__ruLi_ERiAN INHIBΓΠNG SUBSTANCE

This invention was made with government support under CA17393 awarded by the National Cancer Institute of the National Institutes of Health. The government has certain rights in the invention.

FIELD OF THE INVENTION

The present invention relates to a process for the purification of biological materials, especially the purification of Miillerian Inhibiting Substance (MIS).

BACKGROUND OF THE INVENTION

The efficient and rapid purification of biological materials such as proteins, nucleic acids or polysaccharides, has been of great interest. Immunoaffinity chromatography is advantageous since, under the proper conditions, purification of 1,000-10,000 fold can be attained. Cf. Harlow, E. et al, Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory (1988).

One protein for which a rapid and efficient purification system is desirable is the biological modifier Miillerian Inhibiting Substance (MIS)

produced in vivo by the ovary and testis. MIS is a testicular protein responsible for regression of the anlagen of the female reproductive tract in the fetal urogenital ridge, as well as other differentiative functions such as inhibition of oocyte meiosis and lung surfactant. Previous studies in A431 cells and more recently in fetal lung suggest that MIS is a potent inhibitor of epidermal growth factor receptor autophosphorylation. Cf. Cigarroa et al, Growth Factors 1:179-91 (1989); Coughlin et al., Mol. and Cell. Endocrinol. 49:15-86 (1985); and Catlin et al, Metabolism.

Human Miillerian Inhibiting Substance has been cloned and expressed recombinantly (rhMIS) in animal cells as a 140 kDa homodimer (Cat& et al, Cell 45:685 (1986)). By virtue of carboxy-teπninal amino acid homology, MIS is a member of a large gene family that includes TGF-S (Derynck et al, Nature 316:101-5 (1985)), inhibin (Mason et al, Nature 318:659-63 (1985)), activin (Ling et al, Nature 321:119-82 (1986)), Vgl from Xenopus (Weeks et al, Cell 51:861-7 (1987)), the DrosopMa decapentaplegia protein (Padgett et al, Nature 325:81-4 (1987)), and the bone morphogenesis factors (Wozney et al, Science 242:1528-34 (1988)).

RhMIS is a 140 kDa glycoprotein composed of two identical subunits which, under disulfide bond reducing conditions, migrates on polyaciylamide gel electrophoresis at an apparent molecular weight of 70 kDa. The protein can be proteolytically cleaved in approximately one hour by exogenous plasmin into two distinct fragments that migrate electrophoretically as 57 kDa and 12.5 kDa moieties with cleavage at residue 427 of the intact 535 amino acid monomer as demonstrated by Pepinsky (Pepinsky et al, J. Biol. Chem. 26_ (35):18961-4 (1988)). Prolonged exposure to plasmin can result in cleavage of MIS at additional sites. In addition, purification of MIS by known techniques can be contaminated by other proteases that also cleave MIS.

MIS has been proposed as a potential growth inhibitor of epithelial human tumors of Miillerian origin such as endometrial, Fallopian tubal, cervical, and certain ovarian neoplasms. Experimental evidence using purified bovine MIS support this hypothesis (U.S. Patent Application Serial No. 06/792,233, filed October 19, 1985, now allowed; Fuller et al, J. Clin, Endocrinol. Metab. 54:1051-5 (1982); Fuller et al, Gynecol Oncol. 22:135-48 (1985)). Purified rhMIS used in similar in vitro assays, however, demonstrate limited anti-cancer activity (Wallen et al, Cancer Res. 49:2005-11 (1989)).

Miillerian Inhibiting Substance may be obtained by a variety of different methods. U.S. Patent Application No. 06/792,233, filed October 19, 1985, now allowed, and entitled "Purified Miillerian Inhibiting Substance and Process for Treating Human Ovarian Cancer Cells," describes a process for purifying MIS from testes by using aqueous polar dissociative solutions, separation of DNA and RNA, fractionation by gel filtration chromatography, and isolation of the MIS. U.S. Patent No. 4,404,188, filed July 29, 1981 and entitled "Purified Miillerian Inhibiting Substance and Method of Purification" describes a process for purifying MIS from testes which comprises treatment with a protease inhibitor, chromatography on ion exchange, chromatography on wheat germ lectin, on concanavalin A and/or on a supported triazinyl dye. U.S. Patent No. 4,487,833, filed on March 1, 1982 and entitled "Method of Preparing Hybridomas and of Purifying Immunogenic Materials" describes a process for separating MIS using immunoaffinity chromatography. MIS has also been obtained from recombinant DNA techniques as disclosed by Cate (Cate, et al, Cell 45: 685-698 (1986)). None of the references, however, describe a method of recovering substantially pure MIS which retains an antiproliferative activity or is essentially free of enzymes having proteolytic

' . activity against MIS.

A need, therefore, continues to exist for the development of highly efficient techniques for immunoaffinity chromatography, wherein a biological substance can be isolated from a complex biological mixture without interference from contaminating proteolytic enzymes or other factors that impede MIS antiproliferative activity. In particular, the need exists for an immunoaffinity chromatography method which can isolate and purify MIS so that the isolate will be essentially free from undesired proteolysis or other factors which may alter MIS activity.

SUMMARY OF THE INVENTION

The purification of MIS according to the immunoaffinity purification process of this invention results in a MIS product which is substantially free of enzymes having proteolytic activity or inhibitors of MIS antiproliferative activity. The present invention achieves this goal by providing for a method of purifying MIS comprising

(a) binding the MIS to an antibody-chromatography matrix, said antibody being specific to MIS,

(b) substantially removing contaminating enzymes having MIS proteolytic activity or inhibitors of MIS antiproliferative activity by adding to the matrix an effective amount of an alkali metal halide or an alkaline earth metal halide, and

(c) recovering the MIS by eluting with an acid solution having a pH of between about 2.5 and 4.0.

This invention further provides for a composition comprising MIS obtained by

(a) binding the MIS to an antibody-chromatography matrix, said antibody being specific to MIS,

(b) substantially removing contaminating enzymes having MIS proteolytic activity or inhibitors of MIS antiproliferative activity by adding to the matrix an effective amount of an alkali metal halide or an alkaline earth metal halide, and

(c) recovering the MIS by eluting with an acid solution having a pH of between about 2.5 and 4.0.

Also provided by this invention is a composition comprising MIS, wherein said composition is substantially free of enzymes having MIS proteolytic activity or inhibitors of antiproliferative activity of MIS. Such a composition consists essentially of MIS having a molecular weight of 140 kDa or 70 kDa.

BRIEF DESCRIPTION OF THE DRAWING

The present invention will be better understood by reference to the Description of the Preferred Embodiments when taken together with the attached drawing, wherein:

FIG. 1 shows the sequence structure of rhMIS (SEQ. ID. NO:l) compared to bovine (SEQ. ID. NO: 2) and rat (SEQ. ID. NO: 3) MIS.

DESCRIPTION OF THE PREFERRED EMBODIMENTS

The present invention provides for an improved process for the purification of Miillerian Inhibiting Substance (MIS). This process takes advantage of the specificity of antigen-antibody interactions to recover a product having a substantially pure MIS. Specifically, this invention incorporates the use of immunoaffinity chromatography. The added benefit of this invention is that the immunoaffinity chromatography method is improved such that the recovered MIS product is substantially

free of contaminating enzymes having MIS proteolytic activity or inhibitors of antiproliferative activity of MIS.

Immunoaffinity chromatography is a type of chromatography that makes use of a specific affinity between a substance to be isolated, i.e., a ligand, and a molecule that it can specifically bind. The column material is synthesized by covalently coupling a binding molecule to an insoluble matrix. The column material is able to specifically absorb the substance to be isolated from solution. Elution of the product is accomplished by changing the conditions within the column such that the product is released. This procedure either effects the binding site directly or effects the structure of the bound molecule.

If interaction of the ligand to the binder is primarily electrostatic, desorption can be accomplished by a gradient of increasing ionic strength. Ionic strength can be altered by changing pH. Such a change can alter the degree of ionization of charged groups either on the ligand or at the binding site. In desorbing proteins, however, there may be a secondary effect on the protein molecule which results in a confoπnational change. When binding is due to a hydrophobic interaction, solvents with reduced polarity are effective in desorbing the bound molecule.

In purifying nucleic acids and proteins, desorption has been generally accomphshed with the use of chaotropic substances such as CI0 ₄ ^" , CF ₃ COO ^" , ^" SCN, and CCI ₃ COO ^" . These substances are useful because they are able to break down very strong interactions.

As described by Pepinsky et al, supra, MIS has been isolated by an immunoaffinity chromatography method. According to this method, MIS is eluted from the matrix by a standard chaotropic salt, NaSCN, and can be recovered at up to 95% purity. Evaluation of MIS when purified according to previously used immunoaffinity methods indicates that the

protein has an apparent mass of 70 kDa with fractions also existing as 57 kDa and 12.5 kDa.

As demonstrated by the Example herein, the presence of the 57 kDa and 12.5 kDa fragments are the result of proteolysis or inhibition of the activity of the MIS molecule. Using previously described immunoaffinity methods, contaminating proteolytic enzymes or inhibitors of antiproliferative activity of MIS can be eluted with the pure MIS product. This occurs even though these methods are able to achieve an MIS product of up to 95% purity. The proteolytic enzymes initially cleave the MIS molecule into 57 kDa and 12.5 kDa fragments to activate the molecule. However, further cleavage of the 57 kDa fragment to a 34 kDa fragment and to a 22 kDa fragment occurs. Substances which cleave MIS in this manner include serin e proteases, such as plasmin, and endopeptidases. These enzymes are not to be considered as all inclusive or limiting in any manner since other enzymes can also proteolytically cleave MIS and such enzymes can be readily determined by those of ordinary skill in the art.

In order to achieve a substantially pure MIS which will be free of contaminating proteases or inhibitors of antiproliferative activity of MIS, the present invention improves upon the immunoaffinity chromatography method previously employed. This improvement results in a composition which comprises MIS and is substantially free of enzymes having MIS proteolytic activity or inhibitors of antiproliferative activity of MIS.

The immunoaffinity chromatography method of this invention is improved by removing contaminating enzymes having MIS proteolytic activity or inhibitors of antiproliferative activity of MIS from an immunoaffinity chromatography matrix by eluting with an effective amount of an alkali metal halide or an alkaline earth metal halide. The MIS is then recovered by eluting with an acid solution having a pH of

between about 2.5 and 4.0. This elution with halide followed by acid is alternatively referred to as sequential salt/acid elution.

The terms "Miillerian Inhibiting Substance" and "MIS", as alternatively used herein, are intended to include compounds and materials which are structurally similar to MIS. Examples of such included substances and materials are salts, derivatives, and aglycone forms of MIS. Additionally, the present invention is intended to include mutant forms of MIS which have substantially the same biological activity as MIS. Examples of such mutant forms would be MIS molecules carrying at least one deletion, insertion, or alteration in amino acid sequence. MIS may be obtained from any mammalian source or, as indicated above, from non-mammalian sources through the use of recombinant DNA technology, or from the chemical synthesis of the MIS protein. The purification method of this invention is preferably directed toward the recovery of recombinant human MIS.

The term "protein" is meant to include both synthetic and naturally- occurring amino acid sequences derivable from the naturally occurring amino acid sequence of MIS. The protein is said to be "derivable from the naturally-occurring amino acid sequence of MIS" if it can be obtained by fragmenting the naturally-occurring chosen sequence of MIS, or if it can be synthesized based upon a knowledge of the sequence of the naturally occurring amino acid sequence or of the genetic material (DNA or RNA) which encodes this sequence.

The invention further pertains to polypeptides that, in addition to the chosen sequence, may contain or lack one or more amino acids that may not be present in the naturally-occurring sequence, wherein such polypeptides are functionally similar to or possess antagonist activity to the chosen polypeptide. Such polypeptides for the present invention, are

-9-

termed "functional derivatives," provided that they demonstrate activity which is substantially similar to or antagonistic to that of MIS.

The MIS composition may be in the form of the free amines (on the N-terminus), or acid-addition salts thereof. Common acid solution salts are hydro halic acid salts, i.e., HBr, HI, or more preferably, HCl.

The purified MIS of this invention can be obtained in solution at up to 95% purity or greater. While the percent purity is comparable to other immunoaffinity purification processes, the MIS composition of this invention is substantially free of contaminating proteolytic enzymes or inhibitors of MIS antiproliferative activity.

For purposes of this invention, purified MIS is considered to be a MIS composition which is substantially free of contaminating proteolytic enzymes or inhibitors of MIS antiproliferative activity regardless of percent purity. The composition is considered to be substantially free of proteolytic enzymes if gel electrophoresis of the purified MIS product indicates a protein having a molecular weight of 140 kDa or 70 kDa. Gel electrophoresis of such a product will not show time dependent proteolytic fragments which are degradation products of MIS. For example the 57 kDa, 12.5 kDa, 34 kDa and 22 kDa degradation fragments of MIS further described herein will not be readily discernable by standard gel electrophoresis methods.

The MIS composition will be considered to be substantially free of inhibitors of MIS antiproliferative activity if the MIS blocks proliferation of certain tumor cells. Examples of such tumor cells are included herein and in U.S. Patent Application No. 07/683,966, filed April 12, 1991, and which is fully incorporated herein by reference. These examples include tumors selected from the group consisting of vulvar epidermoid carcinoma, endometrical adenocarcenoma, cervical carcenoma, endometrial adenocacenoma, ovarian adenoracenoma, and other ocular

melanoma. The determination of antiproliferation activity of these tumors as well as any other tumor can be achieved by any of the procedures described herein and in U.S. Patent Application No. 07/683,966.

In order to obtain MIS which is substantially free of proteolytic enzymes or inhibitors of MIS antiproliferative activity, the contaminants are separated from the MIS using immunoaffinity chromatography. Separation occurs by eluting the enzymes or inhibitors with an alkali metal halide or an alkaline earth metal halide. Such a compound will generally be in solution and an effective amount of halide will be between about 0.1 M and 2.0 M. As alkali metals, the ions of lithium, sodium and potassium are preferred with sodium being most preferable. As alkaline earth metals, the ions of magnesium and calcium are preferred. As hahdes, the ions of fluorine, chlorine, bromine and iodine are preferred with chlorine being most preferable. When eluting with sodium chloride, a solution of between about 0.1 and 2.0 M is preferred. The concentration of halide can also be varied as elution progresses if desired. This can be accomphshed by increasing molar concentration of halide in a stepwise fashion. It is preferred that each step be altered after about 0.1-2.0 bed volumes of solution have contacted the chromatography matrix, although such steps can be further modified as desired.

The halide can also be accompanied in solution with an effective amount of a chelating agent These agents are capable of binding metal ions which can inactivate enzymes that require the metal ions for activity. Such agents include the compounds ethylenediamine tetraacetate (EDTA), and ethylenebis- (oxyethylenenitrilo)tetraacetic acid (EGTA). Chelants can be effectively added in a range of between 0.1 and 50 mM.

After the contaminating proteolytic enzymes or inhibitors of MIS antiproliferative activity have been separated, the MIS can be recovered

by eluting with an acid solution having a pH of between about 2.0 and 4.0. Although dilutions of strong acids such as HCl can be used, organic acids are preferred because of their relatively mild acid strength. For example, the use of acid amines and i ines can be employed as well as monocarboxylic, dicarboxylic and tricarboxylic acids. Preferred as monocarboxylic acids are acetic, propionic and butyric acid. Preferred as dicarboxylic acids are. succinic, fumaric and malic acid. Preferred as a tricarboxylic acid is citric acid. Preferred among the amines are the acidic amino acids such as aspartic and glutamic acid. The pH of the acid solution can also be incrementally varied as in the application of the halide.

After the purified MIS product has been eluted, it is preferable to neutralize the product to a pH of between 6.8 and 7.6 to guard against acid hydrolysis. This can be accomphshed by various hydroxide compounds such as NaOH or NH OH or by various buffers which can quickly achieve neutralization of the product in the desired pH range. These hydroxide compounds are not to be considered as all inclusive as those of ordinary skill in the art will appreciate.

Preparation of an immunoaffinity chromatography column is well known in the art as demonstrated by Harlow et al, Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory, 1988. For purposes of this invention it is preferable that the chromatography column have a matrix which can be coupled to an antibody specific to MIS. The matrix will generally be solid phase support which binds antibody easily and is commercially available. For example, Protein A can be used as the chromatography matrix. This matrix specifically binds to the Fc domain of antibodies and, after the antibody is bound, the interaction is stabilized by cross-hnking with a bifunctional coupling reagent. In preparing a

protein A bead-antibody column, any bifunctional coupling reagent can be used. The use of methylpimelimidate is generally preferred.

Another method of preparing an immunoaffinity column couples the antibody to an activated bead. The beads are activated chemically to contain reactive groups. Examples of beads which can be used in this method are agarose beads, cross -linked agarose beads, polyacrylamide beads, copolymers of polyacrylamide and agarose, and polyacrylic beads. Compounds which are generally used to activate the beads are carbonyldiimidoxole, cyanogen bromide, glutaraldehyde, hydroxysuccinimide, and tosyl chloride. After the beads are activated, they are then mixed and coupled with purified antibodies.

Yet another method of coupling antibodies to beads is to activate the antibody first Purified antibodies can be activated by treating with a bifunctional reagent, one group binding to an appropriate group on the antibody and the other remaining free to bind to the matrix. Reagents which can generally be used for indirect coupling are the water-soluble carbodiimides such as dicyclohexylcarbodiimide (DCCD), l-ethyl-3-(3- dime__ylaminopropyI)-carbodiimide HCl (EDAC or DECI), or 1- (yclohe_yl-3-(2-morphoIinoethyl)-carbodiimide-metho-β-tolue ne-sulfonate (CMIC or CMLI); the condensing agents for peptide synthesis such as N- ethoxycarbonyl-2-ethoxy-l,2-dihydroquinoline (EEDQ); glutaraldehyde; or periodate. After the antibodies are activated, they are bound to beads.

Having now generally described this invention, the same will be better understood by reference to a certain, specific example which is included herein for purposes of illustration only and is not intended to be limiting of the invention, unless specified.

EXAMPLE I. Materials and Methods

A. RhMIS Purification

Recombinant human MIS (rhMIS) was purified from the conditioned media of Chinese hamster ovary (CHO) cells, transfected with a linear construct of the human rhMIS gene and the DHFR gene, amplified by 30 M Methotrexate selection, grown to confluence in four liter bioreactors on stainless steel coils as described by Epstein (Epstein et al, In Vitro Cell, and Devel Biol 25(2):213-6 (1989)) or modified roller bottles in alpha-Modified Eagle's Medium (α-MEM-), supplemented with 5% female fetal calf serum (FFCS), 10 mg/ml Amikacin, 1.3 g/1 1- glutamine, 2.0 g/1 d-glucose, and 0.1 g/1 sodium pyruvate, in the absence of nucleosides. The medium was collected every 3-4 days, and stored at -20 °C. Media were thawed and filtered through Whatman #4 filter paper to remove debris, concentrated 20x an a Minitan (Millipore) with a 30 kDa exclusion ultrafilter, and stored at -70 °C, until purification. A 5 ml immunoaffinity column was constructed using approximately 50 mg of a Protein A-Sepharose (BioRad) purified monoclonal anti-human rhMIS antibody [6E11], as described by Hudson (Hudson et al, J. Clin. Endocrinol. 70:16-22 (1990)), and covalently attached to Affigel-10 agarose resin (BioRad), per the manufacturer's instructions (approximately 80% coupling efficiency). The column was equilibrated with 100 is of 20 mM HEPES, pH 7.4, and 200 ml of the concentrated media loaded at 1 column volume/hour at 4°C. After loading, the column was washed with 20 mM HEPES, pH 7.4, until the absorbance at 280 nm returned to baseline (60-100 mis).

Elution of rhMIS bound to this column was achieved using 2.0 M sodium thiocyanate (NaSCN); or 1 M Acetic acid, 20 mM HEPES, pH 3.0, with and without a pre-elution step containing 0.5 M NaCl, 1 mM

EDTA, 0.001% NP-40, 20 mM HEPES, pH 7.4. The majority of the rhMIS protein eluted in a single 2 ml fraction, which was immediately desalted by G-25 size exclusion chromatography in 0.02 M HEPES, 0.15 M NaCl, 10% glucose, pH 7.4, in the case of chaotropic salt elution, or immediately neutralized with either NaOH or NH OH to a pH between 7.0 and 7.4, when eluted with acid. Depending on the initial pH of the fraction and technique, of neutralization, dilutional effects ranged from 10- 50%. The resultant rhMIS was examined for total protein according to the method of Bradford (Bradford, Anal Biochem. 72:248-54 (1976)), for rhMIS concentrations by enzyme-linked immunosorbant assay [ELISA] according to the method of Hudson (Hudson et al, J. Gin. Endocrinol 70:16-22 (1990)), on polyacrylamide gel electrophoresis [PAGE] according to the method of Weber (Weber et al, J. Biol Chem. 244(16): A06-12 (1969)), in western blot analysis using polyclonal (anti-homo, and anti -N- and C-terminal peptide) antibodies to rhMIS according to the method of Towbin (Towbin et al, PNAS 76:4350-4 (1979)), for NH ₂ -terminaI sequencing, in organ culture bioassay for Miillerian duct regression, and in tumor antiproliferative assays.

B. RhMIS Bioassay

The standard organ culture bioassay for MIS was performed as described by Donahoe (Donahoe el al, Biol. Reprod. 1(5:238-43 (1977)). Briefly, 14V_ day female fetal rat urogenital ridges were placed on agar coated stainless steel grids above fortified CMRL 1066 media to which test preparations were added at concentrations less than 20% (v v). After incubation for 72 hours in humidified 5% C0 ₂ at 37 °C, the specimens were ahgned, fixed in 15% foimalin, embedded in paraffin, cut in 8 mm cross sections from cephalic to caudal, and then stained with hematoxylin and eosin. The sections were then graded from 0 (no regression) to 5

(complete regression). Female fetal calf serum was used to avoid contamination of the assay with bovine MIS. This change required the addition of 10 ^"9 M testosterone to aid the expression of the Wolffian duct for morphologic comparison according to the method of Ikawa (Ikawa et al, J. Ped. Surg. 17:453 (1982)).

C. RhMIS ELISA

The enzyme linked immunosorbent assay (ELISA), employing anti- rhMIS monoclonal and polyclonal antibodies, was used to measure the rhMIS content in all preparations. This assay, described by Hudson (Hudson et al., /. Gin. Endocrinol 70:16-22 (1990)), detects intact rhMIS with a sensitivity of 1-2 ng/ml, with minimal cross reactivity with N- and C-terminal fragments of rhMIS, and no recognition of Transforming Growth Factor β (another member of the same gene family as MIS), the humor gonadotropin, follicle stimulating hormone, and luteinizing hormone, or proteins contained in conditional medium of wild type CHO cells.

D. Antiproliferative Assays

Immunoaffinity purified rhMIS was assayed for anti turn or activity as described by U.S. Patent Application No. 07/683,966 using a double layer agarose colony inhibition assay as described by Fuller (Fuller el al, Gynecol Oncol. 22:135-48 (1985)). Human cancer cell lines, including A431, Hep3b, HEC1, OM431, OM467, and RT4, were plated on an underlayer of 0.6% agarose in α-MEM-. and FFCS. After addition of rhMIS or vehicle control, plates were incubated for 14 days at 37°C, 5% C0 ₂ , and colonies of more than 30 cells were counted. A liquid media colony inhibition assay that requires fewer cells and 5-7 day incubation

period was also used. Colonies were counted using a computer aided automated program.

E. RhMIS Antibody Preparations

Rabbit polyclonal antiserum against 140 kDa homo rhMIS (MGH- 1), electroeluted from polyacrylamide gels according to the method of Hunkapillar (Hunkapillar et al, Meth. Enzymol 91:486 (1983)), was prepared as previously described by Hudson (Hudson et al, J. Gin, Endocrinol 70:16-22 (1990)). Epitope specific rabbit polyclonal antibodies were raised to regions of the rhMIS molecule NH ₂ -terminal (MGH-N1) or COOH-terminal (MGH-C1) to the monobasic consensus cleavage site at position 427. Synthetic peptides corresponding to residues 411-424 and 471-482 in rhMIS, respectively, constructed from the predicted amino acid sequence of human rhMIS (FIG. 1) were used as antigens. These sequences were chosen for their conserved homology among human, bovine and rat MIS, their differences from other members of the supergene family, and their antigenicity and surface probability (i.e., the likelihood that a specific region will present itself for antigen recognition), as predicted by the sequence analysis software package of the Genetics Computer Group at the University of Wisconsin [version 5]. This program measures hydrophobicity as outlined by Chou and Fasman (Chou et al, Advances in Enτymology 47:45-141 (1978)), and antigenicity and surface probability by the method of Wolf et al (Wolf et al, Comput. Appl Biosci. 4(1):187-91 (1988)).

New Zealand white rabbits were injected in the popliteal lymph nodes with 25-50 μg of relevant peptide, conjugated 1:1 with keyhole limpet hemocyanin (Calbiochem) by 0.25% glutaraldehyde crosslinking, in complete Freund's adjuvant These animals were boosted by subcutaneous injection in the back, 4-6 weeks later, with 20-30 μg of

unconjugated peptide in incomplete Freund's adjuvant. Animals were bled through an ear vein and the serum stored at -20°C. Polyclonal antiserum was purified by 50% (NH ₄ ) ₂ S0 ₄ precipitation, followed by Protein A-Sepharose chromatography. The monoclonal antibody 6E11 was raised according to the method of Kohler (Kohler, G, Immunol. Methods 2:285-98 (1981)) and purified by Protein-A Sepharose affinity chromatography. Control antisera were purchased from DAKO.

F. Temperature, pH, and Storage Effects on rhMIS Preparations

Immunoaffinity purified rhMIS was sterile filtered using a Millex-

GV 0.22 μm filter unit (MILLIPORE), divided into 200 μl aliquots and incubated at -80°C, -20°C, +4°C, +25°C, +36°C, and +70°C to determine temperature stability of the rhMIS molecule. To observe the effects of storage at various temperatures, aliquots of rhMIS at 3, 7, 11,

14 and 31 days, were subjected to Bradford protein assay (Bradford,

M.M., Anal Biochem 72:248-54 (1976)), ELISA polyacrylamide gel electrophoresis in sodium dodecyl sulfate (SDS-PAGE), western blot analysis, organ culture bioassay, and selected amino-terminal analysis.

Additionally, preparations of rhMIS were stored at pH 3.0 for 1, 2, and

4 hours, and 2, 3, 5, and 8 days at + 4°C and +36°C. For comparison, a non-cleavable mutant of rhMIS, produced by site-directed mutagenesis of

Arg ⁴²⁷ to Thr ⁴²⁷ , was tested after a days storage at 37°C.

G. Enzyme Treatments

RhMIS was treated with plasmin for 2 hours, as described by Pepinsky et al, supra, for 1, 3, and 7 days, and with 0.1% trypsin for 1 hr at 4°C. The reactions were quenched with 10% fetal bovine serum. The effect of protease inhibitors was assessed with phenylmethyl sulfonyl fluoride, 0.1 M; soybean trypsin inhibitor, 1 mM; leupeptin, 1 M;

pepstatin, 1 mM; all used together as a mixture, or tosyllysine chloromethyl ketone, 1 mM, alone. Additionally, rhMIS was assayed for intrinsic enzyme activities. Protease activity was determined using azocasein (2 mg ml in 0.05 M Tris-Cl, pH 8.5) as a substrate (Worthington manual 1970). 0.9 ml was brought to 37°C in a water bath prior to addition of 0.1 ml of enzyme (1-20 mg/ml Subtilisin) or rhMIS. The reaction was allowed to proceed for 15 minutes at 37°C, and 0.25 ml 70% perchloric acid was added. The samples were placed on ice for 15 minutes, then centrifuged at 4°C to pellet the precipitate. Absorbance of the supernatant was measured spectrophotometrically at 405 nm. Acid and alkaline phosphatase activities were examined with Sigma chemical kit #104, which measures the ability to hydrolyze p-nitrophenyl phosphate.

H. Sequencing

Acid eluted, immunoaffinity purified rhMIS was submitted as "homo" rhMIS for NH ₂ -terminal sequencing. A similar preparation was heated to 37°C for 8 days to allow cleavage of 70 kDa intact rhMIS to 57 kDa, 34 kDa, 22 kDa, and 12.5 kDa as determined by SDS-PAGE. These bands were then electroeluted from the gel, according to the method of Hunkapillar (Hunkapillar el al, Meth. Enzymol. 91:486 (1983)), and analyzed for N-terminal sequence. The sequential salt/acid eluted rhMIS was similarly submitted for sequence analysis except the cleavage was done by exogenous plasmin. The 34 kDa fragment, which required concentration by lyophilization prior to PAGE, was electrophoretically transferred to an Immobilon PVDF membrane (Millipore), stained, and sequenced. Edman degradation was performed on a model 470A Applied Biosystems gas phase sequencer; PTH-amino acids were analyzed on an on-line microbore HPLC (ABI model 120A).

I. Electrophoresis

Polyacrylamide gel electrophoresis (PAGE) was carried out by the procedure of Laemmli (Laemmli, Nature 227:680-5 (1970)), as follows: 10 or 15% homogeneous gels were prepared in Hoefer gel casting stands to produce 160 x 160 x 1.5 mm slabs, and gels run at 150 V, 30 mA constant current. Proteins in the gels were stained with 0.1% Coomassie Brilliant Blue R250 (Sigma) in 50% methanol, 10% acetic acid for 1 hour before destaining in 50% methanol, 10% acetic acid. As appropriate, samples were reduced using 0.75 M 2-mercaptoethanol with heating to boiling for 10 min. Molecular weight standards were obtained from BioRad.

For western analysis, gels were incubated in transfer buffer (3 g Tris, 14.4 g/1 glycine) prior to electrophoretic transfer to nitrocellulose or Immobilon-PVDF (Milligen Corp.) sheets according to the BioRad technical bulletin no. 83-0050. After the transfer, the sheets were used directly for microsequencing, or blocked by incubation with 30 mg/ml bovine serum albumin for 30 minutes at room temperature, with shaking. Thereafter, the blots were incubated with 1:500 to 1:1000 fold dilutions of rabbit polyclonal anti-rhMIS or anti-rhMIS peptide antisera for 2 hrs, then washed with 0.O5 M Tris-Cl, 0.15 M NaCl prior to addition of a 1:1000 dilution of goal anti-rabbit horseradish peroxidase conjugate (BioRad). Antibody complexes were visualized by the addition of BioRad color reagent for 1-4 minutes, prior to quenching the reaction with water.

For microsequencing, proteins were transferred according to the BioRad technical bulletin No. 83-0050 into Immobilon PVDF sheets and stained with Coomassie blue as described above for gels except that acetic acid was omitted.

J. Labelling of RhMIS

RhMIS, prepared by either sequential salt/acid or acid only elution, was iodinated by the chloramine T method of Hunter (Hunter, Proc. Soc. Exp. Biol. Med. 133(3):989-92 (1970)). Briefly, to 0.5 ml (0.3 mg/ml) of rhMIS, 10 μl of Na ¹²⁵ I (Amersham) and 10 μl of chloramine T (2.5 mg/ml; Sigma) were added. After 90 seconds, the reaction was quenched with 25 μl of NaHS0 ₃ (7.6 mg/ml). The mixture was desalted on a 10 ml Sephadex G-25 column, equilibrated in 20 mM HEPES, pH 7.4.

Biotinylation (Pierce Chemical Co. Bulletin No.21335) was carried out using water soluble N- hydroxysuccinimide-long chain alkyl-biolin. 0.5 ml of rhMIS (0.3 mg ml) was dialyzed overnight at 4°C vs PBS. 5 μl of 4.5 mg/ml NHS-LC-Biotin was added and the mixture incubated at 4°C with gentle agitation for 4 hours, then dialyzed overnight at 4°C vs PBS.

Fluorescent rhMIS was prepared with fluorescein isothiocyanate [FITC] according to the method of Johnson (Johnson el al, J. Biol. Chem. 257(10):5632-6 (1982)), as follows. Approximately 200 μg of rhMIS was dialyzed, overnight at 4°C, vs 2x4 1 O.05 M TRIS, pH 8.8. One tenth volume of fluorescein isothiocyanate (FITC) in TRIS 8.8 was added (50λ F_TC/500λ MIS), and the reaction allowed to proceed for 30 minutes at 4°C. The reaction mixture was desalted with a 10 ml Sephadex G-25 column equilibrated in PBS. Protein was monitored at A _2g0 and fluorescence at A ₉₃ . The protein and fluorescence peaks co-eluted with the void volume.

II. RESULTS

Immunoaffinity purified rhMIS migrates on SDS-PAGE under reducing conditions, at an apparent molecular weight of 70 kDa regardless of the method of elution. A 57 kDa minor band was also seen. These two bands, which represented approximately 90% of the Coomassie

Brilliant Blue stained protein on reducing gels, as well as the 140 kDa dimeric species in non-reducing gels, bind polyclonal antibody against rhMIS dimer. RhHIS subjected to PAGE without SDS, or under conditions of reverse polarity, failed to enter the gel. The acid elution of rhMIS from the immunoaffinity column resulted in higher yields and greater purity than did chaotropic elution (Table 1). Sequential salt/acid elution resulted in rhMIS being substantially free from enzymes having MIS proteolytic activity.

Table 1

RhMIS prepared by single step elution with either chaotropic salt or acid alone undergoes a time and temperature dependent cleavage that is blocked by proteolytic inhibitors. RhMIS prepared by sequential salt and acid elution does not undergo endogenous proteolytic processing. The major species after cleavage are monomers of 57 kDa and 12.5 kDa, consistent with cleavage at residue 427. Amino acid sequence of freshly purified rhMIS yields the known amino terminal sequence LRAEEPAVGT (SEQ. ID. NO: 4). After cleavage, a second sequence (AAGATAADGP SEQ. ID. NO: 5) is found which, except for the first

amino acid, matches the carboxy-terminal sequence of rhMIS beginning at residue 428.

The 34 kDa moiety, seen with prolonged incubation of acid only eluted rhMIS at +36 °C, was sequenced successfully after lyophilization and electrophoretic transfer to PVDF membrane. This species yielded the NH ₂ -term_naI sequence of rhMIS (i.e., LRAEEPAVGT, SEQ. ID. NO. 4), indicative of a second processing event N-terminal to that at residue 427. Sequential salt/acid eluted material showed this same band and the 57 and 12.5 kDa bands after treatment with exogenous plasmin. Occasionally, an additional fragment with apparent molecular weight of 22 kDa was seen, but this fragment was not sequenced. Furthermore, the polyclonal antibodies raised to peptides constructed from the predicted amino acid sequence, both C-terminal or N-terminal to the monobasic protease cleavage site (residue 427), recognized 140 kDa "homo" rhMIS on western blots. Antibody MGH-N1, raised to the peptide corresponding to residues 411-424, recognized only 70 kDa and 57 kDa species. C-terminal anti-peptide (471-482) antibody MGH-C1 recognized the 12.5 kDa species seen in 36 °C treated samples, as well as uncleaved, reduced rhMIS (70 kDa). Additionally, MGH-C1 weakly recognized cleaved N-terminal 57 kDa rhMIS, indicating possible crossreactivity, due to the polyclonal nature of the antibody which was raised to KLH conjugated peptide. A polyclonal antibody, termed MGH-1, which was raised to intact immunoaffinity purified rhMIS, recognized the 140 kDa, 70 kDa, and 57 kDa species, as well as the 34 kDa and 22 kDa fragments. These antibodies recognized similar bands on western blots of a less homogeneous, dye affinity purified rhMIS.

Although protein levels measured by Bradford analysis do not change with the time of incubation, the ELISA values decrease as cleavage increases. In spite of the proteolytic cleavage, however, rhMIS

remains biologically active even if the protein is stored at neutral pH for 1 month at temperatures up to 36°C. RhMIS activity is lost, however, by heating to 70 °C for 4 days, by treatment with 0.1% Trypsin, and by storage for greater than 24 hours in acid (1 M HAc), at 36°C. Acid storage at 4°C loses activity more slowly (30-50% at 8 days).

Iodinated, biotinylated, and fluoresceinated rhMIS were all active in the organ culture bioassay at approximately 50% of their prelabelling levels. FITC labelled rhMIS was immunoreactive in the ELISA at only 20% of its original level, while iodinated or biotinylated material retained virtually all of its immunoreactivity. All labelled preparations showed the characteristic rhMIS bands on PAGE.

To determine if rhMIS acts as an enzyme, i.e., an auto protease, it was assayed in a standard subtilisin azocasein assay. RhMIS eluted with acid alone showed subtilisin-like activity of 1.3 μg/mg rhMIS, in the azocasein assay. Sequential salt/acid eluted material had no activity in this assay, indicating that the co-purifying protease had been effectively separated from rhMIS. By effectively separated, it is to be understood that the protease had been actually removed or inactivated such that it was no longer effective in cleaving the MIS molecule.

Sequential salt/acid eluted rhMIS, treated with plasmin for 2 and 24 hours, yielded the predicted cleavage at Arg ⁴²⁷ , to generate the 57 kDa and 12.5 kDa moieties; these were also seen after prolonged processing of acid-only eluted rhMIS at 36°C. Reaction of sequential salt/acid eluted rhMIS with plasmin for longer periods (3-7 days) shows that after the initial cleavage, the 57 kDa fragment is further cleaved to the 34 kDa species. This species was also observed after prolonged storage at 36 °C of rhMIS purified without the salt pre-elution step. The 34 kDa moiety, generated from the sequential salt/acid eluted rhMIS by prolonged plasmin treatment, was sequenced to yield the NH ₂ -terminal sequence of

MIS, thus indicating a secondary cleavage event to yield the 34kDa fragment.

To address the frustratingly variable or absent response of rhMIS purified without the salt elution step in antiproliferative assays reported by Wallen (Wallen et al, Cancer Res. 49:2005-11 (1989)), the further refined rhMIS preparations (sequential salt/acid eluted MIS) were tested in similar assays. The rhMIS produced following the salt pre-elution step retains all previous activities, and further is consistently inhibitory in colony inhibition, cell cycle, and subrenal capsule protocols. Examination of the proteins eluted by the 0.5 M salt wash shows 3 low molecular weight bands on reducing gels, which were not recognized by either MGH-1 (polyclonal anti-"homo"rhMIS) or MGH-C1 (polyclonal anti-C- terminal peptide).

Analysis of the sequence structure of rhMIS (FIG. 1, SEQ. ID. NO: 1) revealed a string of nine leucine repeats beginning at residue 350, followed by a basic region immediately preceding the Arg cleavage site. This string of repeats strongly resembles a leucine zipper as reported by Vinson (Vinson et al, Science 246 4932):911-6 (1989)). Additionally, there are several other leucine rich regions upstream of this zipper, probably representing the hydrophobic core of the molecule. One of the stretches, beginning at Leu ²⁶⁶ , shows similarities to the S4 domain of the gated ion channels (Tempel et al, Nature 332:831 (1988)). There was also present at positions 194-8, the sequence RGEDS, strikingly similar to the RGDS site for fibronectin binding, which may account for the recognition of a basement membrane binding site for rhMIS by antibody to NH2- terminal rhMIS peptide.

Having now fully described this invention, it will be appreciated by those skilled in the art that the same can be performed within a wide range of equivalent parameters of composition and conditions without

departing from the spirit or scope of the invention or of any embodiment t th-ioer-eeo-*f-.

SEQUENCE LISTING

(1) GENERAL INFORMATION:

(i) APPLICANT: Donahoe, Patricia K Ragin, Richard C HacLaug lin, David T

(ii) TITLE OF INVENTION: Purification of Mullenan Inhibiting Substance

(iii) NUMBER OF SEQUENCES: 5

Civ) CORRESPONDENCE ADDRESS:

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

(B) STREET: 1225 Connecticut Ave. NU Suite 300 CO CITY: Washington

(D) STATE: DC

(E) COUNTRY: USA CF) ZIP: 20036

(v) COMPUTER READABLE FORM:

CA) MEDIUM TYPE: Floppy disk

CB) COMPUTER: IBM PC compatible

CC) OPERATING SYSTEM: PC-DOS/MS-DOS

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

(vϊ) CURRENT APPLICATION DATA:

CA) APPLICATION NUMBER: US 07/683,957

CB) FILING DATE: 12-APR-1991

CC) CLASSIFICATION:

Cviti) ATTORNEY/AGENT INFORMATION:

(A) NAME: Jordan, Richard D

CB) REGISTRATION NUMBER: 33,519

CC) REFERENCE/DOCKET NUMBER: 0609.3060000

(ix) TELECOMMUNICATION INFORMATION:

CA) TELEPHONE: C202) 466-0800

CB) TELEFAX: C202) 833-8716

(2) INFORMATION FOR SEQ ID NO:1:

Ci) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 533 amino acids

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

(ii) MOLECULE TYPE: protein

Cxi) SEQUENCE DESCRIPTION: SEQ ID NO:1:

Ala Glu Glu Pro Ala Val Gly Thr Ser Gly Leu lie Phe Arg Glu Asp 1 5 10 15

Leu Asp Trp Pro Pro Gly He Pro Gin Glu Pro Leu Cys Leu Val Ala 20 25 30

Leu Gly Gly Asp Ser Asn Gly Ser Ser Ser Pro Leu Arg Val Val Gly 35 40 45

Ala Leu Ser Ala Tyr Glu Gin Ala Phe Leu Gly Ala Val Gin Arg Ala 50 55 60

Arg Trp Gly Pro Arg Asp Leu Ala Thr Phe Gly Val Cys Asn Thr Gly 65 70 75 80

Asp Arg Gin Ala Ala Leu Pro Ser Leu Arg Arg Leu Gly Ala Trp Leu 85 90 95

Arg Asp Pro Gly Gly Gin Arg Leu Val Val Leu His Leu Glu Glu Val

100 105 110

Thr Trp Glu Pro Thr Pro Ser Leu Arg Phe Gin Glu Pro Pro Pro Gly 115 120 125

Gly Ala Gly Pro Pro Glu Leu Ala Leu Leu Val Leu Tyr Pro Gly Pro 130 135 140

Gly Pro Glu Val Thr Val Thr Arg Ala Gly Leu Pro Gly Ala Gin Ser 145 150 155 160

Leu Cys Pro Ser Arg Asp Thr Arg Tyr Leu Val Leu Ala Val Asp Arg 165 170 175

Pro Ala Gly Ala Trp Arg Gly Ser Gly Leu Ala Leu Thr Leu Gin Pro 180 185 190

Arg Gly Glu Asp Ser Arg Leu Ser Thr Ala Arg Leu Gin Ala Leu Leu 195 200 205

Phe Gly Asp Asp His Arg Cys Phe Thr Arg Met Thr Pro Ala Leu Leu 210 215 220

Leu Leu Pro Arg Ser Glu Pro Ala Pro Leu Pro Ala His Gly Gin Leu 225 230 235 240

Asp Thr Val Pro Phe Pro Pro Pro Arg Pro Ser Ala Glu Leu Glu Glu 245 250 255

Ser Pro Pro Ser Ala Asp Pro Phe Leu Glu Thr Leu Thr Arg Leu Val 260 265 270

Arg Ala Leu Arg Val Pro Pro Ala Arg Ala Ser Ala Pro Arg Leu Ala 275 280 285

Leu Asp Pro Asp Ala Leu Ala Gly Phe Pro Gin Gly Leu Val Asn Leu 290 295 300

Ser Asp Pro Ala Ala Leu Glu Arg Leu Leu Asp Gly Glu Glu Pro Leu 305 310 315 320

Leu Leu Leu Leu Arg Pro Thr Ala Ala Thr Thr Gly Asp Pro Ala Pro 325 330 335

Leu His Asp Pro Thr Ser Ala Pro Trp Ala Thr Ala Leu Ala Arg Arg 340 345 350

Val Ala Ala Glu Leu Gin Ala Ala Ala Ala Glu Leu Arg Ser Leu Pro 355 360 365

Gly Leu Pro Pro Ala thr Ala Pro Leu Leu Ala Arg Leu Leu Ala Leu 370 375 380

Cys Pro Gly Gly Pro .Gly Gly Leu Gly Asp Pro Leu Arg Ala Leu Leu

385 390 395 400

Leu Leu Lys Ala Leu Gin Gly Leu Arg Val Glu Trp Arg Gly Arg Asp 405 410 415

Pro Arg Gly Gly Arg Ala Gin Arg Ser Ala Gly Ala Thr Ala Ala Asp 420 425 430

Gly Pro Cys Ala Leu Arg Glu Leu Ser Val Asp Leu Arg Ala Glu Arg 435 440 445

Ser Val Leu lie Pro Glu Thr Tyr Gin Ala Asn Asn Cys Gin Gly Val

450 455 460

Cys Gly Trp Pro Gin Ser Asp Arg Asn Pro Arg Tyr Gly Asn His Val 465 470 475 480

Val Leu Leu Leu Lys Met Gin Ala Arg Gly Ala Ala Leu Ala Arg Pro 485 490 495

Pro Cys Cys Val Pro Thr Ala Tyr Ala Gly Lys Leu Leu He Ser Leu 500 505 510

Ser Glu Glu Arg lie Ser Ala His His Val Pro Asn Met Val Ala Thr 515 520 525

Glu Cys Gly Cys Arg

530

(2) INFORMATION FOR SEQ ID H0:2:

Ci) SEQUENCE CHARACTERISTICS:

CA) LENGTH: 550 amino acids

CB) TYPE: amino acid CD) TOPOLOGY: linear

(ii) MOLECULE TYPE: protein

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

Glu Glu Val Phe Ser Thr Ser Ala Leu Pro Arg Glu Gin Ala Thr Gly 1 5 10 15

Ser Gly Ala Leu He Phe Gin Gin Ala Trp Asp Trp Pro Leu Ser Ser 20 25 30

Leu Trp Leu Pro Gly Ser Pro Leu Asp Pro Leu Cys Leu Val Thr Leu 35 40 45

His Gly Ser Gly Asn Gly Ser Arg Ala Pro Leu Arg Val Val Gly Val 50 55 60

Leu Ser Ser Tyr Glu Gin Ala Phe Leu Glu Ala Val Arg Arg Thr His 65 70 75 80

Trp Gly Leu Ser Asp Leu Thr Thr Phe Ala Val Cys Pro Ala Gly Asn 85 90 95

Gly Gin Pro Val Leu Pro His Leu Gin Arg Leu Gin Ala Trp Leu Gly 100 105 110

Glu Pro Gly Gly Arg Trp Leu Val Val Leu His Leu Glu Glu Val Thr 115 120 125

Trp Glu Pro Thr Pro Leu Leu Arg Phe Gin Glu Pro Pro Pro Gly Gly 130 135 140

Ala Ser Pro Pro Glu Leu Ala Leu Leu Val Val Tyr Pro Gly Pro Gly 145 150 155 160

Leu Glu Val Thr Val Thr Gly Ala Gly Leu Pro Gly Thr Gin Ser Leu 165 170 175

Cys Leu Thr Ala Asp Ser Asp Phe Leu Ala Leu Val Val Asp His Pro 180 185 190

Glu Gly Ala Trp Arg Arg Pro Gly Leu Ala Leu Thr Leu Arg Arg Arg 195 200 205

Gly Asn Gly Ala Leu Leu Ser Thr Ala Gin Leu Gin Ala Leu Leu Phe 210 215 220

Gly Ala Asp Ser Arg Cys Phe Thr Arg Lys Thr Pro Ala Leu Leu Leu 225 230 235 240

Leu Leu Pro Ala Arg Ser Ser Ala Pro Met Pro Ala His Gly Arg Leu 245 250 255

Asp Leu Val Pro Phe Pro Gin Pro Arg Ala Ser Pro Glu Pro Glu Glu 260 265 270

Ala Pro Pro Ser Ala Asp Pro Phe Leu Glu Thr Leu Thr Arg Leu Val 275 280 285

Arg Ala Leu Ala Gly Pro Pro Ala Arg Ala Ser Pro Pro Arg Leu Ala

290 295 300

Leu Asp Pro Gly Ala Leu Ala Gly Phe Pro Gin Gly Gin Val Asn Leu

305 310 315 320

Ser Asp Pro Ala Ala Leu Glu Arg Leu Leu Asp Gly Glu Glu Pro Leu

325 330 335

Leu Leu Leu Leu Pro Pro Thr Ala Ala Thr Thr Gly Val Pro Ala Thr

340 345 350

Pro Gin Gly Pro Lys Ser ^' Pro Leu Trp Ala Ala Gly Leu Ala Arg Arg

355 360 365

Val Ala Ala Glu Leu Gin Ala Val Ala Ala Glu Leu Arg Ala Leu Pro

370 375 380

Gly Leu Pro Pro Ala Ala Pro Pro Leu Leu Ala Arg Leu Leu Ala Leu

385 390 395 400

Cys Pro Gly Asn Pro Asp Ser Pro Gly Gly Pro Leu Arg Ala Leu Leu

405 410 415

Leu Leu Lys Ala Leu Gin Gly Leu Arg Ala Glu Trp Arg Gly Arg Glu

420 425 430

Arg Ser Gly Ser Ala Arg Ala Gin Arg Ser Ata Gly Ala Ala Ala Ala

435 440 445

Asp Gly Pro Cys Ala Leu Arg Glu Leu Ser Val Asp Leu Arg Ala Glu

450 455 460

Arg Ser Val Leu lie Pro Glu Thr Tyr Gin Ala Asn Asn Cys Gin Gly

465 470 475 480

Ala Cys Gly Trp Pro Gin Ser Asp Arg Asn Pro Arg Tyr Gly Asn His

485 490 495

Val Val Leu Leu Leu Lys Met Gin Ala Arg Gly Ala Thr Leu Ala Arg

500 505 510

Pro Pro Cys Cys Val Pro Thr Ala Tyr Thr Gly Lys Leu Leu He Ser

515 520 525

Leu Ser Glu Glu Arg He Ser Ala His His Val Pro Asn Met Val Ala

530 535 540

Thr Glu Cys Gly Cys Arg

545 550

(2) INFORMATION FOR SEQ ID H0:3:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 528 amino acids

(B) TYPE: amino acid

CD) TOPOLOGY: linear Cii) MOLECULE TYPE: protein

Cxi) SEQUENCE DESCRIPTION: SEQ ID NO:3:

Glu Glu Leu Thr Asn Thr Arg Gly Leu He Phe Leu Glu Asp Gly Val 1 5 10 15

Trp Pro Pro Ser Ser Pro Pro Glu Pro Leu Cys Leu Val Ala Val Arg 20 25 30

Gly Glu Gly Asp Thr Ser Lys Ala Ser Leu Thr Val Val Gly Gly Leu 35 40 45

His Ser Tyr Glu Gin Ala Phe Leu Glu Ala Val Gin Glu Ser Arg Trp 50 55 60

Gly Pro Gin Asp Leu Ala Thr Phe Gly Val Cys Ser Thr Asp Ser Gin 65 70 75 80

Thr Thr Leu Pro Ala Leu Gin Arg Leu Gly Ala Trp Leu Gly Glu Thr 85 90 95

Gly Glu Gin Gin Leu Leu Val Leu His Leu Ala Glu Val He Trp Glu 100 105 110

Pro Gin Leu Leu Leu Lys Phe Gin Glu Pro Pro Pro Gly Gly Ala Ser 115 120 125

Arg Trp Glu Gin Ala Leu Leu Val Leu Tyr Pro Gly Pro Gly Pro Gin 130 135 140

Val Thr Val Thr Gly Ala Gly Leu Gin Gly Thr Gin Ser Leu Cys Pro 145 150 155 160

Thr Arg Asp Thr Arg Tyr Leu Val Leu Thr Vel His Phe Pro Ala Gly 165 170 175

Ala Trp Ser Gly Ser Gly Leu Ala Leu Thr Leu Gin Pro Ser Lys Glu 180 185 190

Gly Ala Thr Leu Thr He Ala Gin Leu Gin Ala Phe Leu Phe Gly Ser 195 200 205

Asp Ser Arg Cys Phe Thr Arg Lys Thr Pro Thr Leu Val Leu Leu Pro 210 215 220

Pro Thr Gly Pro Thr Pro Gin Pro Ala His Gly Gin Leu Asp Thr Val 225 230 235 240

Pro Phe Pro Gin Pro Gly Leu Ser Leu Glu Pro Glu Asp Leu Pro His 245 250 255

Ser Ala Asp Pro Phe Leu Glu Thr Leu Thr Arg Leu Val Arg Ala Leu 260 265 270

Arg Gly Pro Leu Thr Arg Ala Ser Asn Thr Arg Leu Ala Leu Asp Pro 275 280 285

Gly Ala Leu Ala Ser Phe Pro Gin Gly Leu Val Asn Leu Ser Asp Pro 290 295 300

Val Ala Leu Gly Arg Leu Leu Asp Gly Glu Glu Pro Leu Leu Leu Leu 305 310 315 320

Leu Ser Pro Ala Ala Ala. Thr Val Gly Glu Pro Met Arg Leu His Ser 325 330 335

Pro Thr Ser Ala Pro Trp Ala Ala Gly Leu Ala Arg Arg Val Ala Val 340 345 350

Glu Leu Gin Ala Ala Ala Ser Glu Leu Arg Asp Leu Pro Gly Leu Pro 355 360 365

Pro Thr Ala Pro Pro Leu Leu Ser Arg Leu Leu Ala Leu Cys Pro Asn 370 375 380

Asp Ser Arg Ser Ala Gly Asp Pro Leu Arg Ala Leu Leu Leu Leu Lys 385 390 395 400

Ala Leu Gin Gly Leu Arg Ala Glu Trp Arg Gly Arg Glu Gly Arg Gly

405 410 415

Arg Ala Gly Arg Ser Lys Gly Thr Gly Thr Asp Gly Leu Cys Ala Leu 420 425 430

Arg Glu Leu Ser Val Asp Leu Arg Ala Glu Arg Ser Val Leu He Pro 435 440 445

Glu Thr Tyr Gin Ala Asn Asn Cys Gin Gly Ala Cys Gly Trp Pro Gin 450 . 455 460

Ser Asp Arg Asn Pro Arg Tyr Giy Asn His Val Val Leu Leu Leu Lys 465 470 475 480

Met Gin Ala Arg Gly Ala Ala Leu Gly Arg Leu Pro Cys Cys Val Pro

485 490 495

Thr Ala Tyr Thr Gly Lys Leu Leu He Ser Leu Ser Glu Glu His He 500 505 510

Ser Ala His His Val Pro Asn Met Val Ala Thr Glu Cys Gly Cys Arg 515 520 525

C2) INFORHATIOfl FOR SEQ ID N0:4:

Ci) SEQUENCE CHARACTE ISTICS:

(A) LENGTH: 10 βmino acids

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

(ii) MOLECULE TYPE: peptide

Cxi) SEQUENCE DESCRIPTION: SEQ ID N0:4:

Leu Arg Ala Glu Glu Pro Ala Val Gly Thr 1 5 10

(2) INFORMATION FOR SEQ ID N0:5:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 10 amino acids CB) TYPE: amino acid CD) TOPOLOGY: linear

(ii) MOLECULE TYPE: peptide

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

Ala Ala Gly Ala Thr Ala Ala Asp Gly Pro 1 5 10