METHOD OF TREATING SEPTIC SHOCK

Abstract: A method of treating septic shock is provided. The method comprises administering an effective amount of an antagonist to human IL-6. Preferably, the antagonist is a blocking monoclonal antibody specific for human IL-6, or a fragment or binding composition derived therefrom.

Description: METHQD OF TREATING SEPTIC SHOCK FIELD OF THE INVENTION The invention relates to a method of treating septic shock by administering antagonists to interleukin-6 (IL-6). BACKGROUND Severe infections, particularly with gram negative bacteria, can result in profound physiological and metabolic alterations including fever, hypotension, metabolic acidosis, widespread organ disfunction and ultimately death. Such alterations are frequently referred to as septic shock. The host- derived protein, tumor necrosis factor (TNF), has recently been demonstrated to induce many of the deleterious effects of gram negative septicemia, and passive immunization with antisera to TNF can prevent the lethal effects of gram negative bacteremia or endotoxemia: e.g. Tracey et al., Science. Vol. 234, pg. 470 (1986); Beutler et al., Nature. Vol. 320, pg. 584 (1986); and Tracey et al., Nature. Vol. 330, pg. 662 (1987). High serum levels of TNF have been observed in several infectious diseases including meningococcal meningitis, malaria, and leishmaniasis, and patients with high circulating levels of TNF have increased organ dysfunction along with higher mortality: e.g. Waage et al., Lancet, pg. 355 (1987); Girardin et al., New Engl. J. ed., Vol. 319, pg. 397 (1988); Scuderi et al., Lancet, pg. 1364 (1988); and Kern et al., Am. J. Med.. Vol. 87, pg. 139 (1989). It has been reported that administration of TNF either to animals or to human subjects resulted in detectable IL-6 serum levels two to six hours later: Mclntosh et al., J. Immunol.. Vol. 143, pg. 162 (1989); Jablons et al., J. Immunol.. Vol. 142, pg. 1542 (1989); and Brouckaert et al., J. Exp. Med.. Vol. 169, pg. 2257 (1989). IL-6, also known as B cell stimulating factor-2, interferon-β2, and hepatocyte stimulating factor, has been identified as a T cell-derived glycoprotein that causes B cell maturation, e.g. Kishimoto, BJQOJI, Vol. 74, pg. 1 (1989). More recently, IL-6 has been demonstrated to possess pleiotropic biological activities, including induction of acute phase proteins in hepatocytes and actions on hematopoietic progenitor cell and T cells: e.g. Geiαer et al.. Eur. J. Immunol.. Vol. 18, pg. 717 (1988); Marinjovic et al., J. Immunol.. Vol. 142, pg. 808 (1989); Morrone et al., J. Biol. Chem.. Vol. 263, pg. 12554 (1988); and Perlmutter et al., J. Clin. Invest.. Vol. 84, pg. 138 (1989). Presently, septic conditions, such as bacteremia and the like, are treated with antimicrobial compounds. However, when such conditions are associated with shock, there are no effective adjunctive measures in the therapy for ameliorating the shock syndrome: e.g. Young, pgs. 468-470, in Mandell et al., eds., Principles and Practice of Infectious Diseases, 2nd Ed. (John Wiley & Sons, New York, 1985). In view of this, the availability of techniques for treating shock associated with sepsis could have important clinical utility. SUMMARY OF THE INVENTION The invention provides a method of treating septic shock by administering an effective amount of an antagonist to human IL-6. Preferably, the antagonists to IL-6 are monoclonal antibodies, or binding compositions derived therefrom by standard techniques. BRIEF DESCRIPTION QF THE DRAWINGS Figure 1 illustrates survival data of male BALB/c mice which were injected intraperitoneally with live E. coli and treated with various monoclonal antibodies. DETAILED DESCRIPTION OF THE INVENTION The invention is based on the discovery that increased production of IL-6 is a link in a causal chain of physiological events leading to shock in massive microbial infections, such as septicemia. The method of the invention comprises administering to a patient an effective, or disease-ameliorating, amount of an antagonist to human IL-6. Preferably, antagonists of the invention are derived from antibodies specific for human IL-6. More preferably, the antagonists of the invention comprise fragments or binding compositions specific for IL-6. Antibodies comprise an assembly of polypeptide chains linked together by disulfide bridges. Two major polypeptide chains, referred to as the light chain and the heavy chain, make up all major structural classes (isotypes) of antibody. Both heavy chains and light chains are further divided into subregions referred to as variable regions and constant regions. Heavy chains comprise a single variable region and three different constant regions, and light chains comprise a single variable region (different from that of the heavy chain) and a single constant region (different from those of the heavy chain). The variable regions of the heavy chain and light chain are responsible for the binding specificity of the antibody. As used herein, the term "heavy-chain variable region" means a polypeptide (1) which is from 110 to 125 amino acids in length, and (2) whose amino acid sequence corresponds to that of a heavy chain of a monoclonal antibody of the invention, starting from the heavy chain's N-terminal amino acid. Likewise, the term "light-chain variable region" means a polypeptide (1) which is from 95 to 115 amino acids in length, and (2) whose amino acid sequence corresponds to that of a light chain of a monoclonal antibody of the invention, starting from the light chain's N-terminal amino acid. As used herein the term "monoclonal antibody" refers to homogeneous populations of immunoglobulins which are capable of specifically binding to human IL-6. As used herein the term "binding composition" means a composition comprising two polypeptide chains, (1 ) which, when operationally associated, assume a conformation having high binding affinity for human IL-6, and (2) which are derived from a hybridoma producing monoclonal antibodies specific for human IL-6. The term "operationally associated" is meant to indicate that the two polypeptide chains can be positioned relative to one another for binding by a variety of means, for example, association in a native antibody fragment, such as Fab or Fv, or by way of genetically engineered cysteine-containing peptide linkers at the carboxyl termini. Normally, the two polypeptide chains correspond to the light-chain variable region and heavy- chain variable region of a monoclonal antibody specific for human IL-6. Preferably, antagonists of the invention are derived from monoclonal antibodies specific for human IL-6. Monoclonal antibodies capable of blocking, or neutralizing, IL-6 are selected by their ability to inhibit IL-6-induced effects in standard IL-6 bioassays: e.g. as described in Sehgal et al., Science. Vol. 235, pgs. 731-732 (1987); Billiau, Immunol. Today. Vol. 8, pgs. 84-87 (1987); and Wong et al., Immunol. Today. Vol. 9, pgs. 137-139 (1988). Preferably, inhibition of the following biological activities is used to screen for monoclonal antibodies capable of neutralizing IL-6: (i) antiviral activity, (ii) enhancement of immunoglobulin secretion, and (iii) stimulation of growth of certain EBV- transformed cell lines. Inhibition of antiviral activity can be measured in standard cytopathic effect inhibition assays, such as those described by Armstrong, Meth. Enzvmol.. Vol. 78, pgs. 381-387 (1981), and Familletti et al., Meth. Enzvmol.. vol. 78, pgs. 387-399 (1981 ), both of which are incorporated by reference. Neutralizing antibodies are selected on the basis of their ability to counteract, or negate, inhibition of cytopathic effects of a standard concentration of IL-6. Neutralizing antibodies can also be selected by their ability to prevent IL-6-stimulated growth of certain plasmacytoma cell lines, such as MOPC-315, e.g. Chiu et al., Proc. Natl. Acad. Sci. USA. Vol. 85, pgs. 7099-7103 (1988). MOPC-315 is available from the American Type Culture Collection under accession number TIB 23. Another means for selecting neutralizing antibodies is the inhibition of IL-6-stimulated immunoglobulin secretion of EBV-transformed cell lines, such as CESS and SKW6.4 (available from the ATCC under accession numbers TIB 190 and TIB 215, respectively), e.g. Hirano et al., Proc. Natl. Acad. Sci. USA. Vol. 82, pgs. 5490-5494 (1985). Antibody secretion is conveniently measured by an isotype-specific ELISA using commercially available isotype-specific monoclonal antibodies. For example, (4-6) x 10 3 CESS or SKW 6.4 cells are cultured in 200 μl of culture medium, e.g. RPM1 1640, with about 3-30 pM of IL-6. After about 3 days the supernatants are assayed for IgG concentration (CESS) or for IgM concentration (SKW 6.4). Hybridomas of the invention are produced by well known techniques. Usually, the process involves the fusion of an immortalizing cell line with a B-lymphocyte which produces the desired antibody. Alternatively, non-fusion techniques for generating immortal antibody-producing cell lines are possible, and come within the purview of the present invention, e.g. virally induced transformation: Casali et al., "Human Monoclonals from Antigen- Specific Selection of B Lymphocytes and Transformation by EBV," Science. Vol. 234, pgs. 476-479 (1986). Immortalizing cell lines are usually transformed mammalian cells, particularly myeloma cells of rodent, bovine, and human origin. Most frequently, rat or mouse myeloma cell lines are employed as a matter of convenience and availability. Techniques for obtaining the appropriate lymphocytes from mammals injected with the target antigen are well known. Generally, either peripheral blood lymphocytes (PBLs) are used if cells of human origin are desired, or spleen cells or lymph node cells are used if non-human mammalian sources are desired. A host mammal is injected with repeated dosages of the purified antigen, and the mammal is permitted to generate the desired antibody-producing cells before these are harvested for fusion with the immortalizing cell line. Techniques for fusion are also well known in the art and, in general, involve mixing the cells with a fusing agent, such as polyethylene glycol. Hybridomas are selected by standard procedures, such as HAT selection. From among these hybridomas, those secreting the desired antibody, i.e. one specific for human IL-6, are selected by assaying their culture medium by standard immunoassays, such as Western blotting, ELISA, RIA, IL-6 neutralizing capability, or the like. Antibodies are recovered from the medium using standard protein purification techniques, e.g. Tijssen, Practice and Theory of Enzyme immunoassays (Elsevier, Amsterdam, 1985). Many references are available for guidance in applying any of the above techniques: e.g. Kohler et al., Hybridoma Techniques (Cold Spring Harbor Laboratory, New York, 1980); Tijssen, Practice and Theory of Enzyme Immunoassays (Elsevier, Amsterdam, 1985); Campbell, Monoclonal Antibody Technology (Elsevier, Amsterdam, 1984); Hurrell, Monoclonal Hybridoma Antibodies: Techniques and Applications (CRC Press, Boca Raton, FL, 1982); and the like. Hybridomas producing monoclonal antibodies specific for human IL-6 are then subjected to a second screen using the IL-6 assays described above to select ones capable of blocking, or neutralizing, the biological activity of IL-6. The use and generation of fragments of antibodies is also well known, e.g. Fab fragments: Tijssen, Practice and Theory of Enzyme Immunoassays (Elsevier, Amsterdam, 1985); and Fv fragments: Hochman et al. Biochemistry. Vol. 12, pgs. 1130-1135 (1973), Sharon et al., Biochemistry. Vol. 15, pgs. 1591-1594 (1976), and Ehrlich et al., U.S. Patent 4,355,023; and antibody half molecules: Auditore-Hargreaves, U.S. Patent 4,470,925. Hybridomas and monoclonal antibodies of the invention are produced against either glycosylated or unglycosylated versions of recombinantly produced mature human IL-6. Generally, unglycosylated versions of human IL-6 are produced in E. coli. and glycosylated versions are produced in mammalian cell hosts, e.g. CV1 or COS monkey cells, mouse L cells, or the like. Recombinantly produced mature human IL-6 is produced by introducing an expression vector into a host cell using standard protocols: e.g. Maniatis et al., Molecular Cloning: A Laboratory Manual (Cold Spring Harbor Laboratory, New York, 1982); Okayama and Berg, Mol. Cell. Biol.. Vol. 2, pgs. 161-170 (1982), and Vol. 3, pgs. 280-289 (1983); Takebe et al., Mol. Cell. Bώ Vol. 8, pgs. 466-472 (1988); U.S. patent 4,599,308; U.S. patent 4,675,285; Kaufman et al., Mol. Cell. Biol., Vol. 2, pgs. 1304-1319 (1982); or the like. Construction of bacterial or mammalian expression vectors is well known in the art, once the nucleotide sequence encoding a desired protein is known or otherwise available: e.g., DeBoer in U.S. Patent 4,551 ,433 discloses promoters for use in bacterial expression vectors; Goeddel et al., in U.S. Patent 4,601 ,980, and Riggs, in U.S. Patent 4,431 ,739, disclose the production of mammalian proteins by E. coli expression systems; and Riggs (cited above), Ferretti et al., Proc. Natl. Acad. Sci. USA. Vol. 83, pgs. 599-603 (1986), Sproat et al., Nucleic Acids Research. Vol.13, pgs. 2959-2977 (1985), and Mullenbach et al., J. Biol. Chem.. Vol. 261 , pgs. 719-722 (1986) disclose how to construct synthetic genes for expression in bacteria. Accordingly, these references are incorporated by reference. The amino acid sequence of mature human IL-6 is disclosed by Hirano et al., Nature. Vol. 324, pgs. 73-76 (1986) and Zilberstein et al., EMBO J.. Vol. 5, pgs. 2529-2537 (1986); synthetic genes encoding human IL-6 are available commercially from Beckman Instruments (Fullerton, CA); and purified human IL-6 is commercially available from Genzyme Corp. (Boston, MA). Many bacterial expression vectors and hosts are available commercially and through the ATCC. Antibodies and antibody fragments characteristic of hybridomas of the invention can also be produced by recombinant means by extracting messenger RNA, constructing a cDNA library, and selecting clones which encode segments of the antibody molecule: e.g. Wall et al., Nucleic Acids Research. Vol. 5, pgs. 3113-3128 (1978); Zakut et al., Nucleic Acids Research. Vol. 8, pgs. 3591-3601 (1980); Cabiliy et al., Proc. Natl. Acad. Sri. USA. Vol. 81 , pgs. 3273-3277 (1984); Boss et al., Nucleic Acids Research. Vol. 12, pgs. 3791 -3806 (1984); Amster et al., Nucleic Acids Research. Vol. 8, pgs. 2055-2065 (1980); Moore et al., U.S. Patent 4,642,334; and Skerra et al., Science. Vol. 240, pgs. 1038-1041 (1988). In particular, such techniques can be used to produce interspecific monoclonal antibodies, wherein the binding region of one species is combined with non-binding region of the antibody of another species to reduce immunogenicity, e.g. Liu et al., Proc. Natl. Acad. Sci.. Vol. 84, pgs. 3439-3443 (1987). Antagonists of the invention are administered as a pharmaceutical composition. Such compositions contain a therapeutic amount of such an antagonist, e.g. at least one of the monoclonal antibodies of the invention, or fragments thereof, in a pharmaceutical carrier. A pharmaceutical carrier can be any compatible, non-toxic substance suitable for delivering the compositions of the invention to a patient. Sterile water, alcohol, fats, waxes, and inert solids may be included in a carrier. Pharmaceutically accepted adjuvants (buffering agents, dispersing agents) may also be incorporated into the pharmaceutical composition. Generally, compositions useful for parenteral administration of such drugs are well known, e.g. Remington's Pharmaceutical Science, 15th Ed. (Mack Publishing Company, Easton, PA 1980). Alternatively, compositions of the invention may be introduced into a patient's body by implantable or injectable drug delivery system: e.g. Urquhart et al., Ann. Rev. Pharmacol. ToxicoL Vol. 24, pgs. 199-236 (1984); Lewis, ed. Controlled Release of Pesticides and Pharmaceuticals (Plenum Press, New York, 1981 ); U.S. patent 3,773,919; U.S. patent 3,270,960; and the like. Antagonists of the invention that are derived from antibodies are normally administered parentally, preferably intravenously. Since such protein or peptide antagonists may be immunogenic, they are preferably administered slowly, either by a conventional IV administration set or from a subcutaneous depot, e.g. as taught by Tomasi et al., U.S. patent 4,732,863. When administered parenterally the antibodies or fragments will be formulated in a unit dosage injectable form (e.g., solution, suspension or emulsion) in association with a pharmaceutically acceptable parenteral vehicle. Such vehicles are inherently nontoxic and nontherapeutic. Examples of such vehicles are normal saline, Ringer's solution, dextrose solution, and Hank's solution. Nonaqueous vehicles such as fixed oils and ethyl oleate may also be used. A preferred vehicle is 5% dextrose/saline. The vehicle may contain minor amounts of additives such as substances that enhance isotonicity and chemical stability, e.g., buffers and preservatives. The antibody is preferably formulated in purified form substantially free of aggregates, other proteins, endotoxins, and the like, at concentrations of about 5 to 30 mg/ml, preferably 10 to 20 mg/ml. Preferably, the endotoxin levels are less than 2.5 EU/ml. Selecting an administration regimen for an antagonist depends on several factors, including the serum turnover rate of the antagonist, the serum level of IL-6 associated with the septecemia, the immunogenicity of the antagonist, the accessibility of the target IL-6 (e.g. if non-serum IL-6 is to be blocked), the affinity of IL-6 to its receptor(s) relative to that of IL-6 to the antagonist, and the like. Preferably, an administration regimen maximizes the amount of antagonist delivered to the patient consistent with an acceptable level of side effects. Accordingly, the amount of antagonist delivered depends in part on the particular antagonist and the severity of the condition being treated. Guidance in selecting appropriate doses is found in the literature on therapeutic uses of antibodies: e.g. Bach et al., chapter 22, in Ferrone et al., eds., Handbook of Monoclonal Antibodies (Noges Publications, Park Ridge, NJ, 1985); and Russell, pgs. 303-357, and Smith et al., pgs. 365-389, in Haber et al., eds., Antibodies in Human Diagnosis and Therapy (Raven Press, New York, 1977). Preferably, whenever the antagonist comprises monoclonal antibodies or Fab-sized fragments thereof (including binding compositions), the dose is in the range of about 1-20 mg/kg per day. More preferably the dose is in the range of about 1 -10 mg/kg per day. EXAMPLES The following examples serve to illustrate aspects of the present invention. The selected vectors, hosts, fusion partners, concentration of reagents, temperatures, and the values of other variables are only to exemplify the invention and are not to be considered limitations thereof. Example I. Production of IL-6-Neutralizing Monoclonal Antibody A male Lewis rat was immunized with semi-purified preparations of COS7-cell expressed mouse IL-6 (this expression system being described by Lee et al., Annals N.Y. Acad. Sci.. Vol. 557, pgs. 215-229 (1989) and by Chui et al., Proc. Natl. Acad. Sci. USA. Vol. 85, 7099-7103 (1988)). The rat was first immunized with approximately 50 μg of mouse IL-6 in Freund's Complete Adjuvant, and boosted twice with the same amount of material in Freund's Incomplete Adjuvant. Test bleeds were taken. The animal was given a final boost of 25 μg in phosphate-buffered saline, and four days later the spleen was obtained for fusion. Approximately 3 x 10 8 rat splenocytes were fused with an equal number of P3X63-AG8.653 mouse myeloma cells (available from the ATCC under accession number CRL 1580). 3840 microtiter plate wells were seeded at 5.7 x 10 4 parental myeloma cells per well. Half of these wells were initially cultured with approximately 22 units/well of recombinant human IL-6. Units are defined in relation to the growth of the cell line NFS-60 in response to human IL-6, described by Chiu et al. (cited above). Another 576 wells were seeded at 1.9 x 10 5 parents per well. Standard protocols for the fusion and subsequent culturing of hybrids were followed, and were substantially the same as those described by Chretien et al., J. Immunol. Meth.. Vol. 117, pgs. 67-81 (1989). Twelve days after fusion, supernatants were harvested and screened (1 ) by indirect ELISA on PVC plates coated with COS7-produced mouse IL-6, and (2) by their ability to inhibit growth of NFS-60 cells, which are IL-6 dependent. The above screening protocol was used because of the hybridoma growth factor activity attributed to IL-6. It was reasoned that neutralizing anti-IL-6 antibodies initially produced by these hybridomas might prevent their growth by depleting the IL-6 in the culture medium. Thus, some wells were seeded with human IL-6 (hlL-6) which is active on mouse cells. In every case about the same fraction of neutralizing antibodies was produced, as shown in the Table below. TABLE I Initial seeding Number of wells Number of Percent of seeded condition containing wells seeded wells with neutralizing neutralizing Mabs anti-IL-6 No hlL-6; 1920 0.36% 5.7x10 4 cells/well 22 U/well hlL-6; 8 1920 0.41% 5.7x10 4 cells/well No hlL-6; 576 1.0% 1.9x10 5 cells/well Monoclonal antibody from a hybridoma designated MP5-20F3 was used in the experiments of Example II. Example II. Prophylactic effect of neutralizing anti-IL-6 Mab on mice injected with a lethal dose of E. coli Male BALB/c mice (19-21 g) were randomly assigned to five different treatment groups A - E, and experiments involving the groups were set up on five different days. In Group A (30 mice), each mouse was given by intraperi- toneal injection 0.75 to 1.00 mg of monoclonal antibody (Mab) against recom- binant mouse IL-6 (anti-recombinant mouse IL-6 monoclonal antibody). In Group B (30 mice), each mouse was given by intraperitoneal injection 0.1 to 1.0 mg rabbit anti-recombinant mouse TNFα IgG. In Group C (30 mice), each mouse was given by intraperitoneal injection 0.5 to 1.0 mg isotype control Mab GL113. In Group D (5 mice), each mouse was given by intraperitoneal injection an isotype control Mab with a 2-fold higher level of endotoxin than that of the anti-IL-6 Mab. Finally, in Group E (6 mice), each mouse was given by intraperitoneal injection a 100-fold lower dose of anti-recombinant mouse IL-6 than given to mice in Group A. For each group, two hours after the initial injection, each mouse was given by intraperitoneal injection 1.2 x 10 8 live E. coli (obtained from the ATCC under accession number 25922) in 0.2 to 0.3 ml of saline. The E. coli were cultured on trypticase soy agar plates with 5% sheep blood and were counted by spectrophotometric determination. The effects of the various experimental regimens on the survival of the mice are shown in Figure 1 , where the y-axis shows the percent of surviving mice and the x-axis shows the time in hours after the mice were injected with E. coli. Only the groups of mice receiving a high dose of anti IL-6 antibody or rabbit anti-recombinant mouse TNFα IgG showed any survival more than 24 hours after injection with E. coli: these two groups of mice showed a significant survival rate even 72 hours after injection with E. coli. The descriptions of the foregoing embodiments of the invention have been presented for purpose of illustration and description. They are not intended to be exhaustive or to limit the invention to the precise forms disclosed, and obviously many modifications and variations are possible in light of the above teaching. The embodiments were chosen and described in order to best explain the principles of the invention to thereby enable others skilled in the art to best utilize the invention in various embodiments and with various modifications as are suited to the particular use contemplated. It is intended that the scope of the invention be defined by the claims appended hereto.

Claims: CLAIMS: 1. A method of treating septic shock in a person comprising administering an effective amount of an antagonist to human IL-6. 2. The method of claim 1 wherein said antagonist to human IL-6 is selected from the group consisting of a monoclonal antibody capable of blocking the biological activity of human IL-6, a fragment of a monoclonal antibody capable of blocking the biological activity of human IL-6, and a binding composition comprising the heavy-chain variable region and light- chain variable region of a monoclonal antibody capable of blocking the biological activity of human IL-6. 3. The method of claim 2 wherein said fragment of said monoclonal antibody is an Fab fragment. 4. The method of claim 2 wherein said monoclonal antibody is produced by a human-human hybridoma. 5. The method of claim 4 wherein said fragment of said monoclonal antibody is an Fab fragment. 6. The method of claim 2 wherein said step of administering further includes intravenous delivery of an amount of said antagonist in the range of about 1-20 mg/kg body weight of said individual per day. 7. A pharmaceutical composition comprising, as active ingredient, an antagonist against human IL-6, together with a pharmaceutical carrier or excipient. 8. The pharmaceutical composition of claim 7 wherein the antagonist is a monoclonal antibody, a fragment of a monoclonal antibody capable of blocking the biological activity of human IL-6, especially an Fab fragment, or a binding composition comprising the heavy-chain variable region and light- chain variable region of a monoclonal antibody capable of blocking the biological activity of human IL-6. 9. Monoclonal antibodies against human IL-6. 10. Hybridomas producing monoclonal antibodies against human IL-6, especially the hybridoma designated MP5-20F3 herein. 11. Use of an antagonist against human IL-6 for the preparation of pharmaceutical compositions useful in the treatment of septic shock. 12. Use as claimed in claim 11 wherein the antagonist is a monoclonal antibody, a fragment of a monoclonal antibody capable of blocking the biological activity of human IL-6, especially an Fab fragment, or a binding composition comprising the heavy-chain variable region and light-chain variable region of a monoclonal antibody capable of blocking the biological activity of human IL-6.

Inventors: ABRAMS JOHN S, STARNES H FLETCHER, ABRAMS, JOHN, S., STARNES, H., FLETCHER
Applicants: FLETCHER STARNES H, SCHERING CORP, SCHERING CORPORATION, STARNES, H., FLETCHER
Application year: 1990
Main classification: A61K39
IPC codes: A61K38, A61K39, A61K45, A61P9, A61P31, C07K14, C07K16, C12N5, C12N15, C12P21, C12R1