Classifications

• • B—PERFORMING OPERATIONS; TRANSPORTING
  • B82—NANOTECHNOLOGY
  • B82Y—SPECIFIC USES OR APPLICATIONS OF NANOSTRUCTURES; MEASUREMENT OR ANALYSIS OF NANOSTRUCTURES; MANUFACTURE OR TREATMENT OF NANOSTRUCTURES
  • B82Y5/00—Nanobiotechnology or nanomedicine, e.g. protein engineering or drug delivery
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
  • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
  • C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
  • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
  • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
  • C07K16/2896—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against molecules with a "CD"-designation, not provided for elsewhere
• • G—PHYSICS
  • G01—MEASURING; TESTING
  • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
  • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
  • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
  • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
  • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K2319/00—Fusion polypeptide
  • C07K2319/01—Fusion polypeptide containing a localisation/targetting motif
  • C07K2319/035—Fusion polypeptide containing a localisation/targetting motif containing a signal for targeting to the external surface of a cell, e.g. to the outer membrane of Gram negative bacteria, GPI- anchored eukaryote proteins
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K2319/00—Fusion polypeptide
  • C07K2319/40—Fusion polypeptide containing a tag for immunodetection, or an epitope for immunisation
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K2319/00—Fusion polypeptide
  • C07K2319/70—Fusion polypeptide containing domain for protein-protein interaction
  • C07K2319/74—Fusion polypeptide containing domain for protein-protein interaction containing a fusion for binding to a cell surface receptor
  • C07K2319/75—Fusion polypeptide containing domain for protein-protein interaction containing a fusion for binding to a cell surface receptor containing a fusion for activation of a cell surface receptor, e.g. thrombopoeitin, NPY and other peptide hormones
• • G—PHYSICS
  • G01—MEASURING; TESTING
  • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
  • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
  • G01N2333/435—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans
  • G01N2333/705—Assays involving receptors, cell surface antigens or cell surface determinants

Definitions

• Decay accelerating factor 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- ⁇ onvertases (C4b2a and C3bBb) and C5-convertases (C4b2a3b and C3bBb3b) : Medof, M.E., et al., J. Exp. Med. 160:1558 (November, 1984).
• 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.
• 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.
• Yet another object is to provide a method for the diagnosis and staging of PNH by using monoclonal antibodies to DAF.
• 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 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.
• 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.
• 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.
• Example 10 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.
• FACS fluorescence activated cell sorter
• DAF purified from red cells spon ⁇ taneously reinserts into the membrane of these cells in vitro.
• DAF or a domain of DAF (or a 0 peptide patterned after such a domain)
• DAF 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.
• 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.
• DAF on the lymphocyte surface was also affected.
• 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.
• 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).
• Ham test acidified fresh serum
• DAF will be to enhance killing of cells (e.g. tumor cells) by complement.
• cells e.g. tumor cells
• 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.
• 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.
• use of human complement would be preferable.
• 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.
• EAC14 lim (DAF) and EAC14 lim (Buffer) EACl4 lim cells 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
• EACl4 lim 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 ⁇ C to give 300Z and 1-2Z of C1 and C4 respectively.
• DAF was purified from pooled human erythrocyte (E ) stroma. The fractionation sequence described by
• 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.
• EAC142 o cells 100 microliters of a 1 x 10 cell/ml suspension
• EAC142 o cells 100 microliters of a 1 x 10 cell/ml suspension
• DGVB DGVB
• 1.3 ml of C3-9 was added to the mixtures, followed by incubation at 37 ⁇ C 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.
• 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.
• 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
• Nonidet P-40 a nonionic detergent from Sigma Chemical Co., St. Louis, Mo.
• NP40 a nonionic detergent from Sigma Chemical Co., St. Louis, Mo.
• 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%
• 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.
• IA10 subsequently characterized as IgG2a
• IIH6 and VIIIA7 both subsequently characterized as IgGl
• 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.,
• 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 Protein A-Sepharose (Pharmacia, Piscataway, N.J.) by incubating culture supernatants with the Sepharose beads at pH 8.6. Further incubation of the
• Example 4 Preparation of Peripheral Blood and Tonsil Cells 35 Citrated blood was centrifuged at 500 x g for 10 min at 4 ⁇ C. 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).
• Percoll a colloidal suspension of silica particles that forms a density gradient upon centri ⁇ fugation; available from Pharmacia
• 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).
• Ficoll-Paque a solution of synthetic polymers having a density of 1.077 g/ml; available from Pharmacia
• 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).
• 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 Pieris-Bassham
• PMSF ImM phenylmethyl sulfonyl fluoride
• the supernatant was either used immediately or frozen at -70°C.
• the con ⁇ centration of erythrocytes was determined by measuring the 0D 54i ° ⁇ 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.
• 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.
• 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 ⁇ C.
• 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 1 1 2 2 5 5 II--llaabbeelleedd IA10, IIH6, or VIIIA7 followed by radioautography.
• erythrocytes, mononuclear cells and PMN were surface-labeled with 125I using Iodogen (from Pierce Chemical Company, Rockford, IL;
• 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.
• 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.
• 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.
• Washed erythrocytes from PNH patients were incu ⁇ bated with 60% acidified human serum (10 E %1) for 30 min at 37 ⁇ C. Unlysed erythrocytes were pelleted and washed twice with PBS. DAF-negative cells were selectively lysed by this procedure.
• 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
• 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).
• 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
• 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.
• 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.
• 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.
• 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 125 I-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
• DAF contents were measured in NP40 extracts of cells using a two-site immunoradiometric assay, performed as described in Example 7.
• 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 +_
• the unseparated mononuclear cells and purified monocytes had (3.6 + 0.44)x10 4 , and (6.8 + 1.50)
• 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.
• 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.
• 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.
• 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.
• the red cells were separated on the basis of density in four fractions by centrifugation into a Percoll gradient.
• 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.
• 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.
• 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.
• the acidified-serum lysed preferentially the DAF-negative cells.
• the red cells from these patients expressed widely different amounts of DAF.
• 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.
• GC third
• DAF-negative only the DAF-negative population was lysed by complement in the Ham test.
• the role of DAF as protecting red cells from lysis is confirmed.
• 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-Deficien ⁇ y in Platelets and Leukocytes from PNH Patients
• DAF 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 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.
• Nonidet P40 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
• DAF 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.
• 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)).
• Lymphocytes normal very low or very low undetectable

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