WO1985001298A1 - Monoclonal anti-interleukin-2 antibody useful for immunopurification for interleukin-2 - Google Patents
Monoclonal anti-interleukin-2 antibody useful for immunopurification for interleukin-2 Download PDFInfo
- Publication number
- WO1985001298A1 WO1985001298A1 PCT/US1984/001493 US8401493W WO8501298A1 WO 1985001298 A1 WO1985001298 A1 WO 1985001298A1 US 8401493 W US8401493 W US 8401493W WO 8501298 A1 WO8501298 A1 WO 8501298A1
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- WIPO (PCT)
- Prior art keywords
- antibody
- human interleukin
- interleukin
- dms
- monoclonal anti
- Prior art date
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Classifications
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
- C07K14/52—Cytokines; Lymphokines; Interferons
- C07K14/54—Interleukins [IL]
- C07K14/55—IL-2
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
- C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
- C07K16/24—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against cytokines, lymphokines or interferons
- C07K16/244—Interleukins [IL]
- C07K16/246—IL-2
Definitions
- This invention is in the fields of immunology and cell biology.
- the invention pertains to mono ⁇ clonal anti-interleukin-2 antibodies useful for im unopurification of interleukin-2.
- Interleukin 2 (IL-2) , derived from activated human T-lymphocytes, is a 15,500 (Mr) sialoglyco- protein that initiates mitosis of antigen and lectin-activated cytolytic T-cells.
- IL-2 shares many properties with polypeptide hormones, it was anticipated that antibodies reactive with IL-2 would be advantageous in much the same fashion that anti- hormone antibodies have been so useful.
- anti-IL-2 would make possible definitive studies of IL-2 biologic activity, immunopurifica- tion, the development of IL-2 immunoassays, the identification of the active site of the molecule, and an array of in vivo experiments exploring its physiologic role in the immune response.
- the peptide is generally present and detectable in lymphocyte culture medium in only picomolar concentrations. Consequently, a major problem relates to the practical difficulty of accumulating enough IL-2 protein in a partially- purified and concentrated form so that immunization becomes feasible. Another difficulty concerns the sensitivity and efficiency of detection of anti-Il-2 activity once immunization is accomplished. Hereto ⁇ fore, neutralization of IL-2 biologic activity has been the only assay available to screen for anti- IL-2 in the plasma of immunized mice or hybridoma culture supernatants.
- the invention pertains to monoclonal anti-human interleukin-2 (IL-2) antibodies which are useful for immunopurification of human interleukin-2, and to antibody-producing cell lines which produce the IL-2 antibodies.
- IL-2 monoclonal anti-human interleukin-2
- the antibodies of this invention provide for the isolation of human interleukin-2 in a single step.
- the anti-IL-2 antibodies may be attached to a solid phase to form an immuno- adsorbent which is capable of specifically adsorbing human IL-2.
- Immunoadsorbents so formed may be used to adsorb in erleukin-2 from a biological fluid, a cell culture medium or other liquid. Thereafter, IL-2 may be recovered from the immunoadsorbent in milligram amounts. The recovered IL-2 retains its biological activity and is substantially pure.
- the antibody-producing cell lines which produce the IL-2 antibodies are formed from the fusion of an IL-2 antibody producing cell and an "immortalizing" cell. Alternatively, the anti-IL-2 antibody- producing cell lines may be formed by transformation of an IL-2 antibody-producing cell.
- Figure 1 shows IL-2 production by normal lymphocytes ( • ) versus a high producer subclone (6.8) of the JURKAT human T-leukemia cell line (o) . Serial two-fold dilutions of supernatants were
- FIG. 2 shows plasma anti-IL-2 activity of IL-2 immunized mice, as detected by the neutraliza ⁇ tion of biological activity (A) and by ELISA (B) .
- Figure 3 shows a typical IL-2 titration plot of
- Figure 4 shows neutralization of IL-2 biologi- cal activity by the DMS monoclonal antibodies.
- DMS-1 *
- DMS-2 ⁇
- DMS-3 •
- control mouse Ig o. Miles Laboratories Inc., Elkhart, IN
- FIG. 5 shows DMS-1 monoclonal antibody
- FIG. 6 shows the effect of DMS-1 monoclonal antibody on the biological activity of IL-2 (20 pM) derived from JURKAT subclone 6.8 cells (o) , rat splenocytes ( • ) , normal human tonsil cells ( * -*- ) and mouse splenocytes ( ⁇ ) .
- Figure 7 shows the effect of IL-2 on DMS-1 monoclonal antibody-mediated inhibition of cytolytic
- T-lymphocyte line cell proliferation as monitored by
- Figure 8 shows the lack of effect of DMS-1 monoclonal antibody on the proliferation of IL-2 independent cells.
- HL-60 cells •
- K562 cells o
- BALB/c 3T3 cells A were cultured (1 x 10 5 cells/ml) for 44 h prior to 4 h incubation with [ 3 H]-TdR.
- Figure 9 is a comparison of anti-IL-2 activity of DMS-1 (o) and DMS-3 ( • ) monoclonal antibodies as detected by ELISA.
- Figure 10 shows reversed-phase liquid chromato- graphy elution profile of im unoaffinity-purified human IL-2.
- Figure 11 shows sodium dodecyl sulfate poly- acrylamide gel electrophoresis of immunoaffinity- purified human IL-2.
- IL-2 100 ng is detected by silver stain.
- the anti-human interleukin-2 antibodies of this invention are produced by antibody-producing cell lines.
- the anti-IL-2 antibody-producing cell lines may be hybrid cell lines known as hybridomas.
- the hybridoma cells are formed from the fusion of an • anti-IL-2 antibody-producing cell and an immortali ⁇ zing cell, that is, a cell line which imparts long term tissue culture stability of the hybrid cell.
- the first fusion partner - the anti-IL-2 antibody-producing cell - may be a spleen cell of an animal immunized against human IL-2.
- the anti-IL-2 antibody- producing cell may be an anti-IL-2 generating B lymphocyte obtained from the spleen, peripheral blood, lymph nodes or other tissue.
- the second fusion partner - the immortalizing cell - may be a plasmacytoma cell such as a myeloma cell, itself an antibody-producing cell but also malignant.
- Murine hybridomas which produce monoclonal anti-human IL-2 antibodies are formed from the fusion of mouse plasmacytoma cells and spleen cells from mice immunized against human IL-2. In order to evoke an immune response against human IL-2 in mice, it is important to immunize the mice with at least microgram amounts of human IL-2. Thus, a source of sufficient IL-2 is required.
- One such source is the JURKAT T leukemia cell described by Gillis and Watson. See J. Exp. Med. 152:1709(1980).
- spleen cells are removed for fusion.
- the fusions may be accom ⁇ plished by standard procedures such as those de ⁇ scribed by Fazekas de St. Groth and Scheidegger. - See J. Immunol. Meth. 35:1(1980).
- the hybridomas are then screened for production of antibody reactive with human IL-2, and those which produce reactive antibody are cloned.
- the screening procedure should not be limited to assays for the neutralization of IL-2 biologic activity by the hybridoma cell products. Under a number of
- a neutralization assay would not be able to detect products reactive with IL-2.
- an antibody which reacts with IL-2 may not effectively neutralize its biological activity because while highly specific for IL-2, the antibody does not bind adjacent to the active site of the molecule. Therefore, to screen the hybrids, assays by which the reactivity of the hybridoma products with IL-2 may be directly assessed, such as those described herein, should be employed.
- anti-human interleukin-2 antibody-producing cell lines is by transformation of antibody-producing cells.
- an anti-IL-2 antibody-producing B lymphocyte obtained from an animal immunized against human IL-2 may be infected and transformed with a virus such as the Epstein-Barr virus to give an immortal, anti-IL-2 antibody-producing cell.
- a virus such as the Epstein-Barr virus
- an anti-IL-2 B lymphocyte may be transformed by a transforming gene or transforming gene product to yield an immortal anti-IL-2 antibody-producing cell line.
- the monoclonal anti-IL-2 antibodies are pro- prised in large quantities by injecting the anti-IL-2 antibody-producing hybridomas into the peritoneal cavity of mice and, after an appropriate time, harvesting the ascites fluid which contains a very high titer of homogenous antibody and isolating the monoclonal anti-IL-2 antibodies therefrom.
- the antibodies may be produced by culturing anti-IL-2 producing cell lines in vitro and isola ⁇ ting secreted monoclonal anti-IL-2 antibody from the cell culture medium.
- the anti-IL-2 antibodies of this invention are useful for immunopurification of IL-2.
- the antibodies may be attached to a solid phase to form an immunoadsorbent which specifically binds IL-2.
- Such immunoadsorbents may be constructed to bind milligram amounts of IL-2.
- the IL-2 may be recovered from the immunoadsorbent in biologically active and substantially pure form.
- the solid phase may be a gel material such as agarose or a bead material such as a Sepharose bead or other solid support.
- the antibodies may be coupled to the solid support by conventional proce ⁇ dures for attaching proteins to solid phases.
- the immuno ⁇ adsorbent In order to purify interleukin-2, the immuno ⁇ adsorbent is brought into contact with the biologi ⁇ cal fluid, cell culture medium or other liquid containing IL-2. After an appropriate time, the immunoadsorbent is separated from the liquid, and the interleukin-2 recovered. The interleukin-2 may be eluted from the immunoadsorbent by a mildly acidic aqueous solution.
- Three hybridoma cell lines, designated DMS1, DMS2, and DMS3 which produce anti-IL-2 antibodies useful for the immunopurification of interleukin-2 have been deposited at the American Type Culture Collection in Rockville, Maryland on September 20, 1983.
- Murine IL-2-dependent cytolytic (subclone 15H) T-lymphocyte lines were cultured as previously described at population levels between 1 x 10 4 and 5 x 105 cells/ml in Iscove's Modified Dulbecco's Minimum Essential Medium (IMDMEM) supplemented with 10% heat-inactivated (56°C for 30 minutes) fetal calf serum (FCS, Sterile Systems, Inc., Logan, Utah), 50 U/ml penicillin G, 50 ug/ml gentamycin, 300 ug/ml L-glutamine and 1.0 u/ml human IL-2 derived from JURKAT high producer subcloned (6.8) cells partially purified by gel filtration.
- IMDMEM Iscove's Modified Dulbecco's Minimum Essential Medium
- FCS fetal calf serum
- the P3-NS-1-1 cell line [ref 13] obtained from Dr. Ellis Reinherz, The Dana-Farber Cancer Center, Boston, MA
- BALB/c 3T3 cells and all resulting hybridoma cell lines were maintained in IMDMEM, supplemented with 10% FCS, 50 u/ml penicillin G, 50 ug/ml gentamycin, and 300 ug/ml L-glutamine.
- the JURKAT high IL-2-producer subclone 6.8 See Smith, K.A. ,”T-cell growth factor, a lymphocyto- trophic hormone" In:Proceedings of the 55th Nobel Symposium, Genetics of the Immune Response, Ed. G.
- cell lines were maintained in RPMI 1640 medium [Grand Island Biological Company (GIBCO) , Grand Island, N.Y.] supplemented with 10% FCS, 50 u/ml penicillin G, 50 ug/ml gentamycin, and 300 ug/ml fresh L-glutamine at
- IL-2 biologic activity was determined as previously described by the IL-2 concentration-dependent stimulation of proliferation of a cloned murine cytotoxic T lymphocyte line (CTLL-2, subclone
- OMPI dilutions yielding 50% of the maximum CTLL [ H]-TdR incorporation were determined, and the value for the sample divided by that of the standard to give the concentration of IL-2 in units per milliliter.
- the standard IL-2 preparation which was arbitrarily assigned a value of l ⁇ /ml, consistently yielded 50% of the maximal incorporation at a dilution of 1:10.
- an experimental sample containing 100 U/ml would yield 50% maximal CTLL [ H]-TdR incorporation at a dilution of 1:1000.
- JURKAT subclone 6.8 cells were routinely harvested from the exponential phase of cell growth (0.8 - 1.0 x 10 cells/ml), centrifuged (250 x g, 10 min) , placed into serum-free DMEM (GIBCO) , and cultured at 4.0 x 10 cells/ml for 14-18h in the presence of phytohemagglutinin (1.5 ug/ml, Wellcome Reagents, Beckingham, England) and phorbol myristic acetate (50 ng/ml. Consolidated Midland Corp. , Brewster, N.Y.).
- IL-2 was partially purified from the conditioned medium as described previously by concentration, using an Amicon Cartridge (Amicon Corp. , Lexington, MA) and a YM-5 membrane, followed by gel filtration and isoelectric focusing (IEF) [Robb, R. J. and Smit. h, K.A., Mol. Immunol. 18:1087(1981). Concentrated supernatant was applied to a Sephadex G-1000 superfine column (Pharmacia, Piscataway, NJ) ) and eluted with 0.5 M NaCl, 10 mM Tris-HCl, pH 7.5,
- mice Female BALB/c mice (The Jackson Laboratories, Bar Harbor, Maine) 8-12 weeks of age were inoculated with biochemically purified IL-2 as follows: 1) Primary immunization; 2000 U (equivalent to 15-16 ug IL-2 protein) in 0.1 ml + 0.1 ml complete Freund's Adjuvant (CFA) intraperitoneally (IP) and 2000 U in CFA distributed subcutaneously (SQ) to the limbs; 2) Secondary immunization at week 10; 2000 U in incomplete Freund's Adjuvant (IFA) IP and 2000 u in IFA SQ to the limbs. 3) Tertiary immunization at
- P3-NS-1-1 cells were harvested from the exponential phase of cell growth, washed twice in serum-free DMEM (GIBCO) , combined with the immune splenocytes and exposed to 1 ml 50% polyethylene glycol 4000 ( ⁇ . Merck, Darmstadt, Germany) , 5% dimethyl sulfoxide in saline for 90 sec at 37°C.
- the cells were centrifuged (150 x g, 5 min) and resuspended in 12.5 ml IMDMEM supplemented with 10% FCS, 50 u/ml penicillin G, 50 ug/ml gentamycin, and 300 ug/ml L-glutamine. Serial twofold dilutions of the cell suspension were made and 50 ul of cell suspension distributed to 96-well microtiter plates (Costar) , containing 6 x 10
- BALB/c peritoneal cells/well BALB/c peritoneal cells/well. Plates were routinely seeded at initial mixed-cell concentrations ranging from 4 x 10 5 cells/well to 2.5 x 104 cells/well, as preliminary experiments indicated these cell concentrations favored the initial clonal derivation of hybridoma cells. After 24 hours, selection medium consisting of hypoxanthine (1 x 10-4M) , aminopterin (8 x 10 —8M) , and thymidine (1.6 x 10—5M) was added. The plates were wrapped in aluminum foil and incubated at 37°C, in a humidified atmosphere of 5% C0 2 in air for 10 days without further medium addition.
- hypoxanthine 1 x 10-4M
- aminopterin 8 x 10 —8M
- thymidine 1.6 x 10—5M
- Plasma from IL-2-immunized mice, hybridoma-cell culture supernatants and purified monoclonal anti ⁇ bodies were tested for anti-IL-2 activity by means of three assays: 1) Neutralization of Biologic Activity
- Results are expressed as a percentage of the
- Plasma from IL-2-immunized and control mice, and monoclonal antibody preparations were assayed for anti-IL-2 activity by examining binding to 3 [ H]-leu, lys-IL-2. Robb et al., J. Exp. Med.
- Ice-cold RPMI 1640 (GIBCO) (1 ml) was added, and following centrifugation (9000 x g, 1 min) the supernatant was removed and counted (15 ml Biofluor, New England Nuclear, Boston, MA) via liquid scintil- lation. The Staph-A pellet was then washed twice with 1 ml cold RPMI 1640 and counted by liquid scintillation.
- Plasma from immunized mice and purified mono- clonal antibodies were tested for anti-IL-2 activity by measuring binding to plastic-adsorbed IL-2.
- IL-2 purified by gel filtration and IEF, or by immuno- affinity absorption (see immunoaffinity purification of IL-2) was distributed to 96-well microtiter plates (20 u/well) . After 18h at 25°C the wells were rinsed with Tris-HCl buffered saline (TBS) (pH 8.5) and incubated with 300 ul bovine serum albumin (BSA, 10 mg/ml in PBS) for 1 h at 37°C.
- TBS Tris-HCl buffered saline
- BSA bovine serum albumin
- test plasma or antibody were added for 2 h at 37°C.
- the wells were then washed three times with TBS-0.5% Tween-20 followed by the addition of 50 ul Sheep F (ab) ⁇ anti-mouse Ig-alkaline phosphatase conjugate (New England Nuclear, 650 ng/ l) .
- 50 ul Sheep F (ab) ⁇ anti-mouse Ig-alkaline phosphatase conjugate New England Nuclear, 650 ng/ l
- the wells were washed three times with TBS- Tween 20, (pH-8.0) and twice with distilled water.
- Substrate paranitrophenyl phosphate, 10 mM was then added and after 30-60 min at 37°C the wells were observed visually or read in a spectrophoto- meter (410 nm) .
- mice Twenty female Pristane-primed BALB/c g mice were inoculated with 5 x 10 hybridoma cells from midlog growth phase. After 10-14 days, the ascitic fluid was aspirated repeatedly from each mouse, clarified of cells and debris by centrifuga- tion (1000 x g, 15 min) and stored at -20°C in the presence of 0.02% NaN_.
- IL-2-specific antibody activity was tested by ELISA. Protein concentration was estimated by the approximation that 1 mg of protein yields an absorbence of 1.2 at 280 nm in a cuvette with a path length of 1.0 cm. Ascitic fluids with high levels of antibody contained 30-35 mg/ml of protein and 6-7 mg/ml (20%) of specific antibody.
- the ascitic fluid Prior to ammonium sulfa e precipitation, the ascitic fluid was rendered lipid free by centrifuga- tion (25,000 x g, 30 min, 0°C) followed by filtra ⁇ tion (Whatman No. 1) at 0°C.
- the fluid was diluted with 50 mM Tris-HCl, pH 7.8, 0.14 M NaCl to 10 mg.ml protein, and to 100 ml of diluted solution, 90 ml of room temperature saturated ammonium sulfate solution (adjusted to pH 7.8) was added slowly with vigorous stirring at 0°C. The solution was stirred on ice for 60 min then centrifuged at 1500 x g for 15 min at 4°C.
- the antibody-containing protein pellet was resuspended in 20 mM Tris-HCl, pH 7.8, 40 mM NaCl (2 ml per 100 ml original volume) and dialyzed over ⁇ night against 100 volumes of 20 mM Tris-HCl, pH 7.9, 40 mM NaCl with at least one change of buffer.
- the solution was clarified (27,000 x g, 10 min at 4°C) adjusted to pH 8.2 (IgG_ ) or pH 8.5 (IgG,) , and 200 mg applied to a Protein A-Sepha- rose 4B column (Sigma Chemical Company, St. Louis, MO, 10 ml gel volume) .
- Affi-Gel 10 (Bio-Rad Laboratories, Richmond, CA) was washed on a sintered glass funnel with three volumes of ice-cold isopropanol, followed by three washes with ice cold distilled H 2 0.
- the packed gel was mixed with an equal volume of 10 mg/ml purified antibody solution (equilbrated with 0.2M NaHCO,, pH 8.0, 0.3M NaCl by dialysis) and rotated end over end for 5h at 4°C. After reaction, the gel was centri- fuged and washed twice with 0.1M NaHCO-, 0.15 M NaCl to remove unbound antibody. Protein determination of the combined washes routinely revealed that more than 95% of antibody was coupled to the gel.
- the gel was mixed with an equal volume of 0.1 M ethanolamine-HCl, pH 8.0 and rotated end over end at room temperature for 60 min.
- the gel slurry was washed free of reactants with 10 mM Tris-HCl, pH 7.5 and stored in the presence of 0.02% NaN 3 at 4°C.
- the immunoabsorbent (1 ml gel in a 1 x 10 cm column) was washed with 10 ml 0.2 N acetic acid, pH 3.5.
- the column pH was equili ⁇ brated with 50 mM Tris-HCl (pH7.5) followed by the passage of IL-2-containing supernatants (50 ml/h) .
- the column was washed with 20 ml each of: 1 M NaCl, 10 mM Tris-HCl, (pH 7.5); 0.5% (w/v) Nonidet P-40, 10 mM Tris-HCl (pH 7.5); 10 mM Tris-HCl (pH 7.5); distilled H 2 0.
- Bound IL-2 was eluted from the column by applying five 1 ml aliquots of 0.2 N acetic acid, each time draining the fluid to the bed volume. Eluted fractions were neutralized with 1 M Tris-HCL (pH 8.0) and tested for biological activi ⁇ ty, protein content (Bio-Rad) and analyzed by SDS-PAGE by the method of Laemmli [Nature (Lond.) 227: 680 (19700] [12% acrylamide, reducing conditions, silver stain (Bio-Rad) ] .
- RPLC Reversed-Phase Liquid Chromatography
- IL-2 Monoclonal Antibodies Listed in Table I are the steps that were found to be essential for the development of IL-2 mono ⁇ clonal antibodies. These include the identification of an adequate source of starting material for purification, the development of a purification scheme that provides high recovery of material for immunization, and the development of immunoassays
- T-leukemia cell line JURKAT
- IL-2 T-leukemia cell line
- Figure 1 100-fold greater quantities of IL-2 could be recovered from this cell line compared to other sources of human IL-2.
- PHA-stimulated normal human tonsil cells generally produce IL-1-containing conditioned medium with titers of 1:10 (50% of the maximum CTLL
- JURKAT cells sustain a comparable biologic effect at titers of 1:1000 (range 1:300 to 1:1500) or 100
- IL-2 activity was recover- 10 able in sufficient yield (50% of recovery of star ⁇ ting material) such that mice could be immunized with 20-30 ug of IL-2 protein.
- IL-2 derived from JURKAT cells (the immunogen) , normal human tonsil cells and mouse splenocytes, whereas even at high antibody concentrations, there
- IL-2 competed with the antibody effect in a concentration-dependent manner, such that 1.0 U/ml IL-2 (5 x 10 °M) completely overcame the suppressive effect of 500 ug/ml (3.3 x 10 ⁇ M) DMS-1 antibody.
- 1.0 U/ml IL-2 5 x 10 °M
- the suppressive effect 500 ug/ml (3.3 x 10 ⁇ M) DMS-1 antibody.
- the immunoaffinity-purified IL-2 is homogeneous (see following sections) these results are only interpretable as demonstrating a competitive relationship between IL-2 and the monoclonal antibody.
- DMS-3 proved much more effective than DMS-1 as an immunoabsorbent of human IL-2.
- Table II A representative experiment where solid phase-DMS-1 and DMS-3 were compared as immunoabsorbents for IL-2 derived from several different sources is shown in Table II. Of particular interest was the observa ⁇ tion that DMS-3 absorbed human IL-2 more effectively than did DMS-1, while the DMS-1 antibody absorbed murine IL-2 to a greater extent than human IL-2. It is also notable that neither antibody absorbed rat-derived IL-2.
- Conditioned media containing IL-2 (1 ml) were incubated with 1 ml antibody-coupled Aphi-gel 10 for 30 min at 37°C, eluted and then assayed for IL-2 biologic activity.
- OMPI for the preparative purification of human IL-2.
- JURKAT conditioned medium 1 ml column bed volumes were utilized (10-20 mg of coupled antibody) .
- iJURKAT-derived conditioned medium containing 1.7 mg of IL-2 were passed through a 1 ml DMS-3 immunoaffinity column, followed by extensive washing (20 ml each of: 1.0M NaCl, 10 mM Tris-HCl, pH 7.5; 0.5% Nonidet P-40, 10 mM Tris-HCl pH 7.5; 10 mM Tris-HCl pH 7.5; and H 2 0) to remove nonspecifi- cally-bound contaminants.
- the bound IL-2 activity could be acid-eluted so that all of the activity was concentrated in 5 ml of eluate. Moreover, greater than 90% of the bound activity appeared in the first 2 ml.
- the key aspects include develop ⁇ ment of a rapid, unambiguous, quantitative bioassay to monitor biochemical purification, the identifica ⁇ tion of cellular sources capable of producing the desired lymphokine in large quantities in protein- free medium, and the development of immunoassays utilizing highly purified lymphokine to facilitate the identification of anti-lymphokine secreting hybridomas.
- monoclonal antibodies reactive to human IL-2 thus far produced and charac ⁇ terized using these methods, two have proven to be effective neutralizing antibodies. While all are useful for immunopurification of IL-2, the third, DMS-3, appears best-suited for the immunopurifica ⁇ tion of IL-2.
- IL-2 The production and partial purification of sufficient quantities of IL-2 was advanced consider- ably by the selection of high producer clones and subclones of JURKAT T-leukemia cells. Since JURKAT cells produce IL-2 under serum-free conditions, the conditioned medium contained only 60 mg/L protein. Thus, as IL-2 accounts for 0.5% to 1.5% of the total protein present in the conditioned medium, only a
- JURKAT-derived IL-2 streamlined the purification procedure to only two steps and made for the recovery of 50% of the initial activ- ity. Consequently, milligram quantities of suffi ⁇ ciently purified material could be readily generated for use in immunization, by comparison, Welte et al. recently reported a purification procedure for human IL-2 derived from peripheral blood cells.
- the conditioned medium utilized by these investigators contained 3600 mg/L protein.
- the conditioned medium utilized by these investigators contained 3600 mg/L protein.
- a 37,000-fold purification was achieved, but five separative procedures were required and a total of only 55 ug of product was finally recovered.
- mice immunized with IL-2 only partially purified, but known to contain 15-30 ug of IL-2 protein (calculated on the basis of the units of IL-2 activity inoculated) , developed plasma anti-IL-2 activity after only two inoculations.
- monoclonal hybridomas secreting immunoglobulin reactive with immunopurified IL-2 by ELISA have recently been derived, using splenocytes from mice immunized in this fashion.
- DMS-1 and DMS-2 neutralize IL-2 activity and inhibit radiolabeled IL-2 receptor binding with equal efficiency, the fact that only DMS-2 binds to IL-2 after it is bound to the receptor, suggests that DMS-2 may bind to an epitope that is adjacent to the active site, whereas DMS-1 is likely to bind the active site itself.
- DMS-1-3 monoclonal antibodies should facilitate the identification of the active site from peptide fragments derived from IL-2.
- the immunoaffinity purification of IL-2 described herein permits a rapid and efficient purification of the IL-2.
- the affinity column can be washed extensively with various buffers so that nonspecifically bound proteins are virtually elimi ⁇ nated. Because of the remarkable stability of IL-2 to low pH, acid elution of IL-2 permits recovery of all of the bound activity.
- CMPI S that the purified product be freed of contaminating peptides.
- This is an especially critical consider ⁇ ation for lymphokines, since most appear to be biologically active at picomolar concentrations. Accordingly, to be confident that immunoaffinity- purified IL-2 was free of contaminants, aliquots were examined by SDS-PAGE, RPLC, and amino acid sequence analysis. Within the limits of detection by these procedures, the immunoaffinity-purified product contains only a single protein. Therefore, it has been possible for the first time to obtain relatively large quantities of IL-2 in a state of purity appropriate for detailed studies of structureactivity relationships. Moreover, a lymphokine preparation that meets these criteria assures that _in vitro and ⁇ n vivo biological studies with this IL-2 can be interpreted unambiguously.
Abstract
Monoclonal anti-human interleukin-2 antibodies useful for immunopurification of human interleukin-2 and to antibody-producing cell lines which generate the anti-interleukin-2 antibodies.
Description
ONOCLONAL ANTI-INTERLEUKIN-2 ANTIBODY USEFUL FOR IMMUNOPURIFICATION FOR INTERLEUKIN-2
Description
Field of the Invention
This invention is in the fields of immunology and cell biology. The invention pertains to mono¬ clonal anti-interleukin-2 antibodies useful for im unopurification of interleukin-2.
Background of the Invention Interleukin 2 (IL-2) , derived from activated human T-lymphocytes, is a 15,500 (Mr) sialoglyco- protein that initiates mitosis of antigen and lectin-activated cytolytic T-cells. As IL-2 shares many properties with polypeptide hormones, it was anticipated that antibodies reactive with IL-2 would be advantageous in much the same fashion that anti- hormone antibodies have been so useful. In this respect, anti-IL-2 would make possible definitive studies of IL-2 biologic activity, immunopurifica- tion, the development of IL-2 immunoassays, the identification of the active site of the molecule, and an array of in vivo experiments exploring its physiologic role in the immune response. Although
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recεnt reports suggest that it is possible to develop monoclonal antibodies reactive with IL-2, the hybridoma products reported thus far have only been shown to be useful for neutralization of biologic activity.
Two reports have recently described putative monoclonal antibodies reactive with IL-2. However, in each instance, the data reported fall short of providing convincing evidence that the hybridoma products are in fact antibodies that react with IL-2. Although neutralizing activity by the pro¬ ducts was demonstrated, no experiments on a compe¬ titive relationship between IL-2 and the antibodies were offered. Moreover, experiments to exclude any suppressive effects of the hybridoma products on general cellular metabolism were not included. Thus, the specificity of the reported antibody- mediated reduction of T-cell proliferation is in doubt, since non-specific effects on cellular metabolism have yet to be convincingly excluded.
Further, the reported antibodies have not been shown to be useful in the immunopurification of biologically active IL-2. For example, Gillis and Henney found that solid-phase antibodies absorbed IL-2 activity, but the bound activity could not be successfully eluted. Gillis, S and Henney, C. S., •J. Immunol. 126:1978(1981) .
In part, the difficulties encountered in attempts to develop antibodies reactive to IL-2 relate to the unique hormonal properties of this lymphokine. Since the membrane receptor has a
re arkably high affinity for IL-2 (K, = 5-20 x
-12 10 M) , the peptide is generally present and detectable in lymphocyte culture medium in only picomolar concentrations. Consequently, a major problem relates to the practical difficulty of accumulating enough IL-2 protein in a partially- purified and concentrated form so that immunization becomes feasible. Another difficulty concerns the sensitivity and efficiency of detection of anti-Il-2 activity once immunization is accomplished. Hereto¬ fore, neutralization of IL-2 biologic activity has been the only assay available to screen for anti- IL-2 in the plasma of immunized mice or hybridoma culture supernatants. However, as the IL-2 receptor binds IL-2 so avidly and rapidly (the- half-time for IL-2-receptor association is 84 s) the neutraliza¬ tion assay does not readily lend itself to the detection of anti-IL-2, since monoclonal antibodies exhibit considerably lower antigen-binding affini- ties: reported equilibrium dissociation constants for monoclonal antibody-antigen reactions differ from that of IL-2-receptor binding by several orders of magnitude. Moreover, neutralization assays preclude the identification of antibodies that do not bind adjacent to the active site of the mole¬ cule. Two stage assays involving the precipitation of antibody-bound IL-2, followed by testing for the removal of IL-2 biologic activity circumvent these problems to some extent, however, non-specific inhibition of cellular proliferation by the
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hybridoma supernatants still impairs accurate interpretation of the bioassay results. Summary of the Invention
The invention pertains to monoclonal anti-human interleukin-2 (IL-2) antibodies which are useful for immunopurification of human interleukin-2, and to antibody-producing cell lines which produce the IL-2 antibodies.
The antibodies of this invention provide for the isolation of human interleukin-2 in a single step. To accomplish this, the anti-IL-2 antibodies may be attached to a solid phase to form an immuno- adsorbent which is capable of specifically adsorbing human IL-2. Immunoadsorbents so formed may be used to adsorb in erleukin-2 from a biological fluid, a cell culture medium or other liquid. Thereafter, IL-2 may be recovered from the immunoadsorbent in milligram amounts. The recovered IL-2 retains its biological activity and is substantially pure. The antibody-producing cell lines which produce the IL-2 antibodies are formed from the fusion of an IL-2 antibody producing cell and an "immortalizing" cell. Alternatively, the anti-IL-2 antibody- producing cell lines may be formed by transformation of an IL-2 antibody-producing cell.
Brief Description of the Drawings
Figure 1 shows IL-2 production by normal lymphocytes ( • ) versus a high producer subclone (6.8) of the JURKAT human T-leukemia cell line (o) . Serial two-fold dilutions of supernatants were
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tested for the promotion of murine cytolytic T- ly phocyte line proliferation as monitored by [ H]-TdR incorporation during the last 4 h of a 24 h culture. Figure 2 shows plasma anti-IL-2 activity of IL-2 immunized mice, as detected by the neutraliza¬ tion of biological activity (A) and by ELISA (B) . Control mouse plasma (o) ; IL-2 immune mouse plasma ( • ). Figure 3 shows a typical IL-2 titration plot of
IL-2-dependent cytolytic T-lymphocyte line cell
3 proliferation monitored by [ H]-TdR incorporation during the last 4 h of a 24 h culture.
Figure 4 shows neutralization of IL-2 biologi- cal activity by the DMS monoclonal antibodies.
Antibodies, DMS-1 ( *) , DMS-2 ( ■ ) , DMS-3 ( • ) and control mouse Ig (o. Miles Laboratories Inc., Elkhart, IN) were admixed in serial two-fold dilu¬ tions with 16.5 pM IL-2 just prior to the addition of cytolytic T-lymphocyte line cells.
Figure 5 shows DMS-1 monoclonal antibody
3 inhibition IL-2 biological activity ( • ) and [ H]- leu, lys-Il-2 binding (o) of 20 pM IL-2. The effect of control mouse Ig (Miles) is shown by { .*-- ) for both assays.
Figure 6 shows the effect of DMS-1 monoclonal antibody on the biological activity of IL-2 (20 pM) derived from JURKAT subclone 6.8 cells (o) , rat splenocytes ( • ) , normal human tonsil cells ( *-*- ) and mouse splenocytes ( α ) .
Figure 7 shows the effect of IL-2 on DMS-1 monoclonal antibody-mediated inhibition of cytolytic
T-lymphocyte line cell proliferation as monitored by
[ H]-TdR incorporation. The antibody concentration = 500 ug/ml (3.3 x lθ"6M) .
Figure 8 shows the lack of effect of DMS-1 monoclonal antibody on the proliferation of IL-2 independent cells. HL-60 cells ( • ) , K562 cells (o) and BALB/c 3T3 cells (A) were cultured (1 x 105 cells/ml) for 44 h prior to 4 h incubation with [3H]-TdR.
Figure 9 is a comparison of anti-IL-2 activity of DMS-1 (o) and DMS-3 ( • ) monoclonal antibodies as detected by ELISA. Figure 10 shows reversed-phase liquid chromato- graphy elution profile of im unoaffinity-purified human IL-2.
Figure 11 shows sodium dodecyl sulfate poly- acrylamide gel electrophoresis of immunoaffinity- purified human IL-2. IL-2 (100 ng) is detected by silver stain.
Detailed Description of the Invention
The anti-human interleukin-2 antibodies of this invention are produced by antibody-producing cell lines. The anti-IL-2 antibody-producing cell lines may be hybrid cell lines known as hybridomas. The hybridoma cells are formed from the fusion of an • anti-IL-2 antibody-producing cell and an immortali¬ zing cell, that is, a cell line which imparts long term tissue culture stability of the hybrid cell.
In forming the hybridomas, the first fusion partner - the anti-IL-2 antibody-producing cell - may be a spleen cell of an animal immunized against human IL-2. Alternatively, the anti-IL-2 antibody- producing cell may be an anti-IL-2 generating B lymphocyte obtained from the spleen, peripheral blood, lymph nodes or other tissue. The second fusion partner - the immortalizing cell - may be a plasmacytoma cell such as a myeloma cell, itself an antibody-producing cell but also malignant.
Murine hybridomas which produce monoclonal anti-human IL-2 antibodies are formed from the fusion of mouse plasmacytoma cells and spleen cells from mice immunized against human IL-2. In order to evoke an immune response against human IL-2 in mice, it is important to immunize the mice with at least microgram amounts of human IL-2. Thus, a source of sufficient IL-2 is required. One such source is the JURKAT T leukemia cell described by Gillis and Watson. See J. Exp. Med. 152:1709(1980).
After immunization of the mice, spleen cells are removed for fusion. The fusions may be accom¬ plished by standard procedures such as those de¬ scribed by Fazekas de St. Groth and Scheidegger. - See J. Immunol. Meth. 35:1(1980).
The hybridomas are then screened for production of antibody reactive with human IL-2, and those which produce reactive antibody are cloned. The screening procedure should not be limited to assays for the neutralization of IL-2 biologic activity by the hybridoma cell products. Under a number of
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circumstances, a neutralization assay would not be able to detect products reactive with IL-2. For example, an antibody which reacts with IL-2 may not effectively neutralize its biological activity because while highly specific for IL-2, the antibody does not bind adjacent to the active site of the molecule. Therefore, to screen the hybrids, assays by which the reactivity of the hybridoma products with IL-2 may be directly assessed, such as those described herein, should be employed.
Another way of forming anti-human interleukin-2 antibody-producing cell lines is by transformation of antibody-producing cells. For example, an anti-IL-2 antibody-producing B lymphocyte obtained from an animal immunized against human IL-2 may be infected and transformed with a virus such as the Epstein-Barr virus to give an immortal, anti-IL-2 antibody-producing cell. See for example, Kozbor, D. and Roder, J. Immunology Today 4(3) :72(1983) . Or an anti-IL-2 B lymphocyte may be transformed by a transforming gene or transforming gene product to yield an immortal anti-IL-2 antibody-producing cell line.
The monoclonal anti-IL-2 antibodies are pro- duced in large quantities by injecting the anti-IL-2 antibody-producing hybridomas into the peritoneal cavity of mice and, after an appropriate time, harvesting the ascites fluid which contains a very high titer of homogenous antibody and isolating the monoclonal anti-IL-2 antibodies therefrom. Alterna¬ tively, the antibodies may be produced by culturing
anti-IL-2 producing cell lines in vitro and isola¬ ting secreted monoclonal anti-IL-2 antibody from the cell culture medium.
The anti-IL-2 antibodies of this invention are useful for immunopurification of IL-2. The antibodies may be attached to a solid phase to form an immunoadsorbent which specifically binds IL-2. Such immunoadsorbents may be constructed to bind milligram amounts of IL-2. The IL-2 may be recovered from the immunoadsorbent in biologically active and substantially pure form.
The solid phase may be a gel material such as agarose or a bead material such as a Sepharose bead or other solid support. The antibodies may be coupled to the solid support by conventional proce¬ dures for attaching proteins to solid phases.
In order to purify interleukin-2, the immuno¬ adsorbent is brought into contact with the biologi¬ cal fluid, cell culture medium or other liquid containing IL-2. After an appropriate time, the immunoadsorbent is separated from the liquid, and the interleukin-2 recovered. The interleukin-2 may be eluted from the immunoadsorbent by a mildly acidic aqueous solution. Three hybridoma cell lines, designated DMS1, DMS2, and DMS3 which produce anti-IL-2 antibodies useful for the immunopurification of interleukin-2 have been deposited at the American Type Culture Collection in Rockville, Maryland on September 20, 1983. These hybridomas have been assigned the following ATCC accession numbers:
DMS1: ATCC-HB8360 DMS2: ATCC-HB8361 DMS3: ATCC-HB8362. Preparation and characterization of these cell lines and the anti-IL-2 antibodies generated by them are described in greater detail below. Exemplification Cell Cultures
All cell cultures were maintained in a humidi- fied atmosphere of 5% C02 in air at 37°C. Murine IL-2-dependent cytolytic (subclone 15H) T-lymphocyte lines (CTLL) were cultured as previously described at population levels between 1 x 10 4 and 5 x 105 cells/ml in Iscove's Modified Dulbecco's Minimum Essential Medium (IMDMEM) supplemented with 10% heat-inactivated (56°C for 30 minutes) fetal calf serum (FCS, Sterile Systems, Inc., Logan, Utah), 50 U/ml penicillin G, 50 ug/ml gentamycin, 300 ug/ml L-glutamine and 1.0 u/ml human IL-2 derived from JURKAT high producer subcloned (6.8) cells partially purified by gel filtration. (Oi, V. T. , and Herzen- berg, L.A. , "Immunoglobulin-producing hybrid cell lines" In: Selected Methods in Cellular Immunology, Ed. B.B. Mishell and S. M. Shiigi, W. H. Freeman & Co., San Francisco, CA, p. 351.
The P3-NS-1-1 cell line [ref 13] (obtained from Dr. Ellis Reinherz, The Dana-Farber Cancer Center, Boston, MA) , BALB/c 3T3 cells, and all resulting hybridoma cell lines were maintained in IMDMEM, supplemented with 10% FCS, 50 u/ml penicillin G, 50 ug/ml gentamycin, and 300 ug/ml L-glutamine.
The JURKAT high IL-2-producer subclone 6.8 [See Smith, K.A. ,"T-cell growth factor, a lymphocyto- trophic hormone" In:Proceedings of the 55th Nobel Symposium, Genetics of the Immune Response, Ed. G. Moller, Plenum Publ. Co., N.Y. , in press; Kaplan, J.J. et al. er. J. Hematol. 1:219(1976); Gillis and Watson, J. Exp. Med. 152:1709 (1980)], K562 [See Lozzio and Lozzio, Blood 45:321(1975) ] and HL-60 [Collins et al.. Nature 270:347 (1977)] cell lines were maintained in RPMI 1640 medium [Grand Island Biological Company (GIBCO) , Grand Island, N.Y.] supplemented with 10% FCS, 50 u/ml penicillin G, 50 ug/ml gentamycin, and 300 ug/ml fresh L-glutamine at
5 6 population levels between 2 x 10 and 1 x 10 cells/ml.
IL-2 Biologic Assay
IL-2 biologic activity was determined as previously described by the IL-2 concentration- dependent stimulation of proliferation of a cloned murine cytotoxic T lymphocyte line (CTLL-2, subclone
15H) . Oi, V.T. and L.A. Herzenberg,
"Immunoglobulin-producing hybrid cell lines" In:
Selected Methods in Cellular Immunology, Ed. B.B.
Mishell and S.M. Shiigi, W. H. Freeman & Co., San Francisco, CA, p.351.CTLL proliferation, as
3 monitored by [ H]-thy idine (TdR) incorporation,
(2.0 uCi/ml, Schwartz Mann, Orangeburg, N.J.,
Specific Activity 1.9 Ci/mM) , was determined during the last 4h of a 24-h culture period in the presence of serial twofold dilutions of a standard IL-2 preparation and the experimental sample. The
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dilutions yielding 50% of the maximum CTLL [ H]-TdR incorporation were determined, and the value for the sample divided by that of the standard to give the concentration of IL-2 in units per milliliter. The standard IL-2 preparation, which was arbitrarily assigned a value of lϋ/ml, consistently yielded 50% of the maximal incorporation at a dilution of 1:10. Thus, an experimental sample containing 100 U/ml would yield 50% maximal CTLL [ H]-TdR incorporation at a dilution of 1:1000.
IL-2 Production and Biochemical Purification
JURKAT subclone 6.8 cells were routinely harvested from the exponential phase of cell growth (0.8 - 1.0 x 10 cells/ml), centrifuged (250 x g, 10 min) , placed into serum-free DMEM (GIBCO) , and cultured at 4.0 x 10 cells/ml for 14-18h in the presence of phytohemagglutinin (1.5 ug/ml, Wellcome Reagents, Beckingham, England) and phorbol myristic acetate (50 ng/ml. Consolidated Midland Corp. , Brewster, N.Y.). The cell-free supernatant was harvested by centrifugation (1000 x g, 15 min) , filtered (0.45 u) and stored at 4°C. IL-2 was partially purified from the conditioned medium as described previously by concentration, using an Amicon Cartridge (Amicon Corp. , Lexington, MA) and a YM-5 membrane, followed by gel filtration and isoelectric focusing (IEF) [Robb, R. J. and Smit. h, K.A., Mol. Immunol. 18:1087(1981). Concentrated supernatant was applied to a Sephadex G-1000 superfine column (Pharmacia, Piscataway, NJ) ) and eluted with 0.5 M NaCl, 10 mM Tris-HCl, pH 7.5,
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-. Λ . IPO
0.02% NaN3. Fractions containing IL-2 activity
[IL-2 elutes as a symmetrical peak in a position corresponding to that of a globular protein of
20,000 (Mr)] were pooled, concentrated by filtration and equilibrated with mpholines (0.5% Pharmolyte, pH 6.5-9.0, Pharmacia). IEF was performed for 10 hours at 1000 V on 0.6 x 10 cm polyacrylamide tube gels (7% acrylamide, 6.25% Pharmalyte) . Recovery from these separative procedures averaged 50% and resulted in IL-2 activity that was uniform with respect to size (Mr = 15,500) as analyzed by sodium dodecyl sulfate polyacrylamide gel electrophoresis
(SDS-PAGE) and charge as analyzed by IEF (pH = 8.2).
3 IL-2 biosynthetically radiolabeled with [ H]-leucine 3 and [ H]-lysine was prepared and purified by identical procedures as previously described. Robb et al., J. Ex. Med. 154:1455(1981). Such material contained biologic activity and yielded one
- radiolabeled protein (Mr = 15,500) detectable by fluorography after SDS-PAGE. Robb et al., supra. Immunization and Cell Fusion Procedures
Female BALB/c mice (The Jackson Laboratories, Bar Harbor, Maine) 8-12 weeks of age were inoculated with biochemically purified IL-2 as follows: 1) Primary immunization; 2000 U (equivalent to 15-16 ug IL-2 protein) in 0.1 ml + 0.1 ml complete Freund's Adjuvant (CFA) intraperitoneally (IP) and 2000 U in CFA distributed subcutaneously (SQ) to the limbs; 2) Secondary immunization at week 10; 2000 U in incomplete Freund's Adjuvant (IFA) IP and 2000 u in IFA SQ to the limbs. 3) Tertiary immunization at
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week 12; 4000 u in 0.2 ml normal saline IP. Ten days after the secondary immunization mice were bled via the retro-orbital plexus and their plasma tested for anti-IL-2 activity. Four days after the tertiary immunization, the spleen was removed for cell fusion.
7 Splenocytes (7.5 x 10 ) from an immunized mouse that exhibited plasma anti-IL-2 activity (see assay
7 procedures) were fused with 2.5 x 10 P3-NS-1-1 plasmacytoma cells using the procedures described by Fazekas de St. Groth and Scheidegger. See J. Immunol. Meth. 35:1(1980). P3-NS-1-1 cells were harvested from the exponential phase of cell growth, washed twice in serum-free DMEM (GIBCO) , combined with the immune splenocytes and exposed to 1 ml 50% polyethylene glycol 4000 (Ξ. Merck, Darmstadt, Germany) , 5% dimethyl sulfoxide in saline for 90 sec at 37°C. Following dilution with 20 ml DMEM and a 5 min equilibration period, the cells were centrifuged (150 x g, 5 min) and resuspended in 12.5 ml IMDMEM supplemented with 10% FCS, 50 u/ml penicillin G, 50 ug/ml gentamycin, and 300 ug/ml L-glutamine. Serial twofold dilutions of the cell suspension were made and 50 ul of cell suspension distributed to 96-well microtiter plates (Costar) , containing 6 x 10
BALB/c peritoneal cells/well. Plates were routinely seeded at initial mixed-cell concentrations ranging from 4 x 10 5 cells/well to 2.5 x 104 cells/well, as preliminary experiments indicated these cell concentrations favored the initial clonal derivation of hybridoma cells. After 24 hours, selection
medium consisting of hypoxanthine (1 x 10-4M) , aminopterin (8 x 10 —8M) , and thymidine (1.6 x 10—5M) was added.. The plates were wrapped in aluminum foil and incubated at 37°C, in a humidified atmosphere of 5% C02 in air for 10 days without further medium addition. After testing for anti-IL-2 specificity by performing ELISA in the presence and absence of adsorbed IL-2, and additionally, using ovalbumin in the place of BSA, cells from wells with anti-IL-2 activity were transferred to 24 well cluster trays, then cloned and subcloned by limiting dilution (0.3 cells/well) . Tests for Anti-IL-2 Activity
Plasma from IL-2-immunized mice, hybridoma-cell culture supernatants and purified monoclonal anti¬ bodies were tested for anti-IL-2 activity by means of three assays: 1) Neutralization of Biologic Activity
Serial twofold dilutions of plasma or mono- clonal antibody preparations were tested in micro- titer plates. IL-2 partially purified by gel filtration was added to each well (final IL-2 concentration = 0.04 U/ml) followed by CTLL cells
(4000 cells/well) . After 20h at 37°C in a hu idi-
3 fied atmosphere of 5% CO, in air, [ H]-TdR (2.0 uCi/ml) was added for 4 h. Cultures were harvested
3 onto glass fiber filters and [ H]-TdR incorporation was determined by liquid scintillation counting.
Results are expressed as a percentage of the
[ 3H]-TdR incorporation in the presence of tes plasma/antibody compared to a medium control.
2) Radioimmunoprecipitation
Plasma from IL-2-immunized and control mice, and monoclonal antibody preparations were assayed for anti-IL-2 activity by examining binding to 3 [ H]-leu, lys-IL-2. Robb et al., J. Exp. Med.
15_4: 1455 (1981) . Serial two-fold dilutions (20 ul) were incubated with 5000 DPM [3H]-leu, lys-IL-2 for
1 h at 37°C. Formalin-fixed Staphylococci (Cowan
Strain A) (20 ul, 10% suspension) was then added and the incubation continued for 30 min at 37°C.
Ice-cold RPMI 1640 (GIBCO) (1 ml) was added, and following centrifugation (9000 x g, 1 min) the supernatant was removed and counted (15 ml Biofluor, New England Nuclear, Boston, MA) via liquid scintil- lation. The Staph-A pellet was then washed twice with 1 ml cold RPMI 1640 and counted by liquid scintillation.
3) Enzyme-linked immunoassay (ELISA)
Plasma from immunized mice and purified mono- clonal antibodies were tested for anti-IL-2 activity by measuring binding to plastic-adsorbed IL-2. IL-2 purified by gel filtration and IEF, or by immuno- affinity absorption (see immunoaffinity purification of IL-2) , that yielded a single band on SDS-PAGE (Mr = 15,500) , was distributed to 96-well microtiter plates (20 u/well) . After 18h at 25°C the wells were rinsed with Tris-HCl buffered saline (TBS) (pH 8.5) and incubated with 300 ul bovine serum albumin (BSA, 10 mg/ml in PBS) for 1 h at 37°C. After washing with TBS, 95 ul of test plasma or antibody were added for 2 h at 37°C. The wells were then
washed three times with TBS-0.5% Tween-20 followed by the addition of 50 ul Sheep F (ab) ~ anti-mouse Ig-alkaline phosphatase conjugate (New England Nuclear, 650 ng/ l) . After a 1 h incubation at 37°C, the wells were washed three times with TBS- Tween 20, (pH-8.0) and twice with distilled water. Substrate (paranitrophenyl phosphate, 10 mM) was then added and after 30-60 min at 37°C the wells were observed visually or read in a spectrophoto- meter (410 nm) .
Preparation and Purification of Monoclonal Anti¬ bodies from Ascitic Fluid
The methods used for the preparation and purification of monoclonal antibodies from ascitic fluid were adapted and modified from those reported by Staehelin et al. Proc. Natl. Acad. Sci.
78:1848(1981). Twenty female Pristane-primed BALB/c g mice were inoculated with 5 x 10 hybridoma cells from midlog growth phase. After 10-14 days, the ascitic fluid was aspirated repeatedly from each mouse, clarified of cells and debris by centrifuga- tion (1000 x g, 15 min) and stored at -20°C in the presence of 0.02% NaN_.
At each step of the antibody purification procedure IL-2-specific antibody activity was tested by ELISA. Protein concentration was estimated by the approximation that 1 mg of protein yields an absorbence of 1.2 at 280 nm in a cuvette with a path length of 1.0 cm. Ascitic fluids with high levels of antibody contained 30-35 mg/ml of protein and 6-7 mg/ml (20%) of specific antibody.
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Prior to ammonium sulfa e precipitation, the ascitic fluid was rendered lipid free by centrifuga- tion (25,000 x g, 30 min, 0°C) followed by filtra¬ tion (Whatman No. 1) at 0°C. The fluid was diluted with 50 mM Tris-HCl, pH 7.8, 0.14 M NaCl to 10 mg.ml protein, and to 100 ml of diluted solution, 90 ml of room temperature saturated ammonium sulfate solution (adjusted to pH 7.8) was added slowly with vigorous stirring at 0°C. The solution was stirred on ice for 60 min then centrifuged at 1500 x g for 15 min at 4°C. The antibody-containing protein pellet was resuspended in 20 mM Tris-HCl, pH 7.8, 40 mM NaCl (2 ml per 100 ml original volume) and dialyzed over¬ night against 100 volumes of 20 mM Tris-HCl, pH 7.9, 40 mM NaCl with at least one change of buffer.
After dialysis, the solution was clarified (27,000 x g, 10 min at 4°C) adjusted to pH 8.2 (IgG_ ) or pH 8.5 (IgG,) , and 200 mg applied to a Protein A-Sepha- rose 4B column (Sigma Chemical Company, St. Louis, MO, 10 ml gel volume) . The column was washed with 10 column volumes of PBS, pH 8.2-8.5, followed by two column volumes of distilled H20, then eluted with 7.5 column volumes of 0.1 M Na Citrate, pH 3.5 Acid eluted fractions (10 ml) were neutralized with IM Tris-HCl, pH 8.1, the protein concentration was determined (A2gQ) and each fraction was assayed for anti-IL-2 activity by ELISA. Preparation of Immunoabsorbents
Affi-Gel 10 (Bio-Rad Laboratories, Richmond, CA) was washed on a sintered glass funnel with three volumes of ice-cold isopropanol, followed by three
washes with ice cold distilled H20. The packed gel was mixed with an equal volume of 10 mg/ml purified antibody solution (equilbrated with 0.2M NaHCO,, pH 8.0, 0.3M NaCl by dialysis) and rotated end over end for 5h at 4°C. After reaction, the gel was centri- fuged and washed twice with 0.1M NaHCO-, 0.15 M NaCl to remove unbound antibody. Protein determination of the combined washes routinely revealed that more than 95% of antibody was coupled to the gel. To block unreacted sites, the gel was mixed with an equal volume of 0.1 M ethanolamine-HCl, pH 8.0 and rotated end over end at room temperature for 60 min. The gel slurry was washed free of reactants with 10 mM Tris-HCl, pH 7.5 and stored in the presence of 0.02% NaN3 at 4°C.
Purification of IL-2 by Monoclonal Antibody Column Immunoabsorption
Prior to each use, the immunoabsorbent (1 ml gel in a 1 x 10 cm column) was washed with 10 ml 0.2 N acetic acid, pH 3.5. The column pH was equili¬ brated with 50 mM Tris-HCl (pH7.5) followed by the passage of IL-2-containing supernatants (50 ml/h) . The column was washed with 20 ml each of: 1 M NaCl, 10 mM Tris-HCl, (pH 7.5); 0.5% (w/v) Nonidet P-40, 10 mM Tris-HCl (pH 7.5); 10 mM Tris-HCl (pH 7.5); distilled H20. Bound IL-2 was eluted from the column by applying five 1 ml aliquots of 0.2 N acetic acid, each time draining the fluid to the bed volume. Eluted fractions were neutralized with 1 M Tris-HCL (pH 8.0) and tested for biological activi¬ ty, protein content (Bio-Rad) and analyzed by
SDS-PAGE by the method of Laemmli [Nature (Lond.) 227: 680 (19700] [12% acrylamide, reducing conditions, silver stain (Bio-Rad) ] . Reversed-Phase Liquid Chromatography (RPLC) Fractions eluted from the DMS-3 immunoaffinity column containing IL-2 activity were analyzed by RPLC as previously described. Versteegen et al., J. Biol. Chem. 257:3007(1982). In brief, samples were acidified with trigluoroacetiσ acid (TFA) and applied to a uBondapak C18 column (Waters Asso¬ ciates, Milford, MA) equilibrated wit 0.05% TFA. Proteins were eluted with an acetonitrile gradient in 0.05% Microsequence Analysis Semiautomated microsequence analysis utilizing stepwise Ed an degradation was performed with a Beckman sequence model 8 0C equipped with a cold trap accessory as described. Copeland et al. J. Virol. 36:115 (1980) . Phenylthiohydantoin derivatives of amino acids were identified and quantitated by high pressure liquid chromatography. Results
Essential Requirements for the Development of IL-2 Monoclonal Antibodies Listed in Table I are the steps that were found to be essential for the development of IL-2 mono¬ clonal antibodies. These include the identification of an adequate source of starting material for purification, the development of a purification scheme that provides high recovery of material for immunization, and the development of immunoassays
O πMαP
• WIP
for IL-2 so that dependence on the biologic neutral¬ ization assay was obviated.
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Table I PRODUCTION OF MONOCLONAL ANTIBODIES TO IL-2
1. Production of IL-2 in Adequate Quantities
JURKAT high producer clones-300-800 ug/L 2. Partial Purification of IL-2 with Sufficient Recovery
Gel filtration, IEF - 50% yield
3. Immunization with a Sufficient 2-_mσunt of -Antigen 20-30 ug IL-2 Protein
4. Selection of Immune Mice
Plasma Screening for .Anti-IL-2 Activity
5. Discriminative Immunoassay for Screening Hybridomas ELISA
A sufficient source of starting material was provided by the T-leukemia cell line, JURKAT, in that as described by Gillis and Watson [J. Exp. Med. 132:1709 (1980)] and displayed in Figure 1, 100-fold greater quantities of IL-2 could be recovered from this cell line compared to other sources of human IL-2. As shown, PHA-stimulated normal human tonsil cells generally produce IL-1-containing conditioned medium with titers of 1:10 (50% of the maximum CTLL
3 ' [ H]-TdR incorporation) or 1 unit/ml. In contrast, selected high producer clones and subclones of
JURKAT cells sustain a comparable biologic effect at titers of 1:1000 (range 1:300 to 1:1500) or 100
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units/ml. Since protein determinations performed on highly purified IL-2 yield values of 3-8 ng as being equal to one unit of biologic activity, a conserva¬ tive estimate of the quantity of recoverable IL-2 5 from one titer of JURKAT-derived conditioned medium is in the range of 300 to 800 ug, compared to 3-8 ug produced by the usual cell sources. Using a puri¬ fication protocol of only two steps, gel filtration and isoelectric focusing, IL-2 activity was recover- 10 able in sufficient yield (50% of recovery of star¬ ting material) such that mice could be immunized with 20-30 ug of IL-2 protein.
It was advantageous to select those immunized mice that manifested circulating anti-IL-2 activity. 15 Since it is well known that normal mouse serum may contain inhibitors of T-cell proliferation that need not represent anti-IL-2 antibodies, it is often difficult to interpret experimental results where dependence is placed on serum-mediated neutraliza- 20 tion of biological activity as the sole assay. Accordingly, to circumvent this problem, and in order to provide substantiation data on the presence of serum anti-IL-2 activity, a radioimmunoprecipita- tion (RIP) assay, and an enzyme-linked immunoassay 25. (ELISA) were developed. Of the two, ELISA proved to be the more sensitive and more readily interpre- table. The kind of anti-IL-2 activity present in the plasma of an immunized mouse compared to a control mouse is illustrated in Figure 2. As 30 displayed, the neutralization assay (Fig. 2B) ; the 50% neutralization titer was 1:20, whereas the
anti-IL-2 titer detected by ELISA, was 10-fold greater at the 50% point. Moreover in ELISA, reactivity of plasma from an immunized mouse was still distinguishable from that of control plasma at a dilution of 1:2000. Alternative and confirmatory data on expression of anti-IL-2 reactivity were obtained by the RIP assay: plasma from an immunized mouse tested with 1 pM radiolabeled IL-2 co- precipitated 60% of the input radioactivity, whereas plasma from control mice did not precipitate any radioactivity discernable over background.
Upon fusion of splenocytes from an immunized mouse with P3-NS-1-1 cells, ELISA also permitted rapid, unambiguous identification of anti-IL-2- producing hybridoma cultures. As a result of the first experiment, three of approximately 1000 hybridoma cultures appeared positive as detected by ELISA (A41Q = 1.2 ± 0.2 for the positive cultures, compared to A41_ = 0.02 ± 0.01 for the negative cultures) . Based on the utilization of the initial cloning procedure described by Fazekas de St. Groth and Scheidegger, supra, subsequent cloning and subcloning experiments pointed towards a clonal derivation of the three initially positive cultures. This impression was substantiated by heavy and light chain typing (by Ouchterlony analysis) : two anti¬ bodies were IgG.. , k and the third was an IgG- , k. Neutralizing Activity of the Monoclonal Antibodies To more fully characterize the antibodies, which were reactive with IL-2 by ELISA, neutraliza¬ tion assays were performed. IL-2 was utilized at a
concentration that promoted approximately one half of the maximum biological activity so that the sensitivity of the neutralization assay would be maximal. A typical titration curve for a laboratory standard IL-2 preparation is shown in Figure 3. The one-half maximum CTLL [ H]-TdR incorporation occurs at an IL-2 concentration of 0.04 U/ml which corre¬ sponds to 20 pM [assuming that 1.0 U/ml = 7.75 ng/ml and the active moiety is 15,500 (Mr)]. The effect of the three monoclonal antibodies
3 (designated DMS 1, 2 and 3) on CTLL [ H]-TdR incor¬ poration in response to 16.5 pM IL-2 is shown in Figure 4. Of particular interest is the finding that the two IgG. antibodies (DMS 1,2) neutralized IL-2 more effectively, i.e., at a much lower concen- tration (ED50 = 8.0 ug/ml, 5.0 x 10 M) than the IgG- (DMS-3) antibody (ED.-n = 250 ug/ml, 1.5 x 10" M) . However, even for the DMS-1 and DMS-2 antibodies, assuming two antigen-binding sites, an approximate 10,000-fold molar excess of antibody to IL-2 was required for neutralization.
The lack of neutralization by control mouse Ig, even at concentrations approaching 1 mg/ml, and the marked difference between the concentrations of DMS-1,2 and DMS-3 required for neutralization, suggested that the DMS 1,2 -mediated inhibition was attributable to anti-IL-2 reactivity. The anti¬ bodies used for these experiments had been purified from ascitic fluid and on SDS-PAGE produced only protein bands that migrated coincident with Ig heavy and light chains. Even so, because potentially
contaminating moieties other than antibodies could elicit nonspecific suppression of cellular prolifer¬ ation, it was decided to examine the mechanism and specific of the antibody effect. Since the IL-2 biologic effect requires binding to specific mem¬ brane receptors, and moreover, because IL-2-receptor interaction is antecedent to cellular proliferation and requires only 15 min to reach equilibrium, the effect of the monoclonal antibodies on radiolabeled IL-2-receptor binding was tested. As displayed in Figure 5, a representative experiment of the effect of DMS-1 on [3H]-leu, lys-IL-2 binding to CTLL cells is compared to this effect on IL-2-mediated cellular proliferation. For the radioreceptor experiment the antibody was allowed to interact with radiolabeled IL-2 for 2 h prior to a 15 min incubation with CTLL cells, whereas in the neutralization assay the antibody and IL-2 were cultured with the cells for the entire 24 hour assay period. The results indicate remarkably coincident antibody concentration- dependent inhibition curves for both receptor binding and neutralization of the biological re¬ sponse. In other experiments, DMS-2 demonstrated a similar effect, whereas DMS-3 was found to inhibit IL-2 receptor interaction only at high concentra¬ tions. These observations thus affirm the impres¬ sion that nonspecific suppression of cellular metabolism is not involved in the observed antibody effect on CTLL proliferation; rather that the suppression is a direct result of intervention by antibody in the IL-2-receptor interaction.
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Further evidence that the antibodies mediated the neutralizing effect by specifically reacting with IL-2 was obtained in assays performed with IL-2 derived from different species. As shown in Figure
5 6, DMS-1 inhibited CTLL proliferation promoted by
IL-2 derived from JURKAT cells (the immunogen) , normal human tonsil cells and mouse splenocytes, whereas even at high antibody concentrations, there
3 was minimal suppression of CTLL [ H]-TdR-incorpor-
10 ation with IL-2 derived from rat splenocytes. The species specificity of the antibody effect is especially persuasive evidence that the suppression of CTLL proliferation is not to be explained by effects unrelated to reactivity to IL-2. If the
15 monoclonal antibodies or other components in the antibody preparations suppress CTLL proliferation by some mechanism other than inhibition of the IL-2- CTLL interaction, suppression would be expected regardless of the source of IL-2.
2.0 Should the DMS-1 and DMS-2 monoclonal anti¬ bodies operate to neutralize IL-2 biologic activity via binding to IL-2, excess IL-2 would be expected to overcome the antibody-mediated suppression. Moreover, such an approach should effectively 5 exclude any effects of the antibodies on the cells or other vital culture constituents that could lead to suppression of cellular proliferation. To test the capacity of added IL-2 to overcome antibody- mediated neutralization of IL-2 activity , a maximal 0 inhibitory concentration of DMS-1 antibody was chosen ('500 ug/ml, see Figure 6) , and increasing
concentrations of IL-2 purified by a DMS-3 immuno- affinity column were added to the cultures. As shown in Figure 7, IL-2 competed with the antibody effect in a concentration-dependent manner, such that 1.0 U/ml IL-2 (5 x 10 °M) completely overcame the suppressive effect of 500 ug/ml (3.3 x 10~ M) DMS-1 antibody. As the immunoaffinity-purified IL-2 is homogeneous (see following sections) these results are only interpretable as demonstrating a competitive relationship between IL-2 and the monoclonal antibody.
A final test for possible nonspecific effects of the antibodies on cellular metabolism was made by investigating effects on the proliferation of cell lines that do not require IL-2. The lack of effect of DMS-1 on the proliferation of two human myeloid leukemia cells (K562, HL-6'0) and a murine fibroblast cell line (BALB/c-3T3) is shown in Figure 8. The Immunopurification of IL-2 Although the DMS-3 antibody demonstrated much lower neutralizing activity than the DMS-1 or DMS-2 antibodies, it appeared to bind to IL-2 with higher efficiency. For example, when equivalent concentra¬ tions of DMS-1 and DMS-3 antibodies were titrated in an ELISA, DMS-3 could be diluted much further (Figure 9) . The greater effectiveness of IL-2 binding demonstrated by the DMS-3 antibody also became evident when the antibodies were coupled to a solid support: DMS-3 proved much more effective than DMS-1 as an immunoabsorbent of human IL-2. A representative experiment where solid phase-DMS-1
and DMS-3 were compared as immunoabsorbents for IL-2 derived from several different sources is shown in Table II. Of particular interest was the observa¬ tion that DMS-3 absorbed human IL-2 more effectively than did DMS-1, while the DMS-1 antibody absorbed murine IL-2 to a greater extent than human IL-2. It is also notable that neither antibody absorbed rat-derived IL-2.
Table II IMMUNOABSORPTION OF IL-2 BY MONOCLONAL ANTIBODY*
IL-2 SOURCE** IL-2 Activity Absorbed(%) DMS-1 DMS-3
JURKAT-Clone 6.2 68 97 Human PBL 69 90 Mouse Splenocytes 90 58 Rat Splenocytes 0 0
* Conditioned media containing IL-2 (1 ml) were incubated with 1 ml antibody-coupled Aphi-gel 10 for 30 min at 37°C, eluted and then assayed for IL-2 biologic activity.
**Pre-absorption IL-2 biological activities:
JURKAT = 7.9 U/ml; Human PBL = 3.75 U/ml; Mouse Splenocytes = 3.75 U/ml; Rat splenocytes = 2.3 U/ml. ■ As a result of these observations, DMS-3 was selected for testing as an i munoaffinity absorbent
OMPI
for the preparative purification of human IL-2. To achieve the greatest concentration of IL-2, which is present at low levels (100 u/ml = 5 x 10 —8M) in
JURKAT conditioned medium, 1 ml column bed volumes were utilized (10-20 mg of coupled antibody) .
Application of several liters of conditioned medium to such a column would therefore not exceed the theoretical binding capacity of the antibody in view of the low concentration of IL-2 protein in the medium, but would suffice to diminish the likelihood of nonspecific binding to the immunoabsorbant. As anticipated, this approach made for an efficient means of purification. In a representative experi¬ ment, 4L of iJURKAT-derived conditioned medium containing 1.7 mg of IL-2 were passed through a 1 ml DMS-3 immunoaffinity column, followed by extensive washing (20 ml each of: 1.0M NaCl, 10 mM Tris-HCl, pH 7.5; 0.5% Nonidet P-40, 10 mM Tris-HCl pH 7.5; 10 mM Tris-HCl pH 7.5; and H20) to remove nonspecifi- cally-bound contaminants. The bound IL-2 activity could be acid-eluted so that all of the activity was concentrated in 5 ml of eluate. Moreover, greater than 90% of the bound activity appeared in the first 2 ml. Since this fraction contained 740 ug of measurable protein and biological activity that titrated greater than 10 in the IL-2 assay, it is evident that the immunoaffinity approach provides for a rapid concentration (4L to 2 ml, 2.8 x 10 —8M to 2.5 x 10 -5M) and purification (250 mg to 740 ug) of IL-2.
To assess the purity of the acid-eluted IL-2, the fractions were examined by SDS-PAGE before and after reversed-phase liquid chromatography (RPLC) . Only one fraction from RPLC contained detectable protein; this fraction contained all of the detect¬ able IL-2 activity applied to the column (Figure 10) . SDS-PAGE analysis of the DMS-3 immunoaffinity column eluate, (Figure 11) revealed a single stained band (Mr 15,500). The purity of the immunoaffinity- separated IL-2 was further assessed by amino- terminal amino acid sequence analysis. In a single microsequence analysis of 2 nmol of fraction 13 from RPLC, unambiguous identification of phenylthiohydan- toin derivatives of amino acids was possible up to residue 15, except at position 3, where no amino acid could be assigned (Table III) .
Table III
N-TERMINAL AMINO ACID SEQUENCE OF IL-2
10 Ala Pro Ser Ser Ser Thr Lys Lys Thr
11 15 Gin Leu Gin Leu Glu
As this amino acid sequence is identical to that predicted from the DNA base sequence obtained from cloned JURKAT-derived IL-2 cDNA, it is evident that
the DMS-3 monoclonal antibody immunoaffinity ap¬ proach permits the purification of large quantities of JURKAT-derived IL-2 to homogeneity in a single step. The experience gained in this investigation on the methodology for developing IL-2 monoclonal antibodies is perhaps broadly relevant to lympho- kines generally. The key aspects include develop¬ ment of a rapid, unambiguous, quantitative bioassay to monitor biochemical purification, the identifica¬ tion of cellular sources capable of producing the desired lymphokine in large quantities in protein- free medium, and the development of immunoassays utilizing highly purified lymphokine to facilitate the identification of anti-lymphokine secreting hybridomas. Of the three monoclonal antibodies reactive to human IL-2 thus far produced and charac¬ terized using these methods, two have proven to be effective neutralizing antibodies. While all are useful for immunopurification of IL-2, the third, DMS-3, appears best-suited for the immunopurifica¬ tion of IL-2.
The production and partial purification of sufficient quantities of IL-2 was advanced consider- ably by the selection of high producer clones and subclones of JURKAT T-leukemia cells. Since JURKAT cells produce IL-2 under serum-free conditions, the conditioned medium contained only 60 mg/L protein. Thus, as IL-2 accounts for 0.5% to 1.5% of the total protein present in the conditioned medium, only a
60-200-fold purification sufficed to render the IL-2
free of most contaminating proteins. Moreover, in contrast to normal cellular sources of IL-2, these JURKAT clones do not produce detectable quantities of other contaminating ly phokines such as B-cell growth factor, interferon burst promoting activity and colony stimulating activity. Accordingly, these characteristics of JURKAT-derived IL-2 streamlined the purification procedure to only two steps and made for the recovery of 50% of the initial activ- ity. Consequently, milligram quantities of suffi¬ ciently purified material could be readily generated for use in immunization, by comparison, Welte et al. recently reported a purification procedure for human IL-2 derived from peripheral blood cells. Owing to the use of 2.5 gm/L BSA during the produc¬ tion phase, the conditioned medium utilized by these investigators contained 3600 mg/L protein. Thus, from 3 L of culture medium containing 10.8 gm of protein, a 37,000-fold purification was achieved, but five separative procedures were required and a total of only 55 ug of product was finally recovered.
It bears reemphasis that although it is not essential to immunize with homogeneous IL-2 it is important to inoculate microgram quantities of IL-2 protein. In experiments where sub-microgram amounts of 11-2 were used for immunization, plasma anti-IL-2 activity remained undetectable despite 6-7 immuniza¬ tions. In contrast, in experiments subsequent to those described herein, mice immunized with IL-2 only partially purified, but known to contain 15-30
ug of IL-2 protein (calculated on the basis of the units of IL-2 activity inoculated) , developed plasma anti-IL-2 activity after only two inoculations. Moreover, several additional monoclonal hybridomas secreting immunoglobulin reactive with immunopurified IL-2 by ELISA have recently been derived, using splenocytes from mice immunized in this fashion.
The development of the RIP and ELISA assays for the detection of anti-IL-2 activity proved to be crucial, especially for screening the hybridoma cell culture supernatants. Had neutraliztion of biologic activity been the only assay available, it is almost certain that all of the antibodies reported herein would have remained undetected: viz., relatively hgh DMS-1 and DMS-2 antibody concentrations subsequently were found necessary for neutralization, whereas DMS-3 demonstrated little if any neutralizing activity. Moreover, several prior attempts failed to identify anti-IL-2-producing hybridomas when the hybridoma supernatants were screened for anti-IL-2 activity by the neutralization assau, or by inhibi¬ tion of radiolabeled IL-2-receptor binding. The relatively high antibody concentrations required for neutralization of biologic activity were anticipated considering the parameters of IL-2-receptor binding obtained from equilibrium and kinetic binding experiments performed with intact cells and with isolated plasma membranes. IL-2 receptors bind radiolabeled with a remarkably high affinity (K, = 5-20 x 10 -12 M) , whereas equilibrium dissociation
constants for individual monoclonal antibodies have generally been reported to be considerably lower, in the range of 1 x 10 to 1 x 10 M.
To insure that the ELISA was specific for IL-2, it was critical to use fractions from IEF that yielded only one protein (Mr = 15,500) detectable by SDS-PAGE, in order to avoid selection of hybridomas secreting immunoglobulin reactive with proteins other than IL-2. Consequently, to generate enough IL-2 antigen for ELISA, it was necessary to process several liters of JURKAT conditioned medium and to select only the most purified fractions after the separative procedures. It is possible that non-IL-2 proteins with a pi and molecular size identical to IL-2 may have contaminated the preparation used for the screening ELISA, since the material was not examined by other methods to insure the presence of only one protein. However, such putative contami¬ nants were most probably present in minor amounts, as the ELISA screening assay did identify the hybridomas that were subsequently found to secrete antibodies that react specifically to IL-2. In this regard, it is almost certain that had less purified IL-2 been used to perform the initial screening ELISA, the assay would have been considerably less effective since the determination of the specificity of IL-2 reactivity ultimately relied upon the more time-consuming and laborious assays for neutraliza¬ tion of biological activity and immunoabsorption: this task would have become exceedingly difficult had several hundred, rather than only three
-W E3
OMPI v^
hybridoma products been selected out by the initial screening assay.
To assure that IL-2 binding was actually responsible for the DMS monoclonal antibody concentration-dependent neutralization of biological activity, several experimental approaches were utilized. The close correlation between the inhibi¬ tion of radiolabeled IL-2-receptor binding and suppression of CTLL proliferation, the species specificity of the antibody-mediated neutralization, the observation that neutralization was competi¬ tively antagonized by IL-2, and finally, the absence of an antibody effect on IL-2-independent cells, all are consonant with the conclusion that the neutra- lizing antibodies (DMS1,2) mediate their effect by reacting specifically with IL-2.
Especially noteworthy is our finding that although DMS-3 bound IL-2 with a much greater efficiency than did DMS-1, it proved to be far less effective in neutralizing biological activity. A lack of correlation between binding affinities and neutralizing capacity of monoclonal antibodies has also been reported in other polypeptide hormone systems, and has been explained by the proximity of the active site of the hormone to the epitope recognized by the antibody. In this regard, we have preliminary evidence suggesting that DMS 1, 2, and 3 recognize different epitopes: DMS-2 and DMS-3 still bind to IL-2 after it has bound to the membrane receptor, whereas DMS-1 does not. Since both DMS-1 and DMS-2 neutralize IL-2 activity and inhibit
radiolabeled IL-2 receptor binding with equal efficiency, the fact that only DMS-2 binds to IL-2 after it is bound to the receptor, suggests that DMS-2 may bind to an epitope that is adjacent to the active site, whereas DMS-1 is likely to bind the active site itself. These observations involve more than conceptual interest, as it can be anticipated that a mixture of monoclonal antibodies that recog¬ nize different epitopes could manifest positive cooperativity. Moreover, the DMS-1-3 monoclonal antibodies, as presently characterized, should facilitate the identification of the active site from peptide fragments derived from IL-2. Finally, the availability of monoclonal antibodies that recognize different epitopes should support the development of new immunoassays for detection of IL-2. Indeed, preliminary experiments indicate that a sandwich ELISA constructed with either DMS 1, 2 and DMS-3 constitutes a rapid, sensitive and unambiguous assay for immunoreactive IL-2.
The immunoaffinity purification of IL-2 described herein permits a rapid and efficient purification of the IL-2. The affinity column can be washed extensively with various buffers so that nonspecifically bound proteins are virtually elimi¬ nated. Because of the remarkable stability of IL-2 to low pH, acid elution of IL-2 permits recovery of all of the bound activity.
It has become evident that while it is desi- rable to obtain by purification procedures, a lymphokine in adequate amounts, it is also essential
CMPI S
that the purified product be freed of contaminating peptides. This is an especially critical consider¬ ation for lymphokines, since most appear to be biologically active at picomolar concentrations. Accordingly, to be confident that immunoaffinity- purified IL-2 was free of contaminants, aliquots were examined by SDS-PAGE, RPLC, and amino acid sequence analysis. Within the limits of detection by these procedures, the immunoaffinity-purified product contains only a single protein. Therefore, it has been possible for the first time to obtain relatively large quantities of IL-2 in a state of purity appropriate for detailed studies of structureactivity relationships. Moreover, a lymphokine preparation that meets these criteria assures that _in vitro and ^n vivo biological studies with this IL-2 can be interpreted unambiguously.
Equivalents
While the invention has been particularly shown and described with reference to a preferred embodi¬ ment thereof, it will be understood by those skilled in the art that various changes in form and details may be made therein without departing from the spirit and scope of the invention as defined by the appended claims. Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, that there are many equivalents to the specific examples in the inven¬ tion described herein.
m Ar-m WIPO
Claims
1. Monoclonal anti-human interleukin-2 antibody useful for the immunopurification of human interleukin-2.
2. Murine monoclonal anti-human interleukin-2 antibody useful for the immunopurification of human interleukin-2.
3. -An immunoadsorbent for the purification of human interleukin-2 comprising monoclonal anti-human interleukin-2 antibody of Claim 2 attached to a solid phase.
4. An immunoadsorbent of Claim 3 wherein the solid phase is agarose gel or sepharose beads.
5. A method of isolating human interleukin-2 from a liquid comprising the steps of: a) contacting said liquid with the immunoadsorbent of Claim 3 under conditions which allow adsorption of the interleukin-2 to the immunoadsorbent; b) separating the immunoadsorbent from the liquid; and • c) recovering the interleukin-2 from the immunoadsorbent.
6. An antibody-producing cell line which produces monoclonal anti-human interleukin-2 antibody
useful for immunopurification of human inter- leukin-2.
7. A hybridoma cell line which produces monoclonal anti-human interleukin-2 antibody useful for immunopurification of human interleukin-2.
A hybridoma cell line, formed from the fusion of a murine plasmacytoma cell and a spleen cell obtained from mouse immunized against human interleukin-2, which hybridoma cell produces monoclonal anti-human interleukin-2 antibody which is useful for immunopurification of human interleukin-2.
9. A hybridoma cell line of Claim 8, wherein said plasmacytoma is an P3-NS-1-1 plasmacytoma cell.
10. A hybridoma cell line of Claim 8 wherein said spleen cell is obtained from a mouse immunized against partially purified human interleukin-2 produced by JURKAT high-interleukin-2 producer cells.
11. The hybridoma cell line DMS-1 having the ATCC number ATCC HB8360.
12. Monoclonal anti-human interleukin-2 antibody produced by the hybridoma cell line of Claim 11.
" JRE OMP
13. The hybridoma cell line DMS-2 having the ATCC number ATCC HB8361.
14. Monoclonal anti-human interleukin-2 antibody produced by the hybridoma cell line of Claim
13.
15. The hybridoma cell line DMS-3 having the ATCC number ATCC HB8362.
16. Monoclonal anti-human interleukin-2 antibody produced by the hybridoma cell line of Claim
15.
17. Monoclonal anti-human interleukin-2 antibody having a high affinity for interleukin-2.
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US53409083A | 1983-09-20 | 1983-09-20 | |
US534,090 | 1983-09-20 |
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WO (1) | WO1985001298A1 (en) |
Cited By (2)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
EP0133551A2 (en) * | 1983-08-16 | 1985-02-27 | Biotest Aktiengesellschaft | Monoclonal antibody which recognizes a structure which is common to the human interleukin-2 (TCGF) and to the light chain lambda of human immunoglobulin, and hybridoma cell lines producing this monoclonal antibody |
EP0238971A2 (en) * | 1986-03-17 | 1987-09-30 | F. Hoffmann-La Roche Ag | Antibodies directed to a lymphokine |
Citations (2)
Publication number | Priority date | Publication date | Assignee | Title |
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EP0102601A2 (en) * | 1982-08-28 | 1984-03-14 | Ajinomoto Co., Inc. | Monoclonal antibody, a process for its production and the use of this monoclonal antibody |
EP0111344A2 (en) * | 1982-12-13 | 1984-06-20 | Sloan-Kettering Institute For Cancer Research | Anti-interleukin-2 monoclonal antibodies |
-
1984
- 1984-09-20 WO PCT/US1984/001493 patent/WO1985001298A1/en unknown
- 1984-09-20 EP EP19840903727 patent/EP0155319A1/en not_active Withdrawn
Patent Citations (2)
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EP0102601A2 (en) * | 1982-08-28 | 1984-03-14 | Ajinomoto Co., Inc. | Monoclonal antibody, a process for its production and the use of this monoclonal antibody |
EP0111344A2 (en) * | 1982-12-13 | 1984-06-20 | Sloan-Kettering Institute For Cancer Research | Anti-interleukin-2 monoclonal antibodies |
Non-Patent Citations (3)
Title |
---|
Chemical Abstracts, vol. 96, 1982 (Columbus, Ohio, US) B.M. Stadler et al.: "Monoclonal antibodies against the interleukins", see page 484, abstract no. 33132e, & Prog. Cancer Res. Ther. 1981, 20 (Lymphokines Thymic Horm.: their potential Util. Cancer Ther.) 69-76 (Eng.) * |
The Journal of Immunology, vol. 128, no. 4, April 1982 (US) B.M. Stadler et al.: "Monoclonal antibody against human interleukin 2 (IL 2). I. Purification of IL 2 for the production of monoclonal antibodies" pages 1620-1625, see the entire article * |
The Journal of Immunology, vol. 131, no. 4, October 1983 (US) K.A. Smith et al.: "Production and characterization of monoclonal antibodies to human interleukin 2: strategy and tactics", pages 1808-1815, see the entire document * |
Cited By (4)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
EP0133551A2 (en) * | 1983-08-16 | 1985-02-27 | Biotest Aktiengesellschaft | Monoclonal antibody which recognizes a structure which is common to the human interleukin-2 (TCGF) and to the light chain lambda of human immunoglobulin, and hybridoma cell lines producing this monoclonal antibody |
EP0133551A3 (en) * | 1983-08-16 | 1987-04-15 | Biotest Aktiengesellschaft | Monoclonal antibody which recognizes a structure which is common to the human interleukin-2 (tcgf) and to the light chain lambda of human immunoglobulin, and hybridoma cell lines producing this monoclonal antibody |
EP0238971A2 (en) * | 1986-03-17 | 1987-09-30 | F. Hoffmann-La Roche Ag | Antibodies directed to a lymphokine |
EP0238971A3 (en) * | 1986-03-17 | 1988-09-14 | F. Hoffmann-La Roche Ag | Antibodies directed to a lymphokine |
Also Published As
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