WO1986007062A1 - Monoclonal antibody to decay accelerating factor (daf), a method for making it, and use - Google Patents

Abstract

A monoclonal antibody immunochemically reactive with decay accelerating factor and a method for making such a monoclonal antibody by purifying decay accelerating factor to homogeneity by a combination of anion-exchange and hydrophobic interaction chromatography, gel filtration, lectin-Sepharose chromatography, immunoaffinity chromatography, anion exchange chromatography and high-performance liquid chromatography; immunizing mice with said purified factor; fusing spleen cells from said mice with mouse myeloma cells to produce hybrid cells; and screening said hybrid cells to identify those secreting said monoclonal antibody.

Description

MONOCLONAL ANTIBODY TO DECAY ACCELERATING FACTOR (DAF), A METHOD FOR MAKING IT, AND USE

The United States Government has rights in this invention by virtue of Grants No. NIH AI-13224 and

P30CA-16087 from the United States Department of Health and

Human Services. BACKGROUND OF THE INVENTION.

Decay accelerating factor (DAF) is a 70,000 Mr protein that has been isolated and characterized from the membrane of erythrocytes. It has been suggested that the function of DAF is to inhibit the assembly of the amplifying enzymes of the complement cascade on the eryth- rocyte surface, and thereby protect the red blood cells from damage by autologous complement. Specifically, there is evidence that DAF interferes with the assembly of C3- σonvertases (C4b2a and C3bBb) and C5-convertases (C4b2a3b and C3bBb3b) : Medof, M.E., et al., J. Exp. Med. 160:1558 (November, 1984).

The precise mechanism of DAF action is not known but several lines of evidence indicate that it binds to C4b or C3b and competitively prevents the interaction with C2 and factor B. Medof, et al., supra.

Erythrocyte DAF functions only in the membrane in which it is located, i.e. DAF does not act on C3- or C5- convertases of neighboring cells or on foreign substrates, such as bacteria or immune complexes. This behavior of DAF lead to the suggestion that DAF protects erythrocytes from damage by autologous complement.

The hypothesis about DAF function is in agreement with the finding of Pangburn et al Proc. Nat'l Acad. Sci. 80:5430 (1983) and Nicholsύn-Weller et al Proc. Nat'l Acad Sci. 80:5066 (1983) that erythrocytes from patients with paroxysmal nocturnal hemoglobinuria (PNH), an acquired syn¬ drome characterized by unusually high susceptibility of red blood cells to complement activation, are DAF-deficient. Platelet and leukocyte abnormalities are also found in PNH, but a role for DAF in these disorders has not been estab¬ lished.

It is clear that further study of the nature and function of DAF is necessary. Not only would such study lead to a better understanding of the function of DAF, it also might help in the management or treatment of PNH.

Unfortunately, DAF occurs in nature in very short supply. One milligram of pure DAF requires elaborate and time-consuming processing of about 7,000 g of erythrocytes. Availability of a monoclonal antibody to DAF (anti-DAF) would greatly simplify its purification (by immunoaffinity chromatography) , help locate other sources of DAF, and make its synthesis possible by recombinant techniques. Accom- plishment of the latter goal would also yield detailed information on the DAF structure.

Prior to the present invention, however, attempts to produce monoclonal anti-DAF had failed because of difficulties in purifying the protein to homogeneity, as tested by SDS-PAGE. Pure DAF is needed for the initial immunization prior to cell fusion. Objects of the Invention

It is accordingly an object of the present invention to provide a monoclonal antibody to DAF. It is also an object of the present invention to provide monoclonal antibodies to DAF that would be useful in locating other sources of DAF, purifying DAF, screening recombinant microorganisms that would produce DAF, and serving as an investigation tool in DAF research. It is another object of the present invention to provide means and methods for producing and purifying monoclonal antibodies to DAF. It is still another object of the present invention to use monoclonal anti-DAF in purifying DAF, locating other cells that contain DAF, and screening recombinant organisms that would produce DAF. It is a further object of the present invention to provide means and methods including monoclonal anti-DAF and use them in further investigation of the structure and function of DAF, in diagnosis, management, and treatment of PNH, in the study of the pathogenesis of PNH and other erythrocyte disorders that arise because of defects in DAF biosynthesis.

It is another object of the invention to provide a method for enhancing the killing of cells (including tumor cells) by complement, especially in vitro. Another object is to provide means and methods for targeting molecules for insertion in cell membranes.

Yet another object is to provide a method for the diagnosis and staging of PNH by using monoclonal antibodies to DAF. SUMMARY OF THE INVENTION

One aspect of the present invention is directed to monoclonal antibodies immunochemically reactive with Decay Accelerating Factor.

Another aspect of the present invention is directed to a method for producing a monoclonal antibody immunochemically reactive with Decay Accelerating Factor, said method comprising: purifying DAF from cells containing it to homo¬ geneity as tested by sodium dodecyl sulfate polyacrylamide gel electrophoresis and Western blotting; immunizing mice with said DAF; fusing spleen cells from said mice with mouse myeloma cells to produce hybrid cells; screening said hybrid cells to identify those secreting said monoclonal antibody using said purified DAF as the screening agent. Yet another aspect of the present invention is directed to hybrid cells secreting monoclonal anti-DAF.

Still another aspect of the present invention is directed to a method for purifying DAF, by subjecting impure preparations of DAF to immunoa finity chromatography using monoclonal anti-DAF as the immunoadsorbent.

Another aspect of the present invention is directed to a method for diagnosing or determining the stage of PNH, said method comprising: determining the amount of DAF contained in erythrocytes of a patient and comparing said DAF amount to that contained in erythrocytes of normal, healthy individuals.

A further aspect of the present invention is directed to a method for locating sources of DAF by sub- jecting cell extracts to the presence of anti-DAF under conditions making possible an immunochemical reaction between DAF and anti-DAF, and identifying the bound anti- DAF and thus the tissues where DAF is present. A related aspect of this invention is directed to a method for extracting DAF from such tissues using monoclonal anti-DAF. Still a further aspect of the present invention is directed to a method for screening recombinant micro¬ organisms that produce proteins having amino acid sequences comprising sequences occurring in native DAF molecules, said method comprising exposing a preparation of such microoganisms to the presence of monoclonal anti-DAF under conditions permitting a DAF-(anti-DAF) immunochemical reaction to occur and determining whether said reaction takes place. The invention is further described below by reference to particularly preferred embodiments. In the following description, reference is made to the appended drawings, in which: BRIEF DESCRIPTION OF THE DRAWINGS: Figure 1 is a plot of the number of DAF molecules extracted per erythrocyte against the number of erythrocytes subject to extraction in accordance with the method of

Example 5. Figure 1 measures the efficiency of erythrocyte extraction.

Figure 2 is a series of radioautographs of Western blotting used to detect anti-DAF in hybridoma culture supernatants.

Figure 3 is a series of radioautographs evidenc¬ ing presence of DAF in different cell types.

Figure 4 is a plot of the results of FACS analysis of erythrocytes of normal individuals and PNH patients.

Figure 5 is a plot of the results of FACS analy¬ sis of normal and PNH-patient platelets using anti-DAF.

Figure 6 is a plot of the results of FACS analy- sis of normal and PNH-patient leukocytes (monocytes, polymorphonuclear cells and lymphocytes).

Figure 7 is a plot of the results of FACS analy¬ sis of PNH-patient erythrocytes lysed with acidified serum. DETAILED DESCRIPTION OF THE INVENTION:

Erythrocyte DAF was initially purified (as des¬ cribed by Nicholson-Weller et al Proc. Nat'l Acad. Sci. 80:5066, 1983, incorporated by reference) by fractionation followed by anion exchange chromatography, hydrophobic affinity chromatography, gel filtration, and lectin affini¬ ty chromatography in that order. The thus purified product was further processed in accordance with the method of Iida et al (J. Exp. Med. 155:1427, 1982 — incorporated by reference) by immunoaffinity chromatography cellulose column using monoclonal antibodies to CR1 (obtained as des¬ cribed below) as the immunoadsorbent to remove contaminat¬ ing CR1. The effluent was again purified by anion-exchange chromatography followed by high performance liquid chroma¬ tography on a sizing column (TSK G 3000 SW from LKB, Bromma, Sweden) to remove remaining impurities, especially glycophorin. The final product was homogeneous by SDS-PAGE and Western blotting. This highly purified DAF was used as the antigen in the immunization of mice for monoclonal anti-DAF production Pure antigen is highly desirable for this purpose to avoid raising antibodies to the impurities of DAF preparations, some of which are much more antigenic than DAF itself.

Since the monoclonal antibodies to DAF have been produced by the present invention, however, the preferred method of purifying DAF will be by immunoaffinity chroma¬ tography using a cocktail of monoclonal anti-DAF as the immunoadsorbent. The DAF preparation subject to this im- munoaffinity chromatography is preferably previously purified by the anion exchange chromatography, as described above, if high purity is desired.

Hybridomas were fused from mouse myeloma cells and spleen lymphocytes of mice immmunized with DAF purified as above. The methods used in monoclonal antibody produc¬ tion are generally known: see, e.g., Goding, F.W., Mono¬ clonal Antibodies: Principles and Practice, Academic Press, Inc. (London 1983). An important modification of this method is described in detail in Examples 2 and 3, below. Briefly, the selection of positive clones was made by Western blotting using an impure preparation of DAF as the antigen. This permitted precise identification of the monoclonals reacting with the 70,000 Mr band. The monoclonal anti-DAF thus isolated were characterized and were found to be DAF-specific. Only a cocktail of these antibodies neutralized DAF activity quantitatively (98% — See Example 10). Specificity of these antibodies was confirmed both by inhibition of DAF activity (Example 10 and Table I) and by immunoblotting. The DAF content of other blood and lymph cells was determined as follows: Peripheral blood and tonsil cells were collected and separated, preferably as described in Example 4, but other known blood and lymph fractionation techniques could have been used. Erythrocyte, platelet, neutrophil, buffy-coat cell, monocyte, T-lymphocyte, and tonsil T- and B-lymphocyte preparations were thus obtained. Cell extracts were formed from each preparation as des¬ cribed in Example 5.

DAF was immunoprecipitated from these extracts using the labeled monoclonal anti-DAF previously prepared (Example 1). The procedure is described in Example 6 in detail. The presence of DAF in cell extracts was further demonstrated by a two-site immunoradiometric assay (Example 12). The surface expression of DAF by these different cell 0 types was measured by fluorescence activated cell sorter (FACS) analysis. The highest number of DAF molecules was found in netrophils, followed by monocytes and lymphocytes, if the method of determination was IRMA. By FACS analysis, monocytes had the highest amount of surface DAF. This may 5 indicate the presence of an internal pool of DAF in neutro- phils.

Most important, DAF purified from red cells spon¬ taneously reinserts into the membrane of these cells in vitro. This implies that DAF, or a domain of DAF (or a 0 peptide patterned after such a domain), may be used as a vehicle for targeting other molecules to cell membranes. This can be achieved in several ways: (a) by chemically conjugating the DAF domain, peptide or molecule to the molecule to be transported to the membrane; (b) by geneti- cally engineering a hybrid molecule containing the DAF domain that seeks the membrane; and (c) by synthesizing the membrane-seeking domain of DAF and coupling it to the target molecule.

The molecular weight of DAF varies among differ- 20 ent cell types, as determined by SDS-PAGE.

The amount of DAF present in erythrocytes of normal individuals was compared to that present in erythro¬ cytes of PNH patients by IRMA and by FACS analysis. All PNH patients had DAF-deficient erythrocytes. _,. Similarly, the platelets and leukocytes from PNH patients were found to be DAF-deficient. The results also indicate a correlation between the extent of DAF deficiency of patient cells and the severity of the disease. Therefore, DAF may be used as a marker for diagnosing and staging PNH. The number of DAF molecules per cell can be determined by any of the tech¬ niques described herein, i.e. immunoprecipitation, IRMA, or FACS analysis and compared to standards established for normal individuals and PNH patients.

In one of the PNH patients, SB, two populations of red cells, monocytes; neutrophils, and platelets were found in the peripheral blood. One population was pro¬ foundly deficient in surface DAF while the other appeared normal. The lymphocytes from this patient had a normal level of surface DAF. In two other patients (GC, VR) the expression of

DAF on the lymphocyte surface was also affected. In the patient with more severe desease (GC), PMN (polymorphonu- clear cells), monocytes and most red cells and lymphocytes were DAF-negative. Small amounts of DAF were detected in the blood elements of patient VR, suggesting that in this case the defect was caused by an abnormal regulation of a gene rather than by a non-functional or deleted gene.

These observations offer additional support for the existing concept that the PNH cells are of monoclonal origin, and arise from the clonal expansion of an abnormal bone marrow progenitor. In patient SB, the erythroid, myeloid as well as megakariocytic lineages were affected, indicating that a mutation had occurred in a common pre¬ cursor. In this patient, both normal and abnormal elements from the three lineages were found in circulation — about 3/4 of them DAF-deficient. This proportion remained un¬ changed in blood samples collected ten weeks apart, re¬ flecting a possible steady state between the proliferation of the abnormal and normal bone marrow precursors, and the removal from circulation of the end cells. Because in patients GC and VR the lymphocytes were also DAF-deficient. a more primitive cell giving rise to all blood elements was probably affected. Such totipotent stem cells have not been isolated or morphologically identified, but there is compelling evidence for their presence in postnatal bone marrow of mammals.

Although the sequence of events leading to the multiple abnormalities found in the membrane of PNH cells remains to be explained, the findings made possible by the present invention are relevant to the pathogenesis of the disease. The DAF defect can explain some of the character¬ istic features of PNH, such as the large accumulation of C3 zragments on the surface of erythrocytes, PMN, and platelets and the abnormal susceptibility of these cells to lysis by complement. Indeed, DAF inhibits the assembly of C3 con- 5 vertases and prevents C3b deposition on cell surfaces.

Fig. 7 presents direct evidence that DAF-deficient cells are preferentially lysed when subjected to acidified fresh serum (Ham test). However, the relationship between the C3b accumulation on leukocytes and platelets and the 0 observed thrombocytopenia, leukopenia, enhanced propensity for thrombosis and infection is not yet clear. Others have suggested that C3b deposition on platelets leads to their aggregation, release reaction and to enhanced clotting, and that C3b deposition on the PNH neutrophils results in a 5 chemotactic defect.

On the basis of current knowledge of DAF function, it is difficult to explain the heightened susceptibility of the cells from some PNH patients to the isolated C5b-9 attack complex. Further studies will be required to verify o whether this is secondary to the DAF-defect or whether an unrelated abnormality of the cell membrane is involved. Availability of anti-DAF and greater quantitites of DAF made possible by the present invention will provide useful tools in further investigation of the DAF origin and function. Another important application of antibodies to

DAF will be to enhance killing of cells (e.g. tumor cells) by complement. For example, one serious obstacle to the successful use of bone-marrow transplants is the presence in bone marrow of mature T lymphocytes, which can mediate graft-host reactions. These T cells can be killed with monoclonal antibodies to T-cell markers in the presence of complement. The treatment of the host bone marrow cells with anti-DAF is expected to facilitate T-cell killing and permit use of complement of human origin, even autologous complement.

Currently rabbit complement is used for this purpose and is effective, most likely because rabbit C3b and C4b are not inhibited by human DAF on the host bone marrow cells. However, use of human complement would be preferable.

Much interest is also currently focused on the use of antibody and complement to eliminate tumor cells from the bone marrow selectively, especially bone marrow that is to be used for autologous transplantation. Anti- DAF antibodies may facilitate the killing of these tumor cells in vitro when used in conjunction with an antibody specific to the tumor and complement. Anti-DAF would be coupled with anti-tumor specific antibody. The antibody conjugate would bind to the DAF on these tumor cells and make them more vulnerable to destruction by complement.

The invention is further described below by reference to specific examples, which are intended to illustrate the invention without limiting its scope.

The following abbreviations have the following meanings:

AZ, sodium azide; CR1 , C3b/C4b receptor; DAF, decay accelerating factor; E , human erythrocytes; EAC14-_j^_m, antibody-sensitized sheep erythrocytes bearing an excess amount of C1 and a limiting amount of C4; EAC14_lim(DAF) and EAC14_lim(Buffer) , EACl4_limcells sensitized with DAF or treated with buffer as a control; FACS, fluorescence-activated cell sorter; IRMA, immuno- radiometric assay; NP40, Nonidet P-40, a non-ionic de¬ tergent; IA10, IIH6 and VIIIA7, three anti-DAF monoclonal antibodies; PMSF, phenyl methyl sulfonyl fluoride; PNH, paroxysmal nocturnal hemoglobinuria; SDS, sodium dodecyl sulfate; Z, number of hemolytic sites per cell. Materials

*PBS: Dulbecco's phosphate buffered saline: from

GIBCO, Grand Island, New York. *DGVB ++: isotonic veronal buffer containing 2.47mM sodium veronal buffer (Fisher Scientific Co., Fair Lawn, N.J.) pH 7.3, 72.7mM NaCl, 2.5% dextrose, 0.1% gelatin, 0.15mM CaCl₂ and 0.5mM MgCl₂

*monoclonal anti-human CRI,57F were prepared as described by Iida, K. et al. J. Exp. Med. 155:1427 (1982) incorporated by reference. Briefly, antibodies were obtained from culture of a hybridoma prepared by fusing myeloma cells with spleen cells of mice immunized with CR1. Antibodies to CR1 are commercially available from Becton-Dickinson, Mountainview, California. *Rabbit anti-glycophorin A were prepared as described by Medof, M.E., et al., J. Exp. Med. 160:1558

(1984) incorporated by reference. Briefly, rabbits were immunized with pure glycophorin A and bled four weeks later. *guinea pig Cl was prepared as described by Nelson, R.A., et al. Immunochem. 3:111 (1966) incorpo¬ rated by reference. It is also available from Cordis Laboratories, Inc., Miami, Florida. *human C4 and C2 were prepared as described by Tack, B.F., et al. Meth. Enzymol. 80:64(1981) incorporated by reference. Alternatively, they can be obtained from Cordis, supra. *antibody-sensitized sheep erythrocytes earring 300 hemo- lytic sites (Z:300) of guinea pig C1 and one to two sites (Z:1-2) of human C4, (hereinafter referred to as EACl4_lim) were prepared as described by Medof, supra. Antibody-sensitized sheep erythrocytes are available from Cordis. These cells were incubated an appropriate amount of Cl for 15 minutes at 30°C, washed with DGVB , and then incubated with an appropriate amount of C4 for 20 minutes at 30^βC to give 300Z and 1-2Z of C1 and C4 respectively. o

EAC14, . cells were sensitized with DAF by incubating 5x10

EAC14,li.m cells/ml in DGVB⁺⁺ for 30 min at 37°C. These cells, hereinafter referred to as EAC14, ._(DAF) were washed twice with DGVB . As a control, EAC14-,. cells were treated in the same way with DGVB++ buffer alone and designated as EACl4_lim(Buffer) .

Example 1: Purification of Antigen (DAF)

DAF was purified from pooled human erythrocyte (E ) stroma. The fractionation sequence described by

Nicholson-Weller et al. J. Immunol. 129:184 (1982) and in Proc. Nat'l. Acad. Sci (USA) 80:5066(1983) — both incorpo¬ rated herein by reference, — was initially used.

E ^u ghosts from 4 units of blood were extracted with butanol and the resulting butanol-saturated water phase was subjected to successive chromatographies using DEAE-Sephacel, Phenyl-Sepharose (both from Pharmacia Fine Chemicals, Piscataway, NJ), hydroxyapatite Bio-gel HT (Bio-Rad Laboratories, Richmond, CA) and lentil-lectin- Sepharose (Pharmacia) all according to Nicholson-Weller, supra.

During purification, DAF was assayed by its ability to accelerate the decay of EAC142 as described by Nicholson-Weller, et al. J. Immunol 129:184 (1982). EAC142 o cells (100 microliters of a 1 x 10 cell/ml suspension) were incubated at 30^βC for 5 min. with 100 microliters of samples to be tested or DGVB as a control. Then 1.3 ml of C3-9 was added to the mixtures, followed by incubation at 37^βC for 60 min and centrifugation at 2,500 rpm for 5 min. The degree of cell lysis was determined by measuring the optical density (at 412 nm) of the released hemoglobin in the supernatant. The presence of DAF in the sample was detected and quantified according to how many fewer EAC142 cells were lysed in the samples compared to the controls incubated with DGVB . EAC14 cells were incubated with an appropriate amount of C2 for 5 minutes at 30°C to give 50% lysis in the control tubes.

The preparation of DAF obtained by this procedure was treated with monoclonal antibodies to CR1 (57F) coupled to Sepharose beads to remove contaminating CRl. Analysis of this preparation by sodium dodecyl sulfate (SDS)- polyacrylamide-gel-electrophoresis (PAGE) by the well-known method of Laemmli, U.K. Nature (Lond.) 227:680 (1970) and silver staining (Bio-Rad) showed the presence of several impurities, some of which were identified as glycophorins by Western blotting revealed with specific rabbit anti¬ bodies.

Western blotting was performed as follows: DAF partially purified as described above, was subjected to SDS-PAGE under nonreducing conditions, and was transblotted to a nitrocellulose paper. The paper was incubated with antiglycophorin antiserum and then with radiolabeled, affinity-purified goat anti-rabbit Ig from Becton-Dickinson. The strips were dried and subjected to autoradiography. DAF was further purified using DEAE-Sephacel

(Pharmacia) chromatography and high performance liquid chromatography through a TSKG 3000 SW gel-filtration matrix (LKB, Bromma, Sweden), as follows: The pool of impure DAF from the previous purification steps was dialyzed overnight against 0.01 M Na-phosphate buffer, pH 8.0, containing

0.05% Nonidet P-40 (NP40, a nonionic detergent from Sigma Chemical Co., St. Louis, Mo.) and applied to a 20 ml DEAE- Sephacel (Bio-Rad) column equilibrated in the same buffer. The column was eluted with NaCl/pH gradient formed with 50 ml of the starting buffer and 50 ml of 0.01M Na-phosphate buffer, pH 6.8 containing 0.3M NaCl and 0.05% NP40. The DAF band eluted at 8-10 mS, slightly ahead of the main glycophorin peak. Fractions containing DAF and only trace amounts of glycophorin were pooled, concentrated over an Amicon PM30 ultrafiltration device (Amicon Corp., Lexington, MA)and applied to TSKG 3000 SW equilibrated with 0.1%

NP40-PBS. Four discrete peaks were obtained, but only the first (void volume) contained DAF activity and a band of M 70,000 as evaluated by SDS-PAGE and silver staining. No glycophorin was detected in this preparation by Western blotting and the antigen was homogeneous by SDS-PAGE and W. blotting. Example 2: Production of Monoclonal Anti-DAF

Balb/c mice were immunized by intramuscular in¬ jection with 4 micrograms of purified DAF from Example 1 in complete Freund's adjuvant. The injection was repeated three weeks later. The mice whose sera had antibody titers of 1:200 or more by Western blotting were boosted intra¬ venously with 20 micrograms of pure DAF in 0.1% NP40-PBS. After 3 days, their spleen cells were fused with myeloma cells (X63Ag8.653 from American Type Culture Collection, Rockville, Md) . Culture supernatants of the resulting hybridomas were tested for anti-DAF activity as described in Example 3 below. Three positive monoclonal anti-bodies IA10 (subsequently characterized as IgG2a) , IIH6 and VIIIA7 (both subsequently characterized as IgGl) were thus identi¬ fied.

Monoclonal antibodies were purified from culture supernatants with Protein A-Sepharose (Pharmacia) by the method described by Ey et al. Immunochemistry 15:429 (1978). and labeled with 125I with Iodogen (Pierce Chemical Co.,

Rockland, IL) . ^• Briefly, the Ey method involves specific adsorp¬ tion of antibodies to Protein A-Sepharose (Pharmacia, Piscataway, N.J.) by incubating culture supernatants with the Sepharose beads at pH 8.6. Further incubation of the

^ beads in acetate buffered saline pH 4.3 causes elution of the antibody.

Larger amounts of purified antibodies were ob¬ tained from ascites of mice bearing the hybridoma with precipitation by 50% saturated ammonium sulfate, DEAE-

10 Sephacel chromatography and Sephadex G-200 gel-filtration. Example 3: Detection of Anti-DAF in Hybridoma Supernatants

Purified DAF from Example 1 (1 microgram/ml) was subjected to SDS-PAGE under non-reducing conditions on a 7.5% gel, and then transblotted onto nitrocellulose. The

15 paper was blocked with 5% BSA in PBS containing 0.02% sodium azide (AZ) (5% BSA-PBS-AZ) for 1 hr at 37°C, and cut into 5 x 50 mm strips. A piece of filter paper of the same size was placed onto each strip. 200 microliters of culture supernatants of hybridomas were applied to the

20 filter paper. Controls were conducted with culture super¬ natants from unrelated monoclonals, in HAT medium; immune or normal mouse serum was included. After incubation for 2 hr at room temperature in a humid chamber, the filter papers were removed and the strips were washed individually

25 three times with 5 ml each of 5% BSA-PBS-AZ. Groups of ten washed strips were incubated with 10 ml of 5% BSA-PBS-AZ containing 125I-labeled affinity-purified goat anti-mouse

7

IgG (Cappel Laboratories, West Chester, PA; 10 cpm) at room temperature for 1 hr, and washed three times with 10 30 ml each of 5% BSA-PBS-AZ. Autoradiography was performed by exposing the dried strips to Kodak X-Omat XAR-5 films (Eastman Kodak Co., Rochester, NY).

Example 4: Preparation of Peripheral Blood and Tonsil Cells 35 Citrated blood was centrifuged at 500 x g for 10 min at 4^βC. After removal of plasma and buffy coat, eryth¬ rocytes were washed three times with PBS. Blood samples from 35 normal individuals were obtained from the New York Blood Center (New York City) . Red cells from three indi¬ viduals were fractionated on the basis of their density by centrifugation into a Percoll (a colloidal suspension of silica particles that forms a density gradient upon centri¬ fugation; available from Pharmacia) gradient as described by Lutz, H.V. and Fehr, J., J. Biol. Chem. 254:11177 (1979). Briefly, 15 ml of red cells were mixed with 35 ml of Percoll solution and the mixture was centrifuged at 25,000 rpm for 23 min. The resulting red cell band was divided from top to bottom into 4 fractions of equal volume. The upper fractions contain lighter red cells.

Platelets were collected from platelet-rich plasma by centrifugation for 10 min at 2500 x g and washed three times with PBS containing 10 mM EDTA. Contaminating erythrocytes and leukocytes were removed by centrifugation at 200 x g for 10 min. PMN and mononuclear cells were separated from fresh citrated blood by centrifugation on Ficoll-Paque (a solution of synthetic polymers having a density of 1.077 g/ml; available from Pharmacia) followed by dextran sedimentation as described by Boyum, A. Scand J.Clin. Lab. Invest. 21, Suppl. 97:77 (1967). Human blood 30 ml was layered on 20 ml of Ficoll-Paque and centri¬ fuged at 1500 rpm (400 x g) for 40 min. Mononuclear cells were recovered from the top layer. The pellet containing PMN and red cells was suspended in 1.2% dextran and the suspension was kept for 1 hour to allow cells to sediment. Red cells sediment faster and PMNs were recovered from the top half of the suspension. The contaminating erythrocytres were removed by hypotonic shock.

Monocytes and lymphocytes were obtained from a Percoll gradient according to the method of Wright, S.D. and Silverstein, S.C. J. Exp. Med 156:1149 (1982) as described in detail in the first paragraph of this Example. More than 95% of the cells from the upper band were 0KM5 positive by indirect immunofluorescence staining (mono¬ cytes) . Lymphocytes were obtained from the lower band and contained less than 3% of contaminating monocytes.

Buffy coat cells were collected from the citrated blood and washed twice with PBS by centrifugation at 500 x g for 5 min. Contaminating erythrocytes were removed by hypotonic shock.

T-lymphocytes were obtained by staining blood mononuclear cells with phycoerythrin-conjugated monoclonal anti-Leu 1-positive cells (from Becton-Dickinson, Mountain View, LA) and sorting Leu 1-positive cells with a fluores¬ cence activated cell sorter (FACS, Cytofluorograf 50-H, Ortho Instruments, Westwood, MA).

Tonsil lymphocytes were purified from tonsils by centrifugation on Ficoll-Paque (Pharmacia). T- and B- lymphocytes were isolated as described by Werner, M.S. et al. Blood 42:939 (1973).

Example 5: Preparation of Cell Extracts:

Q

Erythrocytes: 5 x 10 packed, washed erythrocytes were lysed with 120 microliters of 1% NP40 in PBS containing 50 micrograms/ml of the synthetic elastase inhibitor. Sue (OMe)-Ala-Ala-Pro-Val-MCA (Peninsula Laboratories, Inc., San Carlos, CA) and ImM phenylmethyl sulfonyl fluoride (PMSF, Sigma Chemical Co., St. Louis, MO). After 1 hr of incubation at room temperature, 1 ml of 1% BSA-PBS contain¬ ing the same protease inhibitors was added and the mixture was centrifuged at 12,000 x g for 15 min. The supernatant was either used immediately or frozen at -70°C. The con¬ centration of erythrocytes was determined by measuring the ^0D54i °~~ hemolysed sample. The DAF contents obtained by the two-site immunoradiometric assay were multiplied by 1.25 since this procedure solubilized only 80% of extract- able DAF, as determined by the following experiment.

Increasing amounts of packed

to q 2.4 x 10 ) were extracted as above and the DAF contents of the extracts were determined by the two-site immunoradio- metric assay. The amounts of extracted DAF per E were plotted as a function of the number of E ^u initially treated with 1% NP40. Extracted DAF amounts increased slightly but progressively as the number of detergent- solubilized E ^u decreased. The maximum amount of ex- tractable DAF was obtained by extrapolation to zero Ehu used as shown in Fig. 1.

Q

Platelets: The platelet suspension (2 x 10 /ml in PBS) was mixed with the same volume of 1% NP40 in PBS containing 50 micrograms/ml of the elastase inhibitor, 1 M PMSF, 5 micro¬ grams/ml soy bean trypsin inhibitor (Sigma) and 100 units/ml Trasylol (Mobay Chemical Corp., New York, NY). The mixture was incubated for 20 min on ice and centrifuged at 12,000 x g for 15 min to remove the small amounts of remaining insoluble materials.

Q

Leukocytes: The leukocyte suspension (1 x 10 /ml in PBS) was mixed with the same volume of 1% NP40 in PBS containing the protease inhibitors. The mixture was incubated for 20 min on ice, centrifuged at 1500 x g for 15 min to remove intact nuclei and the supernatant was centrifuged at 12,000 x g for 15 min to remove insoluble materials. Example 6: Immunoprecipitation of DAF from Cell Extracts Protein A-Sepharose (100 microliters) was incu¬ bated with 5 ml of culture supernatant containing monoclonal antibody IA10 for 1 hr at room temperature. IAlO-Protein A-Sepharose was washed twice with PBS and then incubated with NP40 extracts of different kinds of cells (20 micro¬ liters of beads per 1 ml of the extract) for 1 hr at 4^βC. The bound DAF molecules were eluted from the beads by incubation for 5 min at 80°C with 50 microliters of sample buffer consisting of 5% SDS-125 mM Tris HCl, pH 6.8 - 10% glycerol - 0.01% Bromphenol blue. The eluates were sub¬ jected to SDS-PAGE using 7.5% gels and transferred electro- phoretically to nitrocellulose paper. DAF was detected iinn tthhee ppaappeerr bbyy 1¹2²5⁵II--llaabbeelleedd IA10, IIH6, or VIIIA7 followed by radioautography. In an alternative procedure, erythrocytes, mononuclear cells and PMN were surface-labeled with 125I using Iodogen (from Pierce Chemical Company, Rockford, IL;

1 mCi Na¹²⁵I per 2 x 10⁸ E^hu or per 1 x 10⁷ leukocytes).

NP40 extracts were prepared and DAF was immunoprecipitated with IAlO or nonrelevant antibodies as a control. The DAF band was analyzed by SDS-PAGE under both reducing and non-reducing conditions followed by radioautography.

Example 7: Two-site Immuno-Radiometric Assay (IRMA) for DAF in Cell Extracts

The wells of plastic plates (96 U-bottom wells: Becton-Dickinson, Oxnard, CA) were coated with anti-DAF monoclonal IAlO (capturing antibody ) by incubation with 50 microliters of 20 micrograms/ml IAlO in PBS containing 0.02% sodium azide (AZ) at room temperature for 2 hr. The wells were filled with 1% BSA - PBS - 0.02%AZ and kept at 4°C overnight to saturate excess binding sites. The wells were washed three times with 1% BSA - 0.05% Tween 20 - PBS - 0.02% AZ (Tween 20 was from ICI Americas, Wilmington, Dela.) and then 25 microliters of cell extracts or serial dilutions of pure DAF in 1% BSA - 0.05% Tween 20 - PBS - 0.02% AZ were added in duplicate. After incubation for

2 hr at room temperature and three washes with the same buffer, 25 microliters of 125I-labeled anti-DAF mono- clonal IIH6 (revealing antibody, 4 ng, 1.2 x 10 cpm) were added to each well. After incubation for 1 hr at room temperature, the wells were washed four times with the same buffer, cut and counted. The amounts of DAF in the cell extracts were calculated from a standard curve obtained with purified DAF. This standard curve, in which the counts of bound revealing antibody were plotted as a func¬ tion of DAF concentration, was linear up to 250 ng DAF per ml. The protein concentration of the pure DAF solution was measured by the well-known method of Lowry et al. J. Biol. Chem. 193:265 (1951), incorporated by reference, using BSA as a reference protein. Example 8: Analysis of Cell Surface DAF by Fluorescence

Activated Cell Sorter (FACS)

Erythrocytes, platelets or buffy coat cells from normal individuals and PNH patients were treated with anti- DAF monoclonal antibodies and fluorescein-conjugated goat

F(ab' )- anti-mouse Ig (H+L) (Cappel Laboratories), and then analyzed by FACS. Erythrocytes (10 6 cells in 25 micro- lites of 1% BSA - PBS - 0.1% AZ) were treated with 25 micro¬ liters of a mixture of three monoclonal antibodies against DAF (5 micrograms of each antibody/ml of medium containing 1% BSA- PBS - 0.1% AZ) or with a mixture of non-relevant monoclonal antibodies of the same subclass as a control. After incubation for 30 min in ice, the red cells were washed twice, resuspended in 25 microliters of the same buffer, incubated with 25 microliters of 1:50 dilution of the second antibody for 30 min in ice, and washed again g with the same buffer. Buffy coat cells (10 cells in 25 microliters of the same buffer) were incubated with 25 microliters of 5 micrograms/ml of IA10 (or control mono- clonal antibodies) for 30 min in ice. This was followed by washing and incubating with the second antibody as above. Platelets were stained in the same way as the red cells, except that 10 mM EDTA was added to the buffer. Example 9: Isolation of DAF-Positive and DAF-Negative Erythrocytes from PNH Patients

Washed erythrocytes from PNH patients were incu¬ bated with 60% acidified human serum (10 E %1) for 30 min at 37^βC. Unlysed erythrocytes were pelleted and washed twice with PBS. DAF-negative cells were selectively lysed by this procedure.

Erythrocytes from PNH patients (10⁸E^hu/ml in PBS - 0.02% AZ) were mixed with an equal volume of 10 micrograms/ml IA10 anti-DAF. After incubation for 60 min on ice, cells were washed once with PBS - 0.02% AZ, resus- pended at 10 E ^u/ml and then loaded to a Protein A-Sepharose CL-4B column (2 x 10 E /ml packed gel) equilibrated in the same buffer. The column was closed and kept for 15 min at room temperature. DAF-negative cells (unbound cells) were collected by washing the column with five column volumes of the same buffer. More than 90% of cells from this fraction were DAF-negative as assessed by FACS analysis. Patients

Three patients with PNH at the NYU Medical Center were studied. Patient GC, a 46 year old Hispanic female, had severe disease of 17 years duration. She was receiv¬ ing norandralone, prednisone, and transfusions of frozen- thawed erythrocytes. Patient SB, a 47 year old Caucasian female, had milder disease of 4 years duration. She was receiving no medications and maintained a stable hematocrit of about 38 vol %, which during episodes of hemolysis de¬ creased to 24%. Patient VR, a 46 year old Hispanic female, presented with pancytopenia, persistent thrombocytopenia

^•5

(platelets 20,000/mm ), and intermittent anemia. She received norandralone and prednisone. All patients had a positive acid serum (Ham) test and sucrose hemolysis test. Example 10:

Characterization of anti-DAF Monoclonal

Antibodies IA10, IIH6 and VIIIA7

The specificity of the Anti-DAF monoclonal anti- bodies was shown by Western blotting experiments using as the antigen either purified DAF or total extract of mem- branes of E (Fig. 2). In the experiments used to generate Fig. 2A, culture supernatants of the anti-DAF hybridomas were incubated with nitrocellulose strips previously blotted with pure DAF that had been subjected to SDS-PAGE under nonreducing conditions, as described in Example 3, above. Each paper strip containing 10ng of DAF, was incubated for 2 hrs at room temperature with

200 microliters of hybridoma culture supernatants. After washing, the strips were incubated with 125I-labeled goat anti-mouse IgG, washed and exposed to autoradiography. All anti-DAF antibodies (IA10 — in lane 1; IIH6 — in lane 2; and VIIIA7 — in lane 3) detected the DAF band. This was not so for HAT medium (lane 4) or monoclonal 57F antibodies to CR1 (lane 5). When the nitrocellulose strips were blotted with membrane extract of E ^u, the results were identical (Fig. 2B). In this experiment, washed E ^u (5 x

Q

10 cells) were lysed with 15mM Tris-HCl buffer, pH 7.8. Erythrocyte ghosts were collected by centrifugation, as described above, and the ghosts were solubilized with nonreducing SDS sample buffer by boiling, and subjected to SDS-PAGE in a 7.5% gel followed by Western blotting.

Each nitrocellulose strip (containing total mem¬ brane proteins derived from 3 x 10 E ) was incubated with culture supernatant from hybridomas IA10 (lane 1), IIH6 (lane 2), VIIIA7 (lane 3) and 57F anti-CRl (lane 5); or with HAT medium alone (lane 4). This was followed by incubation with the second labeled antibody — as described with reference to Figure 2A, above. The strips were then exposed to radioautography. Anti-DAF antibodies detected one band with a molecular weight of 70,000. Control anti-CR1 detected two types of CR1 , but not DAF.

The solid phase two-site IRMA described in Example 7 could be performed with any combination of two different monoclonal antibodies, while negative results were obtained if a single antibody was used as the captur¬ ing and revealing reagent. This means that IA10, IIH6 and VIIIA7 recognized different epitopes on the DAF molecule. The effect of the monoclonal antibodies on the activity of DAF was tested using DAF-treated EAC14, . cells (Table I). Antibody IAlO, IIH6, or VIIIA7 alone had no or only a slight effect on DAF activity. However, when combinations of these antibodies were used, stronger effects were observed, except with the combination of IA10 and VIIIA7. A mixture of three monoclonal antibodies at concentrations of 9 micrograms/ml inhibited 98% of DAF activity. These results suggest that the binding sites of these antibodies do not coincide with the active site of the DAF molecule, and these antibodies do not neutralize DAF. A mixture of these antibodies is therefore suitable for purification of DAF using immunoaffinity chromatography. Example 11 :

Demonstration of the Presence of DAF on Different Cell Populations by Immunoprecipitation

NP40 extracts of platelets, PMN, blood mononu¬ clear cells, tonsil mononuclear cells, tonsil B- and T-lymphocyte fractions, and a solution of pure erythrocyte DAF (1ml each) were incubated with IAlO-bearing Protein A-Sepharose beads (20 microliters of beads) for 1 hr at 4°C. DAF was eluted from the beads (by boiling the beads in nonreducing SDS sample buffer) and subjected to SDS-PAGE followed by Western blotting as described in Example 6.

All the cell types contained DAF as demonstrated in Fig. 3.

DAF bands were detected by incubating the nitro- cellulose paper with 125i-labeled IA10 (107cpm) for 1 hr at room temperature, followed' by washing and radioauto- graphy. As shown in Fig. 3, lanes 1 and 8 contain pure DAF from erythrocytes. Lanes 2-7 contain different cell ex¬ tracts: blood mononuclear cells (lane 2); tonsil mono¬ nuclear cells (lane 3); tonsil B-lymphocytes (lane 4); tonsil T-lymphocytes (lane 5); polymorphonuclear leucocytes (lane 6); and platelets (lane 7). DAF could be detected by ¹²⁵I-labeled IIH6 or VIIIA7 (not shown) as well as IA10. The M of the DAF band was different among cell types (Fig. 3). DAF from platelets and PMN appeared larger (by about 5000 daltons) than DAF from erythrocytes while DAF from mononuclear cells was of an intermediate size. The size difference was also shown by immunoprecipitation of DAF from surface-labeled cells, followed by SDS-PAGE under both reducing and non-reducing conditions (not shown). Example 12:

Quantitation of DAF in Different Cell

Types From Normal Individuals

DAF contents were measured in NP40 extracts of cells using a two-site immunoradiometric assay, performed as described in Example 7. As shown in Table II, erythro¬ cytes from normal individuals had a mean of (3.3 + 0.4) x 10 molecules per cell, ranging between 4.0 and 2.1 x 10 . Platelets and polymorphonuclear cells had (2.1 +_

0.3) x 10³ and (8.5 + 1.5) x 10⁴ molecules per cell, respectively. The unseparated mononuclear cells and purified monocytes had (3.6 + 0.44)x10⁴, and (6.8 + 1.50)

4 x 10 molecules per cell, respectively. Lymphocytes, from peripheral blood had (3.3 + 0.97) x 10 molecules per cell. Purified T-lymphocytes from the peripheral blood of one normal individual had 9.0 x 10 molecules per cell. Unseparated mononuclear cells from a tonsil had

4.2 x 10 molecules per cell. Purified B- and T-lympho- cytes from this tonsil had 5.4 x 10 4 and 1.7 x 104 molecules per cell, respectively.

The surface expression of DAF on different cell types from peripheral blood was studied by indirect i muno- fluorescent staining followed by FACS analysis. Erythro- cytes and platelets were stained with a mixture of the three monoclonal antibodies and FITC-goat F(ab' )₂ anti- mouse IgG (solid lines in Figs. 4 and 5) to increase the fluorescence intensity. Buffy coat cells were stained only with IA10 anti-DAF followed by FITC-goat F(ab')₂ anti- mouse IgG (Fig. 6). These immunoglobulins are available from Cappel Laboratories, Cochranville, Pa. Polymorphonu¬ clear cells, monocytes and lymphocytes were separated according to their light-scattering properties and analyzed separately. Fig. 4A shows the results of analysis of erythro¬ cytes from normal individuals. Fig. 4B shows the results from the three PNH patients. The normal results are similar to those produced in similar tests using cells from yet other normal individuals (not shown). By contrast, the three PNH patients had two different populations of erythrocytes. The dashed lines in both Figs. 4A and 4B represent control staining. Figure 5A shows the results of stained platelet analysis from a normal individual and Figure 5B shows the results with cells of a PNH patient. Again, the PNH patient had two populations of platelets. The dashed lines represent control staining.

Figure 6A shows the results of analysis of stained of leukocytes from a normal individual and Figure 6B shows the results with cells of a PNH patient. The dashed lines represent control staining. As shown in Figs. 4A, 5A and 6A, all the cell types expressed surface DAF. The fluorescence intensity appeared normally distributed in all cells, except in red cells in which the distribution of DAF was very skewed in all individuals examined. For example, the mean relative fluorescence intensity of the sample shown in Fig. 4A was 120, but a sizable proportion of the red cells showed intensities above 300. In three individuals, the red cells were separated on the basis of density in four fractions by centrifugation into a Percoll gradient. We found that DAF expression decreased with red cell density, and that the lightest cells had 19.4 +_ 0.2% higher levels of DAF than the heaviest cells. However, the skewed DAF distribution was still observed in both fractions (results not shown) . By FACS analysis, monocytes showed the highest

DAF surface expression (mean fluorescent intensity = 756). Although, among extracts of blood cells, PMN had the highest amount of DAF (Table II), the surface expression of DAF in PMN (mean fluorescent intensity = 212) was much lower than that of monocytes. Lymphocytes appeared as two distinct populations. The major population, about 65% of the total lymphocytes, expressed less surface DAF (mean fluorescent intensity = 109) than the minor population (mean fluorescent intensity = 349) (Fig. 6A) . When the buffy coat cells were simultaneously stained with phyco¬ erythrin-conjugated anti-Leu 1 (a pan T reagent) and with anti-DAF, more than 90% of the lymphocytes with high DAF expression were Leu-1 negative (not shown). This is in agreement with the IRMA results described in this Example, which showed that an extract of tonsil B cells had higher levels of DAF than an extract of T cells, and that T lymphocytes purified by cell sorting had a very low DAF content.

Example 13:

Quantitation of DAF in Erythrocytes from Patients with PNH DAF levels in extracts of erythrocytes from three

PNH patients were measured with the two-site immunoradio- metric assay of Example 12 and found to be much lower than normal (1,100; 1,000; and 900 molecules per cell, respec¬ tively). The results are shown in Table II. To obtain information about the pattern of surface expression of DAF, the erythrocytes from the same patients were studied by FACS, as in Example 12. In patients SB and VR who had no blood transfusions in the previous six months, (Figs. 4B and 4D), two populations of red cells were detected. One population was DAF-deficient and constituted about 60% of the total in patient SB, and 30% in patient VR. The second population of red cells was positive and contained normal levels of DAF in patient SB but low levels in patient VR. Analysis of the red cells of the third patient was complicated by the fact that she had frequent hemolytic crises and received transfusions. The FACS pattern again revealed two populations of cells, one DAF-negative (49%) and the other bearing lower than normal DAF levels (Fig. 4C). To examine the possibility that the DAF-containing cells originated at least in part from the blood transfusions, we separated the two popula¬ tions by affinity chromatography and acid serum lysis as in Example 8 and determined their reticulocyte contents. Although the unfractionated red cells contained 10% reticu- locytes, the DAF-negative and DAF-positive cells contained 17% and 2% reticulocytes, respectively. In contrast, when the red cells from the nontransfused patients were frac¬ tionated by the same methodology, the reticulocyte content of the unseparated cells and that of the DAF-positive cells were not significantly different (2.1% versus 1.6% in patient SB, and 0.9% versus 0.6% in patient VR) . It appears, therefore, that most or all of patient GC's own red cells were DAF-negative. To confirm this, blood typing was performed on both the DAF-negative and the DAF-positive population of patient GC. The DAF-negative population was devoid of antigens E, N, Kell and Kp^a, while the same antigens were present in the populations of DAF-positive cells.

When SB, GC and VR erythrocytes were subjected to the Ham test, 54%, 39% and 30%, respectively, were lysed by complement. In order to find out which cell population was complement-sensitive, the patients' erythrocytes were first subjected to acidified-serum lysis and the remaining cells then tested for DAF expression by FACS.

Erythrocytes (10 cells) from the patients SB (A-C in Fig. 7) and GC (D - F in Fig. 7), were incubated with 100 microliters of buffer (A,D in Fig. 7), 30% acidi¬ fied serum (B and E in Fig. 7) or 60% acidified serum (C and F in Fig. 7) for 30 min. at 37°C. Unlysed cells were collected by centrifugation, washed three times with PBS, stained with monoclonal antibodies to DAF or with control antibodies (as described in Example 12) and then analyzed by FACS. The solid lines in Fig. 7 represent anti-DAF stained cells. As shown in Fig. 7, the acidified-serum lysed preferentially the DAF-negative cells. In sum, the red cells from these patients expressed widely different amounts of DAF. In two of them (SB and VR) only part of the red cells were DAF-negative and in the third (GC), with the most severe hemolytic episodes, most or all endogenous cells were DAF-negative. In every instance, only the DAF-negative population was lysed by complement in the Ham test. Hence, the role of DAF as protecting red cells from lysis is confirmed. In addition, the DAF content of the patients' red cells decreases with the advance of the disease. It can there¬ fore be used as a marker for the diagnosis and staging of PNH.

Example 14: DAF-Deficienσy in Platelets and Leukocytes from PNH Patients

The DAF surface expression in platelets and leukocytes from the same patients were studied by FACS analysis (Figs. 5B, 6B-D). In patient SB (who had circu¬ lating both DAF-negative and DAF-positive red cells) neutrophils, monocytes as well as platelets showed two distinct populations, one with normal amounts of DAF (23, 20 and 22% of each cell type) and the other DAF-negative. DAF expression on lymphocytes appeared normal (Figs. 5B, 6B) . The patients GC and VR differed from SB in two significant respects: presence of single populations of DAF-deficient neutrophils and monocytes in the peripheral blood, and presence of a DAF-defective lymphocyte popula- tion (Figs. 6C, 6D). Small amounts of DAF were detected in the monocytes and neutrophils from patient VR, and in the lymphocytes from both VR and GC. DAF from lymphocytes and red cells of patient VR was immunoprecipitated as in Example 6 and examined by Western blotting, as in Example 11. DAF bands were detected with M identical to those of DAF from normal individuals (not shown).

DAF levels in extracts of platelets from two PNH patients (SB and GC) were measured with the IRMA as in Example 12 (Table II). Consistent with the results of the FACS analysis, the platelets from patient SB contained about 30% of the normal level of DAF. No DAF was detected in the platelets of patient GC.

Paper Example 15: Purification of DAF from Cell Extracts

Using Monoclonal Anti-DAF DAF is extracted from packed red cells with 0.1%

Nonidet P40 (NP40) in phosphate-buffered saline. The extract is centrifuged at 10,000 x g to remove insoluble materials. The extract is incubated for 60 min. with Sepharose beads coupled to a mixture of monoclonal anti-DAF. The beads are washed until the O.D. of the supernatant is below 0.020 at 280 nm. DAF is eluted with 0.1M triethyla- mine in 0.01% NP40 and filtered through Sephadex G - 25 equilibrated with PBS - 0.01% NP40. The eluate is sub¬ jected to SDS-PAGE and Western blotting and found to be homogeneous by both tests. Paper Example 16

Cloning of DAF

Purified DAF from Example 15 is subjected to microsequencing to obtain the sequence of 20-30 amino acids at the N-terminal in accordance with the method of Henrick, et al. J. Biol. Chem. 256:7990 (1981), incorporated by reference. DAF is also subjected to cleavage by cyanogen bromide or trypsin. Fragments are isolated by high per¬ formance liquid chromatography, and sequenced as above. Sequence data are used to synthesize a mixed deoxynucleo- tide probe as described by Duckworth, M.L. et al Nucleic Acid Res. 9:1981 (1981), incorporated by reference. The probe is used to screen a human DNA library from B lympho¬ cytes and neutrophils. Positive clones are expanded in culture and plasmids purified. The above fragments are excised and sequenced, as described by Sanger, et al.

Proc. Nat'l Acad. Sci. 74:5163 (1977). The sequence of the whole DAF protein is thus revealed.

TABLE I

Effect of the monoclonal antibodies to DAF

* on the inhibitory activity of DAF on C4b hemolytic sites

Concentration of Monoclonal Antibodies % reversal of, IA10 IIH6 VIIIA7 DAF activity

Micrograms/ml

7 , .0

27 , .5

0

1 1 44 , .6

3 3 70 , .3

9 9 78 , .6

1 — 1 1 1 , .7

3 - 3 1 5 , .7

9 — 9 1 8 , .3

— 1 1 29 , .9

- 3 3 56 , .4

— 9 9 72. .0

1 1 1 55 . .8

3 3 3 84 . .5

9 9 9 98 . .0

—'100 microliters of 1 x 10⁸/ml EACl4_lim(DAF) in DGVB⁺⁺ were incubated at 30°C for 15 min with 100 microliters of various combinations of monoclonal antibodies to DAF or DGVB⁺⁺ as controls. EACl4_lim(Buffer) cells were treated with DGVB in the same way, to measure input C4b hemolytic sites. After incubation, the cells were washed once with DGVB and resuspended in 100 microliteas of DGVB . Then the C4b hemolytic sites were developed with C2 followed by C3-9 as described in Medof, M.E. et al, J. Exp. Med. 160:1558 (1984)). The cells were incubated with 150 microliters of C2 at 30°C for 5 min. C3-9 (1.3 ml) was then added and the mixture again incu¬ bated at 37^βC for 60 min, and then centrifuged at 2,500 rpm for 5 min. Degree of lysis was determined by 0D^₁₂ of released hemoglobin in the supernatant. —+ Percent reversal of DAF activity was calcu- lated as f • ooll1*ll1oowwssM :: Z[of anti-DAF treated EAC14_lljn(DAF)] - Z[of EAC14_lin(DAF)]

-x100

Z[of EACl4_lim(Buffer)]- Z[of EACl4_lim(DAF) ] where Z is the number of hemolytic sites per cell. The Z of EACl4_lim( Buffer) and EACl4_lim(DAF) in this experi¬ ment were 2.480 and 0.700, respectively.

TABLE II Distribution of DAF among different cell types from the peripheral blood or normal individuals and PNH patients

_3

Cells DAF Molecules (x 10 ) per cell

+ standard deviation

Normal Erythrocytes 3.3 + 0.4 (35)*

Platelets 2.1 + 0.3 (4)*

PMN 85.0 + 15.0 (4)*

Mononuclear, total 35.7 +_ 4.4 (3)*

Monocytes 67.9 + 15.0 (3)*

Lymphocytes 32.8 + 9.7 (3)*

PNH Erythrocytes

(SB) 1.1

(GC) 1.0

(VR) 0.9 Platelets

(SB) 0.6

(GC) not detectable

*Number of samples examined. TABLE III Distr ibution of DAF in cells from peripheral blood of PNH patients

Expression of DAF in PNH Patients :

Cell type Patient SB Patient GC Patient VR

Erythrocytes 60% undetectable 40% undetectable^ 30% undetectable 40% normal 60% below normal 70% low levels

Platelets 78% undetectable undetectable not determined 22% normal (by IRMA)

Monocytes 80% undetectable undetectable very low 20% normal

PMN 77% undetectable undetectable very low 23% normal

Lymphocytes normal very low or very low undetectable

Most or all cells in this population are transfused.

Claims

What is claimed is:

1. Monoclonal antibody immunochemically reactive with decay accelerating factor.

2. The antibody of claim 1 wherein said factor is human decay accelerating factor.

3. The antibody of claim 2, said antibody being of an isotype selected from the group consisting of IgG2a and IgG1.

4. The antibody of claim 2, said antibody having capacity to bind said decay accelerating factor without neutralizing it.

5. A method for making a monoclonal antibody immuno¬ chemically reactive with decay accelerating factor, said method comprising: providing decay accelerating factor purified to homogeneity as tested by sodium dodecyl sulfate polyacrylamide gel electrophoresis and Western blotting; immunizing mice with said purified factor; fusing spleen cells from said mice with mouse myeloma cells to produce hybrid cells; and screening said hybrid cells to identify those secreting said monoclonal antibody.

6. The method of claim 5 wherein a preparation of partially purified decay accelerating factor is used as a screening agent for screening said hybrid cells.

7. The method of claim 6 wherein the purification of said factor comprises: providing cells comprising said factor; extracting said factor from said cells; subjecting said extract to anion-exchange column chromatography, hydrophobic interaction chromatography, gel filtration and lectin-Sepharose chromatography to obtain a partially purified preparation of said factor; subjecting said preparation to immunoaffinity chromatography using an antibody to CR1 as the immunoadsorbent; subjecting the effluent of said immunoaffinity chromatography to anion exchange column chromatography and eluting said factor using a salt gradient; and further purifying said factor by high performance liquid chromatography whereby the thus purified factor is homo¬ geneous as tested by both sodium dodecyl sulfate polyacrylamide extracting said factor from said cells; subjecting said extract to anion-exchange column chromatography, hydrophobic interaction chromatography, gel filtration and lectin-Sepharose chromatography to obtain a partially purified preparation of said factor; subjecting said preparation to immunoaffinity chromatography using an antibody to CR1 as the immunoadsorbent; subjecting the effluent of said immunoaffinity chromatography to anion exchange column chromatography and eluting said factor using a salt gradient; and further purifying said factor by high performance liquid chromatography whereby the thus purified factor is homo¬ geneous as tested by both sodium dodecyl sulfate polyacrylamide gel electrophoresis and Western blotting.

8. A method of purifying decay accelerating factor from impure preparations thereof comprising: loading an impure, cell-free preparation of said factor to an immunoaffinity chromatography column using, as the immunoadsorbent, a monoclonal antibody to said factor said anti¬ body being capable of reversing binding to said factor; washing said column to remove contaminating impurities while maintaining said factor bound to the column; recovering said purified factor from the column.

9. A method according to claim 8 wherein more than one monoclonal antibody to said factor that binds to said factor is used as the immunoadsorbent.

10. A method according to claim 9, wherein a cocktail of three monoclonal antibodies to said DAF is used as the im¬ munoadsorbent.

11. A method according to claim 8 wherein prior to said immunoaffinity step said preparation of said factor is partially purified by anion-exchange chromatography.

12. A method according to claim 8, wherein said cell- free preparation of said factor is an extract of said factor from red blood cells.

13. A method for enhancing the lysis by complement of cells having surface decay accelerating factor, said method comprising: exposing said cells to a first antibody against a cell surface antigen found in a portion of said cells; exposing said cells to a pool of monoclonal antibodies to decay accelerating factor, whereby said antibodies bind to decay accelerating factor on the cell surface thus rendering said cells vulnerable to lysis by said complement and said first antibody; exposing said cells to the presence of complement; and waiting for said complement to lyse that portion of said cells which bound said first antibody.

14. The method of claim 13, comprising exposing selected cells to the presence of said first antibody.

15. The method of claim 14 comprising exposing said cells to the presence of conjugates of said antibodies to decay accelerating factor and said first antibody.

16. The method of claim 15, wherein said cells are tumor cells.

17. The method for enhancing the resistance of cells to attach by complement, said method comprising the incubation of said cells with purified decay acclerating fac tor _in vitro.