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WO1995006066A1 - Anticorps specifiques contre le regulateur de la conductance transmembranaire de la mucoviscidose et leurs utilisations - Google Patents

Anticorps specifiques contre le regulateur de la conductance transmembranaire de la mucoviscidose et leurs utilisations Download PDF

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Publication number
WO1995006066A1
WO1995006066A1 PCT/US1994/008970 US9408970W WO9506066A1 WO 1995006066 A1 WO1995006066 A1 WO 1995006066A1 US 9408970 W US9408970 W US 9408970W WO 9506066 A1 WO9506066 A1 WO 9506066A1
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Prior art keywords
cystic fibrosis
antibody
fibrosis transmembrane
conductance regulator
transmembrane conductance
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PCT/US1994/008970
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Inventor
Seng H. Cheng
John Marshall
Richard Gregory
Patrick W. Rafter
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Genzyme Corp
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Genzyme Corp
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Priority to AU76306/94A priority Critical patent/AU7630694A/en
Publication of WO1995006066A1 publication Critical patent/WO1995006066A1/fr
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    • 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
    • C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
    • C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
    • C07K14/4701—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals not used
    • C07K14/4712—Cystic fibrosis
    • 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

Definitions

  • Cystic Fibrosis is the most common fatal genetic disease in humans (Boat, T. et al. Cystic fibrosis. In: The Metabolic Basis of Inherited Disease, C. Scriver, A. Beaudet, W. Sly, and D. Valle, eds. (McGraw Hill, New York, 1989), 2649-2860). Based on both genetic and molecular analysis, a gene associated with CF was isolated as part of 21 individual cDNA clones and its protein product predicted (Kerem, B-S. et al. Science 245:1073-1080 (1989); Riordan, J. et al. Science 245:1066-1073 (1989); Rommens, J.H. et al. Science 245:1059-1065 (1989)).
  • USSN 07/488,307 describes the construction of the gene into a continuous strand, expression of the gene as a functional protein and confirmation that mutations of the gene are responsible for CF. ⁇ See also Gregory, R.J. et al. Nature 347:382-386 (1990); Rich, D.P. et al. Nature 347:358-363 (1990)). The copending patent application also discloses experiments which showed that proteins expressed from wild type but not a mutant version of the cDNA complemented the defect in the cAMP regulated chloride channel shown previously to be characteristic of CF.
  • CFTR cystic fibrosis transmembrane conductance regulator
  • CFTR is a member of a class of related proteins which includes the multi-drug resistant (MDR) P-glycoprotein, bovine adenyl cyclase, the yeast STE6 protein as well as several bacterial amino acid transport proteins (Riordan, J. et al. Science 245:1066-1073 (1989); Hyde, S.C. et al. Nature 346:362- 365 (1990). Proteins in this group, characteristically, are involved in pumping molecules into or out of cells.
  • MDR multi-drug resistant
  • CFTR has been postulated to regulate the outward flow of anions from epithelial cells in response to phosphorylation by cyclic AMP-dependent protein kinase or protein kinase C (Riordan, J. et al. Science 245:1066-1073 (1989); Frizzell, R.A. et al. Science 233:558-560 (1986); Welsh, M.J. and Liedtke, CM. Nature 322:467 (1986); Li, M. et al. Nature 331:358-360 (1988); Hwang, T-C. et al. Science 244;1351-1353 (1989); Li, M. et al. Science 244:1353-1356 (1989)).
  • the present invention pertains to antibodies which bind to epitopes of CFTR.
  • the antibodies of the present invention can be monoclonal or polyclonal antibodies which are preferably raised against human cystic fibrosis transmembrane conductance regulator.
  • the present invention also pertains to hybridomas producing antibodies, such as mAb 13-1, mAb 24-1, and mAb 24-2, which bind to an epitope of CFTR.
  • the present invention further pertains to a method of purifying CFTR from an impure solution containing CFTR.
  • the method involves contacting the impure solution with an antibody which binds an epitope of CFTR, allowing the antibody to bind to CFTR to form an immunological complex, and separating the complex from the impure solution.
  • the method of purification can further comprise separating the CFTR from the antibody and recovering the CFTR.
  • the separation is conducted by contacting the immunological complex with a saturating amount of peptide comprising the epitope recognized by the antibody of the immunological complex.
  • the present invention still further pertains to a method for detecting CFTR in a biological sample.
  • the method involves contacting the biological sample with an antibody which binds an epitope of CFTR, allowing the antibody to bind to CFTR to form an immunological complex, and detecting the formation of the immunological complex and correlating presence or absence of the immunological complex with presence or absence of CFTR in the biological sample.
  • Another aspect of the present invention is a method for detecting human cystic fibrosis transmembrane regulator in a biological sample from a nonhuman cystic fibrosis knockout animal wherein the nonhuman cystic fibrosis knockout animal has been subjected to human CFTR replacement therapy.
  • the method involves contacting the biological sample with an antibody which binds an epitope of human CFTR which is different from the corresponding CFTR amino acid sequence in the nonhuman cystic fibrosis knockout animal, allowing the antibody to bind to the human CFTR to form an immunological complex, and detecting the formation of the immunological complex and correlating the presence or absence of the complex with presence or absence of human CFTR.
  • Still another aspect of the present invention is a method for determining the orientation of CFTR in the membrane of a lipid vesicle.
  • the method includes contacting the lipid vesicle with an antibody which binds to a selected surface epitope of CFTR, such as an extracellular or an intracellular surface epitope, allowing the antibody to bind to CFTR to form an immunological complex, and detecting the formation of the immunological complex and correlating presence or absence of the immunological complex with correct or incorrect orientation of the CFTR in the membrane of the lipid vesicle.
  • kits for detecting CFTR in a biological sample includes a container holding an antibody which binds to an epitope of CFTR and instructions for using the antibody for the purpose of binding to CFTR to form an immunological complex and detecting the formation of the immunological complex such that presence or absence of the immunological complex correlates with presence or absence of CFTR.
  • Figure 1 depicts an autoradiograph of a gel showing immunoprecipitation of CFTR by monoclonal antibody mAb 13-1, monoclonal antibody mAb 24-1, monoclonal antibody mAb 24-2, and polyclonal antibody pAb CFTR.
  • the present invention provides antibodies which bind to epitopes of cystic fibrosis transmembrane conductance regulator (CFTR). These antibodies can be used to purify CFTR from an impure solution containing CFTR, to detect CFTR in a biological sample, and to determine the orientation of CFTR in the membrane of a lipid vesicle. In addition, the antibodies can be used in kits for detecting CFTR in a biological sample.
  • CFTR cystic fibrosis transmembrane conductance regulator
  • active fragments can be derived from an antibody of the present invention by a number of techniques.
  • purified monoclonal antibodies can be cleaved with an enzyme, such as pepsin, and subjected to HPLC gel filtration. The appropriate fraction containing Fab fragments can then be collected and concentrated by membrane filtration and the like.
  • an enzyme such as pepsin
  • HPLC gel filtration The appropriate fraction containing Fab fragments can then be collected and concentrated by membrane filtration and the like.
  • the term "antibody” also includes bispecific and chimeric antibodies.
  • the language "monoclonal antibody” is art-recognized terminology.
  • the monoclonal antibodies of the present invention can be prepared using classical cloning and cell fusion techniques.
  • the immunogen (antigen) of interest e.g. different portions of CFTR, is typically administered (e.g. intraperitoneal injection) to mice or rabbits as a fusion protein to induce an immune response.
  • Fusion proteins comprise the peptide against which an immune response is desired coupled to carrier proteins, such as ⁇ -galactosidase, glutathione S-transferase, keyhole limpet hemocyanin (KLH), and bovine serum albumin. In these cases, the peptides serve as haptens with the c-arrier proteins.
  • the spleen is removed and splenocytes are extracted and fused with myeloma cells using the well-known processes of Kohler and Milstein ⁇ Nature 256: 495-497 (1975)) and Harlow and Lane (Antibodies: A Laboratory Manual (Cold Spring Harbor Laboratory, New York 1988)).
  • the resulting hybrid cells are then cloned in the conventional manner, e.g. using limiting dilution, and the resulting clones, which produce the desired monoclonal antibodies, cultured.
  • Examples of monoclonal antibodies raised against human CFTR using this method include mAb 13-1, mAb 24-1, and mAb 24-2.
  • MAb 13-1 recognizes amino acids 729-736 of human CFTR and is produced by the hybridoma deposited under American Type Culture Collection (ATCC) Accession No. ATCC HB 10565. Because the sequence of human CFTR (SEQ ID NO: l and SEQ ID NO: 2, which correspond to the nucleic acid sequence encoding CFTR and the amino acid sequence of CFTR published in Riordan, J.R. et al. Science 245:1066- 1073 (Sept.
  • mAb 13-1 recognized by mAb 13-1 is different from the corresponding sequence of mouse CFTR, it is predicted that mAb 13-1 will not recognize the mouse form of CFTR.
  • This characteristic of mAb 13-1 can be useful in testing for gene or protein transfer of human CFTR into cystic fibrosis "knockout" mice. See Dorin, J.R. et al. Nature 359:211-216 (Sept. 1992); Colledge, W.H. et al. The Lancet 340:680 (Sept. 1992); Snouwaert, J.N. et al. Science 257:1083-1088 (Aug. 1992) for examples of mouse models for cystic fibrosis.
  • MAb 24-1 recognizes amino acids 1477-1480 of human CFTR and is produced by the hybridoma deposited under ATCC Accession No. ATCC . Because the sequence recognized by mAb 24-1 comprises the last four amino acids of human CFTR, the antibody can be used as a probe for the full length CFTR. For example, mAb 24-1 will not recognize CFTR lacking just the carboxy terminal amino acid. Like mAb 13-1, mAb 24-1 does not recognize mouse CFTR and is likewise useful in testing for gene or protein transfer of human CFTR into cystic fibrosis "knockout" mice.
  • MAb 24-2 recognizes amino acids 1433-1439 of human CFTR and is produced by the hybridoma deposited under ATCC Accession No. ATCC . As with mAb
  • mAb 24-2 does not recognize mouse CFTR and can be used in cystic fibrosis mouse "knockout" models for testing gene or protein transfer of human CFTR.
  • polyclonal antibody is art-recognized terminology.
  • polyclonal antibodies raised against human CFTR include pAb ECL1- 18, pAb ECL1-12, and pAb CFTR.
  • the immunogen used to produce pAb ECL1- 18 was a peptide comprising amino acids 102-119 of human CFTR coupled to the carrier keyhole limpet hemocyanin.
  • the epitopes recognized by pAb ECLl- 18 comprise amino acids 103-119 which are predicted to lie on the extracellular surface of the CFTR molecule.
  • These antibodies are, therefore, useful for studying the topology of CFTR.
  • these antibodies can be used to determine the orientation of CFTR reconstituted into artificial liposomes or virosomes.
  • the separation of correctly-oriented from incorrectly-oriented liposomes or virosomes can be achieved using affinity chromatography.
  • the immunogen used to produce pAb ECL1-12 was a peptide comprising amino acids 106-117 of human CFTR, amino acids also predicted to lie on the extracellular surface of the CFTR molecule.
  • the immunogen used to produce pAb CFTR was the entire human CFTR protein purified from insect cells. See George et al. Biochem. Biophys. Res. Comm. 163:1265 (1989) for an example of a system used to express a membrane protein in insect cells. Because its epitopes (over 40) span the entire CFTR, it is likely a useful antibody for immunoprecipitation experiments. In addition, it is likely to cross-react with CFTRs of all species.
  • a common method for preparing polyclonal antibodies to an immunogen of interest includes injecting (e.g. intradermally, intramuscularly) an animal, such as a rabbit, with an the immunogen emulsified in Freund' s complete adjuvant. This process is repeated after two weeks. Two weeks later, monthly subcutaneous booster injections can begin with the immunogen in Freund' s incomplete adjuvant. The animals are bled biweekly by a marginal ear vein technique beginning six weeks after the primary immunization. The collected blood is refrigerated, allowing clots to form, and the supernatant (antiserum) retained.
  • epitope is art-recognized. It is generally understood by those of skill in the art to refer to the region of an antigen, such as CFTR, that interacts with an antibody.
  • An epitope of a peptide or protein antigen can be formed by contiguous or noncontinguous amino acid sequences of the antigen. CFTR, like many large proteins, contains many epitopes.
  • Examples of human CFTR epitopes recognized by antibodies of the present invention include the amino acid sequences 729-736, Asp-Glu-Pro-Leu-Glu-Arg-Arg-Leu (SEQ ID NO:2); 1477- 1480, Asp-Thr-Arg-Leu (SEQ ID NO:2); and 1433-1439, Glu-Arg-Ser-Leu-Phe- Arg-Gln (SEQ ID NO:2). These peptides offer a convenient method for eluting CFTR bound to either mAb 13-1, mAb 24-1, and mAb 24-2 on immunoaffinity columns.
  • CFTR when an antibody which recognizes the epitope for either mAb 13-1, mAb 24-1, or mAb 24-2, is used in an immunoaffinity column to purify CFTR, the peptide recognized by the antibody can be added to the immunoaffinity column to elute the CFTR. See below for a more detailed description of the purification of CFTR.
  • cystic fibrosis transmembrane conductance regulator is intended to include wild-type cystic fibrosis transmembrane conductance regulator and mutant cystic fibrosis transmembrane conductance regulator.
  • sequences of the DNA encoding the human wild-type cystic fibrosis transmembrane conductance regulator and the DNA encoding the mutant cystic fibrosis transmembrane conductance regulator were described previously in the copending applications identified under the Related Applications section.
  • Mutant CFTRs include proteins having mutations introduced at residues known to be altered in CF chromosomes ( ⁇ G508, ⁇ I507, R334W, S5491, G551D) and at residues believed to play an important role in the function of CFTR (e.g., K464N, F508R, N894, 900Q, K1250M). See Example 7 of USSN 07/935,603, filed August 26, 1992. See Tsui, L-C Trends in Genetics 8(l l):392-398 (Nov. 1992) for additional examples of the approximately 300 sequence alterations of CFTR identified to date.
  • extracellular surface is intended to include regions of a protein, such as CFTR, that extend into the extracellular space. For example, it has been predicted that amino acids 103-119 of CFTR lie on the extracellular surface.
  • intracellular surface is intended to include regions of a protein, such as CFTR, that extend into the intracellular space. For example, the amino acids that comprise the nucleotide-binding domain of CFTR as well as the R-domain lie on the intracellular surface of CFTR.
  • CFTR is a protein of approximately 1480 amino acids. Carboxy terminal amino acids of CFTR include amino acids 741-1480.
  • the antibody mAb 24-1 of the present invention was raised against the CFTR carboxy terminal amino acids 1477-1480.
  • the present invention also pertains to hybridomas producing antibodies which bind to an epitope of cystic fibrosis transmembrane conductance regulator.
  • hybridoma is art recognized and is understood by those of ordinary skill in the art to refer to a cell produced by the fusion of an antibody- producing cell and an immortal cell, e.g. a multiple myeloma cell. This hybrid cell is capable of producing a continuous supply of antibody. See the definition of "monoclonal antibody” above and Example 1 below for a more detailed description of the method of fusion.
  • the hybridoma which produces mAb 13-1 is deposited under ATCC Accession Number HB 10565.
  • the hybridoma which produces mAb 24-1 is deposited under ATCC Accession Number .
  • the hybridoma which produces mAb 24-2 is deposited under ATCC Accession Number .
  • the present invention further pertains to a method for purifying CFTR from an impure solution containing CFTR.
  • the method involves contacting the impure solution with an antibody which binds an epitope of CFTR, allowing the antibody to form an immunological complex, and separating the complex from the impure solution.
  • the method of purification can further comprise separating the CFTR from the antibody and recovering the CFTR.
  • the separation is conducted by contacting the immunological complex with a saturating amount of peptide comprising the epitope recognized by the antibody of the immunological complex.
  • purifying is intended to include removal of unwanted constituents of an impure solution containing CFTR such that the concentration of CFTR in the solution after purification is greater that the concentration of CFTR in the solution prior to purification and the concentration of at least one unwanted constituent in the solution after purification is less than the concentration of the unwanted constituent in the solution prior to purification.
  • Unwanted constituents include molecules other than CFTR. It should be understood that the extent of the purification of the solution can depend on the intended use of the CFTR. For example, the CFTR purified for therapeutic use will have to be more pure than CFTR purified for research purposes.
  • impure solution is intended to include a mixture of compounds which includes CFTR and at least one non-CFTR compound.
  • an impure solution can comprise a biological sample containing CFTR as defined below.
  • an impure solution can comprise cell homogenates of cells that contain CFTR.
  • Cell homogenates typically contain the components of the cell culture medium as well as disrupted cellular components, e.g. cellular macromolecules, such as CFTR, organelles, lysosomes. Because CFTR is a membrane protein, it is important to accomplish solubilization of CFTR from its native membrane such as by using detergents. In this case, the cell homogenate containing CFTR is generated by first determining conditions for solubilization of CFTR from its natural lipid environment using whole cells or membrane preparations prepared from cells.
  • the initial solubilization experiments will involve screening a variety of detergents at varying concentrations in order to find conditions that preferably achieve maximal solubilization of CFTR. Briefly, packed membrane pellets are resuspended in detergent solution, gently homogenized, and the insoluble material removed by centrifugation at 100,000 g for one hour. The degree of solubilization achieved is ideally monitored immunologically.
  • Potential detergents include, but are not limited to , CHAPS (3- ⁇ 3- choamidopropyl)dimethylammonio ⁇ -l-pro(anesulfonate) (Borsotto, M. et al. J. Biol. Chem. 260:14255 (1985); Hamada and Tsuro, J. Biol Chem.
  • the initial detergent solubilized CFTR solution can also be diluted into an appropriate concentration of detergent or detergent lipid (Agnew and Raftery, Biochemistry 18:1912 (1979)) to achieve stabilization of the CFTR.
  • Compounds known to stabilize proper folding of membrane proteins sometimes referred to as ozmolytes can also be used. These include polyols such as glycerol, sugars, and amino acids (Ambudkar and Maloney, J. Biol. Chem. 261:10079 (1986)).
  • protease inhibitors against the four major classes of proteases can be advantageously made present throughout these procedures (Hartshorne and Catterall, J. Biol. Chem.
  • an impure solution can comprise culture media which has been used to culture cells containing CFTR and, therefore, can contain CFTR.
  • cultured cells that normally contain CFTR include immortalized airway epithelial cells (e.g. nasal and lung epithelial cells) from cystic fibrosis victims.
  • cells which do not normally contain CFTR but can be engineered to express CFTR include 3T3 fibroblasts, C127, and COS-7 cells. These cells can be transfected with nucleic acid which encodes and directs expression of CFTR.
  • immunological complex is intended to include an antigen, such as CFTR or a fragment thereof, bound to an antibody, such as a monoclonal or polyclonal antibody, or a fragment thereof.
  • the antigen and antibody are typically bound to one another through noncovalent interactions.
  • the immunological complex as well as the antigen alone can be separated from the impure solution by any separation technique known to those of ordinary skill in the art. For example, one commonly used separation method is immunoaffinity chromatography. See Harlow and Lane, Antibodies: A Laboratory Manual (Cold Spring Harbor Laboratory, New York 1988) 511-552.
  • Immunoaffinity purification generally consists of three steps: preparation of an antibody-matrix, binding of an antigen to the antibody-matrix, and elution of the antigen.
  • first step either monoclonal antibodies or affinity-purified polyclonal antibodies can be covalently attached to a solid-phase matrix.
  • An example of covalent attachment of the antibody to the solid-phase matrix is linkage of the antibody to protein A beads. See Harlow and Lane, Antibodies: A Laboratory Manual (Cold Spring Harbor Laboratory, New York 1988) 521-523.
  • the antigen is bound to the antibody and contaminating molecules are removed by washing.
  • the antigen-antibody interaction is broken by treating the immune complexes with strong elution conditions, adding a saturating amount of a small compound (e.g. the peptide comprising the epitope recognized by the antibody used in the column) that mimics the binding site, and/or treating with an agent which induces an allosteric change that releases the antigen, to release the antigen into the eluate.
  • a small compound e.g. the peptide comprising the epitope recognized by the antibody used in the column
  • an agent which induces an allosteric change that releases the antigen to release the antigen into the eluate.
  • Optimal conditions for binding the antigen to the column, washing the column to remove contaminants, and eluting the purified antigen can be determined using conventional parameters as the starting point and testing the effect of varying the parameters. It is recognized that effective wash and elution conditions will significantly impact the degree of purification obtained.
  • Extensive washing in the presence of stabilizers plus higher salt and differing detergents can be utilized to remove nonspecifically adsorbed proteins. Elution can then be carried out most advantageously by lowering the pH followed by immediate pH neutralization of the eluted fractions, by using the above-described specific peptide elution (Courtneige et al. Cold Spring Harbor Conference on Cell Prolif. and Cancer 2:123 (1984)), or chaotropic agents such as potassium thiocyanate.
  • CFTR nucleotide binding domain
  • the accessibility of the nucleotide binding domain in the solubilized form would have to be determined empirically.
  • the amino acid sequence of CFTR contains carbohydrate attachment sites at amino acids 894 and 900. It has also been shown that CFTR is a glycoprotein. Cheng, S.H. et al. Cell 63:827-834 (Nov. 1990). Thus, lectin chromatography can be used to purify CFTR.
  • the present invention still further pertains to a method for detecting CFTR in a biological sample containing CFTR.
  • the method includes contacting the biological sample with an antibody which binds an epitope of CFTR, allowing the antibody to bind to CFTR to form an immunological complex, and detecting the formation of the immunological complex and correlating presence or absence of the immunological complex with presence or absence of CFTR in the biological sample.
  • the language "detecting the formation of the immunological complex” is intended to include discovery of the presence or absence of CFTR in a biological sample.
  • the presence or absence of CFTR can be detected using an immunoassay.
  • immunoassays A number of immunoassays used to detect and/or quantitate antigens are well known to those of ordinary skill in the art. See Harlow and Lane, Antibodies: A Laboratory Manual (Cold Spring Harbor Laboratory, New York 1988) 555-612.
  • Such immunoassays include antibody capture assays, antigen capture assays, and two-antibody sandwich assays. These assays are commonly used by those of ordinary skill in the .art. In an antibody capture assay, the antigen is attached to solid support, and labeled antibody is allowed to bind.
  • the assay is quantitated by measuring the amount of antibody retained on the solid support.
  • an antigen capture assay the antibody is attached to a solid support, and labeled antigen is allowed to bind. The unbound proteins are removed by washing, and the assay is quantitated by measuring the amount of antigen that is bound.
  • a two- antibody sandwich assay one antibody is bound to a solid support, and the antigen is allowed to bind to this first antibody. The assay is quantitated by measuring the amount of a labeled second antibody that can bind to the antigen.
  • These immunoassays typically rely on labeled antigens, antibodies, or secondary reagents for detection.
  • radioactive proteins can be labeled with radioactive compounds, enzymes, biotin, or fluorochromes.
  • radioactive labeling can be used for almost all types of assays and with most variations. Enzyme- conjugated labels are particularly useful when radioactivity must be avoided or when quick results are needed.
  • Biotin-coupled reagents usually are detected with labeled streptavidin. Streptavidin binds tightly and quickly to biotin and can be labeled with radioisotopes or enzymes. Fluorochromes, although requiring expensive equipment for their use, provide a very sensitive method of detection.
  • Antibodies useful in these assays include monoclonal antibodies, polyclonal antibodies, and affinity purified polyclonal antibodies.
  • biological sample is intended to include biological material, e.g. cells, tissues, or biological fluid which, as it exists in nature, contains CFTR, e.g. sputum from cystic fibrosis victims, biological material that does not contain CFTR as it exists in nature but can be engineered to contain CFTR (e.g. cells, tissues) or contains CFTR as a result of its proximity to biological material (e.g. biological fluids from the extracellular space adjacent to cells which naturally contain CFTR or cells that have been engineered to contain CFTR) that naturally contains CFTR or has been engineered to express CFTR, or biological material which has been subjected to CFTR replacement therapy.
  • biological material e.g. cells, tissues, or biological fluid which, as it exists in nature, contains CFTR, e.g. sputum from cystic fibrosis victims, biological material that does not contain CFTR as it exists in nature but can be engineered to contain CFTR (e.g. cells, tissues) or contains CFTR as
  • Biological material such as cells, can be engineered to contain CFTR by transfection with nucleic acid which encodes and directs expression of CFTR.
  • nucleic acid which encodes and directs expression of CFTR.
  • an adenovirus-based gene therapy vector the Ela and Elb regions of the genome of which have been deleted and replaced with CFTR-encoding nucleic acid can be used to deliver nucleic acid encoding CFTR to airway epithelial cells in vivo. See USSN 07/985,478, filed December 2, 1992.
  • Another example of CFTR replacement therapy involves delivering CFTR directly to cells containing defective CFTR. See Example 8 of USSN 08/087,132, filed July 2, 1993.
  • Another aspect of the present invention is a method for detecting human CFTR in a biological sample from a nonhuman cystic fibrosis knockout animal having been subjected to human CFTR replacement therapy.
  • the method involves contacting the biological sample with an antibody which binds an epitope of human CFTR which is different from the corresponding CFTR amino acid sequence in the nonhuman cystic fibrosis knockout animal, allowing the antibody to bind to the human CFTR to form an immunological complex, and detecting the formation of the immunological complex and correlating presence or absence of the immunological complex with presence or absence of human CFTR.
  • nonhuman cystic fibrosis knockout animal is intended to include a nonhuman animal the CFTR gene of which is disrupted or altered such that the animal displays at least one feature common to human cystic fibrosis patients, such as excess mucus in the lungs, intestinal obstruction, or reduced ability to digest and absorb duodenal contents because of pancreatic insufficiency.
  • nonhuman animals in which the CFTR gene can be disrupted or altered include nonhuman animals that contain the CFTR gene.
  • One noted example of such an animal is a mouse. Methods for generating cystic fibrosis knockout mice are described in Dorin, J.R. et al. Nature 359:211-216 (Sept. 1992) and Snouwaert, J.N.
  • cystic fibrosis knockout mice can be used as model systems for the development and testing of therapies for cystic fibrosis patients.
  • These model systems can be used to test targeted gene replacement therapy using, for example, adenovirus-based gene therapy vectors containing nucleic acid encoding functional CFTR. See USSN 07/985,478, filed December 2, 1992.
  • these model systems can be used to test protein therapy. Protein therapy can be accomplished by using CFTR produced by host cells transformed or transfected with CFTR cDNA to correct the cystic fibrosis defect directly by introducing the protein into the membrane of cells lacking functional CFTR. This approach can augment the defective protein by addition of the wild-type molecule.
  • treatment of individuals with cystic fibrosis will comprise the administration of a therapeutically effective amount of replacement CFTR.
  • the CFTR will be administered via aerosol inhalation so that is applied directly to the airway cells.
  • the CFTR can be formulated in a lipid vesicle, such as in a liposome or in a virosome.
  • the final formulation can advantageously comprise a carrier as a vehicle for physically transporting the CFTR and also ideally chemically stabilizing the CFTR.
  • the most preferred embodiment also can comprise a dissolving agent for dissolving the mucus or otherwise assisting the movement of the CFTR through the mucus layer to the airway cellular membrane.
  • a dissolving agent for dissolving the mucus or otherwise assisting the movement of the CFTR through the mucus layer to the airway cellular membrane.
  • Ideal agents in this regard would target the CFTR and/or the delivery vehicle to airway cells and further, promote fusion therewith.
  • Still another aspect of the present invention is a method for determining the orientation of CFTR in the membrane of a lipid vesicle.
  • the method includes contacting the lipid vesicle with an antibody which binds to an extracellular surface epitope of CFTR, allowing the antibody to bind to the CFTR to form an immunological complex, and detecting the formation of the immunological complex and correlating presence or absence of the immunological complex with correct or incorrect orientation of the CFTR in the membrane of the lipid vesicle.
  • orientation is intended to include the position of CFTR in the membrane of a lipid vesicle.
  • CFTR in its functional state in a cell, CFTR is in certain position, e.g. with certain amino acids on the extracellular surface, certain amino acids within the membrane (e.g. transmembrane segments), and other amino acids on the intracellular surface of the molecule (e.g. amino acids comprising the nucleotide binding domain and the R-domain.
  • This is the correct orientation of CFTR within a cellular membrane.
  • it is the correct orientation of CFTR that has been reconstituted into lipid vesicles.
  • CFTR CFTR into artificial liposomes.
  • the incorrect orientation of CFTR within either a cellular membrane or a membrane of a lipid vesicle is one in which the regions of a functional form of CFTR that are normally in one position are no longer in that position.
  • a functional form of CFTR is in an incorrect orientation in the membrane of a lipid vesicle when the amino acid sequences which are normally on the extracellular surface of the vesicle are no longer on the extracellular surface, e.g., they may be in a transmembrane segment or on the intracellular surface of the vesicle.
  • Antibodies which recognize epitopes which are on the extracellular surface or intracellular surface of a functional form of CFTR in its correct orientation in a membrane can be used to determine the orientation of CFTR when it has been reconstituted in a membrane of a lipid vesicle.
  • the separation of correctly-oriented from incorrectly-oriented lipid vesicles can be accomplished using affinity chromatography. Anholt et al. J. Biol. Chem. 256:4377 (1981).
  • the language "lipid vesicle” is intended to include a lipid membrane-bound compartment. Examples of lipid vesicles include liposomes, microsomes, and virosomes.
  • kits for detecting CFTR in a biological sample containing CFTR includes a container holding an antibody which binds an epitope of CFTR and instructions for using the antibody for the purpose of binding to CFTR to form an immunological complex and detecting the formation of the immunological complex such that the presence or absence of the immunological complex correlates with presence or absence of CFTR.
  • containers include multiwell plates which allow simultaneous detection of CFTR in multiple samples.
  • Monoclonal antibodies mAb 13-1, mAb 24-1, and mAb 24-2, specific for predetermined regions or epitopes of the CFTR protein, were prepared using classical cloning and cell fusion techniques.
  • the immunogen used to produce mAb 13-1 was a fusion protein comprising Exon 13 comprising amino acids 591-829 of the human CFTR and ⁇ -galactosidase.
  • the immunogen used to produce mAb 24-1 was a fusion protein comprising glutathione S-transferase and amino acids 1377- 1480 of human CFTR.
  • the immunogen used to produce mAb 24-2 was a fusion protein comprising glutathione S-transferase and amino acids 1377-1480 of human CFTR.
  • the fusion proteins were obtained as described in Mole and Lane, DNA Cloning Volume 111: A Practical Approach (1987), and used to induce an immune response in a mouse.
  • mice (Balb/c females, 6-10 weeks at start of schedule) was to inject each of three with 10 ⁇ g of immunogen (fusion protein or peptide) emulsified in Freund's complete adjuvant (final volume 100-150 ⁇ l) intraperitoneally on day 0. This was repeated on days 14, 28, and 56 except that Freund's incomplete adjuvant was substituted for the complete.
  • 50 ⁇ g of immunogen in PBS 25 ⁇ l was injected intravenously via a tail vein. The mice were sacrificed on day 60 and the spleens taken for fusion.
  • the B-lymphocytes of the immunized mice were extracted from the spleen and fused with myeloma cells using the well known processes of Kohler and Milstein ⁇ Nature 256:495-497 (1975)) .and Harlow and Lane, Antibodies, A Laboratory Manual, Cold Spring Harbor Laboratory, New York (1988), respectively.
  • the resulting hybrid cells were cloned in the conventional manner, e.g., using limiting dilution, and the resulting clones, which produce the desired monoclonal antibodies, cultured.
  • the hybridomas producing monoclonal antibody mAb 13-1, the monoclonal antibody mAb 24-1, and the monoclonal antibody mAb 24-2, prepared according to this procedure have been deposited with the ATCC under the terms of the Budapest Treaty, and assigned Accession Numbers ATCC HB 10565, ATCC , and ATCC , respectively.
  • the peptide immunogens obtained from Multiple Peptide Systems were comprised of the following sequences: (1) amino acids 102-119 (Leu-Gly- Arg-Ile-Ile-Ala-Ser-Tyr-Asp-Pro-Asp-Asn-Lys-Glu-Glu-Arg-Ser-Ile) (used to produce pAb ECL1-18); (2) amino acids 106-117 (Ile-Ala-Ser-Tyr-Asp-Pro-Asp- Asn-Lys-Glu-Glu-Arg) (used to produce pAb ECLl -12); and (3) the entire amino acid sequence 1-1480 of CFTR (SEQ ID NO:2) (used to produce pAb CFTR).
  • Peptide purity as assessed by reverse-phase high-pressure liquid chromatography (HPLC) were judged to be greater than 85% and amino acid composition analysis of the synthetic peptides yielded excellent correspondence between expected and experimental values for all the peptides.
  • the first two peptides were coupled to keyhole limpet hemocyanin (KLH) to increase their antigenicity by utilizing a terminal cysteine residue introduced into all three peptides to facilitate specific coupling to the carrier protein through the thiol moiety using the m- Maleimidobenzoyl-N-hydroxysuccinimide ester (MBS) chemistry (Pierce ImmunoTechnology Catalog and Handbook E-161991) at a ratio of 1:1 (w/w).
  • KLH keyhole limpet hemocyanin
  • the schedule was to immunize each of two (New Zealand White, female) with 100 ⁇ g of immunogen emulsified in Freund's complete adjuvant (final volume 400-500 ⁇ l) intramuscularly on day 0 which was repeated on days 14 and 35 substituting Freund's incomplete adjuvant for complete for these and all subsequent boosts.
  • Blood was taken (20-30 mis) one, three, and five weeks post injection and the rabbits were reboosted 6 weeks after their previous boost before starting the bleeds cycle again.
  • Affinity purification of the polyclonal antibodies was performed on the antisera following a salting out step.
  • the antisera (-20 mis) was made to 45% ammonium sulfate by addition of a 100% saturated solution under gentle mixing. Following one hour equilibration, the precipitate was centrifuged and the pellet washed twice in 50% ammonium sulfate. The final pellet was resuspended in 15 mis PBS and diafiltrated using a Centricon 100 (Amicon, Beverly, MA) yielding 0.5 ml - 1 ml of antisera enriched for immunoglobulins. The enriched antisera was then loaded directly onto the affinity resin.
  • the affinity columns used for the peptide antisera were generated by coupling 10 mg of peptide through the terminal cysteine to an activated thiol resin (Immunopure kit #2, Pierce, Rockford, IL). The antisera was passed over the column. Non-specific proteins were washed away and the specifically bound antibodies were eluted with 0.1M glycine (pH 2.8) and diafiltrated against PBS.
  • the immune complex is allowed to form for one hour at 4°C before capture with Pansorbin (Calbiochem, LaJolla, CA) precoated with rabbit anti-mouse Igs (Cappel) for thirty minutes.
  • Pansorbin Calbiochem, LaJolla, CA
  • rabbit anti-mouse Igs Cappel
  • the washed pellet was resuspended in 50 ⁇ l of PKA buffer (50 mM Tris- HC1 pH 7.5, 10 mM MgCl, BSA 0.1 mg/ml, protein kinase A (Sigma, St. Louis, MO), 100 U/ml 32 P-ATP 20 ⁇ CI/ml) and incubated at 30°C for one hour.
  • the Pansorbin pellet was washed and the eluted immunoprecipitate was loaded onto a 6% polyacrylamide gel.
  • Figure 1 depicts an autoradiograph of a gel showing immunoprecipitation of CFTR with mAb 13-1 (lane 3), mAb 13-2 (lane 5) mAb 24-1 (lane 7), mAb 24-2 (lane 9), and pAb CFTR (lane 11).
  • Lanes 1 and 2 are controls in which no antibody was incubated with the cell lysate from COS-7 cells which express CFTR (lane 1) and COS-7 cells which do not express CFTR (lane 2).
  • Lanes 4, 6, 8, 10, and 12 are controls in which lysates from COS-7 cells which do not express CFTR were incubated with mAb 13-1 (lane 4), mAb 13-2 (lane 6), mAb 24-1 (lane 8), mAb 24- 2 (lane 10), and pAb CFTR (lane 12).
  • TCT GCT GAC AAT CTA TCT GAA AAA TTG GAA AGA GAA TGG GAT AGA GAG 312 Ser Ala Asp Asn Leu Ser Glu Lys Leu Glu Arg Glu Trp Asp Arg Glu 45 50 55 60
  • CAA TCT TTT AAA CAG ACT GGA GAG TTT GGG GAA AAA AGG AAG AAT TCT 2232
  • MOLECULE TYPE protein

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Abstract

L'invention se rapporte à des anticorps capables de se lier à des épitopes du régulateur de la conductance transmembranaire de la mucoviscidose (RCTM) et à des hybridomes qui produisent ces anticorps. Les anticorps de la présente invention peuvent être utilisés dans un procédé visant à détecter le RCTM dans un échantillon biologique et/ou dans un procédé de purification du RCTM à partir d'une solution impure. De plus, la présente invention se rapporte à un procédé de détection du RCTM dans un échantillon biologique provenant d'un animal rendu inconscient, atteint de mucoviscidose, cet animal ayant été soumis à une thérapeutique de substitution avec du RCTM humain. Un autre aspect de la présente invention concerne un procédé de détermination de l'orientation du RCTM dans la membrane d'une vésicule lipidique. Enfin, un autre aspect de l'invention concerne un kit de détection du RCTM dans un échantillon biologique.
PCT/US1994/008970 1993-08-27 1994-08-08 Anticorps specifiques contre le regulateur de la conductance transmembranaire de la mucoviscidose et leurs utilisations Ceased WO1995006066A1 (fr)

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Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5240846A (en) * 1989-08-22 1993-08-31 The Regents Of The University Of Michigan Gene therapy vector for cystic fibrosis

Patent Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5240846A (en) * 1989-08-22 1993-08-31 The Regents Of The University Of Michigan Gene therapy vector for cystic fibrosis

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