BACKGROUND OF THE INVENTION

The present invention relates generally to materials and methods useful in the quantitative detec¬ tion and affinity purification of human immune interferon ("IFN-Ύ"} and structurally related polypeptides euch as analogs of INF-γ produced by recombinant methods. of substantial interest to the background of the present invention is the state of the art with regard to the preparation and use of a class of biologically active substances, the interferons (IFNs). Interferons are secreted proteins having fairly well-defined anti- viral, antitumor and immunomodulatory characteristics. See, e.g., Gray, et al.. Nature, 295, pp. 503-508 (1982) and Edge, et al., Mature, 292, pp. 756-782 (1981), and references cited therein.

On the basis of antigenicity and biological and chemical properties, human interferons have been grouped into three major classes. IFN-α (leukocyte), IFN-0 (fibroblast) and IFN-γ (immune). Considerable information has accumulated on the structures and proper¬ ties of the virus-induced acid-stable interferons (IFN-α and 0). These have been purified to homogeneity and at least partial amino acid sequences have been determined. Analyses of cloned cDNA gene sequences for IFN-Θ and the IFN-α raultigene family have permitted the deduction of

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the complete amino acid sequences of many of the inter¬ ferons. In addition, efficient synthesis of IFH-S and several IFN-αs in E. coli, and subtype IFN-α, , in yeast, have now made possible the purification of large quanti- ties of these proteins in biologically active form.

Much less information is available concerning the structure and properties of IFN-γ, an interferon generally produced in cultures of lymphocytes exposed to various mitogenic stimuli. It is acid labile and does not cross-react with antisera prepared against IFN-α or IFN-β. A broad range of biological activities have been attributed to IFN-γ including pσtentiation of the antiviral activities of IFN-o and -0, from which it differs in terms of its virus and cell specificities and the antiviral mechanisms induced. In vitro studies performed with crude preparations suggest that the primary function of IFN-γ may be as an immunoregulatory agent. The antiproliferative effect of IFN-γ on trans¬ formed cells has been reported to be 10 to 100-fold greater than that of IFN-α or -8, suggesting a potential use in the treatment of neoplasia. Hurine IFN-γ prepara¬ tions have been shown to have significant anti umor activity-against mouse sarcomas.

It has recently been reported (Gray, et al., aupra) that a recombinant plasraid containing a cDHA sequence coding for human IFN-γ has been isolated and characterized. Expression of this sequence in B. coli and cultured monkey cells is reported to give rise to a polypeptide having the properties of authentic human IFN-γ. In the publication, the deduced 146 amino acid sequence of the "mature" polypeptide, exclusive of the putative leader sequence, is as follows:

1 10

Cys-Tyr-Cys-Gln-Asp-Pro-Tyr-Val- ys-Glu-Ala-Glu-Asn-Leu-

20 Lys- yβ-Tyr-Phe-Asn-Ala-Gly-His-Ser-Asp-Val-Ala-Asp-Asn-

/F

30 40

Gly-Thr- eu-Phe- eu-Gly-Ile- eu-Lys-Asn-Trp- ys-Glu-Glu-

50 Ser-Asp-Arg- ys-lle-flet-Gln-Ser-Gln-Ile-Val-Ser-Phβ-Tyr-

60 70 Phe-Lys-Leu-Phe-Lys-Asn-Phe-Lys-Aβp-Asp-Gln-Ser-Ile-Gln-

80 ys-Ser-Val-Glu-Thr-Ile-Lys-Glu-Asp-Met-Aβn-Val-Lyβ-Phe-

90

Phe-Asn-Ser-Asn-Lys-Lys- ys-Arg-Asp-Asp-Phe-Glu-Lys-Leu-

100 110

Thr-Asn-Tyr-Ser-Val-Thr-Asp- eu-Asn-Val-Gln-Arg- ys-Ala-

120 Ile-His-Glu- eu-Ile-Gln-Val-Met-Ala-Glu-Leu-Ser-Pro-Ala-

130 140 Ala- ys-Thr-Gly-Lys-Arg-Lys-Arg-Ser-Gln-Met- eu-Phe-Gln-

146 Gly-Arg-Arg-Ala-Ser-Gln

In a previous publication of the sequence, arginine, rather than glutaraine, was specified at posi¬ tion 140 in the sequence. (Unless otherwise indicated, therefore, reference to "human immune interferon" or, simply "IFH-γ" shall comprehend both the [Ar ] and (Gin ¹⁴⁰ ] forms.) The above-noted wide variations in biological activities of various interferon types makes the con¬ struction of synthetic polypeptide analogs of the inter¬ ferons of paramount significance to the full development of the therapeutic potential of this class of compounds. Co-owned, co-pending U.S. Patent Application Serial Mo. 375,494 by Alton, et al., filed May 6, 1982, relates to procedures for the rapid manufacture of structural genes specifying human interferons and analogs thereof and to the use of these manufactured genes to secure icrobial expression of biologically active polypeptide products.

CVFΓ

Of substantial interest to the background of the invention is the extensive body of information generated over the recent past with respect to the use of immunological procedures for the isolation and quanti- tative detection of complex, biologically active polypep- tides such as the inteferons. Such immunological proce¬ dures have frequently employed polyclonal, serum-derived antibodies directed against the proteinaceous materials of interest. Briefly put, antisera and serum-derived antibodies are obtained by inoculation of an i muno- logically-active animal, such as a rat or rabbit, with the material of interest. The injected material is recognized as a foreign antigenic substance by the immune system of the animal and elicits production of antibodies against the antigen. Differing cells responding to stimulation by the antigenic substance produce and release into circulation antibodies slightly different from those produced by other responding cells. The anti¬ body activity remains in the serum of the animal when its blood is extracted. While unpu ified serum or anti¬ body preparations purified as serum immunoglobulin fractions may then be used in assays to detect and complex with the proteinaceous material in fluids and on gels, the antibody preparations suffer from a major disadvantage. Serum antibodies, composed of all the different antibodies produced by individual cells, are polyclonal in nature and for the most part are reactive with antigens other than the one of Interest. Antibodies specific for the desired product represent a subset of the antibodies present in serum.

Of interest to the background of the present invention are recent advances in the art of developing continuous cultures of cells capable of producing a single species of antibody (a "monoclonal" antibody) which is specifically immunolo ically reactive with a single antigenic determinant of a selected antigen. See,

generally, Chisholra, High Technology, Vol. 3, No. 1, 57-63 (1983) and Todd, et al., T.I.B.S., 7, pp. 212-216 (1982) . Monoclonal antibodies have proven to be invalu¬ able for the characterization, quantitative analysis, and purification of acromolecular antigens, including such biologically active compounds as leukocyte inter¬ ferons. See generally, Secher , et al., Nature, 285, _, pp. 446-450 (1980); Staehelin, et al., P.H.A.S.. £8, pp. 1848-1852 (1981); and ϊmai, et al., J.Immunology, 128, pp. 2824-2825 (1982). To date, however, there have been no published reports of the development of monoclonal antibodies to IFN-γ. The lack of availability of such substances is becoming a more pronounced problem in view of the above-noted advances in the capacity to employ recombinant methods for the production of IFN-γ and its analogs. Antibodies with high specific affinity for these substances would not only be useful for purifica¬ tion of IFN-γ and analogs thereof from raicrobial produc¬ tion systems, but would also be useful in quantitative analytical methods involving IFH-γ. For example, such antibody preparations would be useful in the character¬ ization of cell surface receptors for IFN-γ (including their distribution and modulation) and in characterizing the receptor binding qualities of IFN-γ analogs. Such information, in turn, would have clinical diagnostic and therapeutic significance in interferon management of disease.

Also of interest to the background of the invention are reports of the immunological activity of synthetic peptides which substantially duplicate the amino acid sequence extant in naturally-occurring proteins, glyσoproteins and nuσleoproteins. More specif¬ ically, relatively low molecular weight polypeptideβ have been shown to participate in immune reactions which are similar in duration and extent to the immune reactions of physiolo ically significant proteins such as viral

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antigens, polypeptide hormones and the like. Included among the immune reactions of such polypeptides is the provocation of the formation of specific antibodies in immunologically active animals. See, e.g., erner, et al., Cell, 23,, 309-310 (1981); Ross, et al., Nature, 294, 654-656 (1981) j Walter, et al., P.N.A.S. (USA), 77, 5197-5200 (1980); Lerner, et al., P.N.A.S. (USA), 8, 3403-3407 (1981); Walter, et al., P.N.A.S. (USA) , 78, 4882-4886 (1981); Wong, et al., P.N.A.S. (USA) , 78., 7412- 7416 (1981); Green, et al., Cell, 28., 477-487 (1982); Nigg, et al., P.N.A.S. (USA), 79., 5322-5326 (1982); Baron, et al., Cell, 28, 395-404 (1982); Dreesman, et al., Nature, 295, 158-160 (1982); and Lerner, Scientific American, 248, No. 2, 66-74 (1983). . The advantages of antibody preparations immuno¬ reactive with both a acromolecule and with a readily available synthetic peptide are manifest. Co-owned, co-pending U.S. Patent Application Serial No. 463,724, by Egrie, filed February 4, 1983, discloses the develop- ent and use of monoclonal antibodies which are both immunolo ically reactive with human erythropoi in and reactive with a synthetic polypeptide which is duplica- tive of an amino acid sequence determined to be extant in erythropoietin. This achievement is indeed a rare one, owing to the fact that polypeptide fragments in solutions employed as inoculants cannot ordinarily be expected to assume an "antigenic" configuration similar to that which the polypeptide sequence naturally assumes as part of a macromolecular material. Consequently, monoclonal antibodies to whole raacroraolecules seldom bind peptide fragments well, and vice versa.

To date, there have been no published reports of the development of monoclonal antibodies, raised against interferon peptide fragments, and which have high affinity for interferon-duplicating peptide fragments and high affinity for native interferon species. It has

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quite recently been reported that polyclonal antibody preparations have been raised in rabbits in response to administration of a synthetic peptide corresponding to the first 20 amino acids of the amino {N} terminal of IFN-γ. [Johnson, et al., J.Immunology, 129, pp. 2357- 2359 (1982).] Serum antibodies obtained according to the published procedure were noted to be operative in neutralizing the antiviral biological effects of IFN-γ. While the capacity of antibodies to neutralize biological effects of acromolecules is in some instances considered to be an advantageous characteristic, this is not uni¬ formly the case. As one example, neutralizing antibodies are of limited use in studies directed to elucidation of target cell receptor structure and function. More significantly, use of neutralizing antibodies in affinity purification procedures (wherein products are often isolated by quite harsh elution processes from bound association with fixed antibody preparations) may result in loss of biological activity of the purified material through disruption of "active" sites overlapping sites where binding to the antibody takes place.

There continues to exist a need in the art, therefore, for monoclonal antibodies which are specifi¬ cally immunoreactive with IFN-γ. In order to meet the needs of the art, monoclonal antibody preparations would advantageously be immunoreactive with native and recombi¬ nant IFN-γ and analogs thereof, would also be immuno¬ reactive with synthetic peptides substantially duplica- tive of IFN-γ, and would preferably be non-neutralizing of IFN-γ biological activity.

BRIEF SUMMARY

The present invention provides, for the first time, monoclonal antibody preparations which are specifi¬ cally immunoreactive with IFN-γ and also specifically

immunoreactive with polypeptide substances having amino acid sequences substantially duplicative of sequences extant in IFN-γ. Moreover, preferred antibodies provided by the invention are not operative to neutralize anti- viral activity of IFN-γ.

In one of its aspects, the present invention provides a new mouse-mouse hybridoma cell line A.T.C.C. No. HB8291. This cell line secretes into the media as a product of its growth a ^" highly specific monoclonal, anti-IFN-γ antibody which is also specifically immuno¬ reactive with a polypeptide comprising the following sequence of amino acids: NH ₂ -Lys-Thr-Gly- ys-Arg-Lys- Arg-Ser-Gln-Met-Leu-Phe-Arg-Gly-Arg-Arg-Ala-Ser-Gln-COOH. The antibody produced by A.T.C.C. HB8291 i3 thus the first antibody substance ever demonstrated to be immuno¬ reactive with both IFN-γ and a polypeptide substantially duplicative of the amino acid sequence extant in IFN-γ. Tumor cell line, A.T.C.C. HB8291, is on deposit at the American Type Culture Collection, 12301 Parklawn Drive, Rockville, Maryland 20852.

According to the invention the IgGl antibody produced by A.T.C.C. HB8291 is advantageously employed in immunological procedures for the isolation of large quantities of pure, biologically active, naturally- occurring and recombinant IFN-γ and IFN-γ analogs (here¬ after ⁿ IFN-γ products") and in diagnostic im unoassays for the quantitative detection of IFN-γ products in fluid samples, preparative gels and the like.

The antibody substances provided by the inven- tion facilitate performance of assays for quantitative detection of IFN-γ products in fluid samples (including blood and other body fluids) , especially those assays which require the use of two antibodies which are immuno¬ reactive with two different antigenic determinants of IFN-γ products. In such procedures, a first antibody (e.g., that produced by A.T.C.C. No. HB8291) is immobi-

lized on a solid support and, when contacted with the sample fluid, i munobinds to one antigenic determinant of the IFN-γ product in the fluid. A second antibody (e.g., a polyvalent, serum-derived antibody such as described in Johnson, et al., supra) which may be linked to a detectable label (or is subsequently detected with a labelled material) is contacted with the complex of IFN-γ and the first antibody and binds to a different antigenic determinant thereof. The quantity of IFN-γ products in the sample is thereafter determined through quantification of the bound second antibody,

Antibodies of the present invention also pro¬ vide materials useful in performance of ^• 'compet ti e ^* ' solid or liquid phase assays for detection of IFN-γ products which involve use of two species of antigen (e.g., labelled and unlabelled) forms. (Analytical methods of this type are disclosed in the aforementioned U.S. Patent Application S.N. 463,724 by Egrie and specif¬ ically exemplified by erythropoietin detection procedures. The disclosures of said application are specifically incorporated by reference herein.) In such assays, for example, the quantity of IFN-γ products in a sample may be determined by the extent of competition for binding to the monoclonal antibodies with a labelled or immobi- lized synthetic peptide. Antibodies of the invention can also be expected to find use in affinity purification procedures of the aforementioned Application Serial No. 463,724, wherein differential affinity of a monoclonal antibody for a proteinaceous macro olecule and for a synthetic peptide allows the displacement elution of a desired material from bound association with an immobi¬ lized antibody.

In another aspect of the invention, antibodies of the invention provide for the rapid determination of expression of IFN-γ products by microorganisms.

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- ι -

Other aspects and advantages of the invention will be apparent upon consideration of the following detailed description of preferred embodiments thereof.

DETAILED DESCRIPTION

The following examples illustrate practice of the invention in the production of hybridoma cell line A.T.C.C. No. HB8291 and the isolation of monoclonal antibodies to both ^* IFN-γ products and a nonadβcapeptide (19-mer polypeptide) duplicative of sequences of amino acids extant in IFN-γ at its carboxy terminal. Also illustrated is the characterization, amplification and determination of properties of antibodies produced by A.T.C.C. NO. HB8291.

EXAMPLE 1 Development of Amino Acid Sequences and Synthetic Peptide

Review of the published sequence of IFN-γ noted above revealed the following sequences of amino acids which, according to analysis by the general proce¬ dures of Hopp, et al., P.N.A.S. (U.S.A.), 78 _, , p.- 3824 (1981) appear to have significant antigenic potential independently of association with the entire IFN-γ glycoproteinj

(1) 128 129 130 131 132 133 134 135 136 137 138 139 NH ₂ -Lys-Thr-Gly-Lyβ-Arg-Lye-Arg-Sβr-Gln-Met-Leu-Phe-

140 141 142 143 144 145 146

Arg-Gly-Arg-Arg-Ala-Ser- ln-COOH;

(2) 140 141142 143 144 145 146 NH,-Arg~Gly-Arg -Arg-Ala-8er-Gln-C00ff|

(3) 128 129 130 131 132 133 134 135 136 NH ₂ -Lys-Thr-Gly-Lys-Arg-Lys-Arg-Ser-Gln-COOHj

(4) 87 88 89 90 91 92 93 94 95

NH ₂ -A ^β n-Ser-ABn-Lys-Ly8-Ly8-Arg-Aap-Aaρ-

96 97 98

Phe-Glu-L s-COOHi

(5) ^' 61 62 63 64 65 66 67 68 69 NH2-Lys-Asn-Phe-Lys-Asp-Asp-Gln-Ser-Ile-

70 71 Gln-Lys-COOH; and

(6) 40 41 42 43 44 45 46 NH ₂ -Lys-Glu-Glu-Ser-Λsp-Arg-Lys-COOH.

The sequence of amino acids spanning residues 137 through 139 (Met-Leu-Phe) is significantly hydrophiliσ and hence likely to have substantial antigenic potential and on this basis the 19-mer of sequence No. 1 was selected for initial studies.

A synthetic replica of the above-noted 19-mer sequence lσ. 1 was prepared according to the procedure of [Merrifield, R.B., J.of Am.C em.Soc. , 8^, 2149-2154 (1963) [see also, Stewart, J.M., et al.. Solid Phase Peptide Synthesis, San Francisco: W.H. Freeman & Co. (1969) and covalently σrosslinked using glutaraldehyde to a keyhole limpet he ocyanin (KLH) carrier protein, according to the procedure of Baron, et al,. Cell, 2/3, pp. 395-404 (1982) .

EXAMPLE 2 Hybridoma Production

In the procedure for production of hybridoma cell line A.T.C.C. No. HB8291, BALB/c mice (Si onsen

Laboratories, Gilroy, California) were hyperimmunized to the LH-bound synthetic polypeptide prepared accord¬ ing to Example 1. The immunization and cell fusion procedures were performed according to essentially stan- dard procedures set out in Oi, et al., pp. 351-372 in Selected Methods in Cellular Immunology (Mishell, et al., βds.), W.H, Freeman Publishing, San Francisco (1979). The first inoculation was subcutaneous and contained 10 microgra s of crosslinked 19-mer synthetic peptide plus Difco H37Ra Complete Freund's Adjuvant (Difco Laborato¬ ries), Further inoculations were given intraperitoneally 7, 21 42, and 73 days after the subcutaneous injection, each containing 10 micrograms of crosslinked 19-mer synthetic peptide. Three days prior to cell fusion (on day 94), the mice were inoculated with a final intraperi- toneal injection containing 10 micrograms of crosslinked 19-mer synthetic peptide.

After the third and fifth injections, serum from several mice was assayed by a solid phase binding radioi munoaεsay tested positively for the presence of antibodies to both the synthetic peptide and to a lysate of E. coll cells harboring a vector including a manufac¬ tured DNA sequence coding for IFN-γ products. Briefly put, a solid phase binding assay was developed using the synthetic peptide immobilized in Iromucon wells (Dynatech

Labs., Inc., Alexandria, Va.) , dilutions of mouse anti-

125 synthetic peptide antiserum and I labelled rabbit anti-mouse immunoglobulin. This assay had the advantage of not requiring labelling (and hence possible chemical modification) of the peptide antigen. The above-noted bacterial lysate competed for binding.

Following verification that the inoculated mice were producing serum antibodies to IFN-γ products and the synthetic polypeptide, spleens of the five most responsive immunized BALB/c mice, which contain a small number of antibody-producing lymphocytes, were disrupted

to single cells. In the presence of the fusogen poly¬ ethylene glycol, immune donor spleen cells are fused with a parental BALB/σ myeloma cell line, SP2/0-(HPRT~J ΪSchul an, et al., Nature, 276, 269 (1978)) to produce a variety of hybrids. Briefly described, cell membranes fuse and initially surround a common cytoplasm with two or more nuclei. Several days after that event, the nuclei fuse and become capable of synchronous mitosis. As these fused cells divide, a variable number of chromo- somes of both fused partners are lost until the hybrid cell lines stabilize.

Fused cells are plated into 8 multiple 96-well plates (768 total wells) at from 10 to 10 cells per well. Selection of SP2/0:spleen cell hybrids from the fusion which also produces SP2/Q.SP2/0 and spleen:spleen cell hybrids is accomplished by culturing the fusion mixture in hypoxanthine-aminopterin-thyraidine (IIAT) medium for two weeks. HAT medium prevents SP2/0:SP2/0 hybrids from dividing. The spleen*spleen cell hybrids generally die after two weeks in culture. Thus the HAT medium allows growth of only the SP2/0:spleen hybrid cells.

After 10 days, many of the 760 wells contained multiple, viable cell colonies. Thereafter the indi- vidual cell colonies were screened for the presence of immunoglobulins and IFH-γ-specifiσ antibodies.

EXAMPLE 3 Screening, Cloning and Characterization of Monoclonal Antibodies

A radioimraunoassay as in Example 2 was per¬ formed to reveal specific anti-IFN-γ, anti-polypeptide antibodies in the wells. Based on the results of these two screening assays, 14 of the 768 initial wells, posi¬ tive for anti-polypeptide antibody and IFN-γ, were

selected for minicloning.

Cells from each of the initially screened ^' colonies were further subdivided into several multi-well plates (about ten cells per well), and allowed to grow. These wells were again screened by radioimraunoassay for the presence of both anti-polypeptide and anti-IFN-γ antibody.

Cells from the 6 strongest positive miniclone wells were selected for formal cloning and diluted into new plates at a calculated density of 1 cell per 3 wells. The low density assures that a high proportion of colo¬ nies will derive from a single cell. Monoclonal prolif¬ eration of cells was screened periodically by microscopic examination. Thereafter culture fluids from all wells were assayed by radioimmunoassay in the procedures described above. The results of this cloning procedure produced 130 positive wells, of which the 45 most posi¬ tive were further propagated. From these, ten presump¬ tively equivalent, strongly positive single-cell colonies producing antibody to both the polypeptide and IFN-γ and having satisfactory cell growth rate and density were selected. All colonies produced on the order of 100 micrograms of antibody per πiilliliter of culture fluid. One of these 10 cell lines was selected for deposit as A.T.C.C. Ho. HB8291,

To isolate maximum antibody producing cells, A.T.C.C. No. HB8291 cells are continuously subcultured to avoid deleterious effects of aging. A desirable medium for growth of hybridoma cells is KBlQl (Banna Biologicals) supplemented with 1% fetal calf serum

(Tissue Culture Biologicals) i when cells are in condition for harvesting culture fluid, the medium is preferably changed to HB101 without fetal calf serum.

Monoclonal antibodies may be isolated from culture fluids by AMICON filtration concentration

(A icon) , followed by precipitation with 45% ammonium

sul ate. Alternatively, the concentrated culture fluids may be adsorbed to Protein A-Sepharose columns (Pharmacia Corp.). [See, Coding, J.Imm.Methods, 39., 285-308 (1980)). In these columns, the Protein A attaches to the Fc por- tion of the antibody i munoglobulin, allowing other contaminants in the culture fluid to elute out of asso¬ ciation with the antibody. Although column capacity for mouse IgGl is low, nonetheless, the antibody achieves considerable purification by this method. To obtain a more concentrated antibody than that produced in tissue culture, the monoclonal anti¬ bodies of the present invention may be amplified by the ascites method generally described in Kenneth, et al., (eds.), Monoclonal Antibodiss, Ilybridomas: A New Pinionsion in Biological Analysis, p. 403, New York: Plenum Press (1981). According to this procedure, 10 hybridoma cells may be injected into the peritoneal cavities of 3ALB/c mice, previously treated with 0.50 ml Pristane (Aldriσh Chemical Co.) . Pristane treatment promotes growth of tumor cells in an ascitic form within the peritoneal cavity. Once the ascitic tumor cells grow, the mice are sacrificed and the ascitic fluid containing the monoclonal antibody is harvested from the cells by centrifugation. The monoclonal antibody in the ascites fluid is then assayed for antibody titer. Asciteβ fluids containing on the order of 10 milligrams of IgG antibody per illiliter of culture fluid have been obtained by this method. Ascites fluid antibodies can be further purified from ascites fluid albumin by 45% ammonium sulfate precipitation and ion exchange chroma- tography.

Monoclonal antibody preparations were derived by purification from ascites fluids with Protein-A sepharose as described above for culture fluid purifica- tion. These isolates were radioiodinated using the chloramine T by the general method of Hunter, et al _* ,

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Mature, 194, pp. 495-496 (1962) . The I ¹²⁵ -labelled products retained their prior immunological activity in the radioimraunoassay.

Competitive binding experiments using ascites fluid-derived radiolabelled antibodies were employed to evaluate epitope speci icities of the antibodies and indicated that all ten onoclone products (including that produced by HB8291) recogni2ed the same (or, at least a closely adjacent or overlapping) epitope on the synthetic peptide.

SXAΠPLΓ.4

Western Blot Assays For Microbially-Expressed IFN-γ Products

The value of monoclonal antibody preparations according to the invention as research tools is exempli¬ fied by their use in "Western Blot" analyses [see,Burnett, Anal.Diocheπ ^* ., 112, pp. 195-203 (1981)] of cellular extracts of microorganisms wherein synthesis of recombinant IFN-γ and IFN-γ analogs is attempted.

The following general procedures were employed in those .analyses. Crude extracts of E^ coli cells which had been transformed with plamsid DNA coding for IFN-γ and IFN-γ analogs (which differed IFN-γ in terms of the identity or location of one or more amino acids) were run on a standard SDS-PAGE gel with markers of varying molecular weights in an attempt to determine the presence of the desired polypeptide in the lysate. A nitrocellu- lose filter paper was placed over the gel and the gel was placed in a liquid electrophoretiσ cell in order to transfer the protein bands to the filter paper. The paper was "blocked-' by treatment with 5* bovine serum albumin. The paper was then treated with a monoclonal antibody solution containing approximately 100 miσrogramβ per illiliter of antibody. Specific immunoreactivity

of the antibody with the recombinant IFN-γ or IFN-γ analog allowed autoradiographic visualization of the presence of antibody bound to desired product upon treat¬ ment with radioiodinated rabbit anti-mouse igG antisera.

EXAMPLE 5 Neutralization Studies

A study was conducted for the purpose of comparing the effects of various antibody preparations on the antiviral biological activity of IFN-γ. The general procedure was patterned on Research Reference Reagent Note 22 accompanying sheep antiserum to human leukocyte interferon, N.I.A.I.D. Catalog No. G-026-502- 568. In this procedure, a standard quantity of commer¬ cially prepared IFN-γ (2 x 10 U/τnl) was incubated for three hours prior to incorporation with each of the following antibody preparations: (1) mouse anti-peptide antiserum; (2) rabbit anti-peptide antiserum; (3) mono- clonal antibody of twenty separate formal clones of

Example 3; and rabbit anti-IFN-γ antiserum (Interferon Sciences New Brunswick, M.J.). The results of this procedure, applied to WISH cells challenged with encepha- lo yocarditis virus (EMCV) , are set out in Table I. TABLE 1

FINAL

A TIBODY DILUTION NEUTRALIZATION

Mouse anti-IFN-γ peptidt » lt20 0

Rabbit Anti-IFN-γ peptide 1:20 0

Mouse Anti IFN-γ Peptide Mono- σlones (e.g., HB8291) 1:2 0

Interferon Sciences rabbit Anti-IFN-γ ii0-lι20 92-96 The monoclonal antibodies of the present inven¬ tion, while specifically immunoreactive with naturally-

occurring IFN-γ, do not operate to neutralize the anti¬ viral activity of this interferon.

EXAMPLE 6 Isolation of IFN-γ Products

By Affinity Puri ication

Initial studies are being conducted relating to the utility of monoclonal antibodies of the invention in the affinity purification of IFN-γ products including naturally-occurring IFN-γ, recombinant IFN-γ and analogs of IFN-γ produced by recombinant procedures. In a first series of tests, monoclonal antibodies according to Example 3 were immobilized on Λffi-gel 10 (BIORAD, Richmond, CA.) and employed as an affinity absorbent for crude cell extracts of microorganisms genetically trans¬ formed to express analogs of IFN-γ. Alkaline elution using 0.1M diethylamine, pH 11.5 resulted in the isola- tion of icrogram amounts of highly purified [Lys 81J IFN-γ. A column of cyanogen bromide activated Sepharose

(Pharmacia) containing immobilized monoclonal antibody preparations of the invention for use in large scale isolations was constructed and employed to secure nearly milligram amounts of recombinant IFN-γ analogs from bacterial extracts. As previously noted, it is expected that highly purified biologically active IFN-γ products will be isolatable through use of synthetic peptides to elute desired products from bound association with mono¬ clonal antibodies of the invention by means of differen- tial affinity. Preliminary binding affinity assays indi¬ cate that, e.g., the antibody produced by A.T.C.C. HB8291 has a somewhat higher affinity for the above-noted 19-mer than for naturally-occurring IFN-γ. While the synthetic 19-mer employed in "raising" the antibodies produced by HB8291 is currently the material of choice for use in such procedures, it is anticipated that other polypep-

tides which duplicate only a portion of the 19-mer 's sequence (e.g., polypeptide Nos. 2 and/or 3 of Example 1) may also be useful.

Monoclonal antibodies of the invention may be suitably employed in labelled or unlabelled form for quantitative detection of IFN-γ products in any of the general immunological assay procedures (RIA's, ELISΛ's, and the like) presently employing antibody preparations. As previously noted, the. fact that preferred antibodies of tho invention are specifically iπununoreactive with both IFN-γ products and synthetic peptides makes possible imiuunolo ical quanti ication procedures involving two "antigens". Further, the fact that preferred antibody preparations are known to bo: specifically immunoreactive with a specific region of IFM-γ products allows the development of assays involving two or more antibodies which are individually reactive with differing epitopes. Finally, the fact that preferred antibody preparations of the invention are πou-neutralizing makes possible the practice of IFN-γ detection procedures which do not interfere with biological activity. Such procedures are especially significant to further basic research in the characterization of cell surface IFN—γ receptors.

While the foregoing illustrative examples are specifically directed to the present invention as exem¬ plified by the development of cell line A.T.C.C. 11138291 and monoclonal antibody preparations derived therefrom, it will be understood that the contributions of the present invention are not limited thereto. The mono- clonal antibodies provided by the invention are believed to be the first substances ever isolated which are spe¬ cifically iπuuunoreactive with IFN-γ products. They are also believed to be the first such substances which are specifically immunoreactive with a synthetic polypeptide which duplicates amino acid residues extant in IFN-γ.

Finally, the monoclonal antibodies provided by the inven-

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tion are the first anti-IFN-γ antibody materials ever available which do not neutralize IFN-γ antiviral activity.

Numerous modi ications and variations in praσ- tice of the invention are expected to occur to those skilled in the art upon consideration of the foregoing description of presently preferred embodiments thereof. As one example, while the examples generally refer to the use of synthetic peptides which duplicate the nine- teen carboxyl terminal amino acids of IFN-γ, it will be understood that the use of elongated or abbreviated polypeptideε duplicating all or only an immunologically significant portion of the specified 19-mer are contem¬ plated, as are polypeptides including a [Gin ] residue rather than an [Asp 140] residue. W t respect to the last-mentioned factor, it is noteworthy that the antibody of A.T.C.C. KB8291 appears to be immunoreactive with commercial IFN-v preparations which may include the

[Gin ] species as well as with recombinant IFN-γ and IFN-γ analog products, all of which included an asparagine residue at position 140. Consequently, only such limitations should be placed upon the scope of the invention as appear in the appended claims.