CA1296662C - Hybridoma cell lines and their monoclonal antibodies to human pluripotent granulocyte colony stimulating factor - Google Patents

Abstract

ABSTRACT

"HYBRIDOMA CELL LINES AND THEIR MONOCLONAL ANTIBODIES TO
HUMAN PLURIPOTENT GRANULOCYTE COLONY STIMULATING FACTOR"

Murine-derived hybridoma tumor cell lines and monoclonal anti-human pluripotent Granulocyte Colony Stimulating Factor antibody substances produced by these cell lines. Use of said monoclonal antibody substances, alone or in combination, in immunological procedures for isolation of human pluripotent Granulocyte Colony Stimulating Factor and for quantitative detection of human pluripotent Granulocyte Colony Stimulating Factor in fluid samples.

Description

"HYBRIDOMA CELL LINES AND THEIR MONOCLONAL ANTIBODIES TO
HUMAN PLURIPOTENT GRANULOCYTE COLONY STIMULATING FACTOR
BACKGROUND OF THE INVENTION
The present invention relates generally to materials and methods for use in immunological pro~
cedures for isolation and quantitative detection of hematopoietic growth factors from biological fluids.
Specifically, the invention relates to monoclonal anti-bodies produced by novel hybridoma cell lines (as exemplified by A.T.C.C. HB-8957, A.T.C.C. HB-8958, A.T.C.C. XB-8959, A.T.C.C. ~B-8960, A.T.C.C. HB-8961 and A.T.C.C. HB-8962) which are specifically reactive with hematopoietic growth factors and to uses of these anti-bodies in isolation of hematopoietic growth factors through affinity purification techniques, in assays for detection of hematopoietic growth fàctors and in immuno-logical techniques for study of such growth factors.
More specifically, the invention relates to monoclonal antibodies specifically reactive with human pluripotent granulocyte colony stimulating factor ~hpG-CSF) from natural and recombinant sources.
The human blood-forming (hematopoietic) system replaces a variety of white blood cells (including neutrophils, macrophages, and basophils/mast cells), red blood cells (erythrocytes) and clot-forming cells (megakaryocytes/platelets). The hematopoietic sy~tem of the average human male has been estimated to produce on the order of 4.5 x 1011 granulocytes and erythrocytes every year, which is equivalent to an annual replacement of total body weight. Dexter, et al., BioEssays, 2, 154-158 (19~5).
It is believed that small amounts of certain hematopoietic growth factors account for the dif-ferentiation of a small number of progenitor "stem cells" into the variety of blood cell lines, for the tremendous proliferation of those lines, and for the 2 ~

ultimate differentiation of mature blood cells from those lines. Because the hematopoietic growth factors are present in extremely small amounts, the detection and identification of these factors has relied upon an array oE assays which as yet only distinguish among the different factors on the basis of stimulative effects on cultured cells under artificial conditions. As a result, a large number of names have been coined to denote a much smaller number of factors. For example of the resultant confusion the terms, IL-3, BPA, multi-CSF, HCGF, MCGF and PSF are all acronyms which are now believed to apply to a single murine hematopoietic growth factor. Metcalf, Science, 229, 16-22 (1985).
The application of recombinant genetic techniques has brought some order out of this chaos.
For example, the amino acid and DNA sequences for human erythropoietin, which stimulates the production of erythrocytes, have been obtained. (See, Lin, PCT Pub-lished Application No. 85/02610, published June 20, 1985.) Recombinant methods have also been applied to the isolation of cDNA ~or a human granulocyte-macrophage colony-stimulating factor (GM-CSF) and human macrophage~
speclfic aolony-stimulating factor (CSF~ See, Lee, et al., Proc~ Natl. Acad Sci. (USAl, 82, 4360-4364 _ (1985); Wong, et al~, Science, 228, 810-al4 (1985) and Kowasaki, et al., Science, 230, 291-296 (1985).]
A human hematopoietic growth factor, aalled human pluripotent colony-stimulating factor (hpCSF) or pluripoietin, has been shown to be present in the culture ~edium of a human bladder carcinoma cell line denominated 5637 and deposited under restrictive condi-tions with the American Type Culture Collection, Rock-ville, Maryland as AoT~C~C~ Deposit No. HTB-9. The hpCSF purified from this cell line has been reported to stimulate proliferation and differentiation of pluri-potent proyenitor cells leading to the production of all ~2~ ??

major blood cell types in assays using human bone marrow progenitor cells. Welte, et al., Proc. Natl. Acad. Sci.
(USA), 82, 1$26-1530 (1985).
Preliminary studies indicate that the factor identified as hpCSF has predominately granulocyte colony-stimulating activity during the first seven days in a human CFU-GM assay.
Another factor, designated human CSF-~ has also been isoIated from human bladder carcinoma cell line 5637 and has been described as a competitor of murine 125I-labelled granulocyte colony-stimulating factor (G-CSF) for binding to WEHI-3B D+ cells in a dose-response relationship identical to that of unlabelled murine G-CSF [Nicola, et al., Nature, 314, 625-628 ~1985)]. This dose-response relationship had previously been reported to be unique to unlabelled murine G-CSF and not possessed by such factors as M-CSF, GM-CSF, Il-3 [Nicola, et al., Proc. Natl. Acad. Sci.
(USA), 81, 3765-3769 (1984)]. See also, Metcalf, et Z0 al., Leukemia Research, Vol. 9, No. 5, pp. 521-527 (1985). CSF-~ and G-CSF are also unique among CSF's in that they share a high degree of ability to induce dif-ferentiation of ~EHI-3B D~ cells. Nicola, et al., Immunology Today, 5, 76-80 (1984). At high concentra-~
tion~, &-C5F stimulates mixed granulocyte-macrophage colony-forming cells LNicola, et al., (1984) supra~, which is consistent with preliminary results indicating the appearance of granulocytic, monocytlc, mixed granulocytic/ monocytic and eosinophilic colonies (CFU
GEMM) after 14 days incubation of human bone marrow cultures with hpCSF. CSF-~ has also been described as stimulating formation of neutrophilic granulocytic colonies in assays which employed mouse bone marrow cells, a property which has been a criterion for iden-tification of a factor as a G-CSF. On the basis of these similarities, human C5F-~ has been identified with J~

G-CSF (granulocytic colony stimulating factor). See, Nicola, et al~, Nature, 314, 625-628 (1985).
Based upon their common properties, it appears that human CSF-B of Nicola, et al., supra, and the hpCSF o~ Welte, et al., supra, are the same factor which could properly be referred to as a human pluripotent granulocyte colony-stimulating factor (hpG-C5F).
In co-owned and copending Canadian Patent Application Serial No. 516,737 filed August 25, 1986 for "Production o~
Pluripotent Granulocyte Colony-Stimulating Factor", novel recombinant-produced polypeptides possessing part or all of the primary structural conformation and one or more of the biological properties o~ human pluripotent granulocyte colony stimulating factor are disclosed.~ Also disclosed are DNA
sequences coding for such polypeptides, transformed host cells coding for such polypeptides and processes for the synthesis of such factors by recombinant methods.
Of interest to the background o~ the invention is current research focused on hybridoma technigues for producing tumor cell lines which will manufacture highly specific monoclonal antibodie~ to a ~elected antigenic substance. Techniques ~or the production o~ monoclonal antibodies ara generally well known in the art. Typical descr.iptions of these procedures may be found in Wands, J. R., and Zurawski, V.~., Gastroenterology 80:225 (1981); Marshak-Rothstein, et al., J.
Immunol. 122-2491 (197~); and oi, V~T. and L.A.Herzenberg "Imm~moglobulin Producing Hybrid," Mishell, B.B. and S.M.
Shiigi (eds.) elected methods in Cellular ImmunoloqyL. San Francisco: W. H. Freeman Publishing, lg79 and Godlng, "~ntibody Production by Hybridomas", J. of Immunol. Meth~ 39, 285-308 ~1980). Briefly summarized, lymphocytes remov~d ~rom : the spleen of an animal previously injected wit~ the antigen of interest are induced to ~

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fuse with myeloma cells in the presence of polyethylene glycol. Thousands of "hybrid" myeloma cells are produced from the fusion. The supernatant from growth of each "hybridoma" cell culture is tested for the presence of the desired antibody activity. When such activity is found in the supernatant of one cell culture/ it is cloned by limiting dilutions, and the clones are indi-vidually assayed for supernatant activity.
Due to the highly specific nature of their immunological properties, monoclonal antibodies developed according to hybridoma techniques have been proposed for use as diagnostic reagents, therapeutic agents, and agents for affinity purification of specifically cross-reactive antigenic proteins from crude sources. See, e.g., Trends in Biotechnology, Vol.

3, No. 7 (July, 1985) and U.S. Patent Nos. 4,465,624, 4,SI4,505 and 4,514,507.
While there exists a substantial need for specific monoclonal antibodies for use in detecting, isolating, purifying and studying hematopoietic growth factors such as human pluripotent granulocyte colony-stimulating factor, there have been no reports of the successful use of hybridoma techniques in obtaining monoclonal antibodies to hpG-CSF.
BRIEF SUMM~RY
.

The present invention provides, Eor the first time hybridoma cell lines which produce monoclonal anti-bodies specifically immunoreactive with human pluri-potent granulocyte colony-stimulating factor in an antigen/antibody reaction. Illustratively, the present invention provides new murine-derived hybridoma cell lines, A.T.C.C. HB-8957, A.T.C.C. HB-8958, A.T.C.C. HB-8959, A.T.C.C. HB-8960, A.T.C.C. HB-8961 and A.T.C.C.
HB-8962, each of which produces, as a component of the ~ 66~2 supernatant of its growth in culture, a monoclonal anti-body specifically reactive with hpG-CSF. Hybridoma cell lines, A.T.C.C. HB~8957, A.T.C.C. HB-8958, A.T.C.C. HB-8959, A.T.C.C. HB-8960, A.T.C.C. HB-8961 and A.T.C~C.
HB-8962, are on deposit at the American type culture collection, 12301 Parklawn Drive, Rockville, Md. 20852.
According to the practice of the present invention, a hybrid tumor cell line is produced using a standard immunological technique as described in Oi and Herzenberg, "Immunoglobulin Producing Hybrid'l, supra and Goding, "Antibody Production by Hybridomas", J. of Immunol. Meth. 39 (1980) 285-308. Spleen cells from mice, hyperimmunized with natural hpG-CSF are fused with a mouse myeloma cell line in the presence of polyethy-lene glycol. The supernatant from growth of each"hybridoma" cell culture is tested for the presence of the desired antibody activity. A selected hybridoma cell is cloned to propagate a cell line which produces an antibody in its growth supernatant which has highly specific anti-hpG-CSF activity.
Monoclonal antibodies of the invention may be employed in immunological procedures for affinity purification and isolation of hpG-CSF (as well as other hematopoietic growth factors which may share common or related epitopes) from natural or recombinant sources.
In such a procedure, a selected antibody would be immobilized ~e.g., on a column) and the source material would be contacted with the immobilized antibody. hpG-CSF or antigenically related material would bind to the antibody and would thereafter be eluted from the immobilized antibody in a highly purified form. Anti-bodies of the invention may also be employed alone or jointly with each other or with polyclonal antibodies in immunological procedures (RIA's, ELISA's and the like) for the quantitative detection of hpG-CSF. Antibodies of the invention may also have utility in vivo to bind 66:~

hpG-CSF or related factors in instances of overproduc-tion of such factors. Other aspects of the present invention will become apparent upon consideration of the following detailed description.

DETAILED DESCRIPTION

The following examples illustrate practice of the invention in the production of a number of hybridoma cell lines including A.T.C.C. HB-8957, A.T.C.C. HB-8958, A.T.C.C. HB-8959, A.T.C.C. HB-8960, A.T.C.C. HB 8961 and A.T.C.C. ~B-8962, the isolation of antibodies to hpG-CSF, and the amplification and characterization of the monoclonal antibodies.
More particularly, Example 1 is directed to stimulation of a murine host toward production of poly-clonal serum antibodies to hpG-CSF, Eusion of spleen cells with myeloma cells, and the screenin~, cloning and growth of hybridoma cells and isolation of monoclonal antibody therefrom. Example 2 relates to the char-acterization of the monoclonaI antibodies so produced by ELISA and RIA assayst and competitive inhibition and neutralization assays as well as to the amplification of monoclonal antibody yields by the ascites method.
Example 3 describes procedures for isolatiorl and purification of hpG-CS~ through the use of the mono-clonal antibodies of the invention. Example 4 describes procedures for quantltative detection of hpG-CSF through the use of assay techniques utilizing antibodies of the invention.

EXAMPLE i A. PRODUCTION OF POLYCLONAL SERUM

BALB/C mice each were injected with hpG-CSF
purified by HPLC from human bladder carcinoma cell line ..~
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NoO 5637 (subclone lA6~ ac~Q~dinq to the ~rocedures o~
Example l(b) of United States Patent No. 4,811),643 (Souza).
The material was purified with a C4 ~PLC column to in exces~ of 85~ purity and had a concentration of approximately 60 ~g/ml in a ~olution comprising 50%
propanol and lOOmM ammonium acetate (p~ 7). Each of 10 mice wa~ initially given multiple subcutaneous injec-tions totalling approximately 0.1 ml of a~ inoculant (7.2 ~g hpG-CS~ per mouse) comprising 1.2 ml of the hpG-C~F solution which had been concentrated to 0.6 ml by speed vacuuming and then mixed by sonication with 0.6 ml of BACTO-Freund's complete adjuvant ~31 Ra (DIFCO 3113-60). Eighteen days later the mice were each given multiple subcutaneous booster injection~ totalling approximately 0,15 ml of an inoculant `(7.2 ~9 hpG-CSF
per mou~e) comprising 1.2 ml of the hpG-CSF solution concentrated to 0.75 ml and mi~e~ with 0.75 ml of Freund ~ 9 incomplete adjuvant (BACTO 0639-60-6 from DIFCO).
Four days later the mice wer~ bled and their sera screened by RIA ~or production of anti-hpG-CSF
antibodies. hpG-CS~ used in the s~rum as~ay comprised approximately 0.5 ml o~ 80% pure hpG-CS~ at a concentra-tion o~ approximately lOO~g/ml in a solution comprising lOOmM ammonium sulfate and 50% propanol which had been con~entrated ~our times. Two hundred and fifty ~1 o~
the above solution wa~ concentrated to 150~1 and wa~
mixed with 5 ml of 50mM CO~ 2/HC03-1 bu~fer (pH 9.2).
~ifty ~1 of this material wa~ then applied to each well in a 96 well tray and incubat~d for 2 hour~ at room te~perature and overnight at 4C. A blocking comp~und comprising 5% bovine serum:albumin (BSA) wa3 incuba~d for thirty minute~ in the well~. Te~t serum diluted at ra~io~ of 1:5, 1:25, 1:6~5, and 1:3125 and control ~non-inoculated) serum diluted at ratios o~ 1:5 and 1:125 wa~
appli~d to the w~ at 50~1 p~r well and incubat0d ~or ~`" 12~62 two hours at room temperature. The wells were then washed three times using Wash Solution [(Kirkeguard &
Perry Laboratories, ~KPL) Gaithersburg, Maryland)].
Rabbit anti-mouse IgG antibody labeled with 125I was then applied such that it produced approximately 199,000 counts per minute per 50~1 well and incubated for 1.5 hours at room temperature. The wells were then washed five times with Wash Solution and their radioactivity was determined with a gamma counter.
Twenty-one days after the booster injection, five mice detected by the RIA to be producing anti-hpG-CS~ antibodies were given a third injection. The mice were immunized with the same material as during their booster injection but were given approximately 10~9 hpG-CSF per mouse.

B. CELL FUSION

In the hybridoma production procedure the spleens of the inoculated mice are disrupted to suspend anti-hpG-CSF antibody producing lymphocytes. These spleen cells are fused with cells from an SP2/0 myeloma cell line to form hybrid cells. The cell membranes of spleen and myeloma cells fuse and initially surround a common cytoplasm with two or more nuclei. Several days after fusion of the cell membranes, the nuclei ~use and become capable of synchronous mitosis. As these fused cells divide, a variable number of chromosomes of both fused partners are lost until the hybrid cell lines stabilize. A hypoxanthine aminopterin-thymidine (HAT) media prevents ~P2/O:SP2/0 hybrids produced during the hybridization procedure or unfused SP2/0 cells from growing while spleen:spleen cell hybrids or unfused spleen cells will generally die after two weeks in culture. Thus only the SP2/O:spleen hybrid cells will survive in the cultures.

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Three days after the second booster inocula-tion the mice were sacrificed by cervical dislocation and doused with 70% ethanol. Spleens of the mice were removed asceptically and placed in a sterile petri dish on ice co~taining DMEM (Dulbecco's Modified Eagle's Medium, Catalogue No. 320-1885, lot 18K325~ from GIBCO) supplemented with penicillin G-streptomycin solution (a "lX" solution), hereinafter "lXPS" solution, (catalogue no. 9366 from Irvine Scientific) comprising 100 units of penicillin G and 100 mcg of streptomycin per ml. Fatty tissue adhering to the spleens was removed and the spleens were washed twice with DMEM containing 2XPS (200 units penicillin G and 200 mcg streptomycin per ml~ and placed in a sterile stomacher bag on ice. To the bag was added solution comprising DMEM - 2XPS. The spleens were disrupted in a stomacher apparàtus and then filtered through four layers of sterile gauze into 50 ml centrifuge tubes. The bag and filters were washed with up to 40 ml of solution comprising DMEM - 2XPS solu-tion. The cells were then centrifuged for ten minutesat 1000 rpm in a IEC NH-SII centrifuge (Damon/IEC
Division) and the supernatant was gently aspirated. The cells were then washed twice with a solution comprising DMEM - 2XPS and once with DMEM with no additives. The cells were then re~uspended in DMEM.
Myeloma cells (Sp2/0) were grown in ~B101 medium containing lXPS and 1~ Fetal Bovine 5erum (~BS) (Irvine Scientific Catalogue No. 3000, Lot No. 209608, Heat inactivated) and were expanded in log growth phase for 3 days. The SP2/0 cells were harvested by pelleting in an IEC NH-SII centrifuge at 1000 rpm for ten minutes. The cells were washed in D~EM solution and then suspended in DMEM solution.
The spleen cells~were then combined with the myeloma cells in a 50 ml centrifuge tube at a ratio of 4:1 and centrifuged for ten minutes at 1000 rpm and the ~,~

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supernatant aspirated. The pellet was then placed in a 37~C water bath under a hood. Polyethylene glycol (1500 molecular weight, M.A. Bioproducts, catalogue number 17-780~, lot nu~ber 4J030) was melted and then mixed at a one to one ratio by volume with DMEM solution to make a 50~ solution. The PEG solution was then added to the cells according to the following procedure. During the first minute, 1.0 ml of the 50% PEG solution was added. During the second minute, the solution was stirred gently using a pipette which was stirred con-stantly with a circular motion but which avoided touching the sides or bottom of the tubes. During the third minute, 1.0 ml of solution comprising DMEM, 10~
FCS and lXPS was added. During the fourth minute, 1.0 ml of the same solution was again added. During the fifth and sixth minutes, 8.0 ml of the same solution was again added. The mixture was then centrifuged at 1000 rpm for 10 minutes on the IEC NH-SII centrifuge.
Supernate in centrifuge tube was aspirated and the cells were gently resuspended in 100 to 150 ml of media com-prising DMEM with 10% FBS and lXPS. The cell su~pension was then plated at a rate of 0.1 ml of suspension per well onto approximately fl00 wells on eight and one half 96 well plates which had been pre-equilibrated in a CO2 incubator. The plates were then returned to a CO2 incu-bator with an approximate 7% CO2 content at 37C.
One day later, 100~1 of ~AT medium in DMEM and 10~ FBS was added to each well. On the second day, 100~1 of spent medium was aspirated from each well and replaced with 100ul of fresh HAT medium in DMEM with 10%
FBS. This procedure was repeated on day 2, day 4, day 7 and day ll. On day 14, 100~1 of spent medium was removed from each well and replaced with 100~1 of HT
medium comprising 1.36 mg/dl of hypoxanthine and 0.76 mg/dl of thymidine. The procedure was repeated on day18, day 22 and day 26. On day 28, the cultures were fed .. .

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with a complete medium comprising DMEM with 10~ FBS. The cultures were subsequently re-fed twice a week wikh this complete medium. On day 17 the wells were screened for production of immunoglobulin utilizing anti mouse IgG
antibodies. RIA and ELISA assays showed that approximately 260 out of 800 wells were significantly positivP for mouse IgG.

A. INITIAL CHARACTERIZATION OF MONOCLONAL ANTIBODIES
An ELISA assay was conducted ~or the detection of those clones which were producing anti-hpG-CSF antibodies.
Wells of 96 well trays were coated by applying approximately 50~1 of solution containing 100ng ~f 90% pure hpG-CSF which 15 was diluted in 50mM C03 2/HCO3 buffer solution (pH9.2) at room temperature for two hours then added to each well of the trays and was incubated overnighk at 4C. The wells were then treated with a 5% BSA blocking solution which was incubated for 30 minutes.
Supernatant from the hybridoma cultures was diluted in PBS solution (pH 7.0) containing 1% BSA and was then lncubated against the coated wells ~or 2 hours at room temperature. The wells were then wa~hed three times with Wash Solution and were kreated with horseradish peroxidase 25 labeled anti-mouse IgG antibody (BMB No. 605-250) at a 1:300 dilution ~or 1.5 hours. The wells were then washed ~ive time~ with wash solution and peroxidase substrate ABTS (KPL) was added at a rate of 50 nl per well. The wells were then read for optical density at 414 nm by a Titertek Multiscan*
(Flow Labs, Mchean, VA) spectrophotometer. Twenty-three of the wells (Nos. 3, 4, 5, 20, 21, 28, 35, 37, 39, 40, 58, 61, 63, 64, 66, 68, 72, 74, 75j 98, 151, 182 and 231) were found to significantly react with the reduced hpG-CSF.
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Formal cloning of hybridoma cells obtained from the positive wells was conducted by diluting the cells into additional wells at a ratio such that there was approxlmately 1 cell per 3 wells. Generally, formal cloning from an active well produced formal clones which appeared to be subclones of the same hybridoma cell but in several instances revealed diferent clone popula-tions exhibiting different antibody subtypes and reac-tion properties. Subclones from the same well were labeled with a letter (e.g., clones obtained from well 4 were labeled 4A, 4B, etc.).
An ELISA assay was conducted to determine reactivity of the monoclonal antibodies against natural hpG-CSF as well as reduced natural hpG-CSF. Supernatant obtained from wells found to be producing antibodies reactive with hpG-CSF was tested agàinst both natural hpG-CSF and natural hpG-CSF which had been denatured with SDS/mercaptoethanol. Antibodies from the clones reacted against both materials with approximately equal specificity suggesting that the antibodies may be spe-cifically reactive with antigenic epitopes primarily determined by continuous sequences of amino acids (the primary structure) oE the protein.

B. REACTIVITY OF MONOCLONAL ANTIBODIES
AGAINST MAMMALIAN AND RECOMBINANT hpG-C8E' In this example, solid phase RIA and ELISA
assay~ to measure reactivity of hybridoma supernatant obtained from the active wells were conducted on mammalian hpG-CSF as well as recombinant E. coli produced hpG-CSF. In all but one case, monoclonal anti-body material which reacted with mammalian produced (natural) hpG-CSF reacted equally well with recombinant produced hpG-CSF. That case involved subclones 3SB, 35D
and 35I which produced antibodies which were , ,. ..

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significantly less reactive with the re~ombinant material than with the mammalian derived material in direct binding assays.

5 C. SCREENING FOR ANTIE~ODY SUBTYPES

In this example, formal clones obtained from the 23 wells found to be active originally were screened by RIA for determination of their antibody subtype.
Ninety-six well trays were treated with rabbit anti-mouse-immunoglobulin antibodies of differing subtype specificities. The anti mouse-immunoglobulin antibodies included rabbit anti-mouse IgGl (MILES Laboratories, Naperville, IL, 64-360-1), rabbit anti-mouse IgG2a ~MI~ES, 64-361-1), rabbit anti-mouse IgG2b (MILES, 64-362-1), rabbit anti-mouse IgG3 (MILES, 64-363-1), rabbit anti-mouse IgM (M~LES, 64-365-1) and rabbit anti-mouse IgG tMILES, ~5-157-2). Each well was coated with 50~1 of the anti-mouse-immunoglobulin antibody solution in a CO3 2/~C03 (pH 9.6) buffer solution comprising approxi-mately 0.5~g of antibody and was incubated for 2 hours at room temperature and overnight at 4C. The wells were then blocked by incubation with a 5~ BSA solution for 30 minutes. Each well was then incubated with hybridoma ti~sue culture supernatant for 2 hours at room temperature and washed three times with Wash Solution.
The wells which had been treated with anti-IgG antibody were then incubated for 1.5 hours with 50~1 per well of 125I rabbit anti-mouse-IgG antibody while the wells which had been treated with anti-IgM were then incubated for 1.5 hours with 50~1 per well of 125I rabbit anti-mouse-IgM antibody. The wells were then washed five times with Wash Solutîon and were counted in a gamma counter. The results of analysis of the original 23 clones indicates that 2 wells (Nos. 37 and 182) stopped synthesis of IgG; 1 well was indicated IgG positive but ~ ~Z~6166~

did not demonstrate reactivity in early screening to hpG-CSF (No. 58); 15 wells showed posikive for IgGl (Nos. 4C, 5, 28, 39, 40, 61, 63, 64, 66, 68, 72, 74, 75, 98 and 231). A subclone of one of these, No. 5d was found to be reactive with both anti-IgG3 and anti-IgM
antibodies. Four wells were positive for IgG2a produc-tion (Nos. 3, 4A, 21 and 151) and 1 well for IgG2b production (No. 35).

D. NEUTRALIZATIQN OF hpG-CSF ACTIVITY AS
MEASURED BY ~H-THYMIDINE UPTAKE

In this example, monoclonal antibodies raised against hpG-CSF were tested for the ability to neutralize the biological activity of hpG-CSF of stimulating the incorporation of 3H-thymidine into human bone marrow cells. In order to test for neutralization of 3H-thymidine uptake, recombinant or natural hpG-CSF
was incubated with various concentrations of antibodies according to the invention at 4C for 12 hours. The solutions were then added to low density, non-adherent human bone marrow cells and processed according to methods described in Example 7 of copending U.S. Patent Application Serial No. 768,959~ Antibodies from clone no~. 20A, 35B, 39B, 40A, 61D, 63D, 75A and 151K, among others, were tested with antibodies from clone no. 75A
being the most effective in neutralizing the effect of the hpG-CSF on 3H-thymidlne uptake and with all of the above antibodies demonstrating various degrees of neutralizing, effect on 3H-thymidine uptake with the exception of antibodies produced by clone no. 35B.

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~6662 E. NEUTRALIZATION OF hpG-CSF ACTIVITY AS
MEASURED BY CELL DIFFERENTIATION INDUCTION
In this example antibodies raised against 5 hpG-CSF were tested ~or the ability to neutralize the ability of hpG-CSF to induce differentiation of the murine myelomonocytic leukemic cell line WEHI-3B D+. Various concentrations of the antibodies produced according to the invention (from clones nos. 20A, 39B, 40A, 61D, 63D and 75A) 10 were incubated with hpG-CSF at 4 C for 12 hours. The solutions were then added to WEHI-3B D~ cells in an aghr suspension according to the method described in Example 7 o~
the a~orementioned U.S. Patent No. 4,810,643. Antibodies produced by clone nos. 40A, 61D and 75A showed the ability to 15 block differentiation. Because these monoclonal antibodies have di~ferent precipitating abilities toward hpG-CSF the fact that some block the differentiation inducing ability of hpG-CSF while others do not cannot be explained simply on the basis o~ strength of affinity. Significantly, monoclonal 20 antibody produced by clone 75A not only inhibited differentiation o~ the WEHI-3B D~ cells but also inhibited cell growth suggesting ~hat it may cross-xeac~ with a murine growth factor secreted by WEHI-3B D~ cells and may have antiproliferative ef~ects independently o~ its capacity to 25 bind hpG-CSF.

F. EPITOPE CHARACTERIZATION

In this example, competitive binding studies were conducting utilizing 125I labelled antibody ~rom clones 75A and 151K and immobilized recombinant produced hpG-30 CSF. In these studias, antibodies from okher clones wereallowed to compete against 75A and 151K radio-labelled monoclonal antibodies bound to recombinant produced hpG-CSF.
After incubation and washing ~.r~

96~ 2 according to procedures well known in the art, radiation counts were taken to determine the extent to which either 75A produced antibody or 151K-produced antibody had been displaced from the hpG-CSF.
Results from these assays indicate that antibodies produced by clones nos. 63D, 75A, and 151K
may share overlapping portions of a common antigenic epitope on the hpG-CSF protein. The results also indicate that antibodies produced by clone nos. 4C, 28A, and 35B appear reactive with an epitope or epitopes of the hpG-CSF protein for which the 75A and 151K produced antibodies are not specifically reactive. The results also suggest that the hpG-CSF binding properties of antibodies produced by clones 40A and 61D may be dis-tinguishable from those of antibodies produced by clones 4C, 28A, 35B, 75A and 151K.

G. PROPERTIES OF ANTIBODIES FROM DEPOSITED
REPRESENTATIVE CELL LINES

Representative novel hybridoma cell lines capable o~ producing monoclonal antibodies representa-tive of those provided by the present invention have been deposited with the American Type Culture Collec-25 tion, 12301 Parklawn Drive, Rockville, MD 20852, These cell lines correspond to cell lines developed during khe formal cloning procedure as follows: clone 4C (A.T.C.C.
HB-8962), Clone 28A (A.T.C.C. HB-8957)l clone 35B

(A~ToC~C~ HB-8960), clone 63D (A.T.C.C. HB-8958), clone 30 75A (A.T~C.C. HB-8959) and clone 151K (A.T.C.C. HB-; 8g61). The properties of antibodies produced by these clones are summarized in table 1 below.

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z , . , H. AMPLIFICATIOM OF ANTIBODY YIELDS BY ASCITES METHOD

To obtain a more concentrated antibody than that produced in tissue culturel the monoclonal antibodies of the present invention were amplified by the ascites method generally described in Kenneth, et al. (eds.), Monoclonal Antibodies, Hybridomas: A New Dimension in Biological Analysis, p. 403, New ~ork:
Plenum Press (1981). According to this procedure, mice 10are primed with 0.5 ml of Pristane ~2,6,19,14-tetra methylpentadecane, obtained from Aldrich Chemical Co.) injected into their peritoneal cavities by means of 25 or 27 gauge needles. Pristane treatment permits the growth of tumor cells in an ascitic form within the peritoneal cavity. After approximately 1-2 weeks, approximately I06 hybridoma cells are injected into the peritoneal cavities of the mice in serum-free Dulbecco's modified eagles medium (DMEM) (Irvine Scientific Co.) or HB101 (Hanna Biologicals, Berkeley, Cal.). The set of injections is performed on mice for each of the clones.
Approximately 1-3 weeks after the injection of the hybridoma cells, ascites fluid was obtained from the intraperitoneal cavities of the mice by making small cuts into the skin and pipetting out the ~luid. Ascitic fluid was clarified by centrifugation and then frozen.
Ascites fluid antibodies can be further purified from ascites fluid albumin by precipitation with 45~ ammonium sulfatP and protein A-sepharose chromatography.

ISOLATION AND PURIFICATION OF
HEMATOPOIETIC GROWTH FACTORS

Through its provision of highly specific and highly reactive anti-hpG-CSF monoclonal antibodies, the present invention makes possible Eor the first time the , .. .

~29~;~;~2 isolation of hpG-CSF and hematopoietic growth factors from fermentation cultures as well as from natural mammalian sources according to affinity purification procedures well known in the art. Briefly put, preferred isolation procedures would involve irnmobiliz~
ing an antibody of the invention on a solid support ~e.g., a chromatographic column), contacting the hpG-CSF
or hematopoietic factor-containing fluid with the immo-bilized antibody and thereafter eluting purified hpG-CSF
or hematopoietic growth factor from immune complex asso-ciation with the antibody. By adjusting the particular antibody used, the purification technique may allow the isolation of native hpG-CSF in its correctly folded con-formation from incorrectly folded or denatured hpG-CSF. Analoyues of hpG-CSF could be isolated and studied as could specific antigenic epitope~ of hematopoietic growth factor molecules.

QUANTITATIVE DETECTION OF
HEMATOPOIETIC GROWT~I FACTORS

Through its provision of highly specific anti-hpG-CSF monoclonal antibodies, the present invention also makes possible novel solid or liquid phase assays for quantitative detection oE hpG-CSF and hematopoietic growth factors in a fluid sample which employ more than one antibody. Solid phase assays would typically include the steps of:
(1) contacting the fluid with a first, immobi-lized, antibody which reacts with a first antigenic determinant of hpG-CSF in the fluid to form an immuno-logical complex of hpG-CSF and the first antibody; (2) contactin~ the complex formed in step (1) with a second antibody which reacts with an antigenic determinant of hpG-CSF other than the first antigenic determinant, to :,, .

66~2 form an immunological complex of hpG-CSF and the second antibody, and (3~ quantifying the amount of the second anti-body bound to the immunological complex formed in step (2).
Liquid phase competition assays would involve labelling of hpG-CSF and precipitation of the same with one or more antibodies of the invention. Quaulitation of unlabled hpG-CSF or related factors could then be accomplished in a competative bindlng format. Such assay procedures would preferably include two of the above described monoclonal antibodies, but may also be developed using one of the monoclonal antibodies and a polyclonal serum derived antibody to hpG-CSF.
Numerous modifications and variation in prac-tice of the invention are expected to occur to those skilled in the art upon consideration of the foregoing descriptions of preferred embodiments thereof.
Consequently, only such limitations should be placed on the invention as appear in the following ~laims.

Claims (14)

1. A murine-derived hybridoma cell line capable of producing in the medium of its growth a monoclonal antibody capable of specifically binding with hpG-CSF in an antigen/antibody reaction.

2. A hybridoma cell line according to claim 1 capable of producing a monoclonal antibody of a subtype selected from the group consisting of IgM, IgGl, IgG2a, IgG2b and IgG3-

3. A hybridoma cell line according to claim 1 selected from the group of cell lines consisting of A.T.C.C. HB-8957, HB-8958, HB-8959, HB-8960, HB-8961 and HB-8962.

4. A monoclonal antibody produced by a cell line capable of producing in the medium of its growth a monoclonal antibody capable of specifically binding with hpG-CSF in an antigen/antibody reaction.

5. A monoclonal antibody according to claim 4 of subtype selected from the group consisting of IgM, IgG?, IgG2a, IgG2b and IgG3.

6. A monoclonal antibody according to claim 4 wherein the cell line is selected from the group con-sisting of A.T.C.C. HB-8957, HB-8958, HB-8959, HB-8960, HB-8961 and HB-8962.

7. A monoclonal antibody according to claims 4, 5 or 6 which is capable of neutralizing the capacity of hpG-CSF to induce differentiation of WEHI 3B D+ cells in culture.

8. A monoclonal antibody according to claims 4, 5 or 6 which is capable of neutralizing the capacity of hpG-CSF to stimulate uptake of thymidine by bone marrow cells.

9. In an immunological procedure for isola-tion of biologically active hpG-CSF from a biological fluid on the basis of a selective immunological reaction with an antibody specific for hpG-CSF, the improvement comprising:
employing a monoclonal antibody capable of specifically binding with hpG-CSF in an antigen/antibody reaction.

10. An immunological procedure according to claim 9 wherein the monoclonal antibody is of a subtype selected from the group consisting of IgM, IgGl, IgG2a, IgG2b and IgG3.

11. An immunological procedure according to claim 9. wherein the monoclonal antibody is produced by a cell line selected from the group consisting of A.T.C. C. HB-8957, HB-8958, HB-8959, HB-8960, HB-8961 and HB-8962.

12. In an immunological procedure fox the quantitative detection of hpG-CSF in a biological fluid on the basis of one or more selective immunological reactions with an antibody specific for hpG-CSF, the improvement comprising:
employing one or more monoclonal antibodies capable of specifically binding with hpG-CSF in an antigen/antibody reaction.

13. An immunological procedure according to claim 12. wherein the monoclonal antibodies are of subtypes selected from the group consisting of IgM, IGG1, IgG2a, IgG2b and IgG3.

14. An immunological procedure according to claim 12. wherein the monoclonal antibody is produced by a cell line selected from the group consisting of A.T.C.C. HB-8957, HB-8958, HB-8959, HB-8960, HB-8961 and HB-8962.