The recombinant thioredoxin did not have the biological IL-1 -like activity of the native molecule the group had studied (Wollman et al , J. Biol. Chem. 253: 15506 (1988)).

The material (recombinant ADF) formulated by the Japanese group was reported to have several activities, however all of these activities required very high (> lOOnM) concentrations, and the magnitude of the responses was modest. The activities included: introduction of IL-2 receptors (an activity that was enhanced somewhat by the inclusion of β- mercaptoethanol in the assay medium (Tagaya, Y. et al, EMBO J. 8:757 (1989)), introduction of NF/cB expression in the lymphocyte cell line (Kawabe, T. et al , FASEB J. 5:A 1330 (1991)), and protection of U937 cells from the toxic effects of TNF (Matsuda, M. et al. , J. Immunol. 147:3S-7 (1991)). Since U937 cells are known producers of ECEF, these effects of exogenous ECEF/ADF show that endogenous production does not saturate the signal transduction mechanism. Thus, ECEF has been identified as an important cytokine that is useful for the modulation of eosinophil toxicity. However, the mechanism by which this factor acts is unknown. Active forms of the factor other than that of the native molecule are unknown, and it is not known to separate the ECEF eosinophil stimulating activity and the ECEF dithiol reductase activity. Such forms would allow the retention of the eosinophil-stimulating acitivty without the disadvantage of the proinflammatory dithiol reductase activity.

Lastly, protein species that associate with the active factor in vivo have not been eludicated.

SUMMARY OF THE INVENTION

Recognizing the role that ECEF polypeptides may play in the enhancement of eosinophil cytotoxicity, and cognizant of the need for the elucidation of the role of such action, the inventors investigated the molecular heterogeneity of ECEF. These efforts have culminated in the identification of specific active forms of ECEF that have not previously been known, in the identification of specific protein species that associate with ECEF, and in the identification of unique cell lines for the production of ECEF. Accordingly, the invention is first directed to shortened (truncated) forms of ECEF, such shortened forms being truncated from the carboxy terminal side of the ECEF peptide, and such shortened forms retaining at least two functional activities of ECEF, the eosinophil stimulating activity and the dithiol reductase activity. The invention is further directed to shortened forms of ECEF that possess the enhanced eosinophil stimulating activity but lack dithiol reductase activity.

The invention is further directed to compositions containing specific proteins that form complexes with ECEF, the isolated complexes, and the use of such complexes as targets for therapeutic intervention.

According to the invention, there are further provided ECEF genetic sequences, such genetic sequences providing the recombinant shortened forms of ECEF DNA, cDNA, RNA and anti-sense RNA, such forms retaining the biological activity of the shortened ECEF peptide of the invention.

According to the invention, there are further provided expression vectors containing such genetic sequences, hosts transformed with such expression vectors, and methods for producing the genetically engineered or recombinant ECEF protein The invention is further directed to a novel cell lines for the production of ECEF, such cell line not requiring PMA stimulation for the high production of ECEF, such cell lines being designated U937⁺ cells.

ECEF cDNA, recombinant protein, antisense RNA and antibodies provided by the invention are useful as diagnostic probes for monitoring the activation and involvement of ECEF protein activity and expression in health and disease. In addition, the shoπened ECEF forms that retain the eosinophil stimulating activity but lack the dithiol reductase activity provide the advantage of being useful for eosinphil -stimulation in the absence of proinflammatory dithiol reductase activity.

BRIEF DESCRIPTION OF THE FIGURES

Figure 1. Release of polypeptide precipitated specifically by antibody to 10-kDa ECEF. U937 cells were exposed to [³⁵]methionine/cysteine at time 0, and the accumulation of immunoprecipitable peptides over time is shown for conditioned medium of cultures that were unstimulated, treated with 7.5 μg/ml of LPS, 400 ng/ml of PMA, or LPS and PMA together. The molecular mass of precipitated species is indicated in kDa.

Figure 2. N-terminal amino acid sequence similarities between 14- kDa ECEF, 13-kDa ECEF, 10-kDa ECEF (Silberstein, D.S. et al., J. Immunol. 143:979 (1989)), and rTRX (deduced from cDNA, Wollman E.E. et al, J. Biol. Chem. 253: 15506 (1988)). A question mark indicates that no determination was possible. Only the first 10 positions are shown for the lowest two.

Figure 3. Relative biologic activities with respect to eosinophil cytptoxic function. Data in both panals represent mean values from three experiments, with duplicate determinations for each condition in each experiment. Figure 3 A: purified 14- and 10-kDa ECEF. Purified 14- and 10-kDa ECEF were set at equal concentrations (equal ³⁵S counts) and titrated with three-fold dilutions from left to right. The mock-eluted material from the gel slice between the two species was adjusted to a similar concentration based on volume. ANOVA showed a dose-dependent effect for both the 14-kDa species (p<0.05) and the 10-kDa species (p <0.005). Figure 3B: rTRX. The activity of rTRX at the indicated concentrations is shown. There was a significant dose-dependent effect, as determined by ANOVA (p<0.05). Example of raw date (means of duplicate determinations) from repesentative experiment: percent targets dead in medium alone - <2; percent dead with antibody and unstimulated eosinophils - 14; with addition of 14-kDa ECEF (concentrations from high to low) - 29, 37, 34, 23, 19, 27, 23: slice between - 16, 21, 17, 14, 18, 16, 15, 13; 10-kDa ECEF - 38, 48, 40, 55, 35, 35, 45, 26.

Figure 4. Synthesis of 10-kDa ECEF after PMA stimulus. Methods and display of data are as for Figure 1 , except that contents of washed cell pellets were analyzed.

Figure 5. Presence of ECEF species on the outer cell surface. Cell proteins were labeled chemically or biosynthetically. Figure 5A: U937 cells stimulated with PMA for 24 h were washed and treated with PBS (outer surface block -) or PBS containing sulfosuccinimidyl-3-(4- hydroxyphenyl)propionate (SHPP, outer surface block +) as indicated. Then, either whole cells or broken cell preparations were treated with ¹²⁵I- SHPP. Figure 5B: U937 cells untreated or stimulated with PMA for 24 h and radiolabeled biosynthetically with [³⁵S]methionine/cysteine were washed and treated briefly with trypsin in medium or medium alone. After each of these treatments, the cells were washed and disrupted in lysis buffer. ECEF species were detected by immunoprecipitation and SDS- PAGE.

Figure 6. Sequence of cDNA encoding 14 kDa ECEF/thioredoxin/ADF and the predicted amino acid sequence. The conserved thioredoxin catalytic site is underlined [SEQ ID No.:l: and :2:]. Figure 7. Hopp-Woods hydrophilicity plot for amino acids 71 through 88.

Figure 8. Figure 8A: DNA sequence analysis of inserts in p-MAL expression plasmids, demonstrating the construction of truncated ECEF species. Portions of sequencing gels are shown for the different rECEF species, with the string of 6T's (amino acids 78 and 79) serving as a reference point. Arrows show the insertion of STOP codons at appropriate places to create rECEF-84, rECEF-80, and rECEF-79. The sequences are the sequences of the fragments that were inserted in the pMAL expression plasmid. These fragments contain the coding regions, stop codons, restriction enzyme sites at each end, and a buffer of GGG. Figure 8B: DNA and protein sequence of the full-length rECEF-104 construct [SEQ ID No.:3: and :4:]. Figure 8C: DNA and protein sequence of rECEF-84, the recombinant ECEF 84 amino acid construct [SEQ ID No. :5: and :6:]. Figure 8D: DNA and protein sequence of rECEF-80, the recombinant ECEF 80 amino acid construct [SEQ ID No. :7: and :8:].

Figure 9. SDS-PAGE analysis (15% gel under reducing conditions) of the indicated rECEF preparations. The rECEF polypeptides (note that they are of the expected size), maltose binding protein (MBP), and small amount of Factor Xa are all indicated by arrows.

Figure 10. Stimulation of human eosinophil cytoϊoxic function by rECEF species (concentration ranges from 800 nM to 82 fM). A representative experiment is shown. Eosinophil purity was 88%. Values represent the means of triplicate determinations. Values for rECEF-84 were significantly greater than for "no ECEF" control or comparable concentration of rECEF-104 at all concentrations down to 10 pM (p < 0.05; paired t-test). There was no direct toxicity of preparations to schistosomula targets. A control paramyosin-maltose binding protein fusion protein, prepared in an identical manner, had no effect in the assay at any concentration.

Figure 11. Figure 11A: Dithiol reductase activities of rECEF species. Values represent the means of triplicate determinations. A rECEF's were tested for the ability to reduce intrachain disulfide bonds of insulin in the presence of suboptimal concentrations of dithiothreitol (DTT) over a concentration range from 4 μM to 1.28 nM. Values are shown for the 80 minute time point. Figure 1 IB: The kinetics of the reaction are shown for the indicated materials. Note that DTT alone has a certain effect; rECEF-104 accelerates the rate of the reaction. When plates were allowed to stand for )5 hours, all values attained the same maximum. Figure 12. Figure 12A: Co-precipitation of 16, 18, and 94 kDa species from radiolabeled, PMA-stimulated U937 cells with ECEF NH₂- terminal-specific antibody (SDS-PAGE analysis and fluorography). Molecular weight markers are shown at the left; the remaining lanes show the radiolabeled product precipitated by normal rabbit serum, anti-KLH serum, anti-whole 10 kDa ECEF, and different samples of anti-ECEF NH₂~ terminal. Figure 12B: A longer exposure of the same gel, so that the lower molecular weight species become visible. The 16, 18, and 94 kDa ECEF-associated proteins as well as 10 kDa ECEF are indicated by arrows.

Figure 13. Apparent crosslinking of radiolabeled U937 cell proteins with exogenous rECEF's. PMA-stimulated U937 cells were radiolabeled with ³⁵S-methionine/cysteine, washed in cold PBS, and incubated on ice with or without rECEF's for 4 hours. After this period, cells were washed again and treated with DSS to crosslink neighboring cell-associated proteins. After quenching of the crosslinking reaction and further washing, cell lysates were analyzed by immunoprecipitation, SDS-PAGE, and fluorography. The following conditions were studied: rECEF-84 followed by DSS (lane A), no exogenous cytokine DSS (lane B), and rECEF- 104/DSS (lane C). Please note that there is no treatment-dependent shift in the migration of the 94 kDa species. Note also the new species (arrows) that appear with DSS treatment. The difference in Mr of these new species between the rECEF-84- and rECEF-104-treated samples shows that the rECEF's are crosslinked to a low molecular weight cellular protein. Figure 14. SDS-PAGE analysis of metabolicaly radiolabeled rECEF-104 and rECEF-84. Both species were cleaved from maltose binding protein (MBP), which is also present in each preparation.

Figure 15. Sensitivity of Cell Lines to GSH. In this example, the viability of cells was evaluated, based on the ability to synthesize DNA. 3 x 10⁴ cells were cultured in a volume of 200 μl in RPMI 1640, supplemented with 2% fetal bovine serum, 2 mM glutamine, 100 U/ml penicillin, and 100 μg/ml streptomycin. The indicated concentrations of glutathione (GSH) were added along with 1 μCi of ³H-thymidine per sample. After 16 hours, the cultures were harvested and the incorporation of radiolabel into DNA was measured. Note that the U937ad+ cell line was the most sensitive to GSH, with complete inhibition of DNA synthesis at 125 μM GSH. The other myeloid tumor cell lines were also sensitive. DNA synthesis by COS cells was unaffected.

Figure 16. Selective toxicity of the CDM8-ECEF plasmid. In this example, all cells were transfected with the CDM8 plasmid containing the human growth hormone (HGH) gene. The viability of the cells was evaluated, based on the ability to synthesize HGH from this co-transfected plasmid. 1.4 x 10⁷ cells in a volume of 0.5 ml were transfected with the indicated amount of CDM8-ECEF DNA by electroporation. The cells were then cultured in Iscove's modified Dulbecco's medium, with 10% fetal bovine serum with glutamine and antibiotics, and the samples were taken for the RIA at 22 hours. Note that as little as one μg of DNA inhibited >75% of HGH synthesis by U937 cells, but that as much as 20 μg had little effect on COS cells.

DEFINITIONS

In the description that follows, a number of terms used in recombinant DNA (rDNA) technology are extensively utilized. In order to provide a clear and consistent understanding of the specification and claims, including the scope to be given such terms, the following definitions are provided. Gene. A DNA sequence containing a template for a RNA polymerase. The RNA transcribed from a gene may or may not code for a protein. RNA that codes for a protein is termed messenger RNA (mRNA) and, in eukaryotes, is transcribed by RNA polymerase II. However, a gene containing a RNA polymerase II template wherein a RNA sequence is transcribed which has a sequence complementary to that of a specific mRNA but is not normally translated may also be constructed. Such a gene construct is herein termed an "antisense RNA gene" and such a RNA transcript is termed an "antisense RNA." Antisense RNAs are not normally translatable due to the presence of translational stop codons in the antisense RNA sequence.

A "complementary DNA" or "cDNA" gene includes recombinant genes synthesized by reverse transcription of mRNA lacking intervening sequences (introns). Cloning vehicle. A plasmid or phage DNA or other DNA sequence which is able to replicate autonomously in a host cell, and which is characterized by one or a small number of endonuclease recognition sites at which such DNA sequences may be cut in a determinable fashion without loss of an essential biological function of the vehicle, and into which DNA may be spliced in order to bring about its replication and cloning. The cloning vehicle may further contain a marker suitable for use in the identification of cells transformed with the cloning vehicle. Markers, for example, are tetracycline resistance or ampicillin resistance. The word "vector" is sometimes used for "cloning vehicle." Expression vehicle. A vehicle or vector similar to a cloning vehicle but which is capable of expressing a gene which has been cloned into it, after transformation into a host. The cloned gene is usually placed under the control of (i.e., operably linked to) certain control sequences such as promoter sequences. Expression control sequences will vary depending on whether the vector is designed to express the operably linked gene in a prokaryotic or eukaryotic host and may additionally contain transcriptional elements such as enhancer elements, termination sequences, tissue- specificity elements, and/or translational initiation and termination sites. The present invention pertains both to expression of recombinant ECEF protein, and to the functional derivatives of this protein. Functional Derivative. A "functional derivative" of a ECEF sequence, either protein or nucleic acid, is a molecule that possesses a biological activity (either functional or structural) that is substantially similar to a biological activity of non-recombinant ECEF protein or nucleic acid. A functional derivative of ECEF protein may or may not contain post-translational modifications such as covalently linked carbohydrate, depending on the necessity of such modifications for the performance of a specific function. The term "functional derivative" is intended to include the "fragments," "variants," "analogues," or "chemical derivatives" of a molecule.

As used herein, a molecule is said to be a "chemical derivative" of another molecule when it contains additional chemical moieties not normally a part of the molecule. Such moieties may improve the molecule's solubility, absorption, biological half life, etc. The moieties may alternatively decrease the toxicity of the molecule, eliminate or attenuate any undesirable side effect of the molecule, etc. Moieties capable of mediating such effects are disclosed in Remington 's Pharmaceutical Sciences (1980). Procedures for coupling such moieties to a molecule are well known in the art. Fragment. A "fragment" of a molecule such as ECEF protein or nucleic acid is meant to refer to any portion or the native ECEF amino acid or nucleotide genetic sequence.

Variant. A "variant" of ECEF protein or nucleic acid is meant to refer to a molecule substantially similar in structure and biological activity to either the native ECEF molecule, or to a fragment thereof. Thus, provided that two molecules possess a common activity and may substitute for each other, they are considered variants as that term is used herein even if the composition or secondary, tertiary, or quaternary structure of one of the molecules is not identical to that found in the other, or if the amino acid or nucleotide sequence is not identical. Analog. An "analog" of ECEF protein or genetic sequences is meant to refer to a protein or genetic sequence substantially similar in function to the ECEF protein or genetic sequence described herein. For example, analogs of the ECEF protein described herein include ECEF isozymes and analogs of the ECEF genetic sequences described herein include ECEF alleles.

Substantially Pure. A "substantially pure" ECEF protein is an ECEF protein preparation that is generally lacking in other cellular componants, and especially other non-ECEF proteins. "Substantially pure" ECEF DNA is an ECEF DNA preparation that is generally lacking in other cellular componants, and especially in other non-ECEF DNA.

DESCRIPTION OF THE PREFERRED EMBODIMENTS

I. The Cell Line and Deposit

The parent U937 line is a histiocytic lymphoma cell line that was derived originally from a patient in Sweden and is described in Sundstrom, C. et al, Int. J. Cancer 17:565 (1976). The parent line secretes the 14 kDa form of ECEF, but also secretes the 10 kDa form when it is stimulated with phorbol ester.

Under the theory that a more differentiated U937 cell might be more adherent to tissue culture plastic, a selection process was used in which the cells were cultured in low fetal calf serum (FCS, 1-5% at different times). When fresh medium was required, non-adherent cells were poured off, and the medium was added to the adherent cells in the same vessel. After multiple rounds of selection, a population of adherent cells with a more differentiated morphology emerged. These cells, designated U937ad⁺, released an ECEF activity without PMA-stimulus. Two additional clones of U937ad⁺ have been derived by two rounds of dilution cloning with a feeder population of the U937ad⁺ population on the other side of a semipermeable membrane (Costar, 6.5 mm Transwell). The phenotype of these clones has been stable for at least 5 subsequent passages.

The subclones of the invention, the U937ad⁺ cell line, secrete the 10 kDa form of ECEF constitutively, without phorbol ester stimulation. However, PMA stimulus is still necessary to transport ECEF species to the outer cell surface. The subclones of the invention have a more differentiated (adherent) appearance and are useful as sources of the natural 10 kDa ECEF, the molecules involved in processing the inactive form to the active form, molecules associated with the ECEF associated signal transduction mechanism, and other molecules present in macrophages.

Applicants' subclones of the U937 line are especially useful for the study and isolation of the ECEF and associated proteins of the invention. Applicants' cell line (in the unstimulated form) does not cease to reproduce, unlike the normal U937 cells. Applicants' cell line (U937ad⁺) was deposited at the American Type Culture Collection (ATCC), 12301 Parklawn Drive, Rockville, Maryland, USA on June 26, 1992 and assigned ATCC No. CRL 11080.

II. Cloning of ECEF Genetic Sequences

The process for genetically engineering ECEF protein sequences, according to the invention, is facilitated through the isolation and sequencing of pure ECEF protein and by the cloning of genetic sequences which are capable of encoding the ECEF protein and through the expression of such genetic sequences. As used herein, the term "genetic sequences" is intended to refer to a nucleic acid molecule (preferably DNA). Genetic sequences which are capable of encoding ECEF protein are derived from a variety of sources. These sources include genomic DNA, cDNA, synthetic DNA, and combinations thereof. The preferred source of the ECEF cDNA is a PMA-Stimulated U937 cell human cDNA library. The preferred source of the ECEF genomic DNA is any human cell genomic library.

The ECEF protein recombinant cDNA of the invention will not include naturally occurring introns if the cDNA was made using mature ECEF mRNA as a template. The ECEF protein genomic DNA of the invention may or may not include naturally occurring introns. Moreover, such genomic DNA may be obtained in association with the 5' promoter region of the ECEF protein gene sequences and/or with the 3' tran- scriptional termination region. Further, such genomic DNA may be obtained in association with the genetic sequences which encode the 5' non- translated region of the ECEF protein mRNA and/or with the genetic sequences which encode the 3' non-translated region. To the extent that a host cell can recognize the transcriptional and/or translational regulatory signals associated with the expression of the mRNA and protein, then the 5' and/or 3' non-transcribed regions of the native gene, and/or, the 5'. and/or 3' non-translated regions of the mRNA, may be retained and employed for transcriptional and translational regulation. ECEF protein genomic DNA can be extracted and purified from any cell which naturally expresses ECEF protein by means well known in the art (for example, see Guide to Molecular Cloning Techniques, S.L. Berger et al, eds., Academic Press (1987). Preferably, the mRNA preparation used will be enriched in mRNA coding for ECEF protein, either naturally, by isolation from cells which are producing large amounts of the protein, or in vitro, by techniques commonly used to enrich mRNA preparations for specific sequences, such as sucrose gradient centrifugation, or both. Cell types which are known to be enriched in ECEF protein and which are preferred as a source of ECEF mRNA include human monocytes or U937 cells.

For cloning into a vector, such suitable DNA preparations (either genomic DNA or cDNA) are randomly sheared or eπzymatically cleaved, respectively, and ligated into appropriate vectors to form a recombinant gene (either genomic or cDNA) library.

A DNA sequence encoding ECEF protein or its functional derivatives may be inserted into a DNA vector in accordance with conventional techniques, including blunt-ending or staggered-ending termini for ligation, restriction enzyme digestion to provide appropriate termini, filling in of cohesive ends as appropriate, alkaline phosphatase treatment to avoid undesirable joining, and ligation with appropriate ligases. Techniques for such manipulations are disclosed by Maniatis, T. , (Maniatis, T. et al , Molecular Cloning (A Laboratory Manual), Cold

Spring Harbor Laboratory, second edition, 1988) and are well known in the art.

Libraries containing sequences coding for ECEF may be screened and a sequence coding for ECEF identified by any means which specifically selects for a sequence coding for ECEF such as, for example, a) by hybridization with an appropriate nucleic acid probe(s) containing a sequence specific for the DNA of this protein, or b) by hybridization- selected translational analysis in which native mRNA which hybridizes to the clone in question is translated in vitro and the translation products are further characterized, or, c) if the cloned genetic sequences are themselves capable of expressing mRNA, by immunoprecipitation of a translated ECEF protein product produced by the host containing the clone.

Oligonucleotide probes specific for ECEF protein which can be used to identify clones to this protein can be designed from knowledge of the amino acid sequence of the ECEF protein. The sequence of amino acid residues in a peptide is designated herein either through the use of their commonly employed three-letter designations or by their single-letter designations. A listing of these three-letter and one-letter designations may be found in textbooks such as Biochemistry, Lehninger, A. , Worth Publishers, New York, NY (1970). When the amino acid sequence is listed horizontally, unless otherwise stated, the amino terminus is intended to be on the left end and the carboxy terminus is intended to be at the right end.

Because the genetic code is degenerate, more than one codon may be used to encode a particular amino acid (Watson, J.D., In: Molecular Biology of the Gene, 3rd Ed., W.A. Benjamin, Inc., Menlo Park, CA (1977), pp. 356-357). The peptide fragments are analyzed to identify sequences of amino acids which may be encoded by oligonucleotides having the lowest degree of degeneracy. This is preferably accomplished by identifying sequences that contain amino acids which are encoded by only a single codon.

Although occasionally an amino acid sequence may be encoded by only a single oligonucleotide sequence, frequently the amino acid sequence may be encoded by any of a set of similar oligonucleotides. Importantly, whereas all of the members of this set contain oligonucleotide sequences which are capable of encoding the same peptide fragment and, thus, poten¬ tially contain the same oligonucleotide sequence as the gene which encodes the peptide fragment, only one member of the set contains the nucleotide sequence that is identical to the exon coding sequence of the gene. Because this member is present within the set, and is capable of hybridizing to DNA even in the presence of the other members of the set, it is possible to employ the unfractionated set of oligonucleotides in the same manner in which one would employ a single oligonucleotide to clone the gene that encodes the peptide.

Using the genetic code (Watson, J.D., in: Molecular Biology of the Gene, 3rd Ed., W.A. Benjamin, Inc., Menlo Park, CA (1977)), one or more different oligonucleotides can be identified from the amino acid sequence, each of which would be capable of encoding ECEF. The probability that a particular oligonucleotide will, in fact, constitute an actual ECEF protein encoding sequence can be estimated by considering abnormal base pairing relationships and the frequency with which a particular codon is actually used (to encode a particular amino acid) in eukaryotic cells. Such "codon usage rules" are disclosed by Lathe, R. , et al, J. Molec. Biol. 183: 1-12 (1985). Using the "codon usage rules" of Lathe, a single oligonucleotide sequence, or a set of oligonucleotide sequences, that contain a theoretical "most probable" nucleotide sequence capable of encoding the ECEF protein sequences is identified.

The suitable oligonucleotide, or set of oligonucleotides, which is capable of encoding a fragment of a ECEF gene (or which is complementary to such an oligonucleotide, or set of oligonucleotides) may be synthesized by means well known in the art (see, for example, Synthesis and Application of DNA and RNA, S.A. Narang, ed., 1987, Academic Press, San Diego, CA) and employed as a probe to identify and isolate a cloned ECEF gene by techniques known in the art. Techniques of nucleic acid hybridization and clone identification are disclosed by Maniatis, T., et al. , in: Molecular Cloning, A Laboratory Manual, Cold Spring Harbor Laboratories, Cold Spring Harbor, NY (1982)), and by Hames, B.D., et al, in: Nucleic Acid Hybridization, A Practical Approach, IRL Press, Washington, DC (1985)), which references are herein incorporated by reference. Those members of the above-described gene library which are found to be capable of such hybridization are then analyzed to determine the extent and nature of ECEF encoding sequences which they contain.

To facilitate the detection of a desired ECEF protein DNA encoding sequence, the above-described DNA probe is labeled with a detectable group. Such detectable group can be any material having a detectable physical or chemical property. Such materials have been well -developed in the field of nucleic acid hybridization and in general most any label useful in such methods can be applied to the present invention. Particularly useful are radioactive labels, such as ³²P, ³H, ¹⁴C, ³⁵S, ¹²⁵I, or the like. Any radioactive label may be employed which provides for an adequate signal and has a sufficient half-life. If single stranded, the oligonucleotide may be radioactively labelled using kinase reactions. Alternatively, polynucleotides are also useful as nucleic acid hybridization probes when labeled with a non-radioactive marker such as biotin, an enzyme or a fluorescent group. See, for example, Leary, J.J. et al, Proc. Natl. Acad. Sci. USA 80:4045 (1983); Renz, M. et al, Nucl Acids Res. 12:3435 (1984); and Renz, M., EMBO J. 5:817 (1983). Thus, in summary, the actual identification of ECEF protein sequences permits the identification of a theoretical "most probable" DNA sequence, or a set of such sequences, capable of encoding such a peptide. By constructing an oligonucleotide complementary to this theoretical sequence (or by constructing a set of oligonucleotides complementary to the set of "most probable" oligonucleotides), one obtains a DNA molecule (or set of DNA molecules), capable of functioning as a probe(s) for the identification and isolation of clones containing a ECEF gene.

In an alternative way of cloning a ECEF gene, a library is prepared using an expression vector, by cloning DNA or, more preferably cDNA prepared from a cell capable of expressing ECEF protein into an expression vector. The library is then screened for members which express ECEF. protein, for example, by screening the library with antibodies to the protein.

The above discussed methods are, therefore, capable of identifying genetic sequences which are capable of encoding ECEF protein or fragments of this protein. In order to further characterize such genetic sequences, and, in order to produce the recombinant protein, it is desirable to express the proteins which these sequences encode. Such expression identifies those clones which express proteins possessing characteristics of ECEF protein. Such characteristics may include the ability to specifically bind ECEF protein antibody, the ability to elicit the production of antibody which are capable of binding to ECEF protein, the ability to provide ECEF protein enzymatic activity to a cell, and the ability to provide a ECEF protein-function to a recipient cell, among others. III. Expression of ECEF Protein and its Functional Derivatives

To express ECEF protein and/or its active derivatives, transcriptional and translational signals recognizable by an appropriate host are necessary. The cloned ECEF protein encoding sequences, obtained through the methods described above, and preferably in a double-stranded form, may be operably linked to sequences controlling transcriptional expression in an expression vector, and introduced into a host cell, either prokaryote or eukaryote, to produce recombinant ECEF protein or a functional derivative thereof. Depending upon which strand of the ECEF protein encoding sequence is operably linked to the sequences controlling transcriptional expression, it is also possible to express ECEF protein antisense RNA or a functional derivative thereof. rECEF species may be generated as fusion proteins, for example, with E. coli maltose-binding protein partner and an intervening Factor Xa cleavage site. The construction should be such that cleavage of the fusion protein, for example at the end of the Factor Xa sequence, generates the ECEF NH₂-terminal, without any additional amino acids. DNA species may be prepared for insertion into recombinant vectors, such as the pMAL- c expression vector (New England Biolabs, Beverly, MA; methods were according to the manufacturer's instructions) by polymerase chain reaction using purified ECEF cDNA for a template. The primers should be designed to incorporate an Eco RI site adjacent to the code for the ECEF NH₂-terminal valine, the coding region of the desired length, a stop codon, and an Xbal site. The resulting PCR fragments may then be digested with EcoRl and Xbal ligated into a vector, such as pMAL-c, and used to transform a host cell, preferably, TB1 cells.

Initially, colonies may be selected for production of an IPTG- induced fusion protein of the appropriate size. Subsequently, the production of the desired rECEF structure may be confirmed by DNA -- ").

sequence analysis. The fusion protein may be purified by techniques known in the art. For example, a factor Xa fusion protein may be purified by amylose resin affinity chromatography, after which it may be cleaved cleaved with Factor Xa. Analysis of the cleaved rECEF polypeptides may be performed to show that they are of the expected size. Under non- reducing conditions, rECEF species mirgate as homodimers in SDS-PAGE. This may be expected since the monomer has 5 cysteines. It is not necessary to separate the free rECEF species from the maltose-binding protein, as they migrate together using either size exclusion or anion exchange HPLC, with rECEF and maltose-binding protein co-eluting in all fractions. When rECEF species are separated by preparative SDS-PAGE with electroelution, the polypeptides tend to precipitate from solution in the absence of detergent. This does not occur when the rECEF species were left in the presence of the maltose-binding protein. Therefore, preparations of cleaved, but unseparated fusion proteins may be used for most experiments, with appropriate controls for possible effects of the maltose- binding protein.

Expression of the ECEF protein in different hosts may result in different post-translational modifications which may alter the properties of the protein. Preferably, the present invention encompasses the expression of the ECEF protein or a functional derivative thereof, in eukaryotic cells, and especially mammalian, insect and yeast cells. Especially preferred eukaryotic hosts are mammalian cells either in vivo, or in tissue culture. Mammalian cells provide post-translational modifications to recombinant ECEF protein which include folding at sites similar or identical to that found for the native protein-

A nucleic acid molecule, such as DNA, is said to be "capable of expressing" a polypeptide if it contains expression control sequences which contain transcriptional regulatory information and such sequences are "operably linked" to the nucleotide sequence which encodes the polypeptide- An operable linkage is a linkage in which a sequence is connected to a regulatory sequence (or sequences) in such a way as to place expression of the sequence under the influence or control of the regulatory sequence. Two DNA sequences (such as a ECEF protein encoding sequence and a promoter region sequence linked to the 5' end of the encoding sequence) are said to be operably linked if induction of promoter function results in the transcription of the ECEF protein encoding sequence mRNA and if the nature of the linkage between the two DNA sequences does not (1) result in the introduction of a frame-shift mutation, (2) interfere with the ability of the expression regulatory sequences to direct the expression of the ECEF protein, antisense RNA, or protein, or (3) interfere with the ability of the ECEF protein template to be transcribed by the promoter region sequence. Thus, a promoter region would be operably linked to a DNA sequence if the promoter were capable of effecting transcription of that DNA sequence. The precise nature of the regulatory regions needed for gene expression may vary between species or cell types, but shall in general include, as necessary, 5' non-transcribing and 5' non-translating (non- coding) sequences involved with initiation of transcription and translation respectively, such as the TATA box, capping sequence, CAAT sequence, and the like. Especially, such 5' non-transcribing control sequences will include a region which contains a promoter for transcriptional control of the operably linked gene. Such transcriptional control sequences may also include enhancer sequences or upstream activator sequences, as desired.

Expression of the ECEF protein in eukaryotic hosts requires the use of regulatory regions functional in such hosts, and preferably eukaryotic regulatory systems. A wide variety of transcriptional and translational regulatory sequences can be employed, depending upon the nature of the eukaryotic host. The transcriptional and translational regulatory signals can also be derived from the genomic sequences of viruses which infect eukaryotic cells, such as adenovirus, bovine papilloma virus, Simian virus, herpes virus, or the like. Preferably, these regulatory signals are associated with a particular gene which is capable of a high level of expres¬ sion in the host cell.

In eukaryotes, where transcription is not linked to translation, such control regions may or may not provide an initiator methionine (AUG) codon, depending on whether the cloned sequence contains such a methionine. Such regions will, in general, include a promoter region sufficient to direct the initiation of RNA synthesis in the host cell. Promoters from heterologous mammalian genes which encode a mRNA product capable of translation are preferred, and especially, strong promoters such as the promoter for actin, collagen, myosin, etc., can be employed provided they also function as promoters in the host cell. Examples of eukaryotic promoters include the promoter of the mouse metallothionein I gene (Hamer, D., et al, J. Mol. Appl Gen. 7:273-288 (1982)); the TK promoter of Herpes virus (McKnight, S., Cell 31:355-365 (1982)); the SV40 early promoter (Benoist, C, et al, Nature (London) 290:304-310 (1981)); in yeast, the yeast gal4 gene promoter (Johnston, S.A., et al, Proc. Natl. Acad. Sci. (USA) 79:6971-6975 (1982); Silver, P. A., et al, Proc. Natl. Acad. Sci. (USA) 81:5951-5955 (1984)) or a glycolytic gene promoter may be used. As is widely known, translation of eukaryotic mRNA is initiated at the codon which encodes the first methionine. For this reason, it is preferable to ensure that the linkage between a promoter and a DNA sequence which encodes the ECEF protein, or a functional derivative thereof, does not contain any intervening codons which are capable of encoding a methionine. The presence of such codons results either in a formation of a fusion protein (if the AUG codon is in the same reading frame as ECEF protein encoding DNA sequence) or a frame-shift mutation (if the AUG codon is not in the same reading frame as the ECEF protein encoding sequence). If desired, a fusion product of the ECEF protein with a signal sequence for secretion from a host cell may be constructed. For example. the sequence coding for ECEF protein may be linked to a signal sequence which will allow secretion of the protein from, or the compartmentalization of the protein in, a particular host. Such signal sequences may be designed with or without specific protease sites such that the signal peptide sequence is amenable to subsequent removal. Alternatively, the native signal sequence may be used if that form of ECEF possesses such a sequence. Transcriptional initiation regulatory signals can be selected which allow for repression or activation, so that expression of the operably linked genes can be modulated. Of interest are regulatory signals which are temperature-sensitive so that by varying the temperature, expression can be repressed or initiated, or are subject to chemical regulation, e.g., metabolite. Also of interest are constructs wherein both the ECEF protein mRNA and antisense RNA are provided in a transcribable form but with different promoters or other transcriptional regulatory elements such that induction of ECEF protein mRNA expression is accompanied by repression of antisense RNA expression, and/or, repression of ECEF protein mRNA expression is accompanied by induction of antisense RNA expression. Translational signals are not necessary when it is desired to express ECEF protein antisense RNA sequences. If desired, the non-transcribed and/or non-translated regions 3' to the sequence coding for ECEF protein can be obtained by the above- described cloning methods. The 3 '-non-transcribed region may be retained for its transcriptional termination regulatory sequence, elements; the 3-non- translated region may be retained for its translational termination regulatory sequence elements, or for those elements which direct polyadenylation in eukaryotic cells. Where the native expression control sequences signals do not function satisfactorily host cell, then sequences functional in the host cell may be substituted.

The vectors of the invention may further comprise other operably linked regulatory elements such as DNA elements which confer tissue or cell-type specific expression on an operably linked gene. To transform a host cell with the DNA constructs of the invention many vector systems are available depending upon whether it is desired to insert the ECEF protein DNA construct into the host cell chromosomal DNA, or to allow it to exist in an extrachromosomal form. If the ECEF protein encoding sequence and an operably linked promoter is introduced into a recipient cell as a non-replicating DNA (or RNA) molecule, the expression of the ECEF protein may occur through the transient expression of the introduced sequence. Such a non-replicating DNA (or RNA) molecule may be a linear molecule or, more preferably, a closed covalent circular molecule which is incapable of autonomous replica¬ tion.

In a preferred embodiment, genetically stable transformants may be constructed with vector systems, or transformation systems, whereby ECEF protein DNA is integrated into a stable plasmid or into the host chromosome. Chromosomal integration may occur de novo within the cell or, in a most preferred embodiment, be assisted by transformation with a vector which functionally inserts itself into the host chromosome, for example, with retroviral vectors, transposons or other DNA elements which promote integration of DNA sequences in chromosomes. A vector is employed which is capable of integrating the desired gene sequences into a mammalian host cell chromosome.

Cells which have stably integrated the introduced DNA into their chromosomes are selected by also introducing one or more markers which allow for selection of host cells which contain the expression vector in the chromosome, for example the marker may provide biocide resistance, e.g., resistance to antibiotics, or heavy metals, such as copper, or the like. The selectable marker gene can either be directly linked to the DNA gene sequences to be expressed, or introduced into the same cell by co- transfection. In another embodiment, the introduced sequence is incorporated into a plasmid or viral vector capable of autonomous replication in the recipient host. Any of a wide variety of vectors may be employed for this purpose, as outlined below.

Factors of importance in selecting a particular plasmid or viral vector include: the ease with which recipient cells that contain the vector may be recognized and selected from those recipient cells which do not contain the vector; the number of copies of the vector which are desired in a particular host; and whether it is desirable to be able to "shuttle" the vector between host cells of different species.

Preferred eukaryotic plasmids include those derived from the bovine papilloma virus, vaccinia virus, SV40, and, in yeast, plasmids containing the 2-micron circle, etc., or their derivatives. Such plasmids are well known in the art (Botstein, D., et al , Miami Wntr. Symp. 79:265-274 (1982); Broach, J.R., in: The Molecular Biology of the Yeast Sac- charomyces: Life Cycle and Inheritance, Cold Spring Harbor Laboratory, Cold Spring Harbor, NY, p. 445-470 (1981); Broach, J.R., Cell 28:203- 204 (1982); Bollon, D.P., et al , J. Clin. Hematol. Oncol 70:39-48 (1980); Maniatis, T., In: Cell Biology: A Comprehensive Treatise, Vol. 3, Gene Expression, Academic Press, NY, pp. 563-608 (1980)), and are commercially available. For example, mammalian expression vector systems which utilize the MSV-LTR promoter to drive expression of the cloned gene, and in which it is possible to cotransfect with a helper virus to amplify plasmid copy number, and, integrate the plasmid into the chromosomes of host cells have been described (Perkins, A.S. et al. , Mol Cell Biol. 3:1123 (1983); Clontech, Palo Alto, California). Once the vector or DNA sequence containing the construct(s) is prepared for expression, the DNA construct(s) is introduced into an appropriate host cell by any of a variety of suitable means, including transfection. After the introduction of the vector, recipient cells are grown in a selective medium, which selects for the growth of vector-containing cells. Expression of the cloned gene sequence(s) results in the production of the ECEF protein, or in the production of a fragment of this protein. This expression can take place in a continuous manner in the transformed cells, or in a controlled manner, for example, expression which follows induction of differentiation of the transformed cells (for example, by administration of bromodeoxyuracil to neuroblastoma cells or the like). The expressed protein is isolated and purified in accordance with conventional conditions, such as extraction, precipitation, chromatography, affinity chromatography, electrophoresis, or the like.

The ECEF protein DNA encoding sequences, obtained through the methods above, will provide sequences which, by definition, encode ECEF protein and which may then be used to obtain ECEF protein antisense RNA genetic sequences as the antisense RNA sequence will be that sequence found on the opposite, complementary strand of the strand transcribing the protein's mRNA. An expression vector may be constructed which contains a DNA sequence operably linked to a promoter wherein such DNA sequence expresses the ECEF antisense RNA sequence. Transformation with this vector results in a host capable of expression of a ECEF antisense RNA in the transformed cell. Preferably such expression occurs in a regulated manner wherein it may be induced and/or repressed as desired. Most preferably, when -expressed, antisense ECEF RNA interacts with an endogenous ECEF DNA or RNA in a manner which inhibits or represses transcription and/or translation of the ECEF protein gene and/or mRNA in a highly specific manner. Use of antisense RNA probes to block gene expression is discussed in Lichtenstein, C, Nature 333:801-802 (1988).

IV. Identification of ECEF Derivatives

ECEF-specϊfic antibody may be used to immunoprecipitate metabolically radiolabeled products of U937 cells and, thus, study the synthesis of ECEF, we found that unstimulated U937 cells synthesize a more abundant 14 kDa cross-reacting polypeptide. Stimulus with PMA induced the synthesis of a 10 kDa species (equivalent in size to what had been purified). Limited amino acid sequence data suggest that these two species share the same N-terminal amino acid sequence (Balcewicz- Sablinska et al, J. Immunol 147:2170-2174) (1991)).

The 14 kDa species is detected predominantly in the culture supernatant. In contrast, the 10 kDa species is detected predominantly in the washed cell pellet, within approximately 6 hours after PMA stimulus. Three methods demonstrated that cell-associated 10 (and some 14) kDa ECEF is almost entirely on the outer cell surface: (1) Radioiodination with SHPP (sulfosuccinimidyl-3-[4-hydroxyphenyl] propionate — this is a water soluble, membrane impermeable derivative of Bolton-Hunter reagent that can be iodinated by the chloramine T reaction) permitted detection of immunoprecipitated ECEF series from washed, detergent lysed cells, unless the outer surface of cells was first blocked with cold SHPP. This method of labeling, which is directed at primary amines, was used instead of the more common methods that are aimed at tyrosines, because the amino acid composition analysis bf 10 kDa ECEF showed little or no tyrosine, and gene sequence of 14 kDa ECEF shows only one tyrosine. (2) Brief treatment with trypsin removed essentially all metabolically labeled ECEF from washed cells. (3) It was detected by specific antibody using FACS analysis (Balcewicz-Sablinska et al, J. Immunol. 147:2170-2174 (1991)). ³⁵S-methionine/cysteine labeled 14 and 10 kDa ECEF species were purified by immunoprecipitation, SDS-PAGE, and electroelution. The isolated species were set at equal concentrations (equal ³⁵S counts) and titrated for activity in the assay of eosinophil cytotoxic function. The 10 kDa species stimulated cytotoxicity to a higher maximum and was half- maximally active at >20 times lower concentrations (Balcewicz-Sablinska et al, J. Immunol. 147:2170-2174 (1991)). This finding explains why the sequence of purification methodologies and bioassays utilized herein lead to the 10 kDa species, rather than the more abundant 14 kDa species. It may also explain why other groups have had difficulty demonstrating activity with recombinant 14 kDa ECEF/thioredoxin (see below). Taken collectively, this evidence suggests that the 10 kDa form of the molecule is the active form, and the 14 kDa species is the inactive form. Thus, ECEF/thioredoxin may be like a number of mediators (e.g. , insulin, IL-1) that are activated by proteolytic cleavage of an inactive precursor. It is also possible that the 10 kDa species is an alternative gene product, however there is no evidence for either of these possibilities. As shown herein, a recombinant ECEF polypeptide truncated at amino acid 84 has enhanced activity, equivalent to that of the 10 kDa natural material.

The recombinant 14 kDa molecule has dithiol reductase activity (Wollman et al, J. Biol. Chem. 253: 15506 (1988)). Its activity is increased (although still low) when jS-mercaptoethanol (2-ME) is added to the medium as a donor of reducing equivalents (perhaps this explains the old, observation that addition of 2-ME to culture medium promotes the growth of certain cell types) (Tagaya et al , EMBO J. 8:757 (1989)). Mutation of the Cys at position 31 (in the active site) destroys activity (Kawabe, T. et al., FASEB J. 5.-A1330 (1991)). Cleavage of the disulfide bonds with 2- ME does not destroy the activity of ECEF (Balcewicz-Sablinska et al , J. Immunol. 147:2170-2174 (1991)), but alkylation with iodoacetamide does. The gonadotrophic hormones lutropin and follitropin have a related sequence, CGKC, and thioredoxin-like catalytic activity (Boniface, J.J. et al., Science 247:61 (1990)), suggesting that this mode of signalling may be shared by a number of mediators. However, as shown herein, the hypothesis, such thioredoxin-like catalytic may surprisingly be removed from the shortened ECEF forms of the invention without destroying the ability of such shortened ECEF forms to enhance eosinophil cytotoxicity. The native ECEF has 104 amino acids. Accordingly to the invention, shortened ECEF forms may be prepared that terminate at (including) any amino acid from amino acid 79 (ECEF-79) to amino acid 103 (ECEF-103). According to the invention, shorter ECEF forms may be prepared that retain the ability to stimulate eosinophil cytotoxicity.

Preferably, the shortened ECEF forms of the invention possess deletions of the ECEF COOH-terminal such as, for example, ECEF-79, ECEF-80, ECEF-81 , ECEF-82, ECEF-83, ECEF-84, ECEF-85, ECEF-86, ECEF-87, ECEF-88, ECEF-89, ECEF-90, ECEF-91 , ECEF-92, ECEF-93, ECEF-94, ECEF-95, ECEF-96, ECEF-97, ECEF-98, ECEF-99, ECEF-100, ECEF- 101, ECEF-102, and ECEF-103, consisting of amino acids 1-79, 1-80, 1- 81, 1-82, 1-83, 1-84, 1-85, 1-86, 1-87, 1-88, 1-89, 1-90, 1-91, 1-92, 1-93, 1-94, 1-95, 1-96, 1-97, 1-98, 1-99, 1-100, 1-101, 1-102, 1-103, respectively. Preferably, the shortened ECEF forms of the invention retain eosinophil-stimulating activity but lack the proinflammatory dithiol reductase activity. It is not necessary to retain 100% of the eosinophil- stimulating activity of the native ECEF peptide for a shortened form of the invention to be useful.

V. Identification of ECEF-Associated Proteins

As demonstrated herein, ECEF forms are associated with 16, 18 and 9- kDa molecules in the U937 cell (a cell that both produces and responds to ECEF's) and are thus likely to be involved in the functions of ECEF's. Crosslinking methods may be used to identify such ECEF- associated species. For example, crosslinking demonstrates that certain cellular proteins, probably the 16 and/or 18 kDa species, interact with exogenous ECEF's. Proteins that interact with exogenous ECEF's are candidates as receptor/signal transducer molecules for the ECEF cytokine. The 16, 18, and 94 kDa ECEF-associated proteins may be isolated from U937 cells using techniques known in the art, and specifically affinity chromatography. The presence of such ECEF-associated proteins could also be detected with ECEF-associated protein-specific monoclonal antibodies, especially in human eosinophils which respond to but do not produce ECEF's.

VI. Therapeutic Uses of the Proteins of the Invention The shortened ECEF forms of the invention, and the proteins that associate therewith, are useful in a variety of therapeutic methods. For example, such uses would include any therapy wherein it is desirable to administer to a patient a composition that possesses one of the biological activities of ECEF, including differentiation, stimulation or functional suppression of leukocytes, hemopoietic precursors or tumor cells. For example, such activities would include eosinophil cytotoxicity-stimulating activity, the proliferation of thymocytes and fibroblasts, induced expression of IL-2R from T cells, IL-l-like activity for support of lymphocyte cell growth, induced expression of IL-2 receptors, growth inhibition of transformed cells, induction of NKKB expression in lymphocytes, and protection of ECEF-producing cells from the toxic effects of TNF. Recombinant production of the shortened ECEF forms of the invention provide a cost-effective source for the protein for such therapeutic compositions.

It may also be desired to provide a therapy that does not administer the ECEF of the invention, but rather blocks the active dithiol reductase group of a patient's endogenous ECEF. Any drug that is capable of blocking the active dithiol reductase group in 14 kDa ECEF (and other molecules that have similar dithiol reductase activities such as lutropin and foUitropin) may be useful in such treatment. The only part of the 14 kDa ECEF molecule that is similar to "classical thioredoxin" (that is, thioredoxin characterized from plants and bacteria) is the sequence of amino acids from positions 31 through 34. None of the remaining ECEF molecule is related to classical thioredoxin. In 14 kDa ECEF, at positions 31 and 34, there are cysteine residues. Each of the cysteines has a sulfur molecule that can exist as either as reduced sulfhydryl group or an oxidized disulfide. The exchange between these two forms will alter the function of the molecule. Therefore, drugs that modify either of the cysteines may be administered and used to block the proϊnflammatory dithiol reductase ECEF activity. VII. Construction and Identification of Antibodies to ECEF Protein

In the following description, reference will be made to various methodologies well-known to those skilled in the art of immunology. Standard reference works setting forth the general principles of immunology include the work of Catty, D. (Antibodies, A Practical Approach, Vol. 1, IRL Press, Washington, DC (1988)); Klein, J. (Immunology: The Science of Cell-Noncell Discrimination, John Wiley & Sons, New York (1982)); Kennett, R., et al in Monoclonal Antibodies, Hybridoma: A New Dimension in Biological Analyses, Plenum Press, New York (1980)); Campbell, A. ("Monoclonal Antibody Technology," in:

Laboratory Techniques in Biochemistry and Molecular Biology, Volume 13 (Burdon, R., et al , eds.), Elsevier, Amsterdam (1984)); and Eisen, H.N., in: Microbiology, 3rd Ed. (Davis, B.D., et al , Harper & Row, Philadelphia (1980)). An antibody is said to be "capable of binding" a molecule if it is capable of specifically reacting with the molecule to thereby bind the molecule to the antibody. The term "epitope" is meant to refer to that portion of a hapten which can be recognized and bound by an antibody. An antigen may have one, or more than one epitope. An "antigen" is capable of inducing an animal to produce antibody capable of binding to an epitope of that antigen. The specific reaction referred to above is meant to indicate that the antigen will react, in a highly selective manner, with its corresponding antibody and not with the multitude of other antibodies which may be evoked by other antigens. The term "antibody" (Ab) or "monoclonal antibody" (Mab) as used herein is meant to include intact molecules as well as fragments thereof (such as, for example, Fab and F(ab')₂ fragments) which are capable of binding an antigen. Fab and F(ab')₂ fragments lack the Fc fragment of intact antibody, clear more rapidly from the circulation, and may have less non-specific tissue binding of an intact antibody (Wahl et al , J. Nucl Med. 24:316-325 (1983)).

The antibodies of the present invention are prepared by any of a variety of methods. Preferably, purified ECEF protein, or a fragment thereof, is administered to an animal in order to induce the production of sera containing polyclonal antibodies that are capable of binding ECEF.

Cells expressing ECEF protein, or a fragment thereof, or, a mixture of proteins containing ECEF or such fragments, can also be administered to an animal in order to induce the production of sera containing polyclonal antibodies, some of which will be capable of binding ECEF protein. If desired, such ECEF antibody may be purified from the other polyclonal antibodies by standard protein purification techniques and especially by affinity chromatography with purified ECEF or fragments thereof.

A ECEF protein fragment may also be chemically synthesized and purified by HPLC to render it substantially free of contaminants. Such a preparation is then introduced into an animal in order to produce polyclonal antisera of high specific activity.

Monoclonal antibodies can be prepared using hybridoma technology (Kohler et al, Nature 256:495 (1975); Kohler et al., Eur. J. Immunol. 5:511 (1976); Kohler et al , Eur. J. Immunol. 6:292 (1976); Hammeriing et al., in: Monoclonal Antibodies and T-Cell Hybridomas, Elsevier, N.Y., pp. 563-681 (1981)). In general, such procedures involve immunizing an animal with ECEF protein antigen. The splenocytes of such animals are extracted and fused with a suitable myeloma cell line. Any suitable myeloma cell line may be employed in accordance with the present invention; however, it is preferable to employ the parent myeloma cell line (SP₂O), available from the American Type Culture Collection, Rockville, Maryland. After fusion, the resulting hybridoma cells are selectively maintained in HAT medium, and then cloned by limiting dilution as described by Wands, J.R., et al , Gastroenterology 80:225-232 (1981), which reference is herein incorporated by reference. The hybridoma cells obtained through such a selection are then assayed to identify clones which secrete antibodies capable of binding the ECEF protein antigen.

Through application of the above-described methods, additional cell lines capable of producing antibodies which recognize epitopes of the ECEF protein can be obtained.

Antibodies against both highly conserved and poorly conserved regions of the ECEF protein are useful for studies on the control of biosynthesis and catabolism of ECEF protein in normal and pathologic conditions. Further, these antibodies can be used clinically to monitor the progress of disease states wherein the expression of ECEF protein is aberrant.

The following examples further describe the materials and methods used in carrying out the invention. The examples are not intended to limit the invention in any manner.

EXAMPLES

Materials and Methods

Culture and radiolabeiing of U937 cells. U937 cells were grown to a density of 10⁶/ml as described previously (Siberstein et al , J. Immunol

138:3042 (1987)) (medium - RPMl 1640, antibiotics, glutamine, and 5% FCS) and stimulated as specified with 400 ng/ml PMA (Sigma Chemical Co., St.

Louis, MO) and/or 7.5 μg/ml of LPS (butanol extract of E. coli 0127:B8,

Sigma).

Cells to be radiolabeled biosynthetically with [³²⁵S] methionine/cysteine or [³H]glycine/serine/leucine were washed in serum-free RPMI and cultured for 2 h in RPMI without methionine/cysteine (or other amino acids as appropriate, Select-Amine Kit, GIBCO, Grand Island, NY).

Medium was then supplemented with 100 to 250 μCi/ml of Tran-³⁵S-Label (ICN Biomedicals, Irvine, CA) or a mixture of tritiated glycine, leucine, and serine (Amersham Corp. Arlington Heights, IL). After the period of culture, cells were separated from the conditioned medium by centrifugation at 400 x g for 10 minutes. When cells were to be treated with trypsin before analysis, they were washed in serum-free RPMI and then incubated for 7 minutes at 37°C with 0.05% trypsin. This was followed by 10% fetal calf serum (FCS) to inactivate the trypsin. ^a

For radioϊodination of cell surface polypeptides, U937 cells washed three times in ice cold PBS were treated with [¹²⁵I]SHPP (sulfosuccϊnimidyI-3- (4-hydroxyphenyl)propionate, prepared by chloramine T iodϊnation of SHPP), according to the method of Thompson et al. (Thompson et al. , Biochemistry 25:743 (1987)) in the presence of 100 μg/ml of PMSF. For some experiments, the U937 cells were first treated with unmodified SHPP to block reactive groups on the membrane surface. In parallel with these experiments, aliquots of U937 cells were lysed by three cycles of freezing and thawing, so that intracellular proteins were also iodinated.

FACS analysis. Aliquots containing 2 x 10⁶ unstimulated or PMA- stimulated U937 cells were suspended in ice cold HBSS/0.1 % sodium azide and treated for 30 minutes with 10% human AB⁺ serum. After washing three times in the same buffer, the cells were treated with 400 μg/ml of protein A- purified normal or ECEF-specific rabbit IgG (N-terminal-specificity, concentration known to be saturating). After 30 minutes and subsequent washing, the cells were treated with a saturation concentration of FITC- labeled F(ab')₂ fragments of goat anti-rabbit IgG (absorbed for minimal cross- reactivity with human serum proteins, Jackson ImmunoResearch, West Grove, PA). The cells were washed once more, resuspended in PBS containing 2% paraformaldehyde and analyzed by linear fluorescence amplification in an Ortho 50-H flow cytometer/2150 computer. The net mean channel number of fluorescence was calculated as the increase in mean channel fluorescence of cells treated with ECEF (N-terminal)-specific IgG over cells treated with an equal concentration of normal rabbit IgG. Immunoprecipitation of ECEF-related species. Supematants or cell pellets from 1 ml U937 cell cultures were brought to a volumn of 10 ml with immunoprecipitation/lysis buffer (0.04 M sodium phosphate, 0.5 M NaCl, 0.5% Tween 20, and 100 μg/ml of PMSF: pilot studies with various detergents and protease inhibitors had shown that this composition was optimal for our purposes). This was mixed with 3 μl of ECEF-(10 kDa) specific rabbit antiserum (Silberstein et al, J. Immunol. 143:979 (1989)) or control antiserum for 2 hours at 4°C, and then 40 μl of 50% protein A-Sepharose bead suspension (in PBS 10 mM NaHPO₄, pH 7.2, beads from Sigma) for 3 hours. The beads were pelleted by centrifugation and washed four times in the same buffer. A second antibody was raised by immunizing rabbits with a synthesized polypeptide equivalent to the first 20 amino acids of thioredoxin (TRX) (Wollman et al, J. Biol. Chem. 253: 15506 (1988)) conjugated to keyhole limpet hemocyanin (using kit and methods from Pierce Chemical Co., Rockford, IL).

SDS-PAGE analysis. This method was carried out using Laemmli system buffers (Laemmli, U.K., Nature 277:680 (1970)) and 15 or 20% polyacrylamide gels. Pelleted protein A-Sepharose beads from the immunoprecipitation step were added to sample buffer containing 10% β- mercaptoethanol (2-ME), boiled for 5 minutes, and centrifuged for 1 minute in high speed microfuge. The supernatant was analyzed by SDS-PAGE. ¹⁴C molecular weight (m.w.) markers (BRL, Bethesda, MD) were run in parallel. When the separated polypeptide species were to be visualized, gels run with [³⁵S], [¹⁴C], or [³H]radiolabel were fixed, soaked in Amplify (Amersham) with 5% glycerol, and dried; gels with [^,25I]radiolabel were fixed and dried. For visualization of protein species, dried gels were exposed to Kodak X- OMAT film with an intensifying screen at -70°C. When the separated products were to be recovered for analysis of biologic activity, gel slices were subjected to electroelution overnight at 3 W power, using a solution of 0.05 M ammonium bicarbonate and a dialysis membrane with a m.w. cutoff of 3500 (model 1750 electrophoretic concentrator and sample cups, 1SCO, Lincoln, NE). Eluted material was recovered in the 200 to 500 μl of solution closest to the membrane. When the separated species were to be recovered for amino acid sequence analysis, they were transferred electrophoretically, in the presence of pH 11. 0.01 M 3[cyclohexylamino]-l-propanesulfonic acid buffer with 10% methanol, to a polyvinylidene difluoride membrane (Immobilon-P, Millipore, Bedford, MA).

Polyvinylidene difluoride-bound material to be analyzed for amino acid sequence was stained with Coomassie Blue (10 minutes in 0.1 % Coomassie R-2150 in 50% methanol followed by destaining with 45% methanol/10% acetic acid) to permit precise excision of the polypeptide.

Amino acid sequence analysis. Amino acid sequence analysis was performed using an Applied Biosystems Model 475A Gas Phase Protein Sequencer (Applied Biosystems, Foster City, CA).

Eosinophil purification and assay of eosinophil cytotoxic function. These procedures were carried out as described previously (Siberstein et al , J. Immunol 138:3042 (1987); Silberstein et al, Proc. Natl Acad. Sci. USA 83:1055 (1986); Silberstein et al, J. Immunol. 143:979 (1989)), except for the use of pooled mAb to mediate eosinophil antibody-dependent cell-mediated cytotoxicity. Briefly, human eosinophils were purified from the blood of normal or mildly eosinophilic subjects by dextran sedimentation and discontinuous metrizamide gradient centrifugation (Vadas et al. , J. Immunol 722:1228 (1979)). Erythrocytes were removed by hypotonic lysis.

For cytotoxicity assays, IO⁵ purified eosinophils were cultured in round-bottomed microtiter wells in the presence of schistosomula-specific antibody (6.25% each of cell line E.1 , E.3, E.4 and M.4 culture supematants) (Siberstein et al, Hybridoma? 9:237 (1990)). 100 mechanically-transformed schistosomula (Ramalho-Pinto et al, Exp. Parasirol. 35:360 (1974)), and a control or test source of ECEF. All components were dialyzed, dissolved, or suspended in assay medium (minimal HEPES, 2 mM glutamine, 100 U/ml penicillin G, 10 μg/ml streptomycin, and 10% FCS, all from GIBCO). Samples of ECEF had no direct toxicity to schistosomula targets. rTRX. rTRX was produced in £. coli W3110cl and purified as described previously (Wollman et al, J. Biol Chem. 253:15506 (1988)).

RESULTS

EXAMPLE 1 Identification and in vivo synthesis of Native ECEF Species

Rabbit antiserum raised to purified 10-kDa ECEF (Siberstein et al. , J. Immunol. 138:3042 (1987)) precipitated specifically radiolabeled species of 14, 13, and 10 kDa from 48-hour conditioned medium or washed cell pellets of PMA/LPS-stimulated U937 cells. The relative concentrations of these species varied according to experimental conditions described below. Similar results were obtained with a rabbit antibody specific for the N-terminal 20 amino acids of rTRX This latter reagent also showed some specific reactivity with the 16 and 94 kDa species that associate with ECEF and with an 18-kDa species that has no ECEF activity). Other substances of higher m.w. were precipitated nonspecifically by both preimmune and ECEF-specific antiserum.

U937 cells cultured in medium alone released a 14-kDa immunoreactive polypeptide that was detectable as early as 3 hours and accumulated over 48 hours. When the cells were stimulated with 7.5 μg/ml of LPS, an additional immunoreactive species of 13 kDa was released within the first 6 hours of culture. Stimulation with 400 ng/ml of PMA induced the release of a 10-kDa species that was detectable in the conditioned medium by 48 hours. When the cells were stimulated with both LPS and PMA, all three species were released (Figure 1).

Limited amino acid sequence data suggest that 14-, 13-, and 10-kDa ECEF and rTRX have a common N-terminal (Figure 2). The sequence homology between the 14- and 10-kDa ECEF species (Figure 2) and earlier information that PMA treatment resulted in a great increase of ECEF activity (Siberstein et al, J. Immunol 138:3042 (1987)) raised questions about their relative activities in the in vitro assay of eosinophil cytotoxic function. These activities were evaluated by recovery of the immunoprecipitated radiolabeled forms from SDS gels and titratϊon of each species. Before titration, the two polypeptide preparations were adjusted to equal cpm/ml (in multiple experiments, whether the cells were labeled with [³⁵S] methionine/cysteine or of a pool of [³H]glycine/leucine/serine, the ratio of recovered 14 to 10 kDa was approximately 3:1). Mock-el uted material from the gel slice between these two species was diluted in the same manner as the 10-kDa preparation. The 14-kDa material exhibited weak but statistically significant eosinophil cytotoxicity-enhancing activity at the higher concentrations. However, the 10-kDa material enhanced eosinophil cytotoxic capabilities to a higher maximum and was active at _>_ 20-fold lower concentration (Figure 3A). rTRX also had weak, but statistically significant activity at concentrations _> 300 nM (Figure 3B).

Lysis and immunoprecipitation studies were performed on washed U937 cell pellets, to investigate the intracellular events during the 48-hour period between PMA stimulus and the extracellular appearance of 10-kDa ECEF. There was little or no synthesis of a 10-kDa ECEF species without PMA stimulus, however, substantial amounts of this product were detectable by 6 hours after PMA stimulus (Figure 4). The 14-kDa species was also detectable in cell pellets in most experiments.

Three approaches were used to determine whether cell-associated ECEF was located on the outer cell surface. The first of these involved FACS analysis to detect surface binding of control or ECEF-specific rabbit IgG. In the absence of PMA stimulation, no increase of fluorescence intensity with ECEF-specific antibody as compared to the control antibody. With PMA stimulation of the cells, there was a major increase in fluorescence intensity (net mean channel numbers of 94 and 131.7 in two experiments) indicating the expression of immunoreactive species on the cell surface. The second approach used the compound SHPP, a membrane impermeable, water soluble Bolton-Hunter reagent that attaches covalently to primary amines and can be iodinated. Thus, cell surface proteins were radiolabeled before lysis in detergent and analysis. Other aliquots of PMA- stimulated cells were blocked on the outer surface by treatment with unlabeled SHPP, or broken by freeze-thawing, or blocked and broken before radiolabeling. Immunoprecipitation and SDS-PAGE detected radiolabeled 14- and 10-kDa ECEF species for both surface labeled and broken cells. This labeling of ECEF was abolished or reduced when the cell surface was blocked before radiolabeling (Figure 5A). Analysis of total protein from [¹²⁵I]SHPP- labeled whole and broken cells showed strikingly different patterns, confirming the cell surface specificity of the SHPP-labeling reaction (data not shown).

For the third approach, [³⁵S]methionine/cysteine-labeled cells were exposed briefly to trypsin. This treatment removed biosynthetically labeled ECEF from the cells (Figure 5B), providing additional evidence that cell- associated ECEF is located primarily on the external surface of the plasma membrane.

A variety of pulse-chase experiments were performed using different conditions of stimulation, but no precursor-product relationship between any of the 14-, 13-, and 10-kDa species was found (data not shown). When treated with neuraminidase, N-glycanase, O-giycanase, or combinations of these enzymes, the m.w. of these polypeptides did not change, suggesting the absence of glycosylation (data not shown). The earlier finding that stimulus with PMA induced a major increase in U937 cell release of ECEF activity by 24 to 48 hours of culture (Siberstein et al, J. Immunol. 138:3042 (1987)) suggested that PMA-dependent expression of the 10-kDa ECEF polypeptide would be detected in that time frame. This proved to be the case, however, there were two unexpected findings: 1) without PMA stimulus, U937 cells synthesized immunoreactive polypeptides of 14 and 18 kDa (Figure 1 ), and 2) the 10-kDa species was actually synthesized as early as 3 to 6 hours, but remained primarily cell associated (Figure 4).

Only limited amino acid sequence information was determined for the isolated 14- and 13-kDa forms. There was agreement at positions 3, 4, and 6 between these sequences and those of 10-kDa ECEF and TRX. The only apparent disagreement was at position 1, where the lysine that was detected was probably from the second position (Figure 2).

The rabbit antiserum to ECEF had been generated by injection of an apparently homogeneous material (as judged by the appearance of a single 10- kDa band in silver- or gold-stained SDA-PAGE analysis and by the determination of a single N-terminal amino acid sequence (Silberstein et al. , J. Immunol. 143:979 (1989)). A second antiserum was specific for the N- terminal 20 amino acids of rTRX (see Materials and Methods). Reactivity with these reagents, therefore, provided one indication of a relationship between the 14-, 13-, and 10-kDa species. Further evidence of a relationship came from amino acid sequence data (Figure 2) and from the activity (albeit weak) of the 14-kDa species in the assay of eosinophil cytotoxic function (Figure 3). The precise nature of structural differences between these species is not known, however, the failure to detect a change in electrophoretic migration after treatment with various glycosidases shows that it is probably not due to variable glycosylation.

Two laboratories (Rimsky ft al.J. Immunol 735:3304 (1986); Tagaya et al, J. Immunol 140:2614 (1988)) have purified cytokines with IL-1-like activity, molecular masses of 13.5 or 14 kDa, and, as far as comparison was possible, identical N-terminals to the ECEF species studied here. In terms of biologic function, rTRX and 14-kDa ECEF both had weak activity (Figure 3). Taken together, this evidence suggests that TRX and 14 kDa (or possibly 13 kDa) ECEF are identical.

The recently reported work of Elsas et al. (Elsas et al , Eur. J. Immunol. 20:1143 (1990)) is also relevant to this question. This group prepared mAb that specifically removed ECEF activity from stimulated U937 cell supernatant. Iodination by the iodogen method and immunoprecipitation of affinity purified ECEF revealed several polypeptide species, including one (or perhaps two) species at 13 to 14 kDa and two larger species. Although this is in agreement with the results herein, regarding 13- to 14-kDa ECEF, these investigators did not detect a 10-kDa ECEF species. One possible reason for this is that the iodogen method does not work well for radioiodinating the 10-kDa species. It is known (Silberstein et al, J. Immunol 143:979 (1989)) that this substance has little or no tyrosine and we have not had success labeling the molecule with any of the methods that depend on tyrosine in the target molecule (D. Silberstein, unpublished results). There are no difficulties iodinating the molecule with Bolton-Hunter reagent or, as used here, its sulfated derivative SHPP.

Detection of ECEF by FACS analysis, the ability to modify ECEF chemically with a membrane impermeable reagent (Figure 5A), and the ability to cleave ECEF from intact cells with brief trypsin treatment (Figure 5B) show that the cell-associated forms of ECEF are primarily on the outer cell surface. Several mechanisms are known by which immunologically active molecules may be associated with the outer cell surface. These include lectin- Hke interactions as in the case of IL-1 (Brody et al. , J. Immunol. 743: 1183 (1989)), hydrophobic anchor sequences as in the case of membrane-bound IgM (Rogers et al , Cell 20:303 (1980)) or membrane-bound TNF before cleavage (Kriegler et al , Cell 53:45 (1988)), glycosylphosphatidylinositol ester linkages as in the example of Thy-1 (Low et al, Nature 318:62 (1985); Tso et al. , Science 230: 1003 (1985)), and occupancy of a cell surface membrane receptor as in the example of urokinase (Kirchheimer et al , Blood 74:1396 (1989)). The mechanism of association and release of ECEF species studied here are not known, however, the absence of a detectable m.w. difference between cell-associated and released forms argues against a proteolytic cleavage,. As with the immunologically active molecules mentioned above, the presence of ECEF on the outer cell surface suggests the possibility of cell to cell contact in the regulation of eosinophil function by ECEF.

As compared to 14-kDa ECEF or rTRX, a 10-kDa ECEF stimulated higher levels of eosinophil cytotoxic function and retained significant activity at > 20-fold lower doses. This observed greater activity of the 10-kDa ECEF species explains why, in earlier studies (Siberstein et al, J. Immunol. 138:3042 (1987)), PMA stimulus of U937 cells induced a major increase in release of ECEF-like activity. It also explains why our sequence of separations and bioassays led us to the 10-kDa species rather than the more abundant 14-kDa species. We did not attempt to electroelute and assay the activity of the 13-kDa species, because of concerns about resolving it from the 14-kDa species. Although the induction of the 13-kDa form by LPS may be physiologically important, bioassays of conditioned medium containing this material suggest that it does not have strong activity (Siberstein et al., J. Immunol 138:3042 (1987)).

In summary, 10-kDa ECEF differs from the other related species in the requirement of PMA stimulus for its induction (Figure 1), its greater activity with respect to eosinophil cytotoxic function (Figure 3), and its preferential retention in the cell (comparison of Figures I and 4). Although the molecular basis that underlies these differences is not known, the evidence suggests a complex mechanism for the regulation of eosinophil cytotoxic function by ECEF in vivo.

EXAMPLE 2 Molecular Cloning and Sequence Analysis of 14 kDa ECEF

For initial selection of clones, a commercially prepared (Clontech) cDNA library in phage λ gtlO from PMA-stimulated U937 cells was screened. The probe was generated by a variation on the anchored polymerase chain reaction, in which the primer were 1) a redundant oligonucleotide designed on the basis of the known N-terminal amino acid sequence and 2) phage λ sequences flanking the inserts (Frohman, M.A. et al , Proc. Natl. Acad. Sci. USA 85:8998 (1988)), and the template was total phage library DNA. The product of this PCR consisted of several species from about 300 to 700 base pairs. This mixture was radiolabeled and used to select 20 phage clones (from about 200 positives) for study.

At this time the sequence information for recombinant thioredoxin became available (Wollman et al , J. Biol Chem. 253:15506 (1988)), and similarities with ECEF were noticed. Screening of the library was continued in order to obtain the ECEF gene for expression of the cytokine, and in order to allow for the possibility that alternative cDNA encoding 10 kDa ECEF would be obtained. The coding region of that gene (prepared by PCR from the same total library DNA) was used for secondary screening of clones. This reagent detected 7 different positive subclones from the original 20 picks.

Sequence analysis was performed initially on templates prepared by transfer of the inserts into the phagemid Bluescript (Stratagene). This template was denatured and analyzed by the di-deoxy termination method. Recently, direct sequence analysis of PCR-generated fragments has also been used (Higuchi, R.G. et al , Nucl Acids Res. 77:5865 (1989), and Tung, J.S., et al, in PCR Technology, Henry A. Ehrlich, ed. Stockton Press, 1989, New York). For this procedure, primers complementary to the phage λ sequences flanking the inserts were used to amplify the inserts. Two reactions were performed in parallel, with one primer or the other phosphosphorylated at the 5' end by treatment with T4 polynucleotide kinase and ATP. After the PCR reaction, the product was treated with λ exonuclease to digest the kinased stand, leaving the opposite strand as a sequencing template. Both strands of the sequences were analyzed.

The sequences of 5 clones were determined and found to be identical in the coding region to the sequence reported by Tagaya, Y. et al. , EMBO J. 8:757 (1989) and Deiss, L.P. et al , Science 252: 117 (1991), with some differences in the 3' untranslated region (Figure 6) [SEQ ID No.: l :J. This did not rule out the existence of an alternative ECEF-related cDNA, but that possibility was not pursued further.

EXAMPLE 3 Production of rECEF species

14 kDa ECEF/thioredoxϊn is 104 amino acid polypeptide, with a calculated molecular weight of 12,744. A cleavage event at the COOH terminal yielding a 10 kDa polypeptide would occur in the vicinity of amino acid 82 (calculations based on Mr). Inspection of the sequence in this region revealed that amino acids 72 through 79 are uncharged, while the sequence of amino acids from positions 80 through 84 (KKGQK) is highly charged, containing three lysines. This contrast was reflected in the Hopp-Woods hydrophilicϊty plot (Figure 7). This lysine-rich region does not contain any of the recognized cleavage sequences employed in eukaryotic cells for the processing of pro-hormones or pro-cytokines. Most notably, it contains no arginines (see review, Barr, P.J., Cell 66:1-3 (1991)). Thus, there was no clear candidate for the position of the cleavage site, and it was decided to prepare several truncated recombinant ECEF polypeptides terminating in this region. Recombinant species terminating at (including) amino acid 79 (rECEF-79), amino acid 80 (rECEF-80) and amino acid 84 (rECEF-84) were prepared (Figure 8) . The sequence of rECEF-79 is the same as rECEF-80 but with the terminal AAG (lysine). The full-length 104 amino acid polypeptide (rECEF-104) was also prepared for comparison activities. cDNA encoding ECEF-104. cDNA clones encoding ECEF-104 (full- length) were isolated from a PMA-stimulated U937 λgtlO cDNA library (Clontech, Palo Alto, CA). The probe for screening of the library was generated by PCR, using sequence information from (Wollman et al . J. Biol Chem. 263:15506-15512 (1988)). The primers:

5'-GTGAAGCAGATCGAGAGCAAG-3' (+ strand NH2-terminal) [SEQ ID No.:9:] and 5'-GACTAATTCATTAATGGTGGC-3' (- strand, COOH-terminal) [SEQ ID No.:10:] were used to amplify a sequence from total amplified library phage DNA (0.5 μg), resulting in a single amplified species of the appropriate size (312 base pairs). This fragment was labeled with ³²P by random priming (kit from Boehringer Mannheim, Indianapolis, IN) and used to screen the same U937 cell cDNA library.

From a total of approximately 400,000 phage clones, approximately 200 were positive on initial screening. Six of these were selected for further study and subcloned. The DNA sequence of the inserts was determined, and for all six, the sequence of the coding region was identical to that described in (Tagaya et al, EMBO J. 8:757-764 (1989)). Phage DNA was purified by phenol/chloroform extraction of SDS/EDTA-treated, polyethylene glycol precipitated phage from plaque lysates (Lech et al. , in Current Protocols in Molecular Biology (Ausebel et al , eds.), John Wiley & Sons, New York, pp. 1.13.1-10 (1990)).

Production of rECEF species. DNA species were prepared for insertion in the p-MAL-c expression vector (New England Biolabs, Beverly, MA; except as specified, methods were according to the manufacturer's instructions) by polymerase chain reaction, using purified ECEF-104 cDNA clone as a template. The primers were designed to incorporate an EcoRl site, a factor Xa cleavage site adjacent to the code for the NH₂-terminal valine, the coding region of the desired length, a stop codon, and an Xbal site. Thus, the following oligonucleotide primers were employed:

(1) for all NH₂ terminals, [SEQ ID No.: 12:], 5'-GGGGAATTCGTGAAGCAGATCGAGAG-3';

(2) for the COOH terminal of rECEF-104, [SEQ ID No.: 13:], 5'-GGGTCTAGATTAGACTAATTCATTAATGGTGG-3';

(3) rECEF-84, [SEQ ID No.: 14:], 5'-GGGTCTAGACTACTTTTGTCCCTTCTTAAAAAAGTGC-3'; (4) rECEF-80, [SEQ ID No.: 15:],

5'-GGGTCTAGACTACTTAAAAAACTGGAATGTTGGCG-3' . The PCR fragments generated in the respective reactions were purified on SpinBind columns (FMC, Rockland, ME), digested with EcoKL and Xbal, purified a second time on SpinBind columns, and ligated to EcoRl/Xbal- digested pMAL-c plasmid. The constructs were used to transform TB1 cells. This permitted expression of a fusion protein consisting of E. coli maltose- binding protein, the factor Xa cleavage site, and the ECEF species, and several resulting colonies from each construct were selected for characterization. Initially, colonies were selected for production of an IPTG- induced fusion protein of the appropriate size. Subsequently, the production of the desired rECEF structure was confirmed by DNA sequence analysis of the plasmids and SDS-PAGE analysis of the factor Xa cleaved rECEF species. For certain control studies, a fusion protein consisting of a maltose- binding protein-paramyosin (Schistosoma mansonϊ) was obtained from New England Biolabs.

EXAMPLE 4

Eosinophil-stimulating activity of the Shortened rECEF species

In the assay of eosinophil cytotoxic function, full-length rECEF-104 had a slight, but statistically significant effect, at a concentration of 800 nM. This finding was consistent with earlier results, using a recombinant polypeptide equivalent to ECEF-104 but formulated in a different manner (Balcewicz-Sablinska et al., J. Immunol 147:2170-2174 (1991)). Other studies show that the required dose of this material for its effects on a lymphocyte cell line (Tagaya et al, EMBO J. 8:757 (1989)) or U937 cells (Matsuda, M. et al., J. Immunol 747:38-7 (1991)) is in a very similar range (approx. 100 nM to 1 μM). Higher doses of rECEF-104 did not have a greater effect on cytotoxic function (data not shown). By comparison, rECEF-80 and rECEF-84 enhanced eosinophil cytotoxic function substantially, at concentrations from 10 pM to 800 nM, with the effect of rECEF-84 stronger at most concentrations (Figure 10). The activity of these species was very similar to that determined for purified natural 10 kDa ECEF (half- maximal between 16 pM and 400 pM), as evaluated by the same biological assay (Silberstein et al.. J. Immunol 143:979 (1989)).

None of the rECEF preparations had any direct toxicity to schistosomula targets. In parallel, a fusion protein consisting of maltose- binding protein joined to paramyosin was cleaved with factor Xa, prepared in a similar manner, and tested over the same range of concentrations. This material had no effect on eosinophil cytotoxic function at any concentration. In a separate experiment, it was determined that factor Xa lost its activity after a three-hours incubation (not shown), thus no proteolytic activity would have been carried over to the eosinophil bioassay. This information, taken collectively with the differential activities of the rECEF species, argues that the observed effects in the assay of eosinophil cytotoxic function were due only to the rECEF species, not to the maltose-binding protein or traces of factor Xa. It was also found that rECEF 84, separated from maltose binding protein and factor Xa by preparative SDS-PAGE, enhanced eosinophil cytotoxic function, whereas mock-eluted material from a different region of the same gel did not. However, as mentioned above, rECEF-84 prepared in this matter tended to precipitate from solution (data not shown). These findings demonstrate: 1) the potential for activation of eosinophil-stimulating functions by proteolytic cleavage of the COOH-terminal of full-length ECEF, and 2) that rECEF-84 is functionally similar to 10 kDa ECEF. To date, it has not been possible either to demonstrate a precursor- product relationship between the 14 and 10 kDa ECEF species (if it exists in vt^'vø) by pulse-chase type experiments (Balcewicz-Sablinska et l , J. Immunol. 147:2170-2174 (1991)) or to determine the COOH-terminal amino acid sequence of 10 kDa ECEF. The chief difficulty in these studies is that 10 kDa ECEF is produced only in trace quantities, such that hours of metabolic radiolabeling are required to detect it. It should also be noted that a 13 kDa ECEF species with the same NH-_>-terminal is discernible under certain conditions (Balcewicz-Sablinska et al . J. Immunol. 747:2170-2174 (1991)), so that if proteolytic cleavage occurs, there may be more than one cleavage site.

EXAMPLE 5 Dithiol reductase catalytic activity of the rECEF species

The method of Holmgren (Holmgren, A., /. Biol Chem. 254:9627

(1979)) was used with some modifications. Test samples were added to wells of a microtiter plate in 0.1 M potassium phosphate buffer, pH 7.0, containing 2 mM EDTA, and 1 mg/ml of insulin. The reaction was initiated by the addition of dithiothreitol to a concentration of 1 mM (which is suboptimal in the absence of a catalyst). Free insulin jS-chain precipitates from solution under these conditions, so the increase in turbidity of the reaction mixture correlated with dithiol reductase activity. Absorbance was monitored over time at room temperature at 630 nm, using an automated ELISA plate reader.

Of all rECEF species tested, only rECEF 104 had the dithiol reductase catalytic activity. This property was detectable at concentrations as low as 160 nM (Figure 11A) and was time-dependent (Figure 1 IB). Thus, at least part of the sequence from amino acid position 85 to the COOH-terminal is essential to the dithiol reductase catalytic activity in solution.

It was somewhat surprising to find that strong eosinophil-stimulating activity was not associated with the dithiol reductase activity. This would appear to argue against the hypothesis that the catalytic activity is involved in signal transduction. Clearly, the dithiol reductase activity is not required for the initial interaction of rECEF-84 with the eosinophils. EXAMPLE 6

Co-precipitation of ECEF-associated molecules by ECEF-specific antibody.

In Balcewicz-Sablinska et al , J. Immunol 147:2170-2174 (1991) we described the preparation of an antibody specific for the NH₂-terminal (20 amino acids) of ECEF. This peptide was synthesized, attached to KLH, and used to immunize rabbits. We commented that in addition to the known ECEF species (which had been characterized by an anti-whole 10 kDa ECEF rabbit antibody), this antibody precipitated species of higher molecular weight. Figure 12 demonstrates this phenomenon; the other specifically precipitated species are of 16, 18, and 94 kDa. Trypsin treatment of the intact U937 cells did not reduce the amount of 16/18/94 kDa substances that could be immunoprecipitated from cell lysates. By comparison, trypsin treatment does remove the ECEF species precipitable by anti-whole 10 kDa ECEF. It is also important to note that the NH₂-terminal specific anti-ECEF does not react well with free rECEF species from solution in physiological buffers or nonionic detergents, but that it reacts very well with these species in Western Blot analysis. Thus, it appears that the two antibodies are different and that the NH₂-terminal is only exposed when denatured or in certain circumstances in the cell (such as association with the 16/18/94 kDa molecules).

Explanations for the co-precipitation phenomenon fall into two categories: 1) the 16/18/94 kDa species have the same antigenic determinant as the known ECEF species, and the specific antibody reacts with them directly, or 2) in the cell, ECEF species are associated with the 16/18/94 kDa substances and they are co-precipitated. The following evidence argues for the second possibility.

The cDNA has been cloned in four different labs (Wollman, E.E. et al, J. Biol Chem. 263:2614 (1988), Tagaya, Y. et al , EMBO J. 8:757 (1989), Deiss, L.P. et al , Science 252: 1 17 (1991)), using methods that might well detect alterative species with the same NH₂-terminal, and nobody has found such a structure.

Reduction and alkylation of cellular material before immunoprecipitation abolishes precipitation of the 16, 18, and 94 kDa species but not the ECEF species (not shown).

Crosslinking reagents show that rECEF species associate closely with certain cellular proteins (possibly the 16 and/or 18 kDa species). ³⁵S- methionine/cysteine labeled U937 cells were washed and incubated on ice for 4 hours with excess rECEF-84, or rECEF-104, or no cytokine. After washing, cells from each experimental condition were treated with the cleavable and uncleavable crosslinking reagents DSP and DSS, according to standard methods (Chapter 6: Cytokinesand their cellular receptors. Current Protocols in Immunology, 1991). After quenching of the crosslinking reaction, the cells were washed and proteins were analyzed by imunoprecipitation and SDS-PAGE.

There was no apparent shift in the migration of the 94 kDa species under any of these conditions. However, new species appeared with apparent masses of 51 and 67 kDa in the rECEF-84 treated cells and 54 and 70 kDa in the rECEF-104 treated cells (Figure 13). The molecular composition of these crosslinked immunoprecipitated species is not know. It should be remembered that the rECEF molecules form homodimers in solution, and it may be the dimer that is crosslinked to the cellular molecules. Neither is it known whether the 16 and 18 kDa molecules form homo- or hetero-multimers. Nevertheless, the change in migration, correlating with the size of the rECEF species, makes it clear that the (unlabeled) rECEF species are crosslinked to some radiolabeled cellular protein . The very faint species of higher molecular weight may include the 94 kDa species, but it appears that there are conformational or chemical factors that did not permit crosslinking of the 94 kDa molecule to others. When radiolabeled U937 cells were treated with the cleavable crosslinker DSP and the immunoprecipitated substances were analyzed on SDS-PAGE under reducing conditions, the same species of ECEF and associated proteins were detected. If cells were first incubated with rECEF-84 or rECEF-104, the quantity of 16 kD, and 18 kDa to a lesser extent, was detected in the precipitate. For example, in a representative experiment, the region of the gel containing the 16 and 18 kDa proteins emitted 130 cpm. In a parallel sample derived from cells preincubated with rECEF-84, the gel emitted 808 cpm. This a second indication that at least one of the lower molecular weight associated proteins interacts with exogenous ECEF's.

EXAMPLE 7 Biosynthetic radiolabeling of rECEF species

It was found that addition of ³⁵S-methionine/cysteine to bacteria synthesizing rECEF species at the same times as IPTG induction resulted in recombinant species with an activity of 3 to 9 x 10⁷ cpm/mg (Figure 14). ³⁵S- from SO was not incorporated well. Accordingly, this method may be used to provide readiolabeled rECEF useful in the methods of the invention when it is desired to trace the fate of the rECEF molecule in an assay or host system.

EXAMPLE 8

Characterization of ECEF-Associated Proteins The 16, 18 and 94 kDa molecules are identified as species associated with ECEF species in the U937 cell (a cell that both produces and responds to ECEFs) and thus likely to be involved in the functions of ECEFs based on crosslinking analysis. Crosslinking methods demonstrated that certain cellular proteins, probably the 16 and/or 18 kDa species, interact with exogenous ECEFs.

Any method may be used that allows for the isolation of the desired protein in a purity sufficient for a desired purpose. For example, a series of monoclonal antibodies may be prepared and used as reagents for one-step affinity purification. It is known in the art to isolate 10 kDa ECEF from > 10 liters of U937 cell-conditioned medium by a sequence involving Phenyl- Sepharose chromatography, DEAE anion exchange chromatography, preparative SDS-PAGE, and reversed-phase HPLC. The 14 and 13 kDa species were isolated by immunoprecipitation and preparative SDS-PAGE- In designing a method for the purification of the ECEF-associated proteins, the following considerations should be considered: 1) the ECEF- associated proteins have no known biological activity that can be used to assay for the purification other than their binding to ECEF, 2) other than Mr, there is very little information regarding the physical properties of the molecules, 3) detergent will be required to extract the molecules from U937 cells and may be required to keep them in solution, and 4) the molecules have already been isolated on a small scale by immunoprecipitation and SDS-PAGE. Accordingly, an affinity-based approach toward purification is preferred, using ECEF or antibodies as the affinity capture molecules. However, other methods such as hydrophobic interaction or reversed phase HPLC, (which can be done with detergent in the mobile phase) are also useful.

Abundance of source material for the purification of these proteins is not a problem, since it is possible to grow large quantities of U937 cells. The ECEF-associated proteins appear to be more abundant in cells than 10 kDa ECEF was in conditioned medium.

The conditions for the affinity-based purification are chosen on the basis of pilot studies using a small affinity column and material derived from radiolabeled U937 cells. A first approach is to increase the scale of the immunoprecipitation that has already been successfully performed. Based on experience with 10 kDa ECEF, and on the intensities of protein bands, it may be expected that amino acid sequence analysis or immunization will require 1000 to 10,000 times as much of the protein species as is recovered by immunoprecipitation from one ml of cells (i.e., 1 to 10 liters of cultured cells). Serum sould recognize the NH₂-portion of the ECEF molecule since since that portion is present in all ECEF constructs. The antibody portion of polyclonal serum such as rabbit antiserum is isolated by protein A affinity chromatography (to accomplish the same purpose as protein A in the immunoprecipitation). Affinity purification of specific antibody on Sepharose derivatized with the ECEF-NH₂-terminal synthetic peptide (note: acid-eluted Ig from this column binds peptide in ELISA) may also be used. If there is no yield consideration, this method may be substituted for protein A.

The purified antibody fraction is conjugated to Sepharose beads by the cyanogen bromide method. The beads are packed in a column for use.

Cell-free extracts that are the source material for the isolation of ECEF-associated proteins can be generated from U937 cells stimulated with 100 ng/ml of PMA. Cells (up to 40 liters/batch in roller bottles, as necessary) are grown in RPMI 1640 + 10% FCS, washed, and recultured in serum-free medium with PMA. After two days (optimal time, judged by radiolabeling/immunoprecipitation studies), the cells are washed and treated with lysis buffer containing 1 mM PMSF.

The lysate is centrifuged at high speed to remove the paniculate and passed through the affinity column. After washing with the same buffer, the bound material is eluted by denaturing or competing reagents. Variables that may be considered are the choice of lysis buffer, washing buffer,and eluting reagent. Cells have been successfully lysed and immunoprecipitates washed with a phosphate buffer containing 1 % Tween 80 and 0.5 M sodium chloride. Tween 80 was chosen over NP-40 and Triton X-100 on the basis of preliminary studies, examining only the ECEF species). Recently it has also been found that a pH 8.0 triethanolamine/1 % digitonin buffers gives a better yield of ECEF-associated proteins.

For elution of bound species from the beads, several options are considered. Subsequent handling is simplest if a volatile reagent (e.g., 4M acetic acid) is used so that the sample could be concentrated by evaporation. In the event that detergent is necessary to maintain the ECEF-associated proteins in solution, it should be appropriate to elute the column with 0.1 % SDS (which releases at least the 94 kDa species in the absence of reducing agent). It may also prove effective to elute the bound species by the addition of rECEFs to the washing buffer. The method above depends on the association of endogenously produced ECEF species with the associated protein. Since these are probably not present in saturating quantities (since exogenous ECEF affects U937 functions), the method would seem to be inefficient. There are two main variations on the method described above. The first of these variations is to derivatize the affinity resin with rECEF molecules and to purify the ECEF- associated molecules by affinity for this matrix. Results of the crosslinking experiments suggest either rECEF-84 or 104 would yield the same result. The second variation is to use monoclonal or polyclonal antibodies specific for the 16, 18, or 94 kDa molecules. This method, which is likely to be the more efficient, requires these antibodies.

For further separation, affinity purified ECEF-associated protein preparations are subjected to preparative SDS-PAGE. For amino acid sequence analysis, the proteins are transferred electrophoretically to PVDF membrane. After staining with Coomassie blue, regions of membrane with bound protein are excised and submitted for analysis. For the production of antibodies, mice or rabbits are immunized either with proteins recovered by electroelution from the gel or with protein in crushed, excised gel slices. For the preparation of peptide fragments for sequence analysis, the first choice of method is to cleave the protein while bound to PVDF membrane, for example by exposure to cyanogen bromide vapor, as is known in the art. The fragments are eluted from the membrane with 30 to 70% acetonitrile in 0.1 % trifluoroacetic acid, and the solvent is evaporated. Alternatively, protein species are recovered from the SDS gel by electroelution (with detergent present if necessary) for enzyme cleavage. Peptide fragments of cleaved ECEF-associated proteins may be isolated by reversed phase HPLC- Fragments thai are soluble in 0- 1 % trifluoroacetic acid are dissolved and injected on a reversed-phase C-4 or C-18 column, and eluted with a gradient to acetonitrile in 0.1 % trifluoroacetic acid. If it happens that a peptide fragment is not soluble in 0.1 % TFA and additional sequence information is needed, HPLC is conducted in the presence of SDS. This would alter retention times but would not present resolution or interfere with subsequent sequence analysis. These antibody species are useful for large-scale purification of the ECEF-associated proteins and for further study of the interaction of ECEF with the associated proteins in U937 cells and eosinophils. The production of monoclonal antibodies is performed using techniques known in the art. Mice are immunized with either 16, 18 or 94 kDa proteins purified by immunoaffinity methods and SDS-PAGE or the entire antibody/ECEF/16, 18, 94 kDa protein complex, or both of these in sequence. Sera of the mice is tested for the ability to immunoprecipitate radiolabeled ECEF-associated proteins from lysates of U937 cells. When the sera convert, the mice are given a final boost, and the spleens taken for fusions at 72 to 96 hours. Hybridomas are screened initially for reactivity with the immunogen by ELISA. Initially positive cultures are subcloned, screened by ELISA, and screened by immunoprecipitation of radiolabeled ECEF-associated proteins. Monoclonal antibodies are raised in ascites fluids and isolated by protein A or G chromatography (isotype permitting) or by salting out and anion exchange chromatography.

If monoclonal antibodies are either difficult to raise or unsuitable for our needs for unanticipated reasons, specific rabbit antibodies -are useful. In the best case, purified ECEF-associated proteins and partial amino acid sequence information will be available. An amino acid sequence of 12 to 20 amino acids is selected on the basis of novelty (comparison with sequence databases) and charge (charged and, thus, probably exposed regions will be used). This sequence is synthesized (BWH core facility), conjugated to KLH, and used to immunize with excised fragments of polyacrylamide gel containing each immunoprecipitated ECEF-associated protein. The reactivity of the antisera is evaluated by immunoprecipitation of lysates from radiolabeled U937 cells.

In order to determine the NH₂-terminal amino acid sequence, the various ECEF-associated species are separated by SDS-PAGE, transferred to PVDF membrane, stained with Coomassie blue, excised, and submitted for analysis according to the methods used for ECEF and the serum eosinophil cytotoxicity inhibitor. To obtain additional sequence information (and particularly if the NH₂-terminal proves unsusceptible to Edman degradation), peptide fragments generated by enzyme or CNBr cleavage are separated by reversed-phase HPLC and analyzed.

Amino acid sequence information is used in three ways: 1) comparison with known protein sequences in the PIR and translated Genbank databases to search for identity, partial identity, or similarity to known proteins, 2) design of synthetic peptides for the generation of specific antiserum (section D2b), and 3) design of oligonucleotide probes, for the screening of gene libraries. One would want as much sequence information as possible for each of the three ECEF-associated proteins. For the screening of cDNA libraries, enough sequence information is required to design at least two non- overlapping oligonucleotide probes. However, it is easier and preferable to determine the entire structures by DNA sequence analysis, not by this method . Therefore, there is no need for a series of cleavages to generate series of overlapping peptides.

For the cloning of the ECEF-accosiated proteins, the commercially prepared (Clontech) cDNA library from PMA-stimulated U937 cells is preferable, because PMA-stimulated U937 cells are known to produce the proteins of interest. The library is available and has already been used for the isolation of the ECEF cDNA. In the event that screening procedures do not allow detection of the genes of interest, a new library may be prepared from mRNA of cells that are known to be producing the ECEF-associated proteins. This is done using a commercially available kit, such as the λ-ZAP cDNA synthesis and cloning kits (Stratagene) or the FastTrack/Librarian/μWave system (Invitrogen). The latter of these allows for screening either with nucleic acid or antibody-based screening. This feature may be important, since antibody will be available as well as amino acid sequence information. The first choice for screening method is the use of oligonucleotide probes and the λ library. Redundant oligonucleotide probes are synthesized, based on information from the amino acid sequence. Factors to be considered in the design of the probe are: a) the need to keep redundancy to a minimum (if necessary, multiple probes will be designed based on codon usage tables, and pools with lower redundancy will be used), and 2) the desirability of a relatively G-C rich region, so that the Tmin of hybridization will be >50°C. Oligonucleotide probes will be labeled with ³²P by T4 DNA ligase. For the primary screening procedure, approximately 500,000 plaques are screened. Standard methods are used, however, to confirm that conditions such as washing temperature are reasonable, a phage containing the ECEF cDNA (or a portion of the library mixed with positive phage) is screened with a mixture of redundant oligonucleotide based of the ECEF sequence.

An alternative procedure is an attempt to improve the quality of the probe by the use of the anchored PCR assay. A further alternative is antibody-based screening, which is carried out according to standard protocols (e.g., the Invitrogen system mentioned above, with accompanying protocols).

Plaques (or colonies) corresponding to replicate positive signals are picked and plated for second and third degree screening. In order to confirm the identity of positive clones, these higher degree plating are also screened with a second probe designed from a different peptide fragment. In the event that partial clones (evaluated by sequence analysis) are detected, inserts of these are used to detect complete or overlapping clones.

Methods for the sequence analysis of selected clones are similar to those previously described, with the reservation that the cDNAs (at least for the 94 kDa species) will be much larger than for the ECEF species. Therefore, it is likely that the sequence analysis will require restriction mapping of the insert and subcloning of fragments into Bluescript (for which sequencing primers are available). A second method that may simplify the sequence analysis is the construction of nested deletions in the sequencing vector. This is appropriate for an insert of up to about 5 kb. Information from the restriction analysis would identify enzymes that do not cut the insert but could cut the vector once, so that a double digest would leave a blunt end or 5' overhang near the beginning of the insert and a 3' four base overhang on the other end. This structure is digested over a time course with E. coli exonuclease III, and after trimming the ends would be re-ligated and cloned. The nucleotide sequence is analyzed for information that could encode the proteins under study. If it is decided to construct nested deletions, the Stratagene Exo/Mung deletion kit will be used for this purpose.

Sequence data is analyzed for open reading frames, for agreement with the amino acid sequence information, and for other features, such as a hydrophobic (putative membrane anchoring) region. Nucleic acid sequences are compared by computer search with known sequences. Any indication of similarity with known sequences will contribute to the model of ECEF/cell interactions-

The co-precipitation of the 16, 18 and 94 kDa proteins by ECEF specific antibody does not indicate whether the three proteins are associated together in a complex and whether such a complex is exposed on the cell surface. For example, since the U937 cell both produces and responds to ECEFs, it is possible that certain accessory proteins are associated with production/modification/transport, and others are associated with the response. Reagents specific for the ECEF-associated proteins are used to address this question. The association of the 16, 18 and 94 kDa proteins with each other and ECEFs is studied as previously described by co-precipitation with antibodies specific for each species.

Crosslinking studies are performed using three crosslinking reagents will be used, all with a specificity for primary amines: DSS, BS₃ (a membrane impermeable reagent), and DSP (cleavable by reducing agents). The effects of each of these is tested on radiolabeled cells and also on unlabeled cells exposed to exogenous, radiolabeled rECEFs.

EXAMPLE 9

Role of ECEF in the Death of Tumor Cells

1. High expression of 10 kDa ECEF is associated with sensitivity to glutathione. The U937ad⁺ cell line (described above) expresses high levels of the 10 kDa (high potency) form of the ECEF cytokine. It was considered likely that a reducing agent co-factor would be important to the biology of the molecule as a supplier of reducing equivalents to the dithiol reductase catalytic site. Glutathione (GSH) was considered a likely candidate as the reducing agent, since it is very abundant in cells and biological fluids. Therefore, GSH was added in various doses to cultured U937ad⁺ cells, and the cells were observed for biological effects.

It was found that concentrations of GSH as low as 125 μM were completely toxic to U937ad⁺ cells. Cell death occurred over a period of four hours. Other myeloid tumor cell lines, including THP-1, HL-60, and the parent U937 cells (which express lower levels of ECEF) were sensitive to GSH also, but the effect required a much higher dose (Figure 15). This finding was surprising, since GSH is not known to have toxic properties.

2. Selective toxicity of the ECEF gene to U937 cells. In order to obtain further evidence of the relationship between sensitivity to GSH and ECEF expression, U937 cells (parent line) were transfected with plasmid constructs encoding the full-length ECEF gene. These constructs consisted of the ECEF coding region (see original patent) from positions 1-104, preceded by a HindHL restriction site and an ATG initiation codon and followed by a stop codon and Xbal restriction site. This fragment was prepared by polymerase chain reaction, cleaved with Hindlll and Xbal, and inserted into the CDM8 plasmid that had been cut with HindDI and Xbal. For control studies, the CDM8 plasmid containing the insert for human growth hormone was used. Expression of protein from the CDM8 plasmid is at very high levels (much higher than those of natural ECEF made in the cells) under the control of the cytomegalovirus promoter.

The U937 cells were transfected with 0.5 to 20 μg/ml of the ECEF gene construct by electroporation. At the lowest doses, significant toxicity to the cells was observed. At higher doses, most or all the U937 cells were killed. Death of the cells was evaluated by morphology, the ability to incorporate radiolabeled thymidine into DNA, and by the ability to synthesize growth hormone from the co-transfected control construct. Transfection of COS cells in a similar manner had no effect on viability (Figure 16). Thus, the toxic effects of the transfected gene have cell-lineage specificity.

Taken collectively, the finding that low levels of ECEF expression induce sensitivity to GSH, and that high levels cause cell death, suggest that expression of the ECEF gene (possibly by gene therapy) could be used as a method for selective killing of certain types of tumor cells. Therapy might also involve treatment with GSH or other reducing agents, or with other substances that are reduced by the interaction with glutathione.

Having now fully described the invention, it will be understood by those with skill in the art that the scope may be performed within a wide and equivalent range of conditions, parameters, and the like, without affecting the spirit or scope of the invention or of any embodiment thereof. SEQUENCE LISTING

(1) GENERAL INFORMATION:

(i) APPLICANT: Silberstein, David S.

Balcewicz-Sablinska, Maria Katarzyna

(ii) TITLE OF INVENTION: RECOMBINANT ACTIVE FORMS OF ECEF, PROTEINS THAT ASSOCIATE THEREWITH, AND USES THEREOF

(iii) NUMBER OF SEQUENCES: 15

(iv) CORRESPONDENCE ADDRESS:

(A) ADDRESSEE: Sterne, Kessler, Goldstein & Fox

(B) STREET: 1225 Connecticut Avenue, N.W. , Suite 300

(C) CITY: Washington

(D) STATE: DC

(E) COUNTRY: USA

(F) ZIP: 20036

(v) COMPUTER READABLE FORM:

(A) MEDIUM TYPE: Floppy disk

(B) COMPUTER: IBM PC compatible

(C) OPERATING SYSTEM: PC-DOS/MS-DOS

(D) SOFTWARE: Patentln Release #1.0, Version #1.25

(Vi) CURRENT APPLICATION DATA:

(A) APPLICATION NUMBER: US To be assigned ^'"" FILING DATE:

(Si CLASSIFICATION:

(Viii) ATTORNEY/AGENT INFORMATION:

(A) NAME: Cimbala, Michele A

(B) REGISTRATION NUMBER: 33,851

(C) REFERENCE/DOCKET NUMBER: 0627.0940000

(ix) TELECOMMUNICATION INFORMATION:

(A) TELEPHONE: (202)833-7533

(B) TELEFAX: (202)833-8716

(2) INFORMATION FOR SEQ ID N0:1:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 456 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: both

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: DNA

(ix) FEATURE:

(A) NAME/KEY: CDS

(B) LOCATION: 67..381 (D) OTHER INFORMATION: /note= "Message is polyadenylated.

(xi) SEQUENCE DESCRIPTION: SEQ ID N0:1:

CGGTTTGGTG CAAAGGATCC ATTTCCATCG GTCCTTACAG CCGCTCGTCA GACTCCAGCA 60

GCCAAG ATG CTG AAG CAG ATC GAG AGC AAG ACT GCT TTT CAG GAA GCC 108 Met Leu Lys Gin lie Glu Ser Lys Thr Ala Phe Gin Glu Ala 1 5 10 TTG GAC GCT GCA GGT GAT AAA CTT GTA GTA GTT GAC TTC TCA GCC ACG 156 Leu Asp Ala Ala Gly Asp Lys Leu Val Val Val Asp Phe Ser Ala Thr 15 20 25 30

TGG TGT GGG CCT TGC AAA ATG ATC AAC CCT TTC TTT CAT TCC CTC TCT 204 Trp Cys Gly Pro Cys Lys Met lie Asn Pro Phe Phe His Ser Leu Ser

35 40 45

GAA AAG TAT TCC AAC GTG ATA TTC CTT GAA GTA GAT GTG GAT GAC TGT 252 Glu Lys Tyr Ser Asn Val lie Phe Leu Glu Val Asp Val Asp Asp Cys

50 55 60

CAG GAT GTT GCT TCA GAG TGT GAA GTC AAA TGC ATG CCA ACA TTC CAG 300 Gin Asp Val Ala Ser Glu Cys Glu Val Lys Cys Met Pro Thr Phe Gin 65 70 75

TTT TTT AAG AAG GGA CAA AAG GTG GGT GAA TTT TCT GGA GCC AAT AAG 348 Phe Phe Lys Lys Gly Gin Lys Val Gly Glu Phe Ser Gly Ala Asn Lys 80 85 90

GAA AAG CTT GAA GCC ACC ATT AAT GAA TTA GTC TAATCATGTT TTCTGAAAAC 401 Glu Lys Leu Glu Ala Thr lie Asn Glu Leu Val 95 100 105

ATAACCAGCC ATTGGCTATT TAAAACTTGT AATTTTTTTA ATTTACAAAA ATATA 456

(2) INFORMATION FOR SEQ ID NO:2:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 105 amino acids

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

(ii) MOLECULE TYPE: protein

(xi) SEQUENCE DESCRIPTION: SEQ ID N0:2:

Met Leu Lys Gin lie Glu Ser Lys Thr Ala Phe Gin Glu Ala Leu Asp 1 5 10 15

Ala Ala Gly Asp Lys Leu Val Val Val Asp Phe Ser Ala Thr Trp Cys 20 25 30

Gly Pro Cys Lys Met lie Asn Pro Phe Phe His Ser Leu Ser Glu Lys

35 40 45

Tyr Ser Asn Val lie Phe Leu Glu Val Asp Val Asp Asp Cys Gin Asp 50 55 60

Val Ala Ser Glu Cys Glu Val Lys Cys Met Pro Thr Phe Gin Phe Phe 65 70 75 80

Lys Lys Gly Gin Lys Val Gly Glu Phe Ser Gly Ala Asn Lys Glu Lys

85 90 95

Leu Glu Ala Thr lie Asn Glu Leu Val 100 105

(2) INFORMATION FOR SEQ ID N0:3:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 332 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: both

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: DNA

(ix) FEATURE:

(A) NAME/KEY: CDS

(B) LOCATION: 9..320 (xi) SEQUENCE DESCRIPTION: SEQ ID N0:3:

GGGGAATT CTG AAG CAG ATC GAG AGC AAG ACT ACT TTT CAG GAA GCC TTG 50 Leu Lys Gin lie Glu Ser Lys Thr Thr Phe Gin Glu Ala Leu 1 5 10

GAC GCT GCA GGT GAT AAA CTT GTA GTA GTT GAC TTC TCA GCC ACG TGG 98 Asp Ala Ala Gly Asp Lys Leu Val Val Val Asp Phe Ser Ala Thr Trp 15 20 25 30

TGT GGG CCT TGC AAA ATG ATC AAC CCT TTC TTT CAT TCC CTC TCT GAA 146 Cys Gly Pro Cys Lys Met lie Asn Pro Phe Phe His Ser Leu Ser Glu

35 40 45

AAG TAT TCC AAC GTG ATA TTC CTT GAA GTA GAT GTG GAT GAC TGT CAG 194 Lys Tyr Ser Asn Val lie Phe Leu Glu Val Asp Val Asp Asp Cys Gin 50 55 60

GAT GTT GCT TCA GAG TGT GAA GTC AAA TGC ATG CCA ACA TTC CAG TTT 242 Asp Val Ala Ser Glu Cys Glu Val Lys Cys Met Pro Thr Phe Gin Phe 65 70 75

TTT AAG AAG GGA CAA AAG GTG GGT GAA TTT TCT GGA GCC AAT AAG GAA 290 Phe Lys Lys Gly Gin Lys Val Gly Glu Phe Ser Gly Ala Asn Lys Glu 80 85 90

AAG CTT GAA GCC ACC ATT AAT GAA TTA GTC TAAAGATCTG GG 332

Lys Leu Glu Ala Thr lie Asn Glu Leu Val 95 100

(2) INFORMATION FOR SEQ ID N0:4:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 104 amino acids

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

(ii) MOLECULE TYPE: protein

(Xi) SEQUENCE DESCRIPTION: SEQ ID N0:4:

Leu Lys Gin lie Glu Ser Lys Thr Thr Phe Gin Glu Ala Leu Asp Ala 1 5 10 15

Ala Gly Asp Lys Leu Val Val Val Asp Phe Ser Ala Thr Trp Cys Gly 20 25 30

Pro Cys Lys Met lie Asn Pro Phe Phe His Ser Leu Ser Glu Lys Tyr

35 40 45

Ser Asn Val He Phe Leu Glu Val Asp Val Asp Asp Cys Gin Asp Val 50 55 60

Ala Ser Glu Cys Glu Val Lys Cys Met Pro Thr Phe Gin Phe Phe Lys 65 70 75 80

Lys Gly Gin Lys Val Gly Glu Phe Ser Gly Ala Asn Lys Glu Lys Leu 85 90 95

Glu Ala Thr He Asn Glu Leu Val 100

(2) INFORMATION FOR SEQ ID NO:5:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 272 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: both

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: DNA (ix) FEATURE:

(A) NAME/KEY: CDS

(B) LOCATION: 9..260

(Xi) SEQUENCE DESCRIPTION: SEQ ID N0:5:

GGGGAATT CTG AAG CAG ATC GAG AGC AAG ACT GCT TTT CAG GAA GCC TTG 50 Leu Lys Gin He Glu Ser Lys Thr Ala Phe Gin Glu Ala Leu 1 5 10

GAC GCT GCA GGT GAT AAA CTT GTA GTA GTT GAC TTC TCA GCC ACG TGG 98 Asp Ala Ala Gly Asp Lys Leu Val Val Val Asp Phe Ser Ala Thr Trp 15 20 25 30

TGT GGG CCT TGC AAA ATG ATC AAC CCT TTC TTT CAT TCC CTC TCT GAA 146 Cys Gly Pro Cys Lys Met He Asn Pro Phe Phe His Ser Leu Ser Glu 35 40 45

AAG TAT TCC AAC GTG ATA TTC CTT GAA GTA GAT GTG GAT GAC TGT CAG 194 Lys Tyr Ser Asn Val He Phe Leu Glu Val Asp Val Asp Asp Cys Gin

50 55 60

GAT GTT GCT TCA GAG TGT GAA GTC AAA TGC ATG CCA ACA TTC CAG TTT 242 Asp Val Ala Ser Glu Cys Glu Val Lys Cys Met Pro Thr Phe Gin Phe 65 70 75

TTT AAG AAG GGA CAA AAG TAGAGATCTG GG 272

Phe Lys Lys Gly Gin Lys 80

(2) INFORMATION FOR SEQ ID N0:6:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 84 amino acids

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

(ii) MOLECULE TYPE: protein

(xi) SEQUENCE DESCRIPTION: SEQ ID N0:6:

Leu Lys Gin He Glu Ser Lys Thr Ala Phe Gin Glu Ala Leu Asp Ala 1 5 ^' 10 15

Ala Gly Asp Lys Leu Val Val Val Asp Phe Ser Ala Thr Trp Cys Gly 20 25 30

Pro Cys Lys Met He Asn Pro Phe Phe His Ser Leu Ser Glu Lys Tyr 35 40 45

Ser Asn Val He Phe Leu Glu Val Asp Val Asp Asp Cys Gin Asp Val 50 55 60

Ala Ser Glu Cys Glu Val Lys Cys Met Pro Thr Phe Gin Phe Phe Lys 65 70 75 80

Lys Gly Gin Lys

(2) INFORMATION FOR SEQ ID NO:7:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 260 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: both

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: DNA (ix) FEATURE:

(A) NAME/KEY: CDS

(B) LOCATION: 9..248

(Xi) SEQUENCE DESCRIPTION: SEQ ID N0:7:

GGGGAATT CTG AAG CAG ATC GAG AGC AAG ACT GCT TTT CAG GAA GCC TTG 50 Leu Lys Gin He Glu Ser Lys Thr Ala Phe Gin Glu Ala Leu 1 5 10

GAC GCT GCA GGT GAT AAA CTT GTA GTA GTT GAC TTC TCA GCC ACG TGG 98 Asp Ala Ala Gly Asp Lys Leu Val Val Val Asp Phe Ser Ala Thr Trp 15 20 25 30

TGT GGG CCT TGC AAA ATG ATC AAC CCT TTC TTT CAT TCC CTC TCT GAA 146 Cys Gly Pro Cys Lys Met He Asn Pro Phe Phe His Ser Leu Ser Glu

35 40 45

AAG TAT TCC AAC GTG ATA TTC CTT GAA GTA GAT GTG GAT GAC TGT CAG 194 Lys Tyr Ser Asn Val He Phe Leu Glu Val Asp Val Asp Asp Cys Gin 50 55 60

GAT GTT GCT TCA GAG TGT GAA GTC AAA TGC ATG CCA ACA TTC CAG TTT 242 Asp Val Ala Ser Glu Cys Glu Val Lys Cys Met Pro Thr Phe Gin Phe 65 70 75

TTT AAG TAGAGATCTG GG 260

Phe Lys 80

(2) INFORMATION FOR SEQ ID NO:8:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 80 amino acids

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

(ii) MOLECULE TYPE: protein

(Xi) SEQUENCE DESCRIPTION: SEQ ID N0:8:

Leu Lys Gin He Glu Ser Lys Thr Ala Phe Gin Glu Ala Leu Asp Ala 1 5 10 15

Ala Gly Asp Lys Leu Val Val Val Asp Phe Ser Ala Thr Trp Cys Gly 20 25 30

Pro Cys Lys Met He Asn Pro Phe Phe His Ser Leu Ser Glu Lys Tyr

35 40 45

Ser Asn Val He Phe Leu Glu Val Asp Val Asp Asp Cys Gin Asp Val 50 55 60

Ala Ser Glu Cys Glu Val Lys Cys Met Pro Thr Phe Gin Phe Phe Lys 65 70 75 80

(2) INFORMATION FOR SEQ ID N0:9:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 6 amino acids

(B) TYPE: amino acid

(C) STRANDEDNESS: both

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: peptide

(ix) FEATURE:

(A) NAME/KEY: Peptide

(B) LOCATION: 1..6 (D) OTHER INFORMATION: /note= "First six amino acids of the amino terminus of the 14-kDa ECEF. First amino acid may be substituted for lysine."

(xi) SEQUENCE DESCRIPTION: SEQ ID N0:9:

Val Xaa Gin He Xaa Ser 1 5

(2) INFORMATION FOR SEQ ID NO:10:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 9 amino acids

(B) TYPE: amino acid

(C) STRANDEDNESS: both

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: peptide

(ix) FEATURE:

(A) NAME/KEY: Peptide

(B) LOCATION: 1..9

(D) OTHER INFORMATION: /note= "First nine amino acids of the amino terminus of the 13-kDa ECEF. First amino acid may be substituted for lysine."

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

Val Xaa Gin He Xaa Ser Xaa Xaa Ala

1 5

(2) INFORMATION FOR SEQ ID NO:11:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 11 amino acids

(B) TYPE: amino acid

(C) STRANDEDNESS: both

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: peptide

(ix) FEATURE:

(A) NAME/KEY: Peptide

(B) LOCATION: 1..11

-(D) OTHER INFORMATION: /note= "First ten amino acids of the amino terminus of 10 kDa-ECEF."

(xi) SEQUENCE DESCRIPTION: SEQ ID N0:11:

Val Lys Gin He Glu Ser Lys Thr Ala Phe Leu 1 5 10

(2) INFORMATION FOR SEQ ID NO:12:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 26 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: both

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: DNA

(xi) SEQUENCE DESCRIPTION: SEQ ID N0:12: GGGGAATTCG TGAAGCAGAT CGAGAG 26 (2) INFORMATION FOR SEQ ID NO:13:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 32 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: both

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: DNA

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:13: GGGTCTAGAT TAGACTAATT CATTAATGGT GG 32

(2) INFORMATION FOR SEQ ID NO:14:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 37 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: both

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: DNA

(Xi) SEQUENCE DESCRIPTION: SEQ ID NO:14: GGGTCTAGAC TACTTTTGTC CCTTCTTAAA AAAGTGC 37

(2) INFORMATION FOR SEQ ID NO:15:

(i) SEQUENCE CHARACTERISTICS: LENGTH: 35 base pairs

IS) TYPE: nucleic acid

IS STRANDEDNESS: both TOPOLOGY: linear

(ii) MOLECULE TYPE: DNA

(Xi) SEQUENCE DESCRIPTION: SEQ ID N0:15: GGGTCTAGAC TACTTAAAAA ACTGGAATGT TGGCG 35

INDICATIONS RELATING TO A DEPOSITED MICROORGANISM

(PCT Rule 13bis)

A. The indications made below relate to the microorganism referred to in the description on page , line

B. IDENTIFICATION OF DEPOSIT Further deposits are identified on an additional sheet | |

Name of depositary institution

AMERICAN TYPE CULTURE COLLECTION

Address of depositary institution (including postal code and country)

12301 Parklawn Drive Rockville, Maryland 20852 United States of America

Date of deposit Accession Number

26 June 1992 CRL 11080

C. ADDITIONAL INDICATIONS (leave blank if not applicable) This information is continued on an additional sheet | |

Human cell line, U937ad+

In respect of those designations in which a European Patent is sought a sample of the deposited microorganism will be made available until the publication of the mention of the grant of the European patent or until the date on which the application has been refused or withdrawn or is deemed to be withdrawn, only the issue of such a sample to an expert nominated by the person requesting the sample (Rule 28 (4) EPC) .

D. DESIGNATED STATES FOR WHICH INDICATIONS ARE MADE (if the indications are not for all designated Stales)

E. SEPARATE FURNISHING OF INDICATIONS (leave blankifnot applicable)

The indications listed belowwill be submitted to the International Bureau later (speάfy the general nahtre of the indications e.g., 'Accession Number of Deposit")

For receiving Office use only For International Bureau use only

I j This sheet was received with the international application I I This sheet was received by the Intemationai Bureau on:

Authorized officer Authorized officer

Claims

WHAT IS CLAIMED IS:

1. A shortened ECEF protein possessing eosinophil-stimulating activity.

2. The shortened ECEF protein of claim 1 , wherein said ECEF protein is selected from the group consisting of: ECEF-79, ECEF-80, ECEF-

81, ECEF-82, ECEF-83, ECEF-84, ECEF-85, ECEF-86, ECEF-87, ECEF- 88, ECEF-89, ECEF-90, ECEF-91, ECEF-92, ECEF-93, ECEF-94, ECEF- 95,ECEF-96,ECEF-97,ECEF-98,ECEF-99,ECEF-100,ECEF-101,ECEF- 102, and ECEF-103.

3. The shortened ECEF protein of claim 2, wherein said ECEF protein retains eosinophil-stimulating activity but lacks dithiol reductase activity.

4. The shortened ECEF protein of claim 1 , wherein said ECEF protein is a recombinantly-produced ECEF.

5. A fusion protein, comprising the amino acid sequence of a shortened ECEF selected from the group consisting of: ECEF-79, ECEF-80, ECEF-81, ECEF-82, ECEF-83, ECEF-84, ECEF-85, ECEF-86, ECEF-87, ECEF-88, ECEF-89, ECEF-90, ECEF-91 , ECEF-92, ECEF-93, ECEF-94, ECEF-95, ECEF-96, ECEF-97, ECEF-98, ECEF-99, ECEF-100, ECEF-101 , ECEF-102, and ECEF-103.

6. The fusion protein of claim 4, wherein said fusion protein comprises a factor Xa cleavage site.

7. Substantially pure DNA, wherein said DNA consists essentially of DNA encoding the ECEF protein of claim 1.

8. A recombinant construct which comprises a nucleic acid sequence encoding the shortened ECEF protein of claim 1.

9. A cloning vector, comprisin the recombinant DNA construct of claim 8.

10. The vector of claim 9, wherein said vector is capable of expressing said DNA construct.

11. The vector of claim 9, wherein said vector is capable of expressing antisense RNA to said construct.

12. A host cell transformed with the recombinant DNA construct of claim 8.

13. The host cell transformed with the vector of claim 11.

14. The host cell of claim 12, wherein said vector is capable of expressing said DNA construct.

15. The host cell of claim 13, wherein said vector is capable of expressing said antisense RNA.

16. The host cell of claim 13, wherein said cell is a prokaryotic cell.

17. The host cell of claim 17, wherein said cell is a mammalian cell-

18. A method of producing recombinant ECEF protein, which comprises: a. providing a DNA molecule comprising expressible sequences encoding the ECEF protein of claim 1; b. transforming a host with said molecule; and c. expressing said ECEF protein sequences of said DNA molecule in said host.

19. The method of producing the recombinant ECEF of claim 17, wherein said ECEF is human ECEF.

20. The method of producing the recombinant ECEF of claim 17, wherein said ECEF is expressed as a fusion protein.

21. A method of identifying an inhibitor of ECEF activity which comprises detecting the ability of said inhibitor to inhibit the biological activity of the ECEF protein of claim 2.

22. A method of identifying an inhibitor of ECEF activity which comprises detecting the ability of said inhibitor to lower binding of ECEF to an ECEF-associated protein.

23. A composition comprising an ECEF-associated protein.

24. The composition of claim 23, wherein said ECEF-associated protein is selected from the group consisting of the 9kDa, the 16 kDa and the

18 kDa associated protein.

25. The composition of claim 24, wherein said ECEF-associated protein is produced by expression of a recombinant construct encoding siad ECEF-associated protein.

26. The cultured mammalian cell line, U937ad⁺, having ATCC L 11080.