IE85055B1 - Cytotoxic lymphocyte maturation factor 40kD subunit and monoclonal antibodies directed thereto - Google Patents

Abstract

ABSTRACT CYTOTOXIC LYMPHOCYTE MATURATION FACTOR 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. H599 PATENTS ACT, 1992 ~L.'v.f‘..-"J; .'-‘+1 Rn, .- «.430 fisuo-b-«Lg 4°39. CYTOTOXIC LYMPHOCYTE MA:g?1zATIoN FACTOR z>°'§ ‘PR’ "\ r ‘ 1 ‘' I¢...e'H'«~',r"u - 733 . _ -_ ... ‘fill «‘1]c".’ I ’..?f( .._'1' i’-_'.'_»:1.—_L1_ __ _; WP! '14/. la; -2 I .__.l2 /' .' -1.»; 5 _' r74; Iw .1‘;4»'{"’. . I414 I’ /' "v .’l»‘-"U " HOFFMANN—LA ROCHE AG IE 990006 RAN 4105/129—00A 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 (CMLF). antibodies directed to CLML. The present invention also relates to monoclonal '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. H3/5.12.90 IE 990006 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 it is concomitantly exposed. 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. Cytokines interact in a network by: 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 (l989)]. 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). lymphokine which is produced and secreted by T—lymphocytes- Natural interleukin—2 (IL-2) is a 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 cases to result in regression of established tumors in both experimental animals [J. Exp. Med. l§;:ll69—ll88 (l985)] and in man [N. Engl. J. Med. gig; 1485-1492 (1985) and N. Engl. J. Med. ;;§:889—897 (l987)]. The anti—tumor effects of rIL—2 are thought to be mediated by host cytotoxic effector lymphocytes which are activated by rIL—2 in vivo [J. ;;g:285—294 (l987)]. animal models suggest that rIL—2 might also have value in Immunol. In addition, results from the treatment of certain infectious diseases [J. Immunol. l35:4160-4163 (1985) and J. Virol. §l:2l20—2l27 (l987)] and in ameliorating chemotherapy—induced immunosuppression IE 990006 [Immunol. Lett. ;g:3o7—314 (l985)]. However, the clinical use of rIL—2 has been complicated ‘by the serious side effects which it may cause [N- Engl. J. Med. 3l3:14B5-1492 (1985) and N. Engl. J. Med. 3l6:8B9—B97 (l987)]. therapy while reducing toxicity is to use two or more One approach to improving the efficacy of cytokine cytokines in combination. For example, synergistic antitumor activity has been shown to result when rlL—2 is administered to tumor—bearing mice together with recombinant interferon alpha (rIFN alpha) [Cancer Res. g§:260—264 (1988) and Cancer Res. g§:58l0—5817 (l988)] or with recombinant tumor necrosis factor alpha (rTNF alpha)[Cancer Res. gZ:3948—3953 (l987)]. are thought to be mediated by host cytotoxic effector Since the antitumor effects of IL-2 lymphocytes, it would be of interest to identify and isolate novel cytokines which synergize with rIL—2 to activate cytotoxic lymphocytes in vitro. These novel cytokines would also be useful as antitumor agents when administered in combination with rIL—2 in vivo. Thus, the present invention provides a novel cytokine protein called Cytotoxic Lymphocyte Maturation Factor (CLM) 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. The present invention comprises a process for isolating CLMF in a substantially pure form which process comprises the following steps: IE 990006 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 (CLMF). 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 a 35 kDa subunit which are subunits. a 40 kDa subunit and or more disulfide bonds. The present bonded together via one invention also provides the nucleotide sequence of the CLMF gene and the amino acid sequence of the CLMF protein encoded by the said gene. Based on this sequence information derivatives of the natural CLMF protein may be prepared which CLMF protein derivatives have CLMF activity. Therefore the present invention relates to a protein comprising Cytotoxic Lymphocyte Maturation Factor (CLMF) in a substantially pure form or 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 IE 990006 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 genes encoding the 40 kDa subunit and/or the 35 kDa subunit, to vectors containing these genes to host cells transformed with the vectors containing the said genes and to CLMF proteins and derivatives prepared in such a transformed host cell. In addition the present invention relates to a method for stimulating LAK cells and T—cells which method comprises treating these cells with CLMF alone or with IL—2. 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 125I—labelled immunodepletion of CLMF bioactivity. 3: 5I—CLMF binding to neutralization of CLMF characterized by 1: immunoprecipitation of CLMF, 2: blotting of CLMF, 4: its cellular receptor and 5: western inhibition of bioactivity. Twenty hybridomas secreting anti—CLMF antibodies were identified. The I—1abelled CLMF bioactivity as assessed in antibodies were found to 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 IE 990006 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 l25I—labelled CLMF to PHA activated PBL blast cells in the Of the 20 antibodies l2 antibodies were found to inhibit greater than 60% 25I—labe1led CLMF binding to the blast cells. 7B2 and 4A1. presence and absence of each antibody. tested. neutralize CLMF 8E3. These data confirm that of the inhibitory antibodies. viz. bioactivity while one non—inhibitory antibody, does not neutralize CLMF bioactivity. 51—labelled CLMF binding to its cellular receptor will neutralize CLMF bioactivity as antibodies which block 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 CLME cellular receptor. The monoclonal anti—CLMF antibodies of the present invention 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 tw0—site immunoassay to measure levels of CLMF in biological fluids. cell culture supernatants and human cell extracts. Thus, monoclonal antibodies against CLMF which exhibit a number of the present invention is also directed to utilities including but not limited to: IE 990006 1. Utilizing the monoclonal antibodies as affinity reagents for the purification of natural and recombinant human CLMF: 2. Utilizing the monoclonal antibodies as reagents to configure enzyme—immunoassays and radioimmunoassays to measure natural and recombinant CLMF in biological fluids, cell culture supernatants. cell extracts and on plasma membranes of human cells and as reagents for a drug screening assay; 3. Utilizing the monoclonal antibodies as reagents to construct sensitive two-site immunoassays to measure CLMF in biological fluids, cell culture supernatants and human cell EXCIBCCSZ 4. 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: 5. 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 DRgfl}flGS Figure l 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 IE 990006 B1ue—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 S. 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—mercapt0ethanol) and reducing (lane B; in the presence of B-mercaptoethanol) conditions showing the 75,000 molecular weight CLMF separated into two subunits of 40 kDa and 35 kDa. The remaining lanes in the gel shown in this Figure contain standard proteins comprising the 44 and 68 kDa marker protein. Figure B 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. IE 990006 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: IE 990006 _ 10 _ N-t — N—terminal 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~mercaptoethanol; lane B: Fraction 39 without fl—mercaptoethano1: lane C: Fraction 39 with B—mercaptoethanol; lane D: Standard proteins with B-mercaptoethanol. 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—18 column elution profile shown in Figure 19. S: = protein—standard: F: = f1ow—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 (Fig. about 29. 22B) and after (Fig. 25, 14. 12. and 9 kDa, contain the CNBr fragments having the following sequences: 22A) excising the regions of respectively. The regiones IE 990006 _ 11 _ 1 (P?)—P—K—N-L—Q-L-K—P~L~K—N—?—V—(Q?)- (New sequence from 40 kDa protein) II ?—Q—K—A—(R?)—Q—T—L-E—F—Y—P—?~T— (New sequence starting at residue no. 30 of 35 kDa protein) 111 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. l9O 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 CLMF purified by affinity chromatography using the monoclonal antibody 7B2 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 acid sequence of the 40 kDa subunit of human CLMF. Figure 26 a. acid sequence of the 35 kDa subunit of CLMF b and c show the CDNA sequence and the deduced amino Figure 27 depicts the inhibition of CLMF bioactivity by serum from rats immunized with CLMF and from non—immunized rats (control). IE 990006 _ 12 _ Figure 28 shows a SDS—PAGE analysis of immunoprecipitates of l25I—CLMF by monoclonal antibodies 4A1 (lane 1), 4Dl (lane 2). SE3 (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, 4A1, BE3, 6A3, 9F5 and 2A3 and of rat polyclonal anti—CLMF antibodies (RS1) with the CLMF 75 kDa heterodimer. NRS: = 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. In lanes l to 18 the following mAbs were used: 4A1. 4Dl. 7B2, 7A1, 2A3, 1C1, BE4, 8A2. BE3, 1B8. 4A6. 6A2. 8C4. 9F5, 6A3, 9C8, 8A1 and 22E7, respectively. In lane 19 a control antibody, in lane 20 a fusion rat serum and in lane 21 a normal rat serum was used. Figure 33 shows the binding of l25I—CLMF to PHA—activated peripheral blood lymphocyte (PBL) lymphoblasts. Figure 34 shows the inhibition of l25I—CLMF binding to PHA—activated PBL blast cells by rat anti-CLMF serum. The data are expressed as amount (% bound) of l25I—CLMF binding to the cells in the presence of the indicated IE 990006 - 13 _ concentrations of serum when compared to the total specific binding in the absence of serum. Figure 35 shows the inhibition of the binding of l251—CLMF to PHA-activated PBL blast cells by monoclonal The data are expressed as % inhibition of the binding of 25I—CLMF to the cells in the presence of a 1:1 dilution of supernatant when compared to antibody supernatants. the total specific binding in the absence of antibody supernatant. Figure 36 shows the inhibition of the binding of l25I—CLMF to PHA—activated PBL blast cells by various concentrations of purified monoclonal antibodies. The data l25I—CLMF bound to the cells in the presence of the indicated the total are expressed as the amount (% cpm bound) of concentrations of antibody when compared to 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 fi—mercaptoethanol. ‘IE 990006 . Lane 1 1 ul CLMF 2 3 ul CLMF 3 6 ul CLMF 4 Blank 5 Blank 6 5 ul 7 1 ul prestained molecular weight standards CLMF 3 ul CLMF 6 ul CLMF 10 10 ul prestained molecular weight standards both hereby incorporated herein by reference. All publications mentioned herein. supra and infra. invention as well as CLMF The CLMF active proteins of the present include the homogenous natural CLMF protein active proteins which contain a biologically active fragment of natural CLMF. Furthermore the present invention includes recombinant CLMF proteins as well as fusion proteins, i.e. CLMF protein derivatives comprising the amino acid sequence of natural CLMF or a partial sequence thereof together with amino acid sequences derived from other proteins. The proteins of this invention have the biological activity of CLMF as measured by standard assays such as T—cell growth factor assay as described below in the Example. The CLMF proteins of the present invention also include non~naturally occurring CLMF analogous proteins having an amino acid sequences which is analogous to the amino acid sequence of CLMF or its CLMF active fragments. Such CLM analogue proteins are proteins in which one or more of the amino acids of natural CLMF or its fragments have been replaced or deleted without loss of CLMF activity. Such analogues may be produced by known methods of peptide chemistry or by known methods of recombinant DNA technology. IE 990006 _ 15 _ The CLMF biological activity of all of the proteins of the present invention such as site directed mutagenesis. 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 35 kDa subunit and the 40 kDa subunit of the CLMF protein is depicted in Figures 25 and 26. Based on these sequences, which were obtained in accordance with this invention. biologically active analogues and fragments of the CLMF protein can be obtained. These biologically active proteins may be produced biologically using standard methods of the recombinant DNA technology or may be chemically synthesized in an amino acid synthesizer or by manual synthesis using wel1—known liquid or solid phase peptide synthesis methods. In a similar way analogues, fragments and proteins comprising an amino acid sequence of CLMF together with other amino acids can be produced. All of these proteins may then be tested for CLMF activity. 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 derivative of the said protein which derivative exhibits CLMF activity 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 CLMF and to isolated polynucleotides encoding a protein as defined above, which polynucleotide contains a sequence corresponding to the cDNA encoding CLMF, to recombinant vectors comprising a polynucleotide encoding a CLMF protein, 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 Furthermore the said proteins. vectors and antibodies. IE 990006 F 15 _ present invention relates to methods for stimulating LAK cells, T—cel1s or Natural Killer Cells using the said CLMF protein. As used herein, the term "polynucleotide containing a sequence corresponding to the CDNA encoding CLMF" means that the polynucleotide contains a sequence which is homologous to or complementarity to a sequence in the cDNA encoding CLMF. The degree of homology or complementarity to the CDNA will be approximately 50% or greater. about 70%, correspondence between the CLMF sequences and the CDNA can preferably at least and even more preferably at least about 90%. The be determined by techniques known in the art, including, for example. by direct comparison of the sequenced material with the cDNAs described, stringency conditions which are appropriate to the presumed by hybridization experiments using homology of the sequences. followed by digestion with single strand nucleases and by size determination of the digested fragments. The practice of the present invention will employ, unless otherwise indicated, conventional techniques of molecular bio1oqY. microbiolo9Y. recombinant DNA and immunology. which are within the skills of an artisan in the field. Such techniques are explained fully in the literature. Maniatis, Fitsch & Sambrook, MOLECULAR CLONING; A LABORATORY MANUAL (1982); DNA CLONING. VOLUMES I AND II (D.N Glover ed., 1985): OLIGONUCLEOTIDE SYNTHESIS (M.J- 1984); NUCLEIC ACID HYBRIDIZATION (B.D. Hames & S.J. 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, GENE TRANSFER VECTORS FOR MAMMALIAN CELLS (J.H. Miller and M.P. 1987, Cold See e.g., Gait ed., Higgins eds.. Inc.): Calos eds.. Spring Harbor Laboratory), Methods in Enzymology Vol. IE 990006 _ 17 _ and Vol. and Wu. eds.. IMMUNOCHEMICAL METHODS IN CELL AND MOLECULAR BIOLOGY (Mayer and Walker. 1987, PROTEIN PURIFICATION: PRINCIPLES AND PRACTICE. Edition (1987, Springer—Verlag, N.Y.), and HANDBOOK OF EXPERIMENTAL IMUNOLOGY, VOLUMES I—IV (D.M. Weir and C.C. Blackwell eds., 1986). 155 (Wu and Grossman. respectively); eds., Academic Press, London). Scopes, second The DNA sequences and DNA molecules of the present invention may be expressed using a wide variety of For example, host/vector combinations. useful vectors may consist of segments of chromosomal, non-chromosomal and synthetic DNA sequences. Examples of such vectors are viral vectors. such as the various known derivatives of SV40, such as plasmids from E. coli including phage DNAS, bacterial vectors. pCR1. pBR322. pMB9 and RP4. derivatives of phagex. M13 and other filamentous such as the numerous sing1e—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 adenovirus and/or cells, such as those containing SV40, 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 CLMF proteins are characterized by comprising at least one expression control sequence which is operatively linked to the CLMF DNA sequence inserted in the vector in order to control and to regulate the expression of Examples of useful expression the cloned CLMF DNA sequence. control sequences are the lac system. the trp system, tac system, the trc system, major operator and promoter the control region of fd coat protein, e.g.. the promoters of yeast acid regions of phage K. the glycolytic promoters of yeast. the promoter for 3—phosphoglycerate kinase. IE 990006 - 13 - phosphatase. e.g., Pho 5. the promoters of the yeast a—mating factors. and promoters derived from polyoma virus. adenovirus. retrovirus, and simian virus, e.g., the early and late promoters or SVQO. 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 CLM—related DNA such as in animal and human Mol. sequences in eukaryotic hosts, cells [e.g., P. J. l: 327-41 (1982); S. Subramani et al., Biol. l: 854-64 (1981): R. J. Cell. Biol. lggz 601-64 (1982): S. I. "Expression and Characterization of The Product of_A Human Southern and P. Berg, J. Appl. Genet. Mol. Cell. Kaufmann and P. A. Sharp. M01. Scahill et al., Immune Interferon DNA Gene in Chinese Hamster Ovary Cells", Proc. Natl. Acad. Sci. U.S.A. fig: 4654-59 (1983): G. Urlaub and L. A. Chasin. Proc. Natl. Acad. Sci. USA 11: 4216-20 (1989)]. 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 IE 990006 _ 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 may be selected from a variety of known hosts. 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 SV40 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. IE 990006 - 20 - The host organisms which contain the expression vector comprising the CLMF 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 increase in the number of cells per unit time decreases, the DNA coding for the protein is transcribed and the transcribed the expression of the CLMF protein is induced, i.e. 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 protein 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. gg: 476-556 (197l)], treatment) or by chemical means (e.g. by enzymatic treatment (e.g. lysozyme 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 which organisms. The translation start signal AUG. 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 CLMF produced by fermentation of the prokaryotic and eukaryotic hosts transformed with the DNA sequences of this IE 990006 -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 high ion exchange chromatographic methods such as gel filtration, performance liquid chromatography (HPLC), 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 protein 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 CLMF 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, having the activity of CLMF. Among the known techniques for determining such active sites are x—ray crystallography. spectroscopy and site specific mutagenesis. Accordingly. nuclear magnetic resonance. circular dichroism, fragments obtained in this way may be employed in methods for stimulating T-cells or LAK cells. The CLMF proteins or derivatives prepared in accordance with this invention or pharmaceutical compositions comprising the CLMF protein or derivative 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 IE 990006 _ 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 CLMF protein of the present invention by mixing the said CLMF protein 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., interleukin—2), carriers, adjuvants, excipients, etc., human serum albumin or plasma preparations. 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 CLMF protein or derivative and if desired of interleukin—2 in lyophilized form. The vials containing the CLMF protein or derivative 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. IE 990006 _ 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 p¢rson skilled in the art is in a position to select alternative products from other suppliers. Exgmgts PURIFICATION AND CHARACTERIZATION OF CYTOTOXI§_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% 30 min.) fetal bovine serum. 2 mM and 100 ug/ml heat—inactivated (56°C. L-glutamine. 100 units/ml penicillin, streptomycin (all cell culture media were from GIBCO Laboratories. Grand Island. NY). Higher producer sublines of NC~37 cells were derived by limiting dilution cloning in liquid microcultures. Each well of three Costar 3596 microplates (costar C0,. 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 4 weeks after culture initiation the contents of wells IE 990006 _ 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 10 , in 1 ml cultures containing 3 ng/ml phorbol l2—myristate St. MO) and 100 Supernatants were one million cells were stimulated to produce_CLMF 13-acetate (PMA) (Sigma Chemical Co., Louis, ng/ml calcium ionophore A2318? (Sigma). harvested from the cultures after 2 days, dialyzed against about 50 volumes of Dulbecco's phosphate buffered saline SPECTROPOR® #1 tubing (Fisher Scientific) overnight with one change of buffer and then for (Gibco) using e.g. 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). NC—37.89, NC—37.98. and NC—37.102. identified which routinely produced CLMF at titers ; 4 times Three sublines, were 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 II, Wheaton Millville. NJ). Cell suspensions were prepared containing 1-1.5 x 106 NC-37.89. NC—37.9B or NC—37.102 cells/ml in RPMI 1640 medium supplemented with 1% Instruments. Nutridoma—SP (Boehringer Mannheim Biochemicals, IN), 100 units/ml 100 ug/ml streptomycin. 10 ng/ml PMA and 20-25 Two hundred fifty to three Indianapolis. 2 mM L—g1utamine, penicillin. ng/ml calcium ionophore A23l87. 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 IE 990006 _ 25 _ capped tightly and incubated at 37°C with continuous rolling At the end of this time. the culture EDTA and phenylmethylsulfonyl for three days. supernatants were harvested. fluoride (both from Boehringer Mannheim) were added to the culture supernatants at final concentrations of 1 mM and 0.1 mM, The respectively, to retard proteolytic degradation. supernatants were stored at 4°C. Lympokine Activated Killer Cell Induction (LCI) Assay. (LAK) Culture supernatants and chromatographic fractions were tested for their ability to synergize with r1L—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 concentration of approximately 5 units/ml. The blood was diluted 1:1 with Hanks‘ balanced salt solution (HBSS) without calcium or magnesium (GIBCO). The diluted blood was then layered over 15 ml aliquots of Ficoll/sodium 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 HESS without calcium or magnesium. The resulting cell suspension was then layered over l5 ml aliquots of 20% sucrose (Fisher) in RPMI l64O 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 IE 990006 _ 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. lgl;2282—2290 (1983) for accessory cell depletion by L—leucine methyl ester except that the glutamic acid ester was substituted for the leucine ester. The accessory cel1»dep1eted PBMC were further fractionated by centrifugation on a discontinuous Percoll density gradient (Pharmacia. Piscataway. NJ) as described by l;;:39—54 (1988). from the 38. 41, Wong et al.. Cell Immunol. Mononuclear cells recovered 45, and 58% Percoll layers were pooled and were washed and used as a source of LAK cell precursors in the assay. 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, supplemented with 0.1 mM nonessential amino acids. 60 ug/ml arginine Hcl, 10 mM users buffer, ' 100 2 mM L~glutamine, 100 units/ml penicillin, . . -5 ug/ml streptomycin (all available from GIBCO), 5 x lo M 2-mercaptoethanol (Fisher Scientific, Fair Lawn, NJ), 1 mg/ml dextrose (Fisher), and 5% human AB serum (Irvine Scientific, Santa Ana, CA). These cells were incubated in 24-well tissue culture plates (Costar, Cambridge, MA) in 1 ml cultures (7.5 x 105 cells/culture) to which 1O—4 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 5 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 IE 990006 _ 27 _ 5lCr—1abe1led K562 or Raji cells (both cell lines may be obtained from the ATCC) and tested for Cr and performing 0.1 ml aliquots of their lytic activity in 5 hour 1Cr release assays. method for labelling target cells with the cytolytic assays have been described by Gately et al., [JNCI gg:1245-1254 (l982)]. release was calculated as [(g — g)/(100 — SJ} X 100. . . 51 The percent specific Cr where I(‘D is the percentage of Cr released from target cells released spontaneously from target cells incubated alone. incubated with lymphocytes and Q is the percentage of l . . The total releasable Cr was determined by lysis of the target cells with 2% sodium dodecyl sulfate; JNCI §g:1245—1254 (1982). see Gately et al., All lymphocyte populations were assayed in quadruplicate for lytic activity. 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 Costa: 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 contained l0h4 M hydrocortisone sodium succinate (Sigma) and were brought to a total volume of 0.1 ml by addition of TCM with The cultures were incubated for 3 days at 37°C, 1Cr—1abelled K562 cells (5 x 104 cells/ml in TCM with 5% human AB serum) 5% human AB serum. after which 0.1 ml of were added to each well. 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 VA). Cr released into each supernatant solution was measured collection system (Skatron. Sterling, The amount of IE 990006 _ 23 _ with a gamma counter (Packard, Downer's Grove, IL), and the . . Sl . % specific Cr release was calculated as described above. All samples were assayed in quadruplicate. Qytolytic 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. lgg: 1274-1282 (1986) and by in Cell. ill: 39-54 (1988). peripheral blood mononuclear cells were isolated from the in J. Immunol. Wong et al. lmmunol. Human 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). culture plates (Costar #3424) by incubation of Percoll CTL were generated in MLTC in 24-well tissue 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% For uv—irradiation, HTI44 cells/ml in Hanks‘ balanced salt solution without phenol red (GIBCO) human AB serum (1.2 ml/culture). cells were suspended at a density of l—l.5 x 106 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). (960 uW/cmz (model UVG—54 MINERALIGHT® lamp. Ultra—violet Products, CA). For gamma irradiation, HTl44 cells were suspended at a density of 1-5 x 106 cells/ml in TCM with 5% human AB serum and irradiated (10,000 rad) by use of and the cells were then irradiated for 5 min) by use of a 254 nm uv light Inc., San Gabriel. a cesium source irradiator (model 143, J.L. Shepherd and CA). UV- HTl44 were centrifuged and resuspended in TCM with 5% human Associates. San Fernando, or gamma—irradiated IE 990006 -29.. AB serum at the desired cell density for addition to the MLTC. In addition to lymphocytes and melanoma cells. some MLTC received human rIL—2 and/or purified human CLMF at the concentrations indicated. Hydrocortisone sodium succinate (Sigma) was added to the MLTC at a final concentration of iohq or 10-5 cells) to supress endogenous cytokine production [S. Gillis et al., J. 12;: 1624-1631 (l979)] and to reduce the generation of nonspecific LAK cells in the cultures [L.M. Muul and M.K. Gately, J. lgg: 1202-1207 (l984)]- The cultures were incubated at 37°C in a humidified 2 At the end of this time. lymphocytes from replicate cultures were pooled, M (cultures containing uv—irradiated melanoma cells) M (cultures containing gamma—irradiated melanoma lmmunol. lmmunol. atmosphere of 5% C0 in air for 6 days. centrifuged. resuspended in 1.2 ml TCM containing 5% human and tested for their ability to lyse HTl44 K562 erythroleukemia cells (obtainable from ATCC) in overnight AB serum. melanoma cells. and. as a specificity control, Cr release assays. . 51 Melanoma cells and K562 cells were labeled with Cr sodium chromate as described by Gately et al. [JNCI 63: 1245—1254 (1982)]. Likewise, measurement of lympocyte— mediated lysis of lCr—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 lCr—labeled K562 (2 x 105 cells/ml in in the wells of costar 3696 cells. For assaying the lysis of ul aliquots of TCM with 5% human AB serum) "half—area“ microtest plates. After overnight incubation at 37°C. the plates were centrifuged for 5 min at 1400 x g. and 50 ul of culture medium was aspirated from each well. The 1 _ . amount of Cr in each sample was measured with a gamma . . 51 counter (Packard), and the % specific Cr release was calculated as described above. All assays were performed in quadruplicate. and values in the table (see below) represent IE 990006 -30- the means i l 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 (S X 105 containing 0.1% phytohemagglutinin—P (PHA—P) Laboratories, MI). split 1:1 with fresh TCM. culture to give a final concentration of 50 units/ml. cells/ml) were cultured at 37°C in TCM (Difco Detroit, After 3 days, the cultures were and human rIL—2 was added to each cultures were then incubated for an additional 1 to 2 days. at which time the cells were harvested, washed. and cells/ml. suspension was added heat—inactivated goat anti—human rIL—2 1/200) to block any potential This resuspended in TCM at 4 x 105 To this cell antiserum (final dilution: IL-2-induced cell proliferation in the assay. 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 cultures were incubated for 1 day at 37°C in a humidified atmosphere of 5% CO2 (New England Nuclear. fractions in the wells of costar 3596 microplates. . . 3 . . in air. and 50 ul of H~thym1d1ne MA), 10 uci/ml in TCM, were The cultures were further Boston. then added to each well. incubated overnight. Subsequently, the culture contents were harvested onto glass fiber filters by means of a cell MA). 3 . . . . . H—thym1d1ne incorporation into cellular DNA was measured harvester (Cambridge Technology Inc., Cambridge. and IE 990006 _ 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 and the specific activity of the purified material. 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 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 3596® microplates. The mixtures were 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 3H—thymidine, harvested, and analyzed for 3H-thymidine incorporation as described above. Natural killer (NK) 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 IE 990006 -32- above and were suspended in RPMI 1640 medium supplemented with 10% heat—inactivated fetal bovine serum, loo units/ml penicillin. 100 ug/ml streptomycin, and 2 mM L—glutamine. The PBMC were incubated overnight at 37°C in 1 ml cultures (5 x 106 cells/culture) together with rIL—2 and/or After 18-20 hours. the contents of the cultures were harvested and centrifuged, purified CLMF at various concentrations. 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 Cr release assays as described above. :30 n C 6- r: I r a I i en._.<»L_cc;1_ 1_S_t.u>_<:;r_-=.>..'~ a_LH__e;9,1.1_a.L,i__<::s_:=- 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 PTTK00005; Millipore Bedford. MA). volume of approximately 1.9 liters, performed with 10 mM MES, pH adjusted to 6.0 with 10 N NaOH. Corp.. After concentrating to the desired a buffer exchange was The concentrate was centrifuged at 10,000 x g for 10 minutes at 4°C and the precipitate discarded. Jwn Pxrhunqw -?;«munrwg vh Nunvy E if Wuiumr The concentrated supernatant solution was applied at a flow rate of l20 ml/hr to a Nu—Gel P—SP (Separation Industries. Metuchen. NJ) column (5 X 5 cm). equilibrated in 10mM MES, pH 6.0. 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/10 mM MES. (Fig. 1). Aliquots of fractions were assayed for T cell pH 6.0 at a flow rate of 2 ml/min growth factor (TGF) activity. Fractions containing TGF activity were pooled and dialyzed (Spectra/Por 7, Fisher Scientific) against 50 volumes 20 mM Tris/Hcl. 9% 7.5 in IE 990006 _ 33 - order to reduce the salt concentration of the preparation by 50-fold. 'umn The dialyzed sample was centrifuged at 10,000 x g for 10 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. washed with this same buffer until baseline absorbance The column was 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/HC1. 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. '$£pQ"~‘.:h§ The dialyzed sample was filtered through a 0.45 um NY) and the filtrate applied at a flow rate of 60 ml/hr to a Mono Q cellulose acetate filter (Nalgene Co., Rochester, HR 5/5 (Pharmacia LKB Biotechnology, Inc., Piscataway, NJ) column (5 x 50mm) equilibrated in 20mM Tris/HC1, 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 0 to 0.25 M Nacl/20 mM Tris/HC1, pH 7.5 at a flow rate of 60 ml/hr (Fig. 3). Aliquots of fractions were assayed for absorbance monitoring at 280 nm was obtained. TGF activity and protein purity was assessed without reduction by SDS»PAGE [Laemmli, Nature (London) gg1:6BO—685 (1970)) using 12% slab gels. Anal. _l_lZ:3o7—31o (1981)) to visualize protein (Fig. 4). Fractions 36 and 37 were of greater than Gels were silver stained [MorrisseY. Biochem. IE 990006 -34.. 95% 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 O-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. The gel was silver stained to visualize protein (Fig. and revealed protein of 75.000 molecular weight. Fractions Fractions 86 through 90 were of greater than 95% purity 87 and 88 were pooled and aliquots were analyzed by SDS—PAGE under reducing (in the presence of B—mercaptoethano1) 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 disu1fide— —bonded 40 kDa and 35 kDa subunits. The overall purification of CLMF that was achieved is shown in Table 1. The protein content of the Mono Q— and Vydac dipheny1—purified material was calculated on the basis IE 990006 sea x ~.n oHo.o moo.o mod x m~.m nofi x m.m mov.o Hmo.o nod x mv.n hog x m.m mno.o mno.o oofi M v.o boa x m.~ Ha vm.o moa M v.H ofi K m.~ mm on.o oa M w.H _ W. m _ X: x ma ommm $4 .1: x o.m oz mz oz moa x w.H 3.53 3.5 3595 2: »ufl>fluu< cfiououm cfiououm mafia: ofiufloomm Hmuoe Uwmoom Hmpoh H mqmce HH.m 35>: »;M>Muu< omfioom ovm.H ooo.ow wE:HO> w:+nm zowaumum Hxcmzmfin wvaumm zofluumum 0 0:02 hm cowuomuh 0 0:0: ®moumm<|maw:H: mmum Hwozz wumuuzmucou cwuwufiflumugfiz wucmwmzummzm Hfimu omfioom mmuw IE 990006 of amino acid analysis. A specific activity of 8.5 x lo? units/mq and 5.2 x 107 units/mg for Mono Q— and Vydac diphenyl—purified material respectively, 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 of the molecules of CLMF in the HPLC elution solvents 0.1% trifluoroacetic acid). (i.e., acetonitrile in Chemical Characterization 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). and its Proline, cysteine and tryptophan were not determined (ND). Quantitation of histidine was not possible due to a large artifact peak. associated with Tris, coeluting with His (*). Between 5 and 30 picomoles of dipheny1—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—puritied CLMF. CWO SEQLIBIICGS PEBBEIIE . Data from the first 22 cycles indicated as would be expected from the heterodimeric structure of CLMF. These results may be summarized as follows: _-..._———__..__——.—...._—-:--....___-___.._____. __.__—-_._—-.——.....__.___-._-_.____.__.__._ IE 990006 —-—_———_-_——-.—___._.._,,____,____ —-—-__._.__._...._-_._____________ u-——_—._a._---._..._...__..————¢_.._.__._——_ » --——a—--—_——o.—..—..._—....._.__—.._—.a-.-—_—.-.—_____—..__—_—_.....___ TABLE 2 Amino ggid Aspaztic acid or aspazagine Threonine serine Glucamic acid Proline Glycine Alanine Cysteine Valine Methionine Isoleucine Leucine Tyrcsine Phenylalanine Hiatidine Lysine Arginine Tryptophan glutamine mol % 11.8 14.9 IE 990006 Aspartic acid or ésnaragine Tnreonine Serine Glutamic acid or glutamine Proline Glycine Alanine Cysteine Valine Methionine Isoleucine Leucine Tyrosine Phenylalanine Histidine Lysine Arqinine Tryptophan .n -q ya g.) 1,; ua .1: H as N U1 .1: (.1 u OJ ‘-J O 1 I A 0 u I I 0 I I I I I I I O I .p -4 on -4 as J: M \o N no to x! (D H *0 N (D (_!" IE 990006 Reve:sed—Phaee EPLC ___._____.____.___ by Stern A.S. and Lewis. R.V. (1985) in Research Methods in Neurocnemistry, Eds. Marks. N. and Rodnignt. R. (Plenum. New York) Vol. 6. 153-193. An automated fluorescence detection system using fluoreecamine (Polysciences. PA) monitored the protein in the column ef and Moscnera. J. (1981) Methods Enzymol. Reversed—pnase HPLC was carried diphenyl columns (4.6 x 20mm. CA). Proteins were eluted with gradient in 0.1% TFA. ., warrington, fluents fstein. S. 1g:a35—447J. out using vydac C18 or The Sep/a/ta/tions Group, Hesperia. an acetonitrile and Stein. 5. (1986) in Methods of Protein Microcnaracterization tshively, J.E., Ed.). pp. 105-119. Humana Press. Clifton. NJ]. faqueu,u RuuL}FZL ;xrformed using an Applied Bluff»?-my .u" K;d J A .as phase sequencer (Foster City, _E~wz:<, L.V., Hwnr ;:1.ar, M.W.. Hood, L.E., and Dreyer, w.J.. J. Biol Chem. 256:7990~7997 (1981)). Phenylthionydantoin (PTH) amino acid derivatives were identified “on—line” with an ABI Model 120A PTH analyzer DE'I‘E.“.?-H?‘.’A'."';:’,'*T~.' OF‘ T_jA?\"I‘?_F-.-'_ A!‘-4.21117 .-‘x(.’_i'_‘- S_.“.'.0L'?T?-SITES '1"-IE SU UNITS OF CLMF Purification cf the 40 kDa subpnit of CLMF TDTDI F’ Fm IE 990006 -40- Stored supernatant solutions from NC—37 cells totaling 39.1 liters were pooled and concentrated to approximately 2.4 liters using the Pellicon Cassette System and stored at —20°C. preparation was centrifuged and the precipitate discarded. To clarify this concentrate after thawing, the The supernatant solution was applied to a Nu—Gel P—SP column and protein was eluted with a salt gradient (Fig. fractions were pooled and dialyzed in order to reduce the This after centrifugation to remove particulates, Peak TGF activity was determined and the active salt concentration of the preparation by 50-fold. material. was applied to a Blue—B—Agarose column. Protein was eluted with a salt gradient (Fig. 9). Peak TGF activity was determined and the active fractions were pooled and dialyzed in order to reduce the salt concentration of the preparation by 100—fo1d. Mono Q column. Protein was eluted with a salt gradient (Fig. 10). activity. This material, after filtration. was applied to a Aliquots of fractions were assayed for TGF Fractions 39 and 40 of the previous Mono Q chromato- graphy were pooled and diluted 1:1 vol/vol with 8M urea and pumped onto a Vydac diphenyl column using an 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 O—70% acetonitrile over 7 hrs in 0.1% trifluoroacetic acid (Fig. 11). Aliquots of fractions were assayed for TGF activity. Protein purity of the fractions containing TGF activity was assessed by SDS~PAGE under reducing conditions, i.e. in the presence of B—mercaptoethanol (Fig. 12). Fractions 94 through 97 contained the 40,000 dalton subunit >90% pure. Determination of the amino—terminal sequences of the subunits of CLMF IE 990006 -41- The ability to prepare a highly enriched preparation of the 40,000 dalton subunit of CLMF allowed for its partial sequence analysis. Amino terminal sequence determination was attempted by automated Edman degradation on 20 pmol of the diphenyl— purified 40.000 dalton subunit. The results may be summarized as follows: Cycle 1 2 3 4 5 6 7 Amino Acid I W E L K K D Cycle 8 9 10 11 12 13 14 Amino Acid V Y V V E L D Cycle 15 16 17 18 19 20 21 Amino Acid W Y P D A P G Cycle ;; 23 Amino Acid E M with regard to the sequence analysis of 75.000 dalton CLMF and the sequence analysis of the 40,000 dalton subunit of CLMF, one can deduce the amino terminal sequence of the 35.000 dalton subunit of CLMF. of the 35,000 dalton subunit and the 40,000 dalton subunit The amino terminal sequences can be summarized as follows: ' IE 990006 35.000 dalton subunit: 5 10 NH —?~?—Leu—Pro-Val—Ala—Thr(?)—Pro—Asp—Pro—G1y— 15 20 Met—Phe—Pro—?—Leu—His—His—Ser(?)~Gln- 40,000 dalton subunit: 1 5 10 NH ~I1e—Trp~G1u—Leu—Lys—Lys—Asp—Val—Tyr—Val—Val—G1u 15 20 23 Leu-Asp-Trp-Tyr—Pro-Asp—Ala—Pro—G1y—G1u—Met- where ? represents an undetermined or "best-guessed" Determination of internal amino acid sequence segments of the 40 kDa subunit of CLMF CLMF was purified as described above. The 40,000 dalton subunit was separated and purified from the 35,000 dalton subunit by the method described by Matsudaira [J. Biol. Chem. gggz 10035-10038 (1987)). (in 500 ul of 20 mM Tris. pH 7.5; with 200 pl of a 2 x concentrate of sample buffer [Laemm1i, Nature gglz 6B0~685 (1970)]. concentrated to 400 pl and disulfide bonds broken by the Fifty micrograms of CLMF 0.15 M NaCl) was diluted The sample was addition of 18 ul B—mercaptoethanol followed by exposure to 105°C for 6 minutes. The sample was loaded onto a minigel (1.0 mm thick) containing 12% polyacrylamide and electrophoresed according to Laemmli (supra). After electrophoresis, the gels were soaked in transfer buffer (10 mM 3—cyclohexy1amino—l—pro— panesulfonic acid, 10% methanol, pH 11.0) for 5 minutes to reduce the amount of Tris and glycine. During this time, a polyvinylidene difluoride (PVDF) membrane (Immobilon; Bedford. MA) was rinsed with 100% methanol and The gel. Milliporez stored in transfer buffer. backed with two sheets residue. IE 990006 -43- of PVDF membrane and several sheets of blotting paper, was assembled into a blotting apparatus and electroeluted for 30 The PVDF membrane was The edge of the blot was excised from the PVDF membrane and stained with min at 0.5 Amps in transfer buffer. washed in deionized H20 for 5 minutes. 0.1% Coomassie Blue R-250 in 50% methanol for 5 minutes, and then destained in 50% methanol, 10% acetic acid for 5-10 minutes at room temperature. The 40,000 dalton stained band was then matched to the corresponding region of the unstained blot and the 40,000 subunit was cut from the unstained PVDF. The N—termines of the Coomassie B1ue—stained 40,000 dalton subunit was sequenced to confirm that the N—terminus matched the one previously determined (see above). By this method, the 40,000 dalton protein was identified as the 40,000 subunit of CLMF. Five percent of the PVDF bound 40.000 dalton subunit was remaining 95% of the blotted 40,000 dalton subunit was analyzed for its amino acid composition (Table 4). fragmented with trypsin according to the procedure of Bauw. [Proc. Natl. Acad. Sci. USA gg: 7701-7705 (l989)]. The membrane carrying the protein was cut into pieces of et al. approximately 3 by 3 mm, and collected in an Eppendorf tube. pyrrolidone (40,000 dalton) solution in methanol. They were then immersed in 300 ul of a 2% polyvinyl- After 30 minutes, the quenching mixture was diluted with an equal volume of distilled water and further incubated for 5-10 minutes. The supernatant solution was then discarded and the membrane pieces were washed four times with 300 ul water and once with 300 ul 100 mM Tris HCl (pH 8.5). _Two hundred microliters of this buffer containing 2 ug of trypsin was added. The sample was shaken and incubated for 4 hours at 37°C. transferred into a second Eppendorf tube and the membrane The supernatant solution was then pieces were further washed once with 100 ul of 88% IE 990006 -44- (vol/vol) formic acid and three times with 100 ul of deionized water. All washing solutions were added to the digestion mixture in the second Eppendorf tube. The resultant peptides contained in the pooled digest were separated a YMC C—18 column (2.6 x 50 mm; Morris Plains, NJ). TABLE 4 Amino Acid Residue No. Aspartic acid or asparagine 27.9 Threonine 20.7 Serine 24.6 Glutamic acid or glutamine 44.6 Proline ND Glycine 16.3 Alanine 16.2 Cysteine ND Valine 20.9 Methionine 2.5 Isoleucine 10.3 Leucine 22.9 Tyrosine 12.9 Phenylalanine 9.9 Histidine 5.2 Lysine 24.5 Arginine 12.5 Tryptophan ND Note: The results represent the mean of two analyses. Proline, cysteine. and tryptophan were not determined by narrow bore HPLC (HP1090A. Hewlett Packard) on (23) (23) (34) (35) (14) (15) (14) (10) (23) (12) (22) (12) (26) (12) (10) (ND). Values in parentheses represent the theoretical amino acid composition of the 40,000 dalton subunit based upon the primary structure of the protein deduced from sequence analysis of cloned 40.000 dalton subunit. IE 990006 -45.. The above described procedure is shown schematically in Figures 13 and 14. The tryptic peptide map of the digested 40,000 dalton subunit is shown in Figure 15. Peptides were eluted with a linear gradient of acetonitrile. The peaks which were sequenced are numbered according to their fraction number. The amino acid sequence of these peptides is shown in Table Many cryptic peptides were recovered from all regions of the intact 40,000 dalton subunit (Table 5). 60) was recovered in high yield. The N—terminal hexapeptide (fraction no. The carboxy~termina1 peptide (fraction no. 72) was recovered and is the full length of the predicted C—termina1 peptide although the last two amino acids were not positively confirmed by sequencing. This is probably due to the fact that Cys and Ser residues are not detected well. especially when they occur at the end of a peptide. Four potential Asn—linked carbohydrate sites may be predicted from the CDNA sequence. Two peptides containing two of these sites were sequenced. When peptide 196-208 (fraction no. 70) was sequenced, no peak was detected at residue 200 indicating that this Asn (predicted by the CDNA) is indeed glycosylated. Peptide 103-108 (fraction no. 52) yielded Asn at residue 103. Therefore, this site is not glycosylated. An unknown peak seen in the phenylisothiocyanate (PTH) Biol. Chem. 256: 7990 55 was detected at the position sequence analysis [Hewick et al., J. (l98l)] of fraction no. corresponding to residue no. 148. The site is predicted to be a Cys residue which is normally not detected by sequence analysis unless it is modified. The above PVDF transfer procedure was repeated on a second 50 ug aliquot of CLMF (see Figure 13 and 14 for ?;K2LM;:HH1w TLMF fraction residue 92; £2; 52 103-108 55 139-157 55 57 267—279(?) 57 52-58 57 218-228 60 1-6 67 288-? 67 B5—lO2(?) 70 l96~208 71 85—96(?) 72 28B—306(?) 78 71-85 1-1-r ; - KL} -1‘ D 5 (1 I-1 IE 990006 IA§LE_£ PvQg_ N—te:minal sequence N—K—T—F—L—R G—S—S—D—P—Q~G—V—T—*—G~A~A-T—L—S—A—E—R V—F—T—D—K—T—S—A»T—V—I—?—R T—L—T—I—Q~V—K N—L—Q—L—K-P—L—K—N—S—R I—W-E—L—K—K A—Q—D»R—Y—Y—S—S— K—E—D—G—I~W—S—T—D—I—L—K—D—Q—K—E—P— L—K—Y—E~?—Y—T—S—S—F—F—I—(R?) K~E—D—G—I—?—S—T—D—I—L—K A—Q—D—R~Y—Y—S~S—S—W—E—?—A—S—V—P—?—? (G?)—G—E~V—L—S~H—S—L—L—L-(L?)—H—K—K IE 990006 -47 .. procedure outline). However. the blotted 40,000 dalton subunit was fragmented with the proteolytic enzyme. Staphylococcus aureus V8 protease (Endoproteinase Glu-C, IN). were digested for 6 hours at 37°C with 20 ug of V8. Membrane pieces peptides were extracted with 88% (vol/vol) formic acid and Boehringer Mannheim. Indianapolis. separated on a Phase Separations column (2 x 150 mm, C8 83. England. UK) (Figure 16). with a linear gradient of acetonitrile. Queensferry, Peptides were eluted The peaks which were sequenced are numbered according to their fraction number. The amino acid sequence of these peptides is shown in Table 6. TABLE Q V8 (G1u—C1 40kDa peptides off PVDF fraction no; residue no. N—termina1 sequence 47 1-3 I—W—E 54 4—12 L—K—K—D—V—Y—V—V—E 57 13-22 L—D—W—Y—P—D—A—P—G—E 57 45-59 V-L—G—S-G—K—T—L—T—I—Q—V-K-(E?) Three major peaks of peptide (fraction nos. 47. 54 and 57) containing four peptides were sequenced. All four peptides were from the amino—termina1 region of the 40 kDa subunit indicating that the N—terminus of the protein is most susceptible to VB—digestion. Figure 17 summarizes the protein structural determination of the 40,000 dalton subunit of CLMF. IE 990006 _48 - Direct determination of the amino terminal 35.000 dalton subunit of CLMF sequence of the SDS—PAGE analysis of the Mono Q fraction 39 (see Fig. 3) under reducing (in the presence of B—mercaptoethanol) and non—reducing (in the absence of B—mercaptoethanol) (Fig. 18) demonstrated that the 40,000 dalton molecular weight 40,000 dalton CLMF unassociated with the 35,000 dalton subunit). conditions "contaminant" is "free" subunit (i.e. The evidence which points to this deduction is that without reduction (lane B, Fig. 18) mainly 75,000 dalton CLMF is present with some 40,000 dalton protein. After reduction (lane C. Fig. 18). the 75,000 dalton CLMF is gone yielding the 35,000 dalton subunit and an enriched 40.000 dalton band. Fraction 39 of the previous Mono Q chromatography was reduced in 5% B—mercaptoethanol in the presence of 4 M urea and heated for 5 minute at 95°C. The sample was pumped onto a Vydac C-13 column using an enrichment technique and the column was then washed with 5 ml of 0.1% trifluoroacetic acid. Elution of the proteins was accomplished with a gradient of O—70% acetonitrile over 5 hrs in 0.1% trifluoroacetic acid (Fig. 19). Protein purity of the fractions which were fluorescamine positive was assessed by SDS-PAGE under non—reducing conditions using a 10% slab gel. The gel was silver stained to visualize protein (Fig. 20). 35,000 molecular weight which was greater than 95% pure. Fractions 112 through 117 revealed a diffuse band at The 40,000 dalton subunit and any other proteins present in fraction 39 remained bound to the C—l8 column. These proteins (including the 40.000 dalton subunit) were finally eluted with a solution of 42% formic acid/40% l—propanol. The ability to prepare homogeneous 35,000 subunit allowed for the determination of the amino acid composition and partial sequence analysis of the lower molecular weight subunit of the CLMF protein. Approximately 1 ug of 35 kDa IE 990006 -49 ._ subunit was subjected to hydrolysis. and its amino acid composition was determined (Table 7). Proline. cysteine and tryptophan were not determined (ND). TABLE 7 Amino Acid Mol z Aspartic acid or asparagine 10.9 Threonine 6.7 Serine 8.3 Glutamic acid or glutamine 14.9 Proline ND Glycine 6.1 Alanine 7.7 Cysteine ND valine 6.3 Methionine 2.9 Isoleucine 4.5 Leucine 10.9 Tyrosine 3.2 Phenylalanine 4.4 Histidine 2.3 Lysine 5.6 Arglnine 5.5 Tryptophan ND Amino-terminal sequence determination was attempted by automated Edman degradation on 100 pmol of the C-18 purified 35 kDa subunit. Data from the first 20 cycles confirmed the sequence obtained by deduction as described above.' Furthermore. the second amino acid was obtained in addition to amino acids 21 through 26. These results may be summarized as follows: IE 990006 _ 50 _ Cycle 1 2 3 4 5 6 7*”--0 Amino Acid ? N L P V A T Cycle 8 9 10 11 12 13 14 Amino Acid P D P G M F P Cycle 15 16 17 18 19 20 21 Amino Acid ? L H H S Q N Cycle 22 23 24 25 26 Amino Acid L L R A V Therefore. the amino terminal sequence of the 35,000 dalton subunit can be summarized as follows: 35,000 dalton subunit: 5 10 NH2—?—Asn~Leu—Pro—Va1—Ala—Thr-Pro—Asp—Pro—Gly—Met— 15 20 25 26 Phe—Pro—?—Leu—His—His—ser—Gln-Asn—Leu—Leu—Arg—Ala—Va1 where ? represents an undetermined residue. Determination of the sequence of a tryptic fragment of CLMF Mono Q fractions 36 and 37 from the initial purification of CLMF were pooled (approximately 100 pmol/1.7 ml). A 30 ul sample was removed and the volume of the rest was reduced to 200 ul under a stream of helium. one hundred microliters of 0.1 M ammonium bicarbonate was added. Trypsin (Worthington Biochemical Corp., Freehold, NJ) cleavage was performed at a substrate—to-enzyme ratio of 2:1 (w/w) at 37°C for 20 hours. The resultant peptide fragments IE 990006 were reduced and carboxymethylated. This was accomplished by addition of 160 ul of 0.1 M Tris—HCl, pH 8.5/6 M The volume was reduced to 200 ul under a and 4 ul of dithiothreitol (50 mg/ml) The mixture was incubated at 37°C for 4 hrs. guanidine—HCl. stream of helium. was added. After reductive cleavage of the disulfide bonds. [lqcjiodoacetic acid (4 umol) was added and the resulting solution was incubated in the dark at room temperature for 10 minutes. The resultant peptide fragments were isolated by reversed—phase HPLC (Fig. 21) on an 5-5 120 Rngstrom ODS YMC. NJ). Peptides were eluted with a l—propanol gradient in 0.9 M column (2.6 x 50 mm, Inc., Morris Plains. acetic acid. pH adjusted to 4-0 with pyridine. The amino acid sequence of the peptide found in fraction 46 was found to be: Asp—Ile—I1e—Lys—Pro—Asp—Pro—Pro—Lys (determined by automated Edman degradation). Determination of internal amino acid sequence seqments of CLMF CLMF was purified as previously described. Approximately 80 ug of protein was precipitated with 10% trichloroacetic acid. The precipitate was dissolved in 70% (V/V) aqueous formic acid at room temperature. An approximately 50-fold molar excess over methionine residues of cyanogen bromide (CNBEJ stirring, in a small volume of 70% formic acid was added, with and the mixture was incubated in the dark under oxygen—free helium at room temperature for 48 hrs. The mixture was diluted with 15 volumes of water, divided into two equal portions and dried under a stream of helium. For complete removal of the acid and by—products, the drying was repeated after further addition of water. IE 990006 _ 52... one of the portions (approx. 40 ug) of fragmented CLMF was dissolved with 50 ul Laemmli sample buffer [Laemmli, Nature 227: 680-685 (1970)) containing 4% B—mercaptoethanol followed by exposure to 105° C for 6 minutes. The sample was loaded into 3 wells of a minigel (1.0 mm thick) containing 17.5% polyacrylamide and electrophoresed according to Laemmli (supra). the gels were soaked in transfer After electrophoresis, buffer (10 mM 3—cyclohexy1amino—1—propanesulfonic acid. methanol, pH 11.0) for 30 min. During this time, a polyvinylidene difluoride (PVDF) membrane (Immobilon: Bedford. stored in transfer buffer. MA) was rinsed with 100% methanol and The gel. Millipore: backed with two sheets of PVDF membrane and sandwiched with blotting paper, was assembled into a blotting apparatus and electroeluted for 30 min. at 0.5 Amps in transfer buffer. The PVDF membrane was deionized H20 for 5 min and stained with 0.1% Blue R-250 in 50% methanol for 5 min, washed in Coomassie and then destained in 50% methanol. 10% acetic acid for 5-10 min at room temperature. A number of smeared bands were observed (see Fig. 22B). Five regions of the membrane were excised across the three last lanes containing the CLMF CNBr digest. These A summary of the sequences obtained 22A. regions were sequenced. from the CNBr fragments of CLMF is shown on Fig. The second portion (approx. 40 ug) of fragment CLMF was dissolved in approx. 400—500 ul 88% formic acid 0.1 M Tris/HCl, 0.5 M NaOH. The sample was pH adjusted to pH 4.0 with formic containing 6 M guanidine HC1. 8.0. acid. The peptide fragments were isolated by reversed—phase HPLC (Fig. 23) on a Vydac C4 column (4.6 x 20 mm. The CA). Peptides were eluted Sep/a/ra/tions Group, Hesperia. with a 4.5 hours linear gradient of acetonitrile in 0.1% TFA. One of these peaks was sequenced and the amino acid IE 990006 sequence of this peptide was: Fraction N0. N—Terminal Seguence 47 V—D~A—V-H—K—L—K—Y—E—?~Y-T~S—(S?) -F—F—I—R—D—I—I—K—P- (Starts at residue number 190 of 40 kDa subunit) It is assumed or known that the above sequence is preceded by a Met residue. The residue marked with "?" represents a "best—guessed“ residue. EQRIFIQQTION OF CLMF AND IHE 40.000 DALTON SUBUNIT THEREOF USING AFFINITY CHROMATOGRAPHY An affinity chromatography resin was prepared by covalently attaching the monoclonal antibody 7B2, the preparation of which is described below, to activated agarose. similarly, the below outlined purification could also be carried out by covalently coupling the antibody to silica or thin microporous membranes. The activated agarose was prepared as follows: 1. 100 ml Sepharose CL—6B was washed three times with 100 ml H20. 2. 100 ml of 1% sodium meta—periodate in H20 was added to the resin and the suspension shaken at room temperature for 60 min. 3. The resin was washed with cold H 20 thoroughly. The covalent attachment of 7B2 to the activated agarose was carried out as follows: 1. 9 ml of the activated agarose prepared as described above was suspended in 7 ml of 7B2 (approx. 3.9 mg/ml) in phosphate buffered saline, pH 7.4. IE 990006 _54_ 50.2 mg of cyanoborohydride was added to the gel suspension which was shaken overnight at 4°C. The gel suspension was filtered and added to 7 ml of 1.0 M ethanolamine. pH 7.0 containing 50.2 mg of cyanoborohydride. One milliliter of the above described resin (approx. 2.6 mg lgG/ml gel) was packed in a column and washed extensively with phosphate buffered saline. Fractions from the Mono Q chromatography containing the 75 kDa CLMF protein and additional major contaminating proteins were pooled (approx. 3.5 x 106 U TGF activity) and dialyzed extensively against PBS. This preparation was applied to the 7B2—Sepharose column at a rate of S ml/hr at room temperature. The column was washed with phosphate buffered saline (pH 7.4) until baseline absorbance monitoring at 280 Adsorbed proteins were then eluted with 0.15 M NaCl, nm was obtained. 0.2 N acetic acid, at approx. pH 3. Aliquots of fractions were assayed for TGF activity. Approximately 76% of the starting activity was recovered in the acid eluate. Protein purity was assessed without reduction by SDS—PAGE {Laemm1i, Nature 227: 680-685 (l970)] using a 10% slab gel. Gels were silver stained [Morrissey, Anal. Biochem. ll7:307—3l0 (l98l)] to visualize protein. The acid eluant contained pure CLMF and the "free" unassociated 40 KDa subunit of CLMF (Fig. 24). DETERMINATION OF THE pI OF CLMF Thirty microliters of the pooled Mono Q fractions 36 and 37 (see Fig. 3) were spotted onto a precast ampholine PAGplate gel. determine the pl of CLMF. major band was observed at pl 4.8 and a minor band at pl 5.2. pH 3.5-9.5 (Pharmacia LKB Biotechnology) to Based on pl standard markers. a Based on pH determination. the pl of these bands are IE 990006 4.2 and 4.6 respectively. BIOLOGIC ACTIVITIES OF PURIFIED CLMF Purified CLMF stimulated the proliferation of human PHA-activated lymphoblasts in the T cell growth factor assay (Table 8). purified CLMF recovered from the Mono Q column was compared The T cell growth factor activity of the to that of a standard preparation of human lymphokines in five separate experiments, and the specific activity of the purified CLMF was found to be 8.5 i 0.9 X 107 protein. units/mg In one experiment in which purified CLMF obtained from diphenyl HPLC was compared to the standard lymphokine preparation in the TGF assay. a specific activity of 5.2 X concentrations of purified CLMF and human rIL—2 were tested units/mg protein was observed. When suboptimal in combination in the TGF assay, additive proliferation was observed (Table 8), up to the maximum proliferation caused by rIL—2 could be that the alone. However, proliferation caused by rIL—2 distinguished from proliferation due to CLMF in former was totally inhibited in the presence of a neutralizing goat anti-human IL-2 antiserum but the latter was not affected. The ability of purified CLMF to activate cytotoxic effector cells was examined both in a 4-day LAK cell induction assay and in an overnight NK cell activation assay. In the LCI assay. purified CLMF at concentrations as high as 800 units/ml had little activity in the absence of IL-2 (Table 9). However, CLMF synergized with low concentrations of human rIL—2 in causing LAK cell induction in as much as the lytic activity generated in the presence of both cytokines was significantly greater than the sum of the lytic activities observed in cultures containing either cytokine alone (Table 9). In the presence of rIL-2. purified CLM was active at concentrations as low as 3 units/ml. IE 990006 -56 - TABLE 8 Purified Human CLMF Stimulates the Proliferation of Human PHA~Activated Lymphoblasts Qxtokine Added: 3H—Thymidine Incorporated by Human CLMFC Human rIL-2 PHA-Activated Lymphoblasts Expt. (u/mL1_ (u/ml) (mean cpm + 1 S.E.M.) 13 0 0 10,507 1 596 500 0 70,055 1 1.530 100 0 60,377 1 1,927 20 0 36,018 1 321 4 0 24.996 1 669 0.8 0 17,765 1 790 2b 0 0 9,975 1 374 200 0 60,980 1 1,713 50 0 33.817 1 884 12.5 0 18.835 1 2.132 3.1 0 13,648 1 731 0 15 30.041 1 5.835 0 4 21.282 1 1,145 0 1 11,241 1 898 50 4 62.050 1 2,403 12.5 4 40,628 1 2.196 3.1 4 31,144 1 3,754 ' All cultures in experiment 1 contained goat anti-human rIL—2. “ None of the cultures in experiment 2 contained goat anti-human rIL—2. Purified human CLMF from Mono Q FPLC. IE 990006 _ _ TABLE 9 Purified Human CLMF Synergizes with Human rIL~2 in the Generation of Lymphokine—Activated Killer (LAK) Cells in 4—Day Cultures Cytokine Added: % Specific 5lCr Releasea from: Human CLMFb Human rIL-2 (u/ml) (u/ml) K562 Raji 0 0 3 1 1.7 -l 1 0.5 800 0 7 1 0.3 1 1 0-1 200 0 5 1 1.1 1 1 0.4 50 0 4 1 3.0 0 1 0.9 0 K 10 1 2.4 2 1 0.8 800 H 41 1 4.0 11 1 0.8‘ 200 H 42 1 1.9 11 1 0.3 50 S 36 1 2.7 9 1 0.8 12.5 S 28 1 2.1 7 1_0.7 3.1 S 19 1 0.8 5 1_0-3 0.8 5 14 1 1.2 3 1 0-8 Values represent the means 1 1 S.E.M. of quadruplicate determinations. The spontaneous Cr release values for K562 and Raji were 16% and 14%, respectively- Purified human CLMF from Mono Q FPLC. In contrast to the results in the 4-day LAK induction assay, purified CLMF was effective by itself in activating human NK cells in an overnight assay (Table 10). In this assay, CLMF was active at concentrations as low as 1.6 units/ml. when CLMF was tested in combination with human rIL-2. the two cytokines together had, at best, additive effects in enhancing NK activity (Table 10). IE 990006 - 53 _ IBBLE,l9 Purified Human CLMF Causes Activation of Natural Killer (NK) Cells in Overnight Cultures % Specific 5lCr Releasea from Raji Cells at Effector/Tarqet Ratio=: Cytokine Added: Human CLMFD Human rIL-2 gu/ml} gu/ml) 20/1 5/1 0 0 10 1 0.6 5 1 0.4 40 0 31 1 0.4 14 1 0.5 8 0 23 i 2.1 12 1 0.4 1.6 0 15 1 0.3 10 1 0.6 0.3 0 12 1 1.2 9 1 0.2 0 1 13 1 0.4 6 1 0.5 40 1 33 1 2.0 17 1 0.5 0 1 26 1 0.0 13 1 1.9 1.6 1 19 1 1.1 11 + 2.1 0.3 1 16 1 1.0 10 1 1.5 0 5 20 1 1.3 13 1 0.6 40 5 23 1 2.0 12 1 1.5 8 5 29 1 1.1 16 1 0.7 1.6 5 27 1 1.2 13 1 0.0 0.3 5 24 1 1.0 13 1 1.2 0 25 38 1 1.4 19 1 0.7 Each value represents the mean 1 1 S.E.M. of quadruplicate determinations. The spontaneous 51Cr release was 9%. Purified human CLMF from Mono Q FPLC. IE 990006 _ 59 - In addition to its ability to enhance the lytic activity of nonspecific NK/LAK cells. CLMF also facilitated specific CLMF increased the specific allogeneic CTL response to weakly human cytolytic T lymphocyte (CTL) responses in vitro. immunogenic. gamma—irradiated HTl44 melanoma cells (Table 11). CLMF also facilitated specific allogeneic human CTL responses to In combination with a low concentration of rIL—2. uv—irradiated HTl44 melanoma cells. which did not elicit any detectable CTL response in the absence of added cytokines (Table 11). generated in these studies was demonstrated by their ability to cause substantial lysis of Slcr-labeled HT 144 melanoma cells but little or no lysis of K562 cells. The specificity of the cytolytic effector cells In contrast, LAK cells which were generated in the same experiments by incubating low density lymphocytes with rIL-2 in the absence of hydrocortisone lysed the K562 cells to a much greater extent than HT144 melanoma cells. For further discussion of the specificity and identity of the cytolytic effector cells generated in assays such a those shown in Table 11 [see Gately et al-, J. Immunol. 136: 1274-1282 (l9B6)]. .xHv>HaummmuH .ouumH©muHm:mEEmo Ho 1mumH©mHHH:>: cmca om: :uH:3 mHHuu mEo:mHvE HHHH: ucwmouamu >H: Dam >:H:u .wHOWhDUUhfl HHmo x44 :H zumu mm: m + H :cHuumuu .c:m; Hmzuo may :0 Hmuomusumum x<4 zmu usn muomuzumpm aha om:Huu:ou H :oHuumuu HHouHmm .muw»aH HHouHmm JHH nan inn mzu cwmzumn cam awn can émm mnu cwmzumn mwumuHuu:H oz» Eouu ©wuwm>Hm: mmuxuo:mExH >uHw:mc pw3oH um:Hmu:ou N + H :oHuumHu HHouuom mmmuosz .mummmH HHouHmm awn 0:0 imv wzu cmuzumn mumuHwu:H mzu Eouu cmHm>ouwH mmuxuoLmE>H kgHw:w@ ;mH; cw:Hmu:ou H :oHuuoum HHouHumn .%HH:uuuvmwmH .m can H nu:mEHHmmxm cm inn Ccm amH mm: momx How can .>Hm>Hgummmmu .~ ocm H mu:uEH»wmxo :H aHm can wmm mm: mHHmu HHHH: Eouu mmmmavu hUHm nsomcmucomm wzh .oHnmu wzu cm czozn mum mumo umo:g uzm .mmmmm Umukm o:u ou Umuzamoca woman vuwz mwu»uo:mE>H cwzz >H:o comm mm: mmmmm u:muHuH:mmn .~ ucwemuwmxw :H .H"v xHwumEHxoummm no omumu ummumu “wu>uo:mE»H m ou vcmwcommmuuou .mmmmw umumHoumu wzu cm mmumuo;aE>H no :oHu:HHU mHH wzu mzmma cecmmuno muwz :3o:m mumu uzg .H ucwemummxm :H .:oHu=HHc m"H um Una wmu:HHU:: >um>Huum oHumH non Uommmmm cam .£uH HE ~.H um umozvmwnmmu .cw:moz .nwHoom mum: mmH:uH:u ogmuHHm=c mo wucwucou exp mu:mEHHwmxm nuon :Hm IE 990006 -\ro|||‘l ‘I \|‘ I ' H H HH H H mH H H no H H HH mH H . H H H m H H mm H H H- H H on oH was m-oH H H H H H H oH H H H- H H HH _Q;* m:oH H . H H H- H H HH H H oH H H oH oH m.» »:H: H;oH H m H H H H H H H H H H H m H.» :HH: H1oH H _ H H H- H H HH H H H- H H H oH ;HH: H-cH H H H H H H H- H H H: H H o 32H: H-oH H H H H H H m- H H H- H H H- oH m.» H-oH H H H H H H H- H H H- H H H H-oH H Hoax HHHH= Hon: vvHH= _HaH=_ .HeH=_ _.,H.; Hzv Hm:oHHuH.H HHou.uH. I. ‘KN .uommnl — “pawn mzau Nana» , .;..u ocowmuuououuaz mvuxuozmeaa llm Eouu mmmuHmHH .HuH.w. oHuH.uwnm I ‘I- ‘I 1 7 - {Hmcu.t3.HI:.u|uoIn,.:3::u oxHH> ZH mqqmu <:oz¢Hm: uHmzmuoHH< OH mmmzommum mawuozmzwa H UHHwJOHwu z<::= uHHHumHm mmuzczzm Hzqu zmuo©u>m:Uz Uvwumusmd comgsfiflo m~\H :o_u=~mc m\H coqusfiqc mmxfi comusflflo m\H comusfiflc m~\H comgsamo mxa comusamo mmxfi cofiuznmo mxfi mE\wum:: w.a HE\mUmE: m Hexmumca av Hsxmumcz oo~ nsfi u o~v.vH mwo u mom.¢H mum H n»w.»v oofi H m-.om oafi H amm.mfi amoa H mmm.vH ohm u ovH.~H so» u ~mm.mH men n eoo.m~ mew u omm.am m~o~ H mao.mm emu H mvm.o~ mvm u ~mm.vH n.:.m.m H H Eno cams. mcomumuucousou mummunozmexq owum>muumuu< uouumm zuzouw Hfivu H mm m4mr1 cut fgg c'I.bd!’ Lymphoblasts. monoclonal anti—CLMF antibodies immunoprecipitated 1251-labelled CLMF, bound to the 40 kDa subunit of CLMF. Receptor on Dfl§;Ag;iyated The previous data demonstrated that the immunodepleted CLMF bioactivity and However, the antibodies present in the hybridoma supernatant solutions could not be directly tested for their ability to neutralize CLMF bioactivity in the TGF or LAK cell induction assays due to non-specific inhibitory effects of supernatant solutions containing control antibodies. Our previous work with IL-2 monoclonal antibodies demonstrated that antibodies which would block l25I—IL—2 binding to IL-2 receptor bearing cells would also neutralize IL-2 bioactivity. Since receptor binding assays are usually unaffected by addition of hybridoma supernatant solutions or other substances, a CLMF receptor binding assay was developed to evaluate the anti—CLMF antibodies for inhibitory/neutrali- zation activity. A CLMF receptor binding assay was configured with 5I—1abelled CLMF and the PHA—activated IE 990006 _ 82 _ peripheral blood lymphoblasts (Fig. 33). The binding of l25I—CLMF to the PHA—activated lymphoblasts was saturable and specific (Fig. 33). Scatchard plot analysis [See N.Y. Acad. 5;: 660-672 (l949)] of the eguilbrium binding data indicated that the apparent 5I—CLMF binding to the‘ receptor is approximately 200 pM and that each lymphoblast Scatchard; Ann. Sci. dissociation constant for has about 700-800 receptors. Since the serum from the rat immunized with CLMF showed neutralization of CLMF I—CLMF The rat immune serum bioactivity, it was tested for inhibition of binding to the lymphoblasts (Fig. 34). blocks 50% of l25I—labe1led CLMF binding at approximately a l/500 dilution, while the control rat serum does not show any inhibition at this dilution. with the specificity of the receptor binding assay established. hybridoma supernatant solutions were tested for antibodies which would inhibit 125I—CLMF binding to lymphoblasts. 125I—CLMF binding to the lymphoblasts was determined at a 1/2 dilution of each 35). supernatant solutions inhibited by greater than 60% 125 . . I—CLMF binding to the lymphoblasts. The degree of inhibition of hybridoma supernatant solution (Fig. Twelve hybridoma The antibodies present in these supernatant solutions have been classified as inhibitory/neutralizing antibodies. Six hybridoma 5 . . I—labelled CLMF binding by less than 40% and were classified as supernatant solutions inhibited Control 125I—CLMF non~inhibitory/non—neutralizing antibodies. antibody inhibited by approximately 10% the binding to the lymphoblasts. Three inhibitory antibodies. 7B2. 2A3 and 4A1, 6A3 and BE3, and two non—inhibitory antibodies. were purified from ascites fluid by protein G affinity chromatography on GammaBind G (Genex. 4A1. 125 . . . I—CLMF binding to the lymphoblasts with IC5 Gaithersburg, MD) columns. Antibodies 2A3 and 7B2 inhibit in a dose dependent manner IE 990006 _ 33 _ concentrations of 0.7 ug/ml, 7 ug/ml and 9.5 ug/ml, iggpectively (Fig. 36). Antibodies 6A3 and BE3 do_not block I-CLMF binding at concentrations of 100 ug/ml (Fig. 36). These data demonstrated that the original classification of each antibody as either inhibitory or non-inhibitory was correct. Direct Neutralization of CLMF Bioactivity by Antibodies. To determine if the antibodies classified as inhibitory by the CLMF receptor binding assay would directly neutralize CLMF bioactivity, each inhibitory antibody was tested for 16). dose dependent neutralization of CLMF bioactivity (40 units/ml) from 0.03 to 100 ug/ml. with IC concentrations of approximately 1 ug/ml and 80 pg/ml. neutralizing activity in the TGF assay (Table Two inhibitory antibodies. 4A1 and 7B2, demonstrated a respectively. These data confirmed that antibodies 5I—CLM binding to the CLMF receptor would also neutralize CLMF bioactivity. inhibiting Tab1e_1§: Kssai Contents: CLMFa none CLMF CLMF CLMF CLMF Antibodyb none none 100 pg/ml 200 pg/ml Control 200 pg/ml - 54 - Total 3H-Thymidine IE 990006 Neutralization of CLMF Bioactivity by Monoclonal Anti-CLMF. % Neutralizationc Incorporation 9923 1 439 25752 1 592 12965 1 938 81 12215 1 663 86 12985 1 269 81 19932 1 1016 37 22379 1 410 21 25405 1 1093 2 10763 1 878 96 15083 1 406 67 23690 1 1228 13 25849 1 1408 0 27654 1 1086 0 22221 1 381 22 27335 1 620 0 a Purified CLMF was used in the TGF b Purified antibodies were added at table. assay at a concentration of 40 units/ml. the concentrations indicated in the C Reduction of 3H-thymidine incorporation to the level seen in the absence of added cytokines was considered to be 100% neutralization. IE 990006 Prznesagienrgi.&naibyd3x§_dd2in;1_a,§zugh:Li; rawhide jjragmpxqpgif the jfhrudn da}!rg1:w1buL1L rd VLM§ A peptide. comprising amino acids 3-13 of the NH2-terminal sequence of the 35 kDa CLMF subunit and a COOH—termina1 cysteine (L—P—V~A—T—P—D—P—G-M—F—C). was synthesized by solid—phase peptide methodology. purified by HPLC. and conjugated to keyhole limpet hemocyanin via the methylated bovine serum albumin procedure. Two rabbits were immunized intradermally with the conjugated peptide in Freund's complete adjuvant (300 ug peptide/rabbit). Six weeks after immunization, rabbits were boosted with free peptide (100 pg, (150 us. were prepared from bleedings taken 7 days later. intravenously) and KLH-conjugated peptide subcutaneously) dissolved in PBS. Serum samples boosting and bleeding schedule was repeated every 4-5 weeks. Serum samples from the first and second bleedings from each rabbit were evaluated for reaction with the synthetic peptide in a direct ELISA assay. The synthetic, free peptide was coated on microtiter plates at 4 ng/ml and 20 ng/ml, and the plates were washed and blocked with bovine serum albumin. Serum samples were tested at various dilutions (Table 17), and antibody reactivity was detected with the use of a second antibody (HRP-conjugated goat anti—rabbit IgG) with o-phenylenediamine as substrate. Absorbance values were read at 490 nm after addition of H2804 to stop the reaction. The results indicate that antibody was produced in both rabbits against 35.000 dalton CLMF peptide (Table 17). verified that the antibody was specific for the peptide In separate experiments. we since (a) serum from non—immunized rabbits does not react with the peptide in ELISA. with the synthetic peptide do not react with a peptide from the 40,000 dalton CLMF subunit and (c) (b) sera from rabbits immunized fragment purified IgG from the serum samples also reacts with the IE 990006 — 86 - synthetic peptide. A serum sample from one of the rabbits (first bleed) was tested by Western blot analysis for reactivity with 75 kDa CLMF and with the 35 kDa CLMF subunit (Fig. 37). purified CLMF (approximately 120 ug/ml) was run on SDS-PAGE. 1:500 dilution of the rabbit anti—CLMF peptide antiserum. Partially transferred to nitrocellulose. and treated with a Antibody reactivity was detected by use of biotinylated goat anti—rabbit IgG and alkaline phosphatase—conjugated streptavidin. The anti—CLMF peptide antibody was found to react both with nonreduced 75 kDa CLMF protein and-with the reduced 35 kDa CLMF subunit (Fig. 37). Although the antibodies produced in this example were polyclonal, a similar approach could be used to prepare synthetic peptide used in this example or other synthetic monoclonal antibodies to the 35 kDa subunit of CLMF. peptides based on the amino acid sequence of the 35 kDa CLMF subunit (Fig. 26) could be used to immunize rats. 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Description

Cytotoxic Lymphocyte Maturation Factor 40kD Subunit and Monoclonal Antibodies Directed Thereto F. HOFFMANN-LA ROCHE AG 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 (cnq~)- The present invention also relates to monoclonal antibodies directed to arm.

'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 gr cup 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, endocrine manner. Cytokines are extremely potent, rather than 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 regulator y 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. Cytokines interact in a network by: 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 (l989)].

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. experimental animals [J. in man [N. Engl. lymphocytes which are activated by rIL—2 in vivo [J.

Immunol. l39:2B5—294 (l987)]. In addition, results from animal models suggest that rIL-2 might also have value in the treatment of certain infectious diseases [J. :4l60—4163 (1935) and J. Virol.

[Immunol. Lett. _Q:307-314 (l9B5)].

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

Med. 3l3:l4B5-1492 (1985) and N. Engl. J. Med. 3l6:889—897 (1987)].. One approach to improving the efficacy of cytokine therapy while reducing toxicity is to use two or more cytokines in combination. For example. synergistic antitumor activity has been shown to result when rIL-2 is administered to tumor—bearing mice together with recombinant interferon alpha (rIFN alpha) [Cancer Res; and Cancer Res. g1:394e—39s3 (1987)]. Since the antitumor effects of IL-2 are thought to be mediated by host cytotoxic lymphocytes, effector” it would be of interest to identify and isolate novel cytokines which synergize with rIL-2 to activate cytotoxic lymphocytes in vitro. These novel cytokines would also be useful as antitumor agents when administered in combination with rIL—2 in vivo.

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 40 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. low concentrations of In the presence 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—ce1l 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 protein being Cytolytic Lymphocyte Maturation Factor (CLMF). substantially pure form. said 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 40 kDa subunit of the CLMF gene and the amino acid sequence of the 40 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 CLMT or its biologically active fragments which proteins exhibit CLMF activity.

The ab°V€ 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 40 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 . . . . . . 25 characterized by 1: immunoprecipitation of I I-labelled CLMF, 2: immunodepletion of CLMF bioactivity. 3: . . . . . 125 blotting of CLMF, 4: inhibition of —CLMF binding to its cellular receptor and 5: neutralization of CLMF Twenty hybridomas secreting anti-CLMF antibodies were identified. The 125I—labelled CLMF bioactivity as assessed in WBSEEED. bioactivity. antibodies were found to immunoprecipitate CLMF and to immunodeplete the T~cell proliferation and 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 LAK cell induction assays. 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. tested, Of the 20 antibodies 12 antibodies were found to inhibit greater than 602 of the 1251-labelled CLMF binding to the blast cells. Two inhibitory antibodies. viz. 7B2 and 4A1. neutralize CLMF bioactivity while one non—inhibitoty antibody. BB3, does not neutralize CLMF bioactivity. 1 These data confirm that . . .

I—labelled CLMF binding to its cellular receptor will neutralize CLMF bioactivity as antibodies which block assessed by the T—ce1l proliferation and LAK cell induction assays. The ability of the antibodies specific for the 40 kDa subunit of CLM to neutralize CLMF bioactivity indicates that determinants on the 40 kDa subunit are necessary for binding to the CLMF cellular receptor.

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: _ 7 - l. 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 supernatants, 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 Elue—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—po1yacry1amide 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. refer to the apparent molecular weight of standard proteins of 44 and 68 kDa in lane 8. i.e.

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—mercaptoethano1) and reducing (lane B; in the presence of B-mercaptoethanol) conditions showing the 75,000 molecular weight CLMF separated into two subunits of 40 kDa and 35 kDa. The remaining lanes in the gel shown in this Figure contain 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—Ge1 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 ll.

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 the proteolytic digestion and the complete sequencing of the 40 kDa subunit of the CLMF cytokine. sequencing.

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: _ 10 _ N-t — N—termina1 sequencing on intact protein Tr - tryptic peptides from map HP2383 numbered by ‘fraction number VB - V8 protease peptides from map HP24l2 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—mercaptoethanol; lane B: Fraction 39 without B—mercaptoethanol: lane C: Fraction 39 with B~mercaptoethano1; lane D: Standard proteins with B-mercaptoethanol.

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-18 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 cryptic 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 (Fig. 22B) and after (Fig. 22A) excising the regions of , 14. 12. and 9 kDa. respectively. The regiones contain the CNBr fragments having the following sequences: about 29, _ 11 _ 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. protein) of 35 kDa I11 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—B-?—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 CLMF purified by affinity chromatography using the monoclonal antibody 7B2 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 acid sequence of the 40 kDa subunit of human CLMF.

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). _ 12 _ Figure 28 shows a SDS—PAGE analysis of immunoprecipitates of 125I—CLMF by monoclonal antibodies 4A1 (lane 1), 4Dl (lane 2). BE3 (lane 3) and 9C8 (lane 4), by a control antibody (lane 5), by immune rat serum (lanes 5 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, 4A1, 8B3, 6A3. 9F5 and 2A3 and of rat polyclonal anti—CLMF antibodies (RS1) with the CLMF 75 kDa heterodimer. NR3: = 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. In lanes 1 to 18 the following mAbs were used: 4A1, 4D1, 7B2, 7A1, 2A3. 1C1. 8B4, 8A2. BE3, 1B8, 4A6. 6A2, 8C4, 9P5, 6A3, 9C8, 8A1 and 2237, respectively. In lane 19 a control antibody, in lane a fusion rat serum and in lane 21 a normal rat serum was used. . . . 5 Figure 33 shows the binding of 12 I-CLMF to PHA-activated peripheral blood lymphocyte (PBL) lymphoblasts. . . . . . 125 . .

Figure 34 shows the inhibition of I—CLME binding to PHA—activated PBL blast cells by rat anti—CLMT serum. The 125 data are expressed as amount (% bound) of I—CLMF binding to the cells in the presence of the indicated - 13 _ concentrations of serum when compared to the total specific binding in the absence of serum.

Figure 35 shows the inhibition of the binding of lZ5I—CLMF to PHA-activated PBL blast cells by monoclonal antibody supernatants. The data are expressed as % l25I—CLMF to the cells in the presence of a 1:1 dilution of supernatant when compared to the total specific binding in the absence of antibody supernatant . inhibition of the binding of Figure 36 shows the inhibition of the binding of l25I—CLMF to PHA—activated PBL blast cells by various concentrations of purified monoclonal antibodies. The data are expressed as the amount (% cpm bound) of l25I—CLMF V bound to the cells in the presence of the indicated concentrations of antibody when compared to the total specific binding in the absence of antibody.

Figure 37 shows a Western blot analysis of the eeactivity 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. ul CLMF ul CLMF ul CLMF Blank Blank ul prestained molecular weight standards 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—ce11 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 40 kDa subunit of the said protein which exhibits CLMF activity if combined with the 35 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 a40kDa subunit of CLMF and to isolated polynucleotides encoding a subunit as defined above, which polynucleotide contains a sequence corresponding to the CDNA encoding a 40 kDa subunit of CLMF, to recombinant vectors comprising a polynucleotide encoding a 40 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., 1985)) OLIGONUCLEOTIDE SYNTHESIS (M.J. Gait ed.. 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. 1 _ 17 _ and Vol. 155 (Wu and Grossman, and Wu, eds., respectively); IMMUNOCHEMICAL METHODS IN CELL AND MOLECULAR BIOLOGY (Mayer and Walker. eds., 1987. Academic Press, London). Scopes, PROTEIN PURIFICATION: PRINCIPLES AND PRACTICE, second Edition (1987, Springer-Verlag, N.Y.), and HANDBOOK OF EXPERIMENTAL IMUNOLOGY, VOLUMES I-IV (D.M. Weir and C.C.

Blackwell eds., 1986).

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-chromosomal and synthetic DNA sequences{ Examples of such vectors are viral vectors, such as the various known derivatives of SV40, 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 40 kDa CLMF subunits are characterized by comprising at least one expression control sequence which is operatively linked to the 40 kDa CLMF subunit DNA sequence inserted in the vector in order to control and to regulate the expression of the cloned 40 kDa CLMF subunit DNA sequence. Examples of useful expression control sequences are the lac system, tac system. the trp system, the the trc system, major operator and promoter regions of phage X, the control region of fd coat protein, the glycolytic promoters of yeast, e.g.. the promoter for 3—phosphoglycerate kinase, the promoters of yeast acid phosphatase, e.g.. Pho 5. the promoters of the yeast a—mating factors, and promoters derived from polyoma virus, adenovirus, retrovirus, and simian virus. e.g.. the early and late promoters or sv4o, 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 cells [e.g., P. J. Southern and P. Berg. J. Mol. Appl.

Genet. lz 327-41 (1982); S. Subramani et al., Mol. Cell.

Biol. lz 854-64 (1981): R. J. Kaufmann and P. A. Sharp, Mol. (l989)].

G. Urlaub Sci. 42l6~2O 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 eukaryotic cells. such hosts are prokaryotic or 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 SV40 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. costs and the folding, biosafety. form or any other necessary post—expression modifications of the desired protein. .- 20- The host organisms which contain the expression vector comprising the 40 kDa subunit of CLMF 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 increase in the number of cells per unit time decreases, the expression of the 40 kDa subunit of CLMF protein 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. gg: 476-556 (l971)j. by enzymatic treatment (e.g. lysozyme treatment) or by chemical means (e.g. detergent treatment, urea or guanidine-HC1 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. certain cases.

In depending on the expression system used and possibly depending on the polypeptide to be expressed this N-terminal methionine residue is cleaved off.

The 40 kDa subunit of CLMF 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 40 kDa subunit of CLMF 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, having the activity of CLMF. including synthetic peptides, Among the known techniques for determining such active sites are x-ray crystallography. nuclear magnetic resonance, circular dichroism, 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.

The 40 kDa CLMF subunit prepared in accordance with this invention or pharmaceutical compositions comprising the 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 40 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. interleukin-2), e.g., carriers. adjuvants. excipients. human serum albumin or plasma preparations.

QCC. , 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 40 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 interleukin-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. 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, 100 units/ml penicillin. and 100 ng/ml streptomycin (all cell culture media were from GIBCO Laboratories. Grand Island. NY). mM L—glutamine.

Higher producer sublines of NC—37 cells were derived by limiting dilution cloning in liquid microcultures. Each well of three Costar 3596 microplates (Costar C0,, Cambridge, MA) received 100 pl 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 e 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 CLMF in 1 ml cultures containing 3 ng/ml phorbol 12-myristate l3-acetate (PMA) (Sigma Chemical Co., St. Louis, MO) and 100 ng/ml calcium ionophore A2318? (Sigma). Supernatants were harvested from the cultures after 2 days, dialyzed against about 50 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-37.102, werei identified which routinely produced CLMF at titers > 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 II, Wheaton Millville. NJ). Cell suspensions were prepared containing 1-1.5 X lO6 NC-37.89, NC—37.98 or NC—37.l02 cells/ml in RPMI 1640 medium supplemented with 1% Instruments, Nutridoma—SP (Boehringer Mannheim Biochemicals, Indianapolis. IN). 2 mM L—glutamine. 100 units/ml ng/ml PMA and 20-25 Two hundred fifty to three penicillin, 100 ug/ml streptomycin, ng/ml calcium ionophore A23l87. 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% CO , 95% air.

The roller bottles were then . the following method. _ :5‘?-';"5',.,,“;_..;. . _ 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, respectively, to retard proteolytic degradation. supernatants were stored at 4°C.

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 Blood from normal volunteer donors was drawn into syringes containing sufficient sterile preservative-free heparin (Sigma) to give a final concentration of approximately 5 units/ml. diluted 1:1 with Hanks‘ 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 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 g 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 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—2290 (1983) for accessory cell depletion by L—leucine methyl ester except that the glutamic acid ester was substituted for the leucine ester.

The accessory cell—depleted PBMC were further fractionated by centrifugation on a discontinuous Percoll density gradient (Pharmacia, Piscataway, NJ) as described by Wong et al., Cell Immunol. ;;;:39—54 (1988). cells recovered from the 38, 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) Mononuclear were pooled and The were washed and the assay. _composed of a 1:1 mixture of RPMI 1640 and Dulbecco's modified Eagle's medium, supplemented with 0.1 mM nonessential amino acids) 60 pg/ml arginine Hcl. 10 mM HEPES buffer, 2 mM L-glutamine. ‘ ug/ml streptomycin (all available from GIBCO). 5 x 10' M 2—mercaptoethanol (Fisher Scientific, Fair Lawn, NJ), 1 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 cells/culture) to which lO‘4 M hydrocortisone sodium succinate (Sigma) was added to units/ml penicillin, and 5% human AB serum (Irvine scientific, Santa Ana, minimize endogenous cytokine production. some cultures also received human r1L-2 (supplied by Hoffmann-La Roche, Inc., Nutley, NJ) at a final concentration of 5 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 _ .1 ml aliquots of 5lCr—labelled K562 or Raji cells (both cell lines may be obtained from the ATCC) and tested for . . . . . 5 ‘~ their lytic activity in 5 hour lcr release assays. The method for labelling target cells with Slcr and performing the cytolytic assays have been described by Gately et al., The percent specific 51c: release was calculated as [(g - g)/(100 - g)] X 100, where g is the percentage of Cr released from target cells incubated with lymphocytes and g is the percentage of 51c: released spontaneously from target cells incubated alone.

The total releasable 51C: was determined by lysis of the LAK Cell Induction Microassay. The microassay for measuring synergy between rIL—Z 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. 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 10 cells/well). some of the wells also received rIL—2 (5 units/ml final concentration) and/or purified CLMF_or immunodepleted CLMF—containing solutions. , -4 contained 10 All cultures M hydrocortisone sodium succinate (Sigma) and were brought to a total volume of 0.1 ml by addition of TCM with 5% human AB serum. . 51 3 days at 37°C, after which 0.1 ml of Cr-labelled K562 cells (5 x 104 cells/ml in TCM with 5% human AB serum) were added to each well. 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, Sterling, VA). 51 Cr released into each supernatant solution was measured The amount of Human peripheral blood mononuclear The cultures were incubated for _ 23 - with a gamma counter (Packard, Downer's Grove, IL), Cr release was calculated as described and the 2 specific 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 1274-1282 (1986) and by in Cell. lll: 39-54 (1988). Human peripheral blood mononuclear cells were isolated from the Wong et al. Immunol. 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. HT144 (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-l.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 (Falcon #3001). (960 uw/cmz for 5 min) by use of a 254 nm uv light (model UVG—54 MINERALIGHT® lamp, CA). For gamma irradiation. HTl44 cells were suspended at a density of 1-5 x 106 cells/ml in TCM with 5% human AB serum and irradiated (10,000 rad) by use of J.L. Shepherd and Uv- or gamma-irradiated dishes and the cells were then irradiated Ultra—violet Products, Inc., San Gabriel, a cesium source irradiator (model 143, CA).

HT144 were centrifuged and resuspended in TCM with 5% human Associates, San Fernando, _ 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 lO*4 M (cultures containing uv-irradiated melanoma cells) or 10-5 M (cultures containing gamma—irradiated melanoma cells) to supress endogenous cytokine production [S. Gillis et al.. J. Immunol. 12;: 1624-1631 (l979)] and to reduce the generation of nonspecific LAK cells in the cultures [L.M.

Muul and M.K. Gately, J. Immunol. lggz 1202-1207 (l984)].

The cultures were incubated at 37°C in a humidified for 6 days. At the end of replicate cultures were pooled, .2 ml TCM containing 5% human AB serum, and tested for their ability to lyse HTl44 atmosphere of 5% CO2 in air this time. lymphocytes from centrifuged, resuspended in melanoma cells, and, as a specificity control. K562 erythroleukemia cells (obtainable from ATCC) in overnight 1 Cr release assays.

Melanoma cells and K562 cells were labeled with Slcr sodium chromate as described by Gately et al. [JNCI 63: 1245-1254 (l982)]. Likewise, measurement of lympocyte— mediated lysis of 51 Cr-labeled melanoma cells was performed in a manner identical to that described by Gately et al. (ibid.) for quantitating lysis of glioma target For assaying the lysis of lcr-labeled K562 cells, 0.1 ml aliquots of lymphocyte suspensions were mixed with 25 ul aliquots of 5lCr—labeled K562 (2 x 105 cells. cells/ml in TCM with 5% human AB serum) in the wells of costar 3696 ”half—area“ microtest plates. After overnight incubation at °C. the plates were centrifuged for 5 min at 1400 x g. and ul of culture medium was aspirated from each well. The . amount of Cr in each sample was measured counter (Packard), 51C: All assays with a gamma and the % specific calculated as described above. release was were performed in quadruplicate, and values in the table (see below) represent - 3o _ the means 1 l 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, cells/ml. To this cell suspension was added heat-inactivated goat anti—human rIL-2 the cultures were and resuspended in TCM at 4 x 105 antiserum (final dilution: 1/200) to block any potential IL—2—induced cell proliferation in the assay. This antiserum may be prepared using methods wel1—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 l/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 fractions in the wells of costar 3596 microplates. The cultures were incubated for 1 day at 37°C in a humidified atmosphere of 5% CO2 in air, and 50 ul of 3H-thymidine (New England Nuclear, MA). 10 uci/ml in TCM, were The cultures were further Boston, then added to each well. incubated overnight. Subsequently. the culture contents were harvested onto glass fiber filters by means of a cell harvester (Cambridge Technology Inc., Cambridge. MA), . . . . .

H—thym1d1ne incorporation into cellular DNA was measured _ 31 _ by liquid scintillation counting. in triplicate.

All samples were assayed 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 and the specific activity of the purified material. 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 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 3596® microplates. The mixtures were 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 . . 3H—thymidine, H—thym1d1ne harvested, and analyzed for incorporation as described above.

Natural killerl1NK) 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, loo units/ml penicillin, 100 ug/ml streptomycin, and 2‘mM L—glutamine.

The PBMC were incubated overnight at 37°C in 1 ml cultures (5 x 106 cells/culture) together with rIL—2 and/or purified CLMF at various concentrations. After 18-20 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 NuGe1 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), eguilibrated in lOmM MES, pH 6.0. 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 to 0.5 M NaC1/10 mM MES, pH 6.0 at a flow rate of 2 ml/min (Fig. 1). 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 _ 33 _ order to reduce the salt concentration of the preparation by 50-fold.

Qxe-Affinity Chromatography on Blue B—Aqarose Column The dialyzed sample was centrifuged at 10,000 x g for 10 minutes at 4°C and the precipitate discarded. The supernatant solution was applied at a flow rate of 20 ml/hr 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-Exchange 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 absorbance monitoring at 280 nm was obtained. Absorbed proteins were then eluted with a l hr linear salt gradient from 0 to 0.25 M Nacl/20 mM Tris/Hcl, pH 7.5 at a flow rate of 60 ml/hr (Fig. 3). Aliquots of fractions were assayed for TGF activity and protein purity was assessed without reduction by SDS—PAGE [Laemmli. Nature (London) gg1:680—685 (l970)] using 12% slab gels. Gels were silver stained [Morrissey, Anal. Biochem. ll1:307—3l0 (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). fractions were assayed for TGF activity.

Aliquots of Protein purity of the fractions containing TGF activity was assessed by sDs—PAGE under non-reducing conditions using a 10% slab gel. 6).

The gel was silver stained to visualize protein (Fig.

Fractions 86 through 90 were of greater than 95% purity and revealed protein of 75.000 molecular weight. 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 QC+m.D :omuU...ur.._ nod ~.m oHo.o aoo.o aofi x mm.m mod x v>.m H.a Aacmzmfio L ~vA:om cofiuoaum nod m.o mov.o Hmo.o sea x nv.m cod K om.o m 0 0:0: nm cowuoouu nod m.m mno.o mno.o uoa x v.o ooa N oe.m a 0 0:0: sea m.H ad v~.o wca x v.H ooa x ~H.m mv omo.ao<-mum:Hn oofi c.~ no o>.o moa x m.H moa x oo.~ om mmlm Hmozz mwmuucmocou voa m.m ommm ma.~ mod n o.m moa x nm.H ovo.H cmumuamumuuab mucmumzummsw oz oz oz mod x m.~ meg K am.~ ooo.oo Hawu cwfioom .53 35 32x95 A3 3.53 3.5 >»m>fiuo< cwmuoum cwmuoum mums: hu«>muo< owuwummw fiance cmaoom Ampoh UDHODQ ®EDHO> mmum H mam _ 35 - of amino acid analysis. . 7 . units/mq and 5.2 x lo units/mg for Mono Q- and Vydac dipnenyl-purified material respectively. fact that A specific activity of 8.5 x 107 was obtained. The» the diphenyl—putified protein has a slightly lower specific activity than the Mono O-purified material may be due to inactivation or denaturation of some of the molecules of CLMF in the HPLC elution solvents (i.e., 0.1% trifluoroacetic acid). acetonitrile in Chemical Characterization The ability to prepare homogeneous CLMF allowed for the first time the determination of the amino acid composition and a partial sequence analysis or the naturally occurring CLMF protein. Between 10 and 20 picomoles of Mono—Q—purified CLMT was subjected to hydrolysis, and its amino acid composition was determined (Table 2). cysteine and tryptophan were not determined (ND).

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

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 heterodimeric structure of CLMF. These results may be summarized as follows: ..

Claims (2)

CLAIMS:

1.Factor

(CLMF) protein characterized in that

A subunit of the Cytotoxic Lymphocyte Maturation

(a) the subunit comprises the amino acid sequence

Ile

Ty:

T:p

P:o

Glu

Leu

Pro

Val

Gly

SEE

Asp

His

Th:

Leu

Se:

Phe

Trp T:p

Se:

Ala

Glu

Glu

Leu

Ile

ASH.

Se:

Th:

Ty:

Glu

Lys

Lys

Se:

Th:

Gly

Th:

Arg

Se:

Gln

Lys

Val

Ala Se:

and that

(b)

Glu

Asp

Glu

Gly

Ala

Se:

Asp

Arg

Leu

Arg

Leu

Se:

Se:

Ty:

Pro

A:q

Pro

Lys

Se:

Ala

Val

Leu

Ala

Asp

Se:

Gly

Leu

Ile

Cys

Th:

Gly

Se:

Val

Leu

Glu

Asp

Gln

His

Se:

Ala

Gln

PEO

Lys

P:o

Gly

Gly

Gln

Leu

Leu

Glu

Th:

Se:

Ala

Glu

Pro

Asn

Pro

Val

Se:

Lys

Th:

Asp

Cys

Lys

Gly

Ile

Lys

Ty:

Leu

Lys

Ala

Ile

Se:

Glu

Cys

Ile

Ty:

Pro

Glu

Ty:

Arq

val

Arg

Se:

Asp

Glu

Th:

Th:

Th:

Leu

Asp

Lys

Se:

Asp

Arq

Glu

Glu

Th:

Lys

Val

Phe

Glu

Ile

Ty:

Val

MET

Trp

Leu

Cys

His

Gln

Ash

Th:

Pro

Val

Glu

Val

Se:

Ash

Se:

Se:

Lys

Cys

Ty:

Ty:

Val

Th:

Th:

His

Lys

Lys

Ty:

Asp

Gln

Arq

Asp

MET

Se:

Leu

Trp

Leu

Lys

Arg

Se:

Val

Val

Leu

Ile

Lys

Lys

Glu

Se:

Leu

Gly

Gly

Se:

Val

Phe

Gln

Glu

Th:

Asp

Lys

Se:

Val

Leu

Asp

Gln

Gly

Glu

P:o

Gly

Th:

Val

Asp

Ala

Asp

Phe

Leu

Ty:

Phe

Arg

Ash

Se:

Glu

Th:

Gln

Val

Gly

Asp

Lys

Acg

Phe

Th:

Ash

Cys

Ala

Ile

Lys

Pro

Cys

Val

Ala

Trp

Leu

Cys

Se:

Lys

Glu

Gly

Ash

Phe

Se:

Cys

Lys

Pro

Val

Arq

Pro

Asp

Val

Phe

Se:

Se:

Asp

Asp

Se:

Glu

Val

Ile

Lys

Th:

Val

Gly

Glu

Ala

His

Asp

Leu

Th:

Gln

Th:

Ile

Glu

if combined with the second subunit of the CLMF

protein comprising the amino acid sequence

Trp

Th:

Glu

Phe

Leu

Trp

Th:

Cys

Lys

Ala

Ty:

Ala

Lys

Ile

Lys

Trp

Val

Asp

Se:

Trp

E\)

(J:

-89..

Atg Asn Leu Pro Val Ala Th: Pro

Leu His His Ser Gln Asn Leu Leu

Gln Lys Ala Acg Gln Th: Leu Glu Phe Ty: Pro Cys Th: Se: Glu

Glu Ile Asp His Glu Asp Ire Th: Lys Asp Lys Th: Se: Th: Val

Glu Ala Cys Leu Pco Leu Glu Leu Tht Lys Asn Glu Set Cys Leu

Asn Se: Aug Glu Th: Se: Phe Ile Tar Asn Gly Set Cys Leu Ala

set Aug Lys Th: Se: Phe MET MET Ala

Ty: Glu Asp Leu Lys MET Ty: Gln val

Ala Lys Leu Leu MET Asp Pro Lys

Asn MET Leu Ala val Ile Asp Glu

Asn Sec Glu Th: Val Pro Gln Lys

Phe Ty: Lys Th: Lys IIe Lys Len

Arg Ile Aug Ala Val Thr Tle

Ala Set

Asp Pro Gly MET Phe Pro Cys

Arg Ala Val Se: Asn MET Leu

Leu Cys Len Set Set Ile

Glu Phe Lys Th: MET Asn

Arg Gln Ile Phe Leu Asp Gln

Leu MET Gln Ala Leu Asn Pne

Set Set Leu Glu Glu Pro Asp

Cys IIe Leu Leu His Ala Phe

Asp Atg Val Th: Se: Ty: Leu Asn

the combined CLMF protein is active in a T cell growth

factor assay, and di5P1aYin9 3 Specific 3CtiVitY 0f at

least 5 2 K 107 Units/mg when determined in a T cell growth

” ctor assay and when combined with the protein as defined,

176! I

in claim l(b)-

2 The subunit of claim 1 comprising the amino acid

sequence

Ile Ttp Glu Leu Lys Lys Asp Val Ty: Val Val Glu Leu Asp Trp

Cys Asp Th:

Leu Asp Gln Set Set Glu

Val Leu Gly Set Gly Lys Th: Leu Th: Ile Gln Val Lys Glu Phe

Gly Asp Ala Gly Gln Ty: Th: Cys His Lys sly Gly Glu Val

Set His Se: Leu Leu Leu Leu His Lys

Ty: Pro Asp Ala Pro Gly Glu LET Val Val Leu Th:

Pro Glu Glu Asp Gly Ile Th: Tcp Th:

Leu

Lys Glu Asp Gly Ile Trp

Sen Th: Asp Ile Leu Lys Asp Gln Lys Glu

Phe Len Atq Cys Glu Ala Lys Asa Ty:

T[p'T£p Leu Th: Th: Ile Set Th: Asp

Pto Lys Asn Lys Th:

Se: Gly Atg Phe Th: Cvs

Leu Th: Phe Set Val Lys

Set Set Aug Gly Set Set Asp Pro Gln Gly Val Th: Cys Gly Ala

Ala

Glu

Glu

Leu

Ile

Asn

Se: Th: Pro

Gln Gly Lys

Lys Th: Se:

Val Acq Ala

Ala Se: Val

3.

Ty:

Glu

Lys

Lys

Se:

Th: Leu

Se:

Se:

Ty:

Pro

Arg

Se:

Val

Leu

Glu

Asp

Gln

His

Se:

Ala

Gln

Pro

Ala

Glu

Pro

Asn

P:o

Val

Se:

Lys

Th:

Asp

Cys

Glu

Cys

Ile

Ty:

Pro

Glu

Ty:

Acg

Val

A:q

Se:

Arq

Gln

Glu

Th:

Lys

Val

Phe

Glu

Ile

Ty:

90 -

Se: Se:

Asn Leu

Se: Trp

Se: Leu

Lys Lys

Cys Arg

Ty: Se:

Val Atg

Glu Asp

Val MET

Gly

Se:

Val

Phe

Gln

Glu

Th:

Asp

Lys

Se:

Asp

Ala

Asp

Phe

Len

TY:

Phe

Arq

Asn

Se:

Asn

Cys

Ala

Ile

Lys

Pro

Cys

Val

Ala

Trp

Lys

Pro

Val

Arg

Pro

Asp

Val

Phe

Se:

Se:

Glu

Ala

His

Asp

Leu

Th:

Gln

Th:

Ile

Glu

Ty:

Ala

Lys

Ile

Lys

T:p

Val

Asp

Se:

Tcp

A polynucleotide encoding a subunit as claimed in any

of claims 1 to 2.

4.

A polynucleotide encoding a subunit as claimed in any

one of claims 1 to 3 which polynucleotide comprises the

nucleotide sequence

zaxrc

cm

TAT

C-TG

Ace

ACC

CAC

AAA

TAT

GAT

CAA

TGT

GCA

GTC

GTC

TTG

ATC

AAA

AAG

GAA

TCT

TTG

GGG

CAC

TCT

GTA

CTC

GAC

CAA

GGA

GAA

CCC

GGA

ACA

GTG

CAG

CCC

GAA

ACC

CAG

GTC

GGC

GAT

AAA

CGT

TTC

ACG

CAG

CTC

TTG

TGT

AGC

GAG

GGA

TTC

AGT

TCC

TTG

GTG

GAT

GAC

AGT

GAG

GTT

ATT

AAG

ACC

GTC

GGA

GTC

GCC

TGG

ACC

GAG

TTT

CTA

TGG

ACC

TGC

AAA

GCT

ATC

ATA

TAT

CCT

GTC

GGA

AGC

TCC

TTT

TGG

AGC

GCT

TCT

TG

ccc

GAA

TTA

om,’

CAT

ACT

cm

TC-G

AGC

ACA

TGG

GAA

GAT

GAA

GGC

GCT

TCG

GAT

AGA

CTG

AGA

CTC

TTT

CTG

ccc

GAT

TCT

GGC

CTC

ATT

TGC

ACG

GGC

TCT

TCC

CCT

GGT

GGC

CAG

CTG

TTA

GAG

ACA

TCT

GCA

CTG

GGA

ATC

AAA

TAC

CTG

AAG

GCC

ATC

TCT

GAG

GTT

GAT

GAA

ACC

ACC

ACC

CTT

GAC

AAG

ACT

GAC

AGA

TTT

GTT

ATG

TGG

CTG

TGT

CAC

CAG

AAT

ACT

ccc '

GTC

AGA

GAC

RTG

AGC

TTG

GGG

AGT

GTG

TTC

CAG

TGG GAG

CTG ACA

AAA GAT

CGC

AGC

GAG

GCT

CAC

GAC

TTA

ACC

CAG

ACG

ATT

GAA

TAT

GCT

AAG

ATC

AAG

TGG

GTC

GAC

AGG-

TGG

GAG

GAG

CTC

ATC

AAT

AGT

CAG

TCA

AGT

TAT

CCT

CGG

CCA

GTG

CTG

GAA

GAC

CAG

CAT

AGC

GCC

CAG

CCC

GAG

CCC

TGC

ATT

TAC

CCC

GAG

TAC

AGA

GTC

CGC

AGT.

CAG

GAG

ACC

AAG

GTC

TTC

GAA

ATC

TAC

TGC

GCC

RTC

CCA

GTT

AGG

CCA

GAC

GTT

TTC

AGC

AGC

CCA

GTG

TCC

AAG

ACG

GAC

TGC

CCT

TGC

GTC

GCC

TGG

TCA

GCC

GTG

GTG

TCA GCA

5. A recombinant vector comprising a polynucleotide

encoding a subunit as claimed in any one of claims 1 to 2

or comprising all or parts of the polynucleotide of claim

4.

6. A microorganism transformed with a recombinant vector

comprising a polynucleotide encoding a subunit as claimed

in any one of claims 1 to 2 or all or parts of the

polynucleotide of claim 4.

7. A polyclonal or monoclonal antibody directed to a

subunit as claimed in any one of claims 1 to 2

immunodepleting CLMF bioactivity as assessed in the T cell

proliferation and LAK cell induced assays.

8. A process for producing a subunit according to any one

gcf claims 1 to 2 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.

GAG

GTC

AGC

AAC

RGC

TCC

TGC

I0

9. A process for producing a subunit according to any one

of claims 1 to 2 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.

l0. A process for producing the CLMF protein which

comprises a process as claimed in any one of claims 8 to 9.

ll. A pharmaceutical composition comprising a subunit as

claimed in any one of claims 1 to 2 and a pharmaceutically

acceptable diluent, adjuvant or carrier.

l2. Use of a compound as claimed in any one of claims 1 to

2 for the manufacture of a medicament for antitumor

therapy.

13. Use of a subunit as claimed in any one of claims 1 to

2 for the preparation of a CLMF protein.