IE84906B1 - Cytotoxic Lymphocyte Maturation Factor - Google Patents

Abstract

ABSTRACT The present invention relates to a novel subunit of the cytokine protein called Cytotoxic Lymphocyte Maturation Factor (CLMF) which is produced and synthesized by a human B lymphoblastoid cell line. CLMF synergistically induces in the presence of low concentrations of IL-2 the cytolytic activity of Lymphokine Activated Killer (LAK) cells. CLMF is also capable of stimulating T-cell growth. The present invention also relates to cloned genes coding for the novel proteins and derivatives thereof, to recombinant vectors comprising a polynucleotide encoding said proteins, to microorganisms transformed with the said recombinant vectors, to antibodies directed to the said proteins as well as to processes for the preparation of the said proteins, vectors and antibodies.

Description

The present invention relates to the field of cytokines, in particular to those cytokines which synergize with interleukin-2 (IL-2) to activate cytotoxic lymphocytes such as the cytokine Cytotoxic Lymphocyte Maturation Factor (CHfl‘). The present invention also relates to monoclonal antibodies directed to cunx 'Cytokine' is one term for a group of protein cell regulators. variously called lymphokines, monokines, interleukins and interferons, which are produced by a wide variety of cells in the body. These cytokines play an important role in many physiological responses, are involved in the pathophysiology of a range of diseases, and have therapeutic potential. They are a heterogeneous group of proteins having the following characteristics in common.

They are low molecular weight (580 kDa) secreted proteins which are often glycosylated; they are involved in immunity and inflammation where they regulate the amplitude and duration of a response; and are usually produced transiently and locally. acting in a paracrine or autocrine, rather than endocrine manner. Cytokines are extremely potent, generally acting at picomolar concentrations; and interact with high affinity cell surface receptors specific for each cytokine or cytokine group. Their cell surface binding ultimately leads to a change in the pattern of cellular RNA and protein synthesis, and to altered cell behavior. Individual cytokines have multiple overlapping cell regulatory actions. ‘it is concomitantly exposed.

The response of a cell to a given cytokine is dependent upon the local concentration of the cytokine, upon the cell type it is acting on and upon other cell regulators to which The overlapping regulatory actions of these structurally unrelated proteins which bind to different cell surface receptors is at least partially accounted for by the induction of common proteins which can have common response elements in their DNA. interact in a network by: Cytokines first. inducing each other: second, transmodulating cytokine cell surface receptors and third. by synergistic. additive or antagonistic interactions on cell function. [Immunology Today lg: 299 (1989)).

The potential utility of cytokines in the treatment of neoplasia and as immunoenhancing agents has recently been demonstrated in studies using human recombinant interleukin-2 (rIL—2). Natural interleukin-2 (IL-2) is a lymphokine which is produced and secreted by T—lymphocytes.

This glycoprotein molecule is intimately involved in the induction of virtually all immune responses in which T—cells play a role. B cell responses in vitro are also enhanced by the presence of IL-2. IL-2 has also been implicated as a differentiation inducing factor in the control of B and T lymphocyte responses.

Administration of human rIL—2 has been shown in some of established tumors in both The anti—tumor effects of animal models suggest that rIL-2 might also have value in In addition, results from in ameliorating chemotherapy-induced immunosuppression the treatment of certain infectious diseases [J.

[Immunol. Lett. ;g:307-314 (1985)).

However, the clinical use of rIL—2 has been complicated ‘by the serious side effects which it may cause [N. Engl. J.

Med. 3l3:148S-1492 (1985) and N. Engl. J. Med. 3l6:889—897 Kobayashi et al. (J. Exp. Med. (1989) 170, 827-845) relates to the identification and purification of natural killer cell stimulatory factor (NKSF), a cytokine with multiple biologic effects on human lymphocytes.

Thus, the present invention provides a 35 kDa subunit of a cytokine protein called Cytotoxic Lymphocyte Maturation Factor (CLMF) which is produced and synthesized by cells capable of secreting CLMF. Examples for such cells are mammalian cells particularly human lymphoblastoid cells. In the presence of low concentrations of IL-2 CLMF synergistically induces the cytolytic activity of Lymphokine Activated Killer (LAK) cells. CLMF is also capable of stimulating T—cell growth.

CLMF can be isolated in a substantially pure form by the following steps: a) stimulating B lymphoblastoid cells such as NC—37 cells to produce and secrete cytokines into a supernatant liquid: b) collecting the supernatant liquid produced by the stimulated cells; c) separating the supernatant liquid into protein fractions; d) testing each protein fraction for the presence of CLMF: e) retaining the protein fractions which are able to stimulate T—cell growth, said fractions containing an active protein which is responsible for the T—cell stimulating activity of the protein fractions: f) isolating said active protein into a substantially pure form, said protein being Cytolytic Lymphocyte Maturation Factor (CLM).

The CLMF protein obtained in this way is free from other cytokine proteins. The natural CLMF protein is a 75 kilodalton (kDa) heterodimer comprised of two polypeptide subunits. a 40 kDa subunit and a 35 kDa subunit which are bonded together via one or more disulfide bonds. The present invention also provides the nucleotide sequence.of the 35 kDa subunit of the CLMF gene and the amino acid sequence of the 35 kDa subunit of the CLMF protein encoded by the said gene. The present invention relates to a protein which exhibits CLMF activity and contains a biologically active portion of the amino acid sequence of CLMF or which contains an amino acid sequence of CLMF as well as other amino acids or proteins containing analogous sequences to CLMF or its biologically active fragments which proteins exhibit CLMF activity.

The above process steps c) to f) may be used to purify CLMF from any liquid or fluid which contains CLMF together with other proteins. The present invention relates also to protein fractions having CLMF activity and being capable of stimulating T-cell growth. to a substantially purified active CLMF protein. obtained by the above described process. to the isolated cloned gene encoding the 35 kDa subunit, to vectors containing this gene to host cells transformed with the vector containing the said gene and to CLMF proteins prepared in such a transformed host cell. Furthermore the present invention relates to isolated polyclonal or monoclonal antibodies capable of binding to CLMF.

Monoclonal antibodies prepared against a partially purified preparation of CLMF have been identified and characterized by 1: immunoprecipitation of 125I—labelled CLMF, 2: immunodepletion of CLMF bioactivity. 3: western blotting of CLMF. 4: 12Sl—CLMF binding to its cellular receptor and 5: neutralization of CLMF Twenty hybridomas secreting anti—CLMF antibodies were found to inhibition of bioactivity. antibodies were identified. The I—label1ed CLMF bioactivity as assessed in immunoprecipitate CLMF and to immunodeplete the T—cel1 proliferation and LAK cell induction assays. western blot analysis showed that each antibody binds to the 70 kDa heterodimer and to one of the subunits. Each of the above-mentioned 20 anti—CLMF monoclonal antibodies were specific for CLMF and in particular for the 40 kDa subunit of CLMF. A CLMF receptor binding assay has been developed to evaluate the ability of individual antibodies to inhibit CLMF binding to its cellular receptor.

The assay measures the binding of 125 I—labelled CLMF to PHA activated PBL blast cells in the presence and absence of each antibody. Of the 20 antibodies tested, L2 antibodies were found to inhibit greater than 60% . . of the I-labelled CLMF binding to the blast cells. Two inhibitory antibodies, viz. 7B2 and 4A1, neutralize CLMF bioactivity while one non—inhibitory antibody, SE3, does not neutralize CLMF bioactivity.

I—labelled CLMF binding to its cellular receptor will neutralize CLMF bioactivity as assessed by the T—cell proliferation and LAK cell induction assays. The ability of the antibodies specific for the 40 kDa subunit of CLMF to neutralize CLMF bioactivity indicates that determinants on the 40 kDa subunit are necessary for binding to the CLM cellular receptor.

These data confirm that antibodies which block The monoclonal anti—CLMF antibodies provide powerful analytical, diagnostic and therapeutic reagents for the immunoaffinity purification of natural and recombinant human CLMF, the development of human CLMF immunoassays. the identification of the active site of the 40 KDa subunit of CLMF and may be used in therapeutic treatments of patients which require selective immunosuppression of cytotoxic T cells. such as in transplantation. Monoclonal antibodies which recognize different epitopes on human CLMF can be used as reagents in a sensitive two—site immunoassay to measure levels of CLMF in biological fluids, cell culture supernatants and human cell extracts.

The monoclonal antibodies against CLMF exhibit a number of utilities including but not limited to: . Utilizing the monoclonal antibodies as affinity reagents for the purification of natural and recombinant human CLMF: . Utilizing the monoclonal antibodies as reagents to configure enzyme-immunoassays and radioimmunoassays to measure natural and recombinant CLMF in biological fluids, cell culture supernatahts, cell extracts and on plasma membranes of human cells and as reagents for a drug screening assay; . Utilizing the monoclonal antibodies as reagents to construct sensitive two-site immunoassays to measure CLMF in biological fluids, cell culture supernatants and human cell extracts: . Utilizing the monoclonal antibodies as reagents to identify determinants of the 40 kDa subunit which participate in binding to the 35 kDa subunit and which participate in binding to the CLMF cellular receptor: . Utilizing the intact IgG molecules, the Fab fragments or the humanized IgG molecules of the inhibitory monoclonal antibodies as therapeutic drugs for the selective blockade of proliferation and activation of cytotoxic T cells, such as in transplantation.

BRIEF DESCRIPTION OF THE DRAWINGS Figure 1 is a plot of a supernatant solution obtained from cultured NC37 lymphoblastoid cells applied to a Nu-Gel P-SP column showing the protein fraction containing TGF activity being eluted with a salt gradient.

Figure 2 is a plot of the material containing TGF activity obtained from the separation shown in Figure 1 as it was being eluted with a salt gradient through a Blue-B—Agarose Column.

Figure 3 shows the plot of the material containing TGF activity obtained from the separation shown in Figure 2 as it was being eluted with a Nacl gradient through a Mono Q column.

Figure 4 shows a SDS—polyacrylamide gel electrophoresis (SDS—PAGE) analysis of the fractions 30 to 45, 48 and 50 obtained from the step illustrated in Figure 3. The numbers on the left side, 44 and 68, molecular weight of standard proteins of 44 and 68 kDa in i.e. refer to the apparent lane 5.

Figure 5 shows the elution profile through a Vydac Diphenyl column of fraction 38 from the Mono Q Chromatography separation (reversed—phase HPLC) shown in Figure 3.

Figure 6 shows SDS—PAGE analysis of protein purity of the protein fractions 85-90 recovered from the separation process depicted in Figure 5.

Figure 7 shows a SDS—PAGE analysis of fractions 87 and 88 from the reversed-phase HPLC separation under non—reducing (lane A; without B—mercaptoethanol) and reducing (lane B; in the presence of B-mercaptoethanol) conditions showing the 75,000 molecular weight CLMF The remaining lanes in the gel shown in this Figure contain separated into two subunits of 40 kDa and 35 kDa. standard proteins comprising the 44 and 68 kDa marker protein.

Figure 8 shows the elution pattern of the proteins from the supernatant solution from NC—37 cells applied to a Nu- Gel P-SP column and eluted with a salt gradient.

Figure 9 is a Blue—B—Agarose column salt gradient elution profile of the active fractions obtained from the Nu-Gel P-SP column elution shown in Figure 8.

Figure 10 is a Mono-Q column salt gradient elution profile of the active fractions obtained from the elution shown in Figure 9.

Figure 11 is the elution pattern through a Vydac Diphenyl column of active fractions 39 and 40 obtained from the Mono Q Chromatography shown in Figure 10.

Figure 12 shows a SDS-PAGE analysis under reducing conditions of the active fractions obtained from the separation process shown in Figure 11.

Figure 13 is a schematic diagram depicting the separation of the 40 kDa subunit from the 35 kDa subunit of the CLMF cytokine.

Figure 14 is a schematic diagram depicting the determination of the amino acid composition, the N—terminal sequencing. the proteolytic digestion and the complete sequencing of the 40 kDa subunit of the CLMF cytokine.

Figure 15 shows a separation of the tryptic peptides of the digested 40 kDa subunit of the CLMF cytokine.

Figure 16 shows a separation of the proteolytic peptides of the Staphylococcus aureus V8 protease digested 40 kDa subunit CLMF.

Figure 17 is a chart which summarizes the information on the protein structure obtained from the analysis of the proteolytic peptides of the 40 kDa subunit of CLMF. The following abbreviations and symbols are used: _ lo _ N~t — N—termina1 sequencing on intact protein Tr — tryptic peptides from map HP2383 numbered by "fraction number V8 — V8 protease peptides from map HP2412 numbered by fraction number — indicates probable glycosylation site: boxes indicate potential sites Figure 18 shows the SDS—PAGE analysis of Fraction 39 from the Mono Q FPLC elution profile shown in Figure 3. Lane A: Standardproteins without B—mercaptoethano1: lane B: Fraction 39 without B—mercaptoethanol; lane C: Fraction 39 with B-mercaptoethanolz lane D: Standard proteins with B—mercaptoethano1.

Figure 19 relates to the purification of the 35 kDa subunit by reversed—phase HPLC and depicts the elution pattern through a Vydac C-18 column of fraction 39 of the Mono Q chromatography which was reduced in 5% B-mercapto— ethanol.

Figure 20 shows a SDS—PAGE gel analysis under non—reducing conditions of the fractions which were fluorescamine positive from the Vydac C-l8 column elution profile shown in Figure 19. S: = protein—standard; F:‘= flow-through; numbers refer to the fraction number.

Figure 21 depicts the elution pattern of a tryptic digest of fractions 36 and 37 of the Mono Q Chromatography through a YMC ODS column.

Figure 22 shows the stained PVDF membrane with the smeared bands comprising the CNBr cleaved CLMF before 22B) and after (Fig. 22A) excising the regions of about 29, 25, 14. 12, and 9 kDa. contain the CNBr fragments having the following sequences: (Fig. respectively. The regiones I (P?)—P-K-N-L—Q-L-K-P-L—K-N-?-V-(Q?)— (New sequence from 40 kDa protein) ?—Q—K—A—(R?)—Q—T—L-E—F—Y—P—?—T— (New sequence starting at residue no. 30 of 35 kDa protein) III V—V-L-T-?-D-T-P—E-E—D—G-I—T— (Starts at residue no. 24 of 40 KDa protein) IV V—D—A—V—(H?)—K—L—K—Y—E—?—Y—T-?—?—F—F—I— (Starts at residue no. 190 of 40 kDa protein) note: it is assumed or known that the above sequences are preceeded by a Met residue.

Figure 23 shows a reverse—phase HPLC separation of the peptide fragments obtained by cleaving CLMF with CNBr.

Figure 24 shows an SDS—PAGE of pure CLMF and "free" unassociated 40 kDa subunit of CLMT purified by affinity chromatography using the monoclonal antibody 782 covalently attached to an agarose resin. Lane A: molecular weight marker proteins; lane B: starting material: lane C: flow- through; lane D: acid eluate; lane E: potassium thiocyanate eluate.

Figure 25 a. b, c and d show the DNA sequence and the deduced amino CLMF. acid sequence of the 40 kDa subunit of human Figure 26 a, b and c show the cDNA sequence and the deduced amino acid sequence of the 35 kDa subunit of CLMF Figure 27 depicts the inhibition of CLMF bioactivity by serum from rats immunized with CLMF and from non—immunized rats (control).

Figure 28 shows a SDS-PAGE analysis of immunoprecipitates of l25I—CLMF by monoclonal antibodies A1 (lane 1), 4D1 (lane 2). 8E3 (lane 3) and 9C8 (lane 4), by a control antibody (lane 5), by immune rat serum (lanes 6 and 8) and by normal rat serum (lanes 7 and 9). On the left side the molecular weight in kDa is indicated.

Figure 29 shows the immunodepletion of CLMF bioactivity (TGF activity) by monoclonal anti—CLMF antibodies (a—CLMF).

Figure 30 shows the immunodepletion of CLMF bioactivity (LAK induction activity) by monoclonal anti—CLMF antibodies (a—CLMF).

Figure 31 shows a Western blot analysis of the reactivity of the monoclonal antibodies (mAbs) 7B2. 8B3, 6A3, A1, 9F5 and 2A3 and of rat polyclonal anti—CLMF antibodies (RS1) with the CLMP 75 kDa heterodimer. NR8: — normal rat serum.

Figure 32 shows a Western blot analysis of the reactivity of monoclonal and rat polyclonal anti—CLMF antibodies with the CLMF 40 kDa subunit. the following mAbs were used: 4A1. 4Dl, 7B2, 7A1, 2A3, 1C1, 8B4, 8A2, 8B3. 1B8. 4A6, 6A2. 8C4, 9P5, 6A3. 9C8. QAI and E7, respectively. In lane 19 a control antibody, in lane In lanes 1 to 18 a fusion rat serum and in lane 21 a normal rat serum was used. l25I—CLMF to PHA-activated peripheral blood lymphocyte (PBL) Figure 33 shows the binding of lymphoblasts.

I—CLMF binding to PHA—activated PBL blast cells by rat anti-CLMF serum. The 1 I—CLMF binding to the cells in the presence of the indicated Figure 34 shows the inhibition of data are expressed as amount (% bound) of concentrations of serum when compared to the total specific binding in the absence of serum.

Figure 35 shows the inhibition of the binding of -CLMF to PHA-activated PBL blast cells by monoclonal antibody supernatants. The data are expressed as % .

I—CLMF to the cells in the presence of a 1:1 dilution of supernatant when compared to inhibition of the binding of the total specific binding in the absence of antibody supernatant.

Figure 36 shows the inhibition of the binding of . » .

I-CLMF to PHA—act1vated PBL blast cells by various concentrations of purified monoclonal antibodies. The data -CLMF bound to the cells in the presence of the indicated are expressed as the amount (% cpm bound) of concentrations of antibody when compared to the total specific binding in the absence of antibody.

Figure 37 shows a western blot analysis of the reactivity of a rabbit polyclonal anti-CLMF antibody with the 75 KDa CLMF (nonreduced) and with the 35 kDa CLMF subunit (reduced). The antibody was prepared against a synthetic peptide fragment of the 35 kDa CLMF subunit. Lanes 1 to 5 are without B-mercaptoethanol: lanes 6 to 10 with B-mercaptoethanol.

Lane ul CLMF ul CLMF ul CLMF Blank Blank ul prestained molecular weight standards 7 1 ul CLMF ul CLMF ul CLMF ul prestained molecular weight standards The CLMF biological activity of all of the proteins of the present invention including the fragments and analogues may be determined by using a standard T—cell growth factor assay.

In accordance with the present invention, natural CLMF is obtained in pure form. The amino acid sequences of the kDa subunit and the 40 kDa subunit of the CLMF protein is depicted in Figures 25 and 26.

Thus, the present invention relates to a protein having Cytotoxic Lymphocyte Maturation Factor (CLMF) activity in a substantially pure form, such as the CLMF protein per se, or to a 35 kDa subunit of the said protein which exhibits CLMF activity if combined with the 40 kDa subunit and comprises at least a part of the amino acid sequence of the natural form of CLMF.

The present invention also relates to cloned genes coding for 35 kDa subunit of CLMF and to isolated polynucleotides encoding a subunit as defined above, which polynucleotide contains a sequence corresponding to the CDNA encoding 35 kDa subunit of CLMF, to recombinant vectors comprising a polynucleotide encoding a 35 kDa subunit of the CLMF protein, to microorganisms transformed with the said recombinant vectors, to antibodies directed to the said subunits as well as to processes for the preparation of the said subunits, govectors and antibodies.

The practice of the present invention will employ, unless otherwise indicated, conventional techniques of molecular biology. microbiology, recombinant DNA and immunology, which are within the skills of an artisan in the field. Such techniques are explained fully in the literature. See e.g., Maniatis, Fitsch & Sambrook, MOLECULAR CLONING: A LABORATORY MANUAL (1982); DNA CLONING, VOLUMES I AND II (D.N Glover ed., 1935); OLIGONUCLEOTIDE SYNTHESIS (M.J. 1984); NUCLEIC ACID HYBRIDIZATION (B.D. Hames & S.J. Higgins eds., 1984); TRANSCRIPTION AND TRANSLATION (B.D. Harnes & S.J. Higgins eds., 1984); ANIMAL CELL CULTURE (R.I. Freshney ed.. 1986): IMMOBILIZED CELLS AND ENZYMES (IRL Press, 1986); B. Perbal. A PRACTICAL GUIDE TO MOLECULAR CLONING (1984); the series, METHODS IN ENZYMOLOGY (Academic Press. Inc.): GENE TRANSFER VECTORS FOR MAMMALIAN CELLS (J.H. Miller and M.P. Calos eds.. 1987, Cold Spring Harbor Laboratory), Methods in Enzymology Vol. 154 Gait ed., - 17 _ and Vol. 155 (Wu and Grossman, and Wu. eds., respectively); IMMUNOCHEMICAL METHODS IN CELL AND MOLECULAR BIOLOGY (Mayer and walker. 1987. London), Scopes, PROTEIN PURIFICATION: PRINCIPLES AND PRACTICE. second Edition (1987. Springer—Verlag, N.Y.), and HANDBOOK OF EXPERIMENTAL IMMUNOLOGY, VOLUMES I—IV (D.M. Weir and C.C.

Blackwell eds., 1986). eds., Academic Press, The DNA sequences and DNA molecules of the present invention may be expressed using a wide variety of host/vector combinations. For example, useful vectors may consist of segments of chromosomal, non—chromosoma1 and synthetic DNA sequences[ Examples of such vectors are viral vectors, such as the various known derivatives of SVQO, bacterial vectors. such as plasmids from E. coli including pCRl, pBR322, pMB9 and RP4. phage DNAs, such as the numerous derivatives of phagex, M13 and other filamentous single-stranded DNA phages, as well as vectors useful in yeasts, such as the Zn plasmid, vectors useful in eukaryotic cells more preferably vectors useful in animal cells, such as those containing SV40. adenovirus and/or retrovirus derived DNA sequences. Useful vectors may be also derived from combinations of plasmids and phage DNA's, such as plasmids which have been modified to comprise phage DNA or other derivatives thereof.

Expression vectors which may be used for the preparation of recombinant 35 kDa CLMF subunits are characterized by comprising at least one expression control sequence which is operatively linked to the 35 kDa CLMF subunit DNA sequence inserted in the vector in order to control and to regulate the expression of the cloned 35 kDa CLMF subunit DNA sequence. Examples of useful expression control sequences are the lac system, the major operator and promoter the trp system. tac system, the trc system. regions of phage X, the control region of fd coat protein. the glycolytic promoters of yeast. e.g., the promoter for —phosphoglycerate kinase, the promoters of yeast acid _ 13 _ phosphatase. e.g., Pho 5, a—mating factors. and promoters derived from polyoma the promoters of the yeast virus, adenovirus, retrovirus, and simian virus. e.g., the early and late promoters or SV40. and other sequences known to control the expression of genes of prokaryotic or eukaryotic cells and of their viruses as well as combinations of the said promoter/operator sequences.

Among such useful expression vectors are known vectors that enable the expression of the cloned CLMF—related DNA sequences in eukaryotic hosts, such as in animal and human P. J. Southern and P. Berg, J. Mol. Appl. -41 (1982): S. Subramani et al., Mol. Cell. 854-64 (1981): R. J. Kaufmann and P. A. Sharp, Mol.

Biol. lggz 601-64 (1982); S. I. Scahill et al., "Expression and Characterization of The Product of A Human cells [e.g., Genet. ;: Biol. lz Cell.

Proc. Sci.

Furthermore, within each specific expression vector. various sites may be selected for insertion of the CLMF—related DNA sequences of the present invention. These sites are usually designated by the restriction endonuclease which cut them. They are well recognized by those of skill in the art. It is. of course to be understood that an expression vector useful in this invention need not have a restriction endonuclease site for insertion of the chosen DNA fragment. Instead. the vector could be joined to the fragment by alternative means. The site chosen in the expression vector for the insertion of a selected DNA fragment and the operative linking of the DNA fragment to an expression control sequence is determined by a variety of factors. such as the number of sites susceptible to a particular restriction enzyme. the location of start and stop codons relative to the vector sequence and the desired -19.. selection method for the host transformed with the recombinant vector. The choice of a vector and an insertion site for a DNA sequence is determined by a balance of these factors, not all selections being equally effective for a given case.

The host cell used for the expression of the CLMF-related DNA sequence known hosts. may be selected from a variety of Examples for such hosts are prokaryotic or eukaryotic cells. A large number of such hosts are available from various depositories such as the American Type Culture Collection (ATCC) or the Deutsche Sammlung fur Mikroorganismen (DSM). Examples for prokaryotic cellular hosts are bacterial strains such as E.coli, B.subtilis and others. Preferred hosts are mammalian cells such as the SV4O transformed African Green monkey kidney cell line COS.

Not all host/expression vector combinations function with equal efficiency in expressing a given DNA sequence.

However, a particular selection of a host/expression vector combination may be made by those of skill in the art after due consideration of the principles set forth herein without departing from the scope of this invention. For example, the selection should be based on a balancing of a number of factors, These include, for example, compatibility of the host and vector. susceptibility of the protein to proteolytic degradation by host cell enzymes, possible contamination of the protein to be expressed by host cell proteins difficult to remove during purification, toxicity of the proteins encoded by the DNA sequence to the host, ease of recovery of the desired protein, expression characteristics of the DNA sequence and the expression control sequence operatively linked to them, biosafety. costs and the folding, form or any other necessary post—expression modifications of the desired protein. _ 20 _ The host organisms which contain the expression vector comprising the 35 kDa CLMF subunit DNA are usually grown up under conditions which are optimal for the growth of the host organism. Towards the end of the exponential growth. when the ihcrease in the number of cells per unit time decreases, the expression of the CLMF subunit is induced, i.e. the DNA coding for the subunit is transcribed and the transcribed mRNA is translated. The induction can be effected by adding an inducer or a derepressor to the growth medium or by altering a physical parameter, e.g. by a temperature change.

The CLMF subunit produced in the host organism can be secreted by the cell by special transport mechanisms or can be isolated by breaking open the cell. The cell can be broken open by mechanical means [Charm et al., Meth. Enzmol. ggz 476-556 (1971)). by enzymatic treatment (e.g. lysozyme treatment) or by chemical means (e.g. detergent treatment. urea or guanidine-HCl treatment, etc.) or by a combination thereof.

In eukaryotes, polypeptides which are secreted from the cell are synthesized in the form of a precursor molecule.

The mature polypeptide results by cleaving off the so-called signal peptide. As prokaryotic host organisms are not capable of cleaving eukaryotic signal peptides from precursor molecules. eukaryotic polypeptides must be expressed directly in their mature form in prokaryotic host organisms. The translation start signal AUG, which corresponds to the codon ATG on the level of the DNA. causes that all polypeptides are synthesized in a prokaryotic host organism with a methionine residue at the N-terminus. In certain cases, depending on the expression system used and possibly depending on the polypeptide to be expressed this N—terminal methionine residue is cleaved off.

The 35 kDa CLMF subunit produced by fermentation of the prokaryotic and eukaryotic hosts transformed with the DNA sequences of this _ 21 _ invention can then be purified to essential homogeneity by known methods such as. for example, by centrifugation at different velocities, by precipitation with ammonium sulphate, by dialysis (at normal pressure or at reduced pressure), by preparative isoelectric focusing, by preparative gel electrophoresis or by various chromatographic methods such as gel filtration, high performance liquid chromatography (HPLC), ion exchange chromatography, reverse phase chromatography and affinity chromatography (e.g. on Sepharose” Blue CL-6B or on carrier—bound monoclonal antibodies directed against CLMF).

The purified CLMF subunit of the present invention can be employed for the preparation of LAK cell and T cell activator and antitumor compositions and in methods for stimulating LAK cell, T—cells or Natural Killer Cells.

The 35 kDa CLMF subunit of the present invention can also be analyzed to determine the active sites for CLMF activity. The information from this analysis may be used to predict and produce fragments or peptides, including synthetic peptides.

Among the known techniques for determining such active sites are x—ray crystallography.

UV spectroscopy and site specific mutagenesis. Accordingly. the fragments obtained in this way may be employed in methods for stimulating T-cells or LAK cells. having the activity of CLMF. nuclear magnetic resonance, circular dichroism, The CLMF subunits prepared in accordance with this invention or pharmaceutical compositions comprising the 35 kDa CLMF subunit may be administered to warm blooded mammals for the clinical uses indicated above. The administration may be by any conventional modes of administration of agents which exhibit antitumor activity auch as by intralesional or parenteral application either intravenously. subcutaneously or intramuscularly. Obviously, the required dosage will vary _ 22 _ with the particular condition being treated, the severity of the condition, the duration of the treatment and the method for administration. A suitable dosage form for pharmaceuti- cal use may be obtained from sterile filtered, lyophilized protein reconstituted prior to use in a conventional manner.

It is also within the skill of the artisan in the field to prepare pharmaceutical Compositions comprising 35 kDa CLMF subunit of the present invention by mixing the said CLMF subunit with compatible pharmaceutically acceptable carrier materials such as buffers. stabilizers, bacteriostats and other excipients and additives conventionally employed in pharmaceutical parenteral dosage forms. The present invention also relates to such pharmaceutical compositions.

The preferred form of administration depends on the intended mode of administration and therapeutic application. The pharmaceutical compositions comprising a CLMF protein or peptide derivative of the present invention also will preferably include conventional pharmaceutically acceptable carriers and may include other medicinal agents (e.g. e.g., human serum albumin or plasma preparations. interleukin—2), carriers. adjuvants. excipients, etc., Preferably, the compositions of the invention are in the form of a unit dose and will usually be administered one or more times a day. The unit dose is preferably packed in 1 ml vials containing an effective amount of the 35 kDa CLMF subunit and if desired of interleukin—2 in lyophilized form. The vials containing the CLMF subunit and if desired the interleukin-2 are preferably packed in a container together with written instructions describing the correct use of the pharmaceutical composition. The present invention relates also to such a unit dose packed in a container, preferably together with a separate unit dose of inter1eukin—2, most preferably together with the appropriate instructions. Furthermore the present invention relates to a process for the preparation of the said unit dose. _ 23 _ In order that our invention herein described may be more fully understood, the following examples are set forth. It should be understood that these examples are for illustrative purposes only and should not be construed as limiting this invention in any way to the specific embodiments recited therein. It has to be noted that the specific product names and suppliers mentioned below are not meant to be mandatory. The person skilled in the art is in a position to select alternative products from other suppliers.

EXAMPLE PURIFICATION AND CHARACTERIZATION OF CYTOTOXIC LYMPHOCYTE MATURATION FACTOR (CLMF) Production of Supernatant Liquid Containing_CLMF.

Human NC-37 B lymphoblastoid cells (ATCC CCL 214, American Type Culture Collection. Rockville, MD) were used for production of CLMF. These cells were maintained by serial passage in RPMI 1640 medium supplemented with 5% heat-inactivated (56°C. 30 min.) fetal bovine serum, and 100 ug/ml streptomycin (all cell culture media were from GIBCO Grand Island. NY). mM L—glutamine, 100 units/ml penicillin, Laboratories, Higher producer sublines of NC-37 cells were derived by limiting dilution cloning in liquid microcultures. Each well of three Costar 3596 microplates (Costar Co., Cambridge, MA) received 100 ul of a cell suspension containing five NC—37 cells/ml. The medium used for the cloning was a 1:1 mixture of fresh passage medium and filtered, conditioned medium from stock cultures of the parent NC-37 cells. One week and two weeks after culture initiation each of the microcultures was fed with 50 ul of the 1:1 mix of fresh and conditioned medium. Between 3 and weeks after culture initiation the contents of wells _ 24 _ containing clones of NC—37 cells were harvested and passed into larger cultures. when the number of cells in a given subline exceeded 1.4 X 106, one million cells were stimulated to produce CLMT in 1 ml cultures containing 3 ng/ml phorbol l2—myristate l3~acetate (PMA) (Sigma Chemical Co., St. ng/ml calcium ionophore A23lB7 (Sigma).

Louis, MO) and 100 Supernatants were harvested from the cultures after 2 days. dialyzed against about so volumes of Dulbecco's phosphate buffered saline (Gibco) using e.g. SPECTROPOR® #1 tubing (Fisher Scientific) overnight with one change of buffer and then for 4 hours against 50 volumes of RPMI 1640 medium with 50 ug/ml of gentamicin (both from Gibco) and tested for CLMF by means of the T cell growth factor assay (see below).

Three sublines, NC—37.89, NC—37.98, and NC—37.lO2, werei identified which routinely produced CLMF at titers 3 4 times the titers produced by the parental NC-37 cell line. Since cells from these three sublines produced CLMF at similar titers (3 800 units/ml), culture supernatants derived from the three sublines were pooled for use as starting material for the purification of CLMF.

Bulk production of CLMF was carried out in roller bottle cultures on a roller apparatus set at about 38 rpms (wheaton Cell Production Roller Apparatus Model 11, Wheaton Millville. NJ). containing l-1.5 X 106 NC~37;B9, NC—37.98 or NC—37.l02 Instruments, Cell suspensions were prepared cells/ml in RPMI 1640 medium supplemented with 1% Nutridoma—SP (Boehringer Mannheim Biochemicals.

IN), 100 units/ml penicillin, 100 ug/ml streptomycin. 10 ng/ml PMA and 20-25 ng/ml calcium ionophore A23l87.

Indianapolis, 2 mM L—glutamine, Two hundred fifty to three hundred fifty ml aliquots of the cell suspensions were added to Falcon 3027 tissue culture roller bottles (Becton Dickinson, Lincoln Park. NJ) which had been gassed with a mixture of 5% CO2, 95% air. The roller bottles were then _ 25 - capped tightly and incubated at 37°C with continuous rolling for three days. At the end of this time, supernatants were harvested. the culture EDTA and phenylmethylsulfonyl fluoride (both from Boehringer Mannheim) were added to the culture supernatants at final concentrations of 1 mM and 0.1 mM. to retard proteolytic degradation. supernatants were stored at 4°C. respectively, The Lympokine Activated Killer (LAK) Cell Induction (LCI) Assay.

Culture supernatants and chromatographic fractions were tested for their ability to synergize with rIL-2 to induce the generation of cytolytic LAK cells as follows. Human peripheral blood mononuclear cells (PBMC) were isolated by the following method. Blood from normal volunteer donors was drawn into syringes containing sufficient sterile preservative—free heparin (Sigma) to give a final The blood was balanced salt solution (HBSS) without calcium or magnesium (GIBCO). The diluted blood was then layered over 15 ml aliquots of Ficoll/sodium concentration of approximately 5 units/ml. diluted lzl with Hanks’ diatrizoate solution (Lymphocyte Separation Medium, organon Teknika Corp.. Durham, NC) in 50 ml Falcon 2098 centrifuge tubes. The tubes were centrifuged for 30 minutes at room temperature at 500 x g. Following centrifugation, the cells floating on the Ficoll/sodium diatrizoate layer were collected and diluted by mixing with 3 2 volumes of HBSS without calcium or magnesium. The resulting cell suspension was then layered over 15 ml aliquots of 20% sucrose (Fisher) in RPMI 1640 medium with 1% human AB serum (Irvine Scientific, Santa Ana, CA) in Falcon 2098 centrifuge tubes.

The tubes were centrifuged for 10 minutes at room temperature at 500 x g. and the supernatant fluids were discarded. The cell pellets were resuspended in 5 ml of HBSS without calcium or magnesium, repelleted by centrifugation, and finally resuspended in the appropriate - 25 _ culture medium. Accessory cells were removed from the PBMC by treatment with 5 mM L—glutamic acid dimethyl ester (Sigma) using the same conditions as described by Thiele et al. J. Immunol. l31:2282—229O (1983) for accessory cell depletion by L-leucine methyl ester except that the glutamic acid ester was substituted for the leucine ester.

The accessory ce1l—depleted PBMC were further fractionated by centrifugation on a discontinuous Percoll density gradient (Pharmacia, Piscataway, NJ) as described by Wong et al.. Cell Immunol. ;;l:39—54 (1988). cells recovered 41. 45, and 58% Percoll layers used as a source of LAK cell precursors in cells recovered from the Percoll gradient suspended in tissue culture medium (TCM) composed of a 1:1 mixture of RPMI 1640 and Dulbecco's modified Eagle's medium.

Mononuclear from the 38. were pooled and The were washed and the assay. supplemented with 0.1 mM nonessential amino acids} 60 ug/ml arginine HCl, 10 mM HEPES buffer. 2 mM L—g1utamine. . ng/ml streptomycin (all available from GIBCO), 5 x 10-5 M 2—mercaptoethanol (Fisher Scientific. Fair Lawn, NJ). units/ml penicillin, 100 mg/ml dextrose (Fisher), CA). These cells were incubated in 24-well tissue culture plates (costar, Cambridge, MA) in ml cultures (7.5 x 105 and 5% human AB serum (Irvine Scientific, Santa Ana, , -4 cells/culture) to which 10 M hydrocortisone sodium succinate (Sigma) was added to minimize endogenous cytokine production. some cultures also received human rIL-2 (supplied by Hoffmann-La Roche, Inc., Nutley, NJ) at a final concentration of S units/ml and/or supernatants to be assayed for CLMF activity. All cultures were incubated for 3~4 days at 37°C in a humidified atmosphere of 5% CO2. 95% air.

At the end of this incubation, the contents of each culture were harvested. and the cells were pelleted by centrifugation and resuspended in 0.5 ml of fresh TCM. One tenth ml aliquots of these cell suspensions were mixed with _ 27 - lCr—labelled K562 or Raji cells (both cell lines may be obtained from the ATCC) and tested for .1 ml aliquots of their lytic activity in 5 hour Cr release assays. The . . 51 method for labelling target cells with Cr and performing the cytolytic assays have been described by Gately et a1., release was calculated as [(g - g)/(100 — 5)] X 100, where g . 51 the percentage of Cr released from target cells Slct released spontaneously from target cells incubated alone.

The total releasable 51 incubated with lymphocytes and g is the percentage of see Gately et al..

LAK Cell Induction Microassay. The microassay for measuring synergy between rIL—2 and CLMF-containing solutions in the induction of human LAK cells was similar to the LAK cell induction assay described above but with the following modifications. Human peripheral blood mononuclear cells which had been depleted of accessory cells and fractionated by Percoll gradient centrifugation as described above were added to the wells of Costar 3596 microplates (5 x 104 cells/well). (5 units/ml final concentration) and/or purified CLMF or Some of the wells also received rIL-2 immunodepleted CLMF—containing solutions. All cultures M hydrocortisone sodium succinate (Sigma) and were brought to a total volume of 0.1 ml by addition of TCM with 3 days at 37°C, , -4 contained 10 % human AB serum. The cultures were incubated for lCr—labelled K562 cells (5 x 104 cells/ml in TCM with 5% human AB serum) were added to each well. after which 0.1 ml of The cultures were then incubated overnight at 37°C. Following this, the cultures were centrifuged for 5 minutes at 500 x g. and the supernatant solutions were harvested by use of a skatron supernatant collection system (Skatron.

. . Cr released into each supernatant solution was measured Sterling, VA). The amount of with a gamma counter (Packard, Downer's Grove, IL), and the . . 1 % specific Cr release was calculated as described above. All samples were assayed in quadruplicate.

Cytolytic T Lymphocyte (CTL) Generation Assay.

Methods used for generating and measuring the lytic activity of human CTL have been described in detail by Gately et al. in J. Immunol. lggz l274—l282 (1986) and by Wong et al. in Cell. Immunol. lll: 39-54 (1988). Human peripheral blood mononuclear cells were isolated from the blood of normal volunteer donors, depleted of accessory cells by treatment with L-glutamic acid dimethyl ester. and fractioned by Percoll gradient centrifugation as described above. High density lymphocytes recovered from the interface between the 45% and 58% Percoll layers were used as responder lymphocytes in mixed lymphocyte—tumor cultures (MLTC). CTL were generated in MLTC in 24-well tissue culture plates (Costar #3424) by incubation of Percoll gradient—derived high density lymphocytes (7.5 x 105 culture) together with l x 105 uv—irradiated melanoma cells e.g. HTl44 (obtainable from ATCC) or with 5 x 104 gamma—irradiated melanoma cells e.g. HTl44 in TCM with 5% human AB serum (1.2 ml/culture). For uv—irradiation, HTl44 cells were suspended at a density of l-1.5 x 106 cells/ml in Hanks’ balanced salt solution without phenol red (GIBCO) containing 1% human AB serum. One ml aliquots of the cell suspension were added to 35 x 10 mm plastic tissue culture dishes (Falcon #3001), and the cells were then irradiated (960 uw/cmz for 5 min) by use of a 254 nm uv light (model UVG—54 MINERAL1GHT® lamp, U1tra—violet Products, CA). For gamma irradiation, HTI44 cells were suspended at a density of l—5 x 106 cells/ml in TCM with 5% human AB serum and irradiated (10,000 rad) by use of a cesium source irradiator (model 143, J.L. Shepherd and CA).

HTl44 were centrifuged and resuspended in TCM with 5% human Inc.. San Gabriel, Associates, San Fernando, Uv— or gamma-irradiated _ 29 - AB serum at the desired cell density for addition to the MLTC. In addition to lymphocytes and melanoma cells, some MLTC received human rlL-2 and/or purified human CLMF at the concentrations indicated. Hydrocortisone sodium succinate (sigma) was added to the MLTC at a final concentration of -4 M (cultures containing uv-irradiated melanoma cells) or 1o"S M (cultures containing gamma—irradiated melanoma cells) to supress endogenous cytokine production [s. Gillis et al.. J. 13;: 1624-1631 (1979)) and to reduce the generation of nonspecific LAK cells in the cultures [L.M.

Muul and M.K. Gately, J. 1202-1207 (1984)]. at 37°C in a humidified for 6 days. At the end of replicate cultures were pooled.

Immunol.

Immunol. 132: The cultures were incubated atmosphere of 5% CO2 in air this time, lymphocytes from centrifuged, resuspended in 1.2 ml TCM containing 5% human AB serum, and tested for their ability to lyse HTl44 melanoma cells. and. as a specificity control, K562 erythroleukemia cells (obtainable from ATCC) in overnight Cr release assays.

Slcr [JNCI Q3: measurement of lympocyte— Melanoma cells and K562 cells were labeled with sodium chromate as described by Gately et al. 1245-1254 (l982)]. mediated lysis of Likewise, Slcr-labeled melanoma cells was performed in a manner identical to that described by Gately et al. (ibid.) for quantitating lysis of glioma target Slcr-labeled K562 cells, 0.1 ml aliquots of lymphocyte suspensions were mixed with 25 ul aliquots of 51Cr—labe1ed K562 (2 X 105 cells/ml in TCM with 5% human AB serum) in the wells of costar 3696 cells. For assaying the lysis of “half—area" microtest plates. After overnight incubation at °C. the plates were centrifuged for 5 min at 1400 x g, and 50 ul of culture medium was aspirated from each well. The amount of lcr in each sample was measured with a gamma counter (Packard), and the % calculated as described above. . . 51 specific Cr release was All assays were performed in quadruplicate, and values in the table (see below) represent _ 30 _ the means 1 1 S.E.M. of replicate samples.

T cell growth factor (TGF) assay.

The ability of culture supernatants and chromatographic fractions to stimulate the proliferation of PHA~activated human T lymphoblasts was measured as follows. Human PBMC were isolated by centrifugation over discontinuous Ficoll and sucrose gradients as described above for the LCI assay.

The PBMC (5 x 105 cells/ml) were cultured at 37°C in TCM containing 0.1% phytohemagglutinin—P (PHA-P) (Difco Laboratories, Detroit, MI). After 3 days. split 1:1 with fresh TCM. and human rIL-2 was added to each culture to give a final concentration of 50 units/ml. The cultures were then incubated for an additional 1 to 2 days. at which time the cells were harvested. washed, and resuspended in TCM at 4 x 105 cells/ml. To this cell suspension was added heat—inactivated goat anti-human rlL-2 the cultures were antiserum (final dilution: 1/200) to block any potential lL~2—induced cell proliferation in the assay. This antiserum may be prepared using methods well—known in the art or may be obtained from Genzyme Co.. Boston, MA. The antiserum used was shown to cause 50% neutralization of 2 units/ml rIL—2 at a serum dilution of 1/20,000.

Fifty ul aliquots of the cell suspension containing anti~IL—2 antiserum were mixed with 50 ul aliquots of serial dilutions of culture supernatants or chromatographic The cultures were incubated for 1 day at 37°C in a humidified fractions in the wells of costar 3596 microplates. . . 3 . . atmosphere of 5% CO2 in air. and 50 ul of H—thym1d1ne (New England Nuclear, Boston, MA), 10 uCi/ml in TCM, were then added to each well. The cultures were further incubated overnight, the culture contents Subsequently. were harvested onto glass fiber filters by means of a cell harvester (Cambridge Technology Inc., Cambridge. MA), and . . . . .

H—thymidine incorporation into cellular DNA was measured _ 31 _ by liquid scintillation counting. All samples were assayed in triplicate.

In purifying CLMF it was necessary to define units of activity in order to construct chromatographic elution profiles and to calculate the percent recovery of activity To do this, a partially purified preparation of human cytokines produced by coculturing PHA-activated human PBMC with NC—37 cells was used as a standard. The preparation was assigned an arbitrary titer of 2000 units/ml. Several dilutions of and the specific activity of the purified material. this preparation were included in each TGF or LAK induction assay. The results obtained for the standard preparation were used to construct a dose-response curve from which could be interpolated units/ml of activity in each unknown sample at the dilution tested. Multiplication of this value by the dilution factor yielded the activity of the original sample expressed in units/ml.

For antibody neutralization studies, the TGF assay was modified as follows. Twenty—five ul aliquots of CLMF—containing medium were mixed with 50 ul aliquots of serial dilutions of antiserum or antibody solutions in the wells of COSTAR 3S96® microplates. incubated for 30 minutes at 37°C, and 25 ul aliquots of a suspension of PHA—activated lymphoblasts (8 x 105/ml in TCM plus 1:100 anti—rIL—2) were then added to each well.

The cultures were further incubated, pulsed with The mixtures were H—thymidine, harvested, and analyzed for 3H—thymidine incorporation as described above.

Natural killer LNK) cell activation assay.

Purified CLMF was tested for its ability to activate NK cells when added alone or in combination with rIL-2 as follows. Human PBMC were isolated by centrifugation over discontinuous Ficoll and sucrose gradients as described _ 32 _ above and were suspended in RPMI 1640 medium supplemented with 10% heat—inactivated fetal bovine serum, 100 units/ml penicillin, 100 ug/ml streptomycin, and 2.mM L—glutamine.

The PBMC were incubated overnight at 37°C in 1 ml cultures 6 (5 x 10 purified CLMF at various concentrations. cells/culture) together with rIL-2 and/or After l8—20 hours, the contents of the cultures were harvested and centrifuged, and the cells were resuspended in the same medium used for the overnight cultures. The cytolytic activity of the cultured PBMC was then assessed in 51 described above.

Cr release assays as Concentration of cell supernatant solutions Stored. frozen crude human CLMF supernatant solutions totaling 60 liters prepared from several batches of induced NC-37 cells were pooled and concentrated 30-fold using the Pellicon Cassette System (30,000 NMWL PTTKOOOOS; Millipore Corp.. Bedford. MA). After concentrating to the desired volume of approximately 1.9 liters, a buffer exchange was performed with 10 mM MES, pH adjusted to 6.0 with 10 N NaOH. The concentrate was centrifuged at 10,000 X g for minutes at 4°C and the precipitate discarded.

Ion—Exchange Chromatography on NuGel P—SP Column The concentrated supernatant solution was applied at a flow rate of 120 ml/hr to a Nu—Gel P-SP (separation Industries, Metuchen, NJ) column (5 x 5 cm), equilibrated in l0mM MES, pH 5.0. absorbance monitoring at 280 nm was obtained.

The column was washed until baseline Absorbed proteins were then eluted with a 500 ml salt gradient from O to 0.5 M NaCl/l0 mM MES, pH 6.0 at a flow rate of 2 ml/min (Fig. l). Aliquots of fractions were assayed for T cell growth factor (TGF) activity. Fractions containing TGF activity were pooled and dialyzed (Spectra/Por 7, Fisher Scientific) against 50 volumes 20 mM Tris/Hcl, pH 7.5 in r 33 _ order to reduce the salt concentration of the preparation by 50-fold.

Q1e—Affinity Chromatography on Blue B-Agarose Column The dialyzed sample was centrifuged at 10,000 x g for 10 The supernatant solution was applied at a flow rate of 20 ml/hr minutes at 4°C and the precipitate discarded. to a Blue B~Agarose (Amicon. Danvers, MA) column (2.5 x 10 cm) equilibrated in 20 mM Tris/HCl, pH 7.5. The column was washed with this same buffer until baseline absorbance monitoring at 280 nm was obtained. Absorbed proteins were then eluted with a 500 ml salt gradient from 0 to 0.5 M Nacl/20 mM Tris/Hcl, pH 7.5 at a flow rate of 15 ml/hr (Fig. 2). Aliquots of fractions were assayed for TGF activity. Fractions containing TGF activity were pooled and dialyzed (Spectra/Por 7, Fisher Scientific) against 100 volumes 20 mM Tris/HCl, pH 7.5 in order to reduce the salt concentration of the preparation by 100-fold.

Ion—Exchanqe Chromatography on Mono Q Chromatography The dialyzed sample was filtered through a 0.45 um cellulose acetate filter (Nalgene Co., Rochester, NY) and the filtrate applied at a flow rate of 60 ml/hr to a Mono Q HR 5/5 (Pharmacia LKB Biotechnology, Inc.. Piscataway, NJ) column (5 X 50mm) equilibrated in 20mM Tris/HCl, pH 7.5.

The column was washed with this same buffer until baseline Absorbed proteins were then eluted with a 1 hr linear salt gradient from O to 0.25 M NaC1/20 mM Tris/HCI, pH 7.5 at a flow rate of so ml/hr (Fig. 3). absorbance monitoring at 280 nm was obtained.

Aliquots of fractions were assayed for TGF activity and protein purity was assessed without reduction by SDS—PAGE [Laemm1i. Nature (London) g;1:680—685 (1970)] using 12% slab gels. Gels were silver stained [Morrissey, Anal. Biochem. l;1:307-310 (l98l)] to visualize protein (Fig. 4). Fractions 36 and 37 were of greater than _ 34 _ % purity and revealed a major band at 75.000 molecular weight. Fractions 38 through 41 containing TGF activity, revealed the 75 kDa protein by SDS-PAGE with major contaminants at 55,000 and 40,000 molecular weight.

Therefore. to eliminate these contaminating proteins, fraction 38 of the previous Mono Q chromatography was diluted 1:1 vol/vol with 8 M urea and pumped onto a Vydac diphenyl column using a reversed—phase HPLC enrichment technique. The column was then washed with 5 ml of 0.1% trifluoroacetic acid. Elution of the proteins was accomplished with a gradient of 0—70% acetonitrile over 7 hrs in 0.1% trifluoroacetic acid (Fig. 5). Aliquots of fractions were assayed for TGF activity. Protein purity of the fractions containing TGF activity was assessed by sDS—PAGE under non—reducing conditions using a 10% slab gel. 6). and revealed protein of 75,000 molecular weight.

The gel was silver stained to visualize protein (Fig.

Fractions 86 through 90 were of greater than 95% purity Fractions 87 and 88 were pooled and aliquots were analyzed by SDS—PAGE under reducing (in the presence of B—mercaptoethanol) and non—reducing conditions (in the absence of B-mercapto— ethanol). Under the reducing conditions. the 75,000 molecular weight CLMF was separated into two subunits of 40,000 and 35.000 daltons (Fig. 7). Thus it was concluded that CLMF is a 75 kDa heterodimer composed of disulfide— -bonded 40 kDa and 35 kDa subunits.

The overall purification of CLMF that was achieved is shown in Table l. The protein content of the Mono Q— and Vydac diphenyl—purified material was calculated on the basis boa X ~.m mm+nm zowuucuh oHo.o moo.o meg x -.m mod x vn.m H.H Hzcozaflo ~vAxmm cowuomuu nod x m.m mov.o Hmo.o boa x mv.m mod x oa.o m o ocoz hm :o..3u.our._ nofi x m.m m>o.o mno.o woa x v.o coa x oe.e A 0 ocoz boa x m.H dd vm.o mod x v.~ ooa x HH.m me mmoumm<»m-m:Hn oofi x o.m mo o>.o wed x m.H ooa x oo.~ ca mmsm fimozz munduzmocov vofi x m.m ommm mm.H mod n c.m noa x >m.~ ovm.fi cmumuflfiumuyfis L mpcmuwcummsw oz oz oz aofi x m.~ moa x am.~ ooo.oo Hawo cofioom Amsxav Aoav Adsxosv As. AHE\:v AHEV »u«>«n_u< cflmuoum cwwuoum mu..H:: .3fi>W_u< ofiwommm Hauom. cmfioom Hanan. cofioom ®E:.mO> H mam mmum of amino acid analysis. . 7 . units/mq and 5.2 x 10 units/mg for Mono Q- and Vydac diphenyl—purified material respectively, A specific activity of 8.5 x 107 was obtained. The fact that the diphenyl-purified protein has a slightly lower specific activity than the Mono Q—purified material may be due to inactivation or denaturation of some or the molecules of CLMF in the HPLC elution solvents (i.e.. 0.1% trifluoroacetic acid). acetonitrile in Chemical Characterization j\: The ability to prepare homogeneous CLMF allowed for the first time the determination of the amino acid composition and a partial sequence analysis of the naturally occurring CLMF protein. Between 10 and 20 picomoles of Mono—Q—purified CLMF was subjected to hydrolysis. amino acid composition was determined (Table 2). cysteine and tryptophan were not determined (ND).

Quantitation of histidine was not possible due to a large artifact peak. and its Proline, associated with Tris. coeluting with His (*1.

Between 5 and 30 picomoles of diphenyl—purified CLMF was subjected to hydrolysis with and without pre-treatment with performic acid. Complete amino acid composition was thus obtained (Table 3) with the exception of tryptophan.

Amino—terminal sequence determination was attempted by automated Edman degradation on 100 pmol of the Mono Q-purified CLMF. Data from the first 22 cycles indicated two sequences present. as would be expected from the hetetodimeric structure of CLMF. summarized as follows: These results may be

Claims (14)

Claims: 1 A subunit of the Cytotoxic Lymphocyte Maturation Factor
1. (CLMF) protein characterized in that (a) the subunit comprises the amino acid sequence if combined with comprising the amino the second subunit of acid sequence
2. The subunit of claim 1, wherein the combined CLMF protein displays a specific activity of at least 5.2 x 107 Units/mg when determined in a T cell growth factor assay, and when combined with the protein as defined in claim l(b).
3. 3. The sequence Arg Leu Gln Glu Glu Asn Se: Ty: Ala Asn Asn Phe Arg Ala Asn His Lys IIe Ala Se: AC9 Glu Lys MET Se: Ty: IIe Se: subunit of claim 1 or 2 comprising the amino acid Leu His Ala Asp CY5 Arg Lys Asp Leu Len Glu Lys Arg Pro Se: Arg His Leu Glu Th: Len Len Ala Th: Th: Ala Val Cln Gln Glu Pro Th: Se: Lys Val Val Lys Val Ala Asn Th: Asp Leu Se: Phe MET Asp Ile Pro Ile Th: Th: Leu Leu Ile Glu Phe MET Ty: Pro Asp Gln Lys Ile Pro Leu Glu Th: Leu Ile Gln Lys Glu Lys Leu Asp. Asp Arg Phe Lys Th: Th: Ala Val A:q Leu Se: Cys Arg Pro Ala TY: Asp Lys Asn Leu Glu Gln Se: Ire Val Gly Val Pro Lys Asn Gly Cys Phe Ile Gln Leu Léu Th: MET Se: Cys Th: Glu Se: Leu Lys Phe Ala Glu Leu Se: Phe Asn Th: Se: Pro MET Se: Th: Cys Cys Leu Glu Val Leu Ala IIe Asn Gln Phe Asp Phe Asn Se: Cys Leu Set Set Th: Leu Asp Asn Pro Ala Leu Leu Glu His Ty:
4. 4. A polynucleotide encoding a subunit as claimed in any of claims 1 to 3.
5. 5. A polynucleotide encoding a subunit as claimed in any one of claims 1 to 4 which polynucleotide comprises the nucleotide S €qU€1’1C€ ATG TGT CCA GCC CCC AGC CTC CTC CTT GTG GCT ACC CTG GAC CAC CTC AGT TTG CCC AGA AAC CTC CCC CTG GAC CCA GGA ATG TTC CCA TCC CTT CTG GTC cac CAC TCC cm AAC‘ cm cm AGG GCC GTC AGC AAC ATG CTC CAG AAG GCC AGA CAA ACT CTA GAA TTT TAC CCT TCC ACT TCT GAA GAG ATT CAT CAT GAA GAT ATC ACA AAA CAT AAA ACC AGC ACA GTG GAG GCC TGT TTA CCA TIC GAA TTA ACC AAG AAT GAG AGT TCC CTA AAT TCC AGA GAG ACC TCT TTC ATA ACT AAT GGG ACT TGC CTG GCC TCC AGA AAG ACC TCT TIT ATC ATG GCC CTG TGC CTT ACT ACT ATT TAT GAA GAC TTG AAG ATG TAC CAG CTG GAG TTC AAG ACC ATG AAT GCA AAG CTT CTG ATG GAT CCT RAG AGG CAG ATC TTT CTA CAT CAA AAC ATG CTG GCA GTT ATT CAT GAG CTG ATG CAG CCC CTG AAT TTC AAC ACT GAG ACT GTG CCA CAA AAA TCC TCC CTT GAA GAA CCG CAT TTT TAT AAA ACT AAA ATC RAG CTC TGC ATA CTT CTT CAT GCT TTC AGA ATT CGG GCA CTG ACT ATT GAC AGA GTG ACG AGC TAT CTG AAT GCT TCC.
6. 6. A recombinant vector comprising a polynucleotide encoding a subunit as claimed in any one of claims 1 to 3 or comprising all or parts of the polynucleotide of claim 5.
7. 7. A microorganism transformed with a recombinant vector comprising a polynucleotide encoding a subunit as claimed in any one of claims 1 to 3 or all or parts of the polynucleotide of claim 5.
8. 8. A polyclonal or monoclonal antibody directed to a subunit as claimed in any one of claims 1 to 3.
9. 9. A process for producing a subunit according to any one of claims 1 to 3 which process comprises culturing a microorganism transformed with a recombinant vector comprising a polynucleotide encoding the said subunit in a culture medium under conditions permitting the expression of the encoded subunit.
10. 10. A process for producing a subunit according to any one of claims 1 to 3 which process comprises (a) preparing sub—unit peptides of the said subunit by conventional peptide synthesis methods; and (b) coupling the sub—unit peptides under conditions favouring the formation of peptide bonds.
11. 11. A process for producing the CLMF protein which comprises a process as claimed in any one of claims 9 to 10.
12. 12. A pharmaceutical composition comprising a subunit as claimed in any one of claims 1 to 3 and a pharmaceutically acceptable diluent, adjuvant or carrier.
13. 13. Use of a subunit as claimed in any one of claims 1 to 3 for the manufacture of a medicament for antitumor therapy.
14. 14. Use of a subunit as claimed in any one of claims 1 to 3 for the preparation of a CLMF protein.