Chemistry: molecular biology and microbiology – Micro-organism – tissue cell culture or enzyme using process... – Enzymatic production of a protein or polypeptide
Patent
1995-03-10
1998-03-31
Feisee, Lila
Chemistry: molecular biology and microbiology
Micro-organism, tissue cell culture or enzyme using process...
Enzymatic production of a protein or polypeptide
530413, 530415, C12P 2106, C07K 122
Patent
active
057337420
DESCRIPTION:
BRIEF SUMMARY
This application is a .sctn.371 National Phase Application based upon PCT/GB94/01210, filed Jun. 3, 1994 published as WO94/01210 Dec. 22, 1994.
The present invention relates to the production and use of immunoglobulin Fab fragments.
Antibodies are formed as part of the immune response to a microorganism or foreign macromolecule. They are immunoglobulins (Ig) and are used extensively in clinical practice for the diagnosis, monitoring, prevention and treatment of an increasing number of diseases.
The basic unit from which all antibody molecules are formed was elucidated by Porter (1959) Biochem J. 73, 119-126, using specific proteolytic enzymes. The most important of the immunoglobulins, IgG, comprises two heavy and two light chains with the former being coupled at their hinge region by disulphide linkages. Cleavage with papain above these linkages releases two antibody binding fragments (Fab) and a crystalline fragment (Fc) as shown in FIG. 1. Cleavage with pepsin, below the hinge results in a somewhat smaller Fc fragment and a single F(ab').sub.2 fragment with two binding sites as shown in FIG. 1. Each Fab fragment contains both a light chain and part of a heavy chain, and includes the sequences responsible for specific binding to a microorganism or foreign macromolecule. The Fc consists of the remainder of the two heavy chains; this is the site to which complement, macrophages and polymorphonuclear white blood cells can bind. The two heavy chains (but not the light chains) are different for each class of antibody ie IgG, IgM, IgA and IgE. IgG is the dominant circulating immunoglobulin in terms of concentration. It consists of a single basic immunoglobulin unit and, characteristically, has a high affinity for its specific antigen. Further details of antibody structure and function are disclosed in Roitt (1991) Essential Immunology, 7th Edition, Blackwell Scientific Publications, Oxford.
One of the major problems in preparing immunoglobulins and immunoglobulin fragments for therapeutic use is the possibility of bacterial contamination during manufacture of the product. This risk applies equally to the processing of polyclonal antisera and monoclonal antibodies. The problem is not eliminated by sterile filtration of the end-product because, while this may remove the bacteria themselves, it does not remove the soluble toxins which they may have released at an earlier stage.
The risk to the patient of bacterial and toxin contamination can be obviated by careful testing of the end-product both in vitro (LAL test) and in vivo (rabbits). However, it is clearly preferable that the primary contamination should be avoided, at the very least because this would save the considerable expense of discarding batches of material which did not meet the very stringent quality control parameters. Contamination of product is likely to occur whenever it is transferred from one sterile environment to another, for example from the vessel in which the blood is collected to that in which the initial protein purification is performed.
Classically, the production of Fab fragments relies on papain digestion. The digestion procedure involves incubation of a solution of papain with a substantially pure preparation of immunoglobulins under defined conditions of pH, temperature and co-factors (such as cysteine and ethylene diamine tetra-acetic acid; EDTA) which serve to activate the enzyme and bind its inhibitors, respectively. Although the need to purify the immunoglobulin fraction from blood, plasma or serum before digestion with papain is time consuming and may result in significant losses and impairment of their ability to bind and neutralise antigens, the application of papain to blood, serum or plasma has never been described.
SUMMARY OF THE INVENTION
One aspect of the invention provides a method of preparing polyclonal immunoglobulin Fab fragments comprising cleaving immunoglobulin molecules, characterised in that the cleaving is carried out on immunoglobulins in blood, serum or plasma.
Preferably, the cleaving is carried out, a
REFERENCES:
patent: 4061767 (1977-12-01), Ertel et al.
patent: 4381295 (1983-04-01), Kung et al.
patent: 4603106 (1986-07-01), Cerami et al.
patent: 4684623 (1987-08-01), Larrick et al.
patent: 4731244 (1988-03-01), Talle et al.
patent: 4742159 (1988-05-01), Batz et al.
patent: 4822776 (1989-04-01), Cerami et al.
patent: 4849352 (1989-07-01), Sullivan et al.
patent: 4870163 (1989-09-01), Rubin et al.
patent: 4918163 (1990-04-01), Young et al.
Garvey et al. Methods in Immunology p. 256 1977.
Aggarwal et al. Human Tumor Necrosis Factor Production, Purification, and Characterization, Journal of Biological Chemistry, vol. 260, No. 4, pp. 2345-2354, Aug. 21, 1984.
Beutler et al., Purification of Cachectin, A Lipoprotein Lipase-Suppressing Hormone Secreted by Endotoxin-Induced Raw 264.7 Cells, J. Exp. Med., vol. 161, pp. 984-995, May, 1985.
Beutler et al., Identity of Tumour Necrosis Factor and the Macrophage-Secreted Factor Cachectin, Nature, vol. 316, pp. 552-554 Aug., 8, 1985.
Beutler et al., Cachectin and Tumour Necrosis Factor as Two Sides of the Same Biological Coin, Nature, vol. 320, pp. 584-588 Apr. 17, 1986.
Beutler et al., Passive Immunization Against Cachectin/Tumor Necrosis Factor Protects Mice From Lethal Effect of Endotoxin, Science, vol. 229, pp. 869-871, Aug. 30, 1985.
Beutler et al., Fed. Proc. 44, 1704, Activated macrophages Secrete A Novel Lipolytic Polypeptide Hormone, Abstract No. 7565, 1985.
Bone, A Critical Evaluation of New Agents for the Treatment of Sepsis, JAMA, vol. 266, No. 12, pp. 1686-1691, Sep. 25, 1991.
Buttle et al., The Preparation of Fully Active Chymopapain Free of Contaminating Proteinases, Biol. Chem. Hoppe-Seyler, vol. 371, pp. 1083-1088, Nov., 1990.
Buttle et al., Affinity Purification of the Novel Cysteine Proteinase Papaya Proteinase IV, and Papain from Papaya Latex, Biochem J., vol. 261, pp. 469-476, 1989.
Brennan et al., Inhibitory Effect of TNF.alpha. Antibodies on Synovial Cell Interleukin-1 Production in Rheumatoid Arthritis, The Lancet, pp. 244-247, Jul. 29, 1989.
Carswell et al., An Endotoxin-Induced Serum Factor that Causes Necrosis of Tumors, Proc. Nat. Acad. Sci., vol. 72, No. 9, pp. 3666-3670, Sep., 1975.
Cerami et al., Weight Loss Associated with an Endotoxin-Induced Mediator from Perintoneal Macrophages: The Role of Cachectin (Tumor Necrosis Factor), Immunology Letters, vol. 11, pp. 173-177, 1985.
Elliott et al., Treatment of Rheumatoid Arthritis with Chimeric Monoclonal Antibodies to Tumor Necrosis Factor.alpha., Arthritis & Rheumatism, vol. 36, No. 12, pp. 1681-1690, Dec., 1993.
Erikaku et al., Bioluminescent Immunoassay Using a Monomeric Fab'-Photoprotein Aequorin Conjugate, Biochemical and Biophysical Research Communications, vol. 174, No. 3, pp. 1331-1336, Feb. 14, 1991.
Feldmann et al., Evaluation of the Role of Cytokines in Autoimmune Disease: The Importance of TNF.alpha. in Rheumatoid Arthritis, Progress in Growth Factor Research, vol. 4, pp. 247-255, 1992.
Fransen et al., Molecular Cloning of Mouse Tumour Necrosis Factor cDNA and its Eukaryotic Expression, Nucleic Acids Research, vol. 13, No. 12, pp. 4417-4429, 1985.
Glauser et al., Septic Shock: Pathogenesis, The Lancet, vol. 338, pp. 732-736, Sep. 21, 1991.
Goldstein et al., A Randomized Clinical Trial of OKT3 Monoclonal Antibody for Acute Rejection of Cadaveric Renal Transplants, The New England Journal of Medicine, vol. 313, No. 6, pp. 337-342, Aug. 8, 1985.
Haranaka et al., Purification and Partial Amino Acid Sequence of Rabbit Tumor Necrosis Factor, Int. J. Cancer, vol. 36, pp. 395-400, 1985.
Hinds, Monoclonal Antibodies in Sepsis and Septic Shock, BMJ, vol. 304, pp. 132-133, Jan. 18, 1992.
Hotez et al., Lipoprotein Lipase Suppression in 3T3-L1 Cells by a Haematoprotozoan-Induced Mediator from Peritoneal Exudate Cells, Parasite Immunology, vol. 6, pp. 203-209, 1984.
Lamoyi et al., Preparation of F(ab').sub.2 Fragments from Mouse IgG of Various Subclasses, Journal of Immunological Methods, vol. 56, No. 2, pp. 235-243, Jan. 28, 1983.
Kawakami e
Feisee Lila
Gambel Phillip
Therapeutic Antibodies Inc.
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