METHOD FOR TREATING ENDOTOXIN SHOCK USING ANTI-ADHESION ANTIBODIES

Abstract: Novel method for treating a patient suffering from endotoxin shock which comprises the administration of an anti-CD18 antibody or an anti-ICAM-1 antibody, or fragments thereof.

Description: METHOD FOR TREATING ENDOTOXIN SHOCK USING ANTI-ADHESION ANTIBODIES FIELD OF THE INVENTION This invention relates a novel method for treating endotoxin shock using anti-adhesion antibodies. More particularly, this invention relates to a method for treating endotoxin shock using anti-CD18 and anti-ICAM-1 antibodies. BACKGROUND OF THE INVENTION Cellular adhesion is a process through which leukocytes attach to cellular substrates, such as endothelial cells, in order to migrate from circulation to sites of ongoing inflammation, and properly defend the host against foreign invaders such as bacteria or viruses. An excellent review of the defense system is provided by Eisen, H. . , Microbiology, 3rd Ed., Harper & Row, Philadelphia, PA (1980), pp. 290-295 and 381-418. One of the molecules on the surface of endothelial cells which participates in the adhesion process is the intercellular adhesion molecule ICAM-1. See Rothlein et al, J. Immunol. 137: 1270 (1986) , herein incorporated by reference. This molecule has been shown to mediate adhesion by binding to molecules of the CD18 family of glycoproteins which are present on the cell surfaces of leukocytes [Sanchez- Madrid et al, J. Exper. Med. 158: 1785 (1983) ; Keiser et al, Eur. J. Immunol. 15: 1142 (1985)]. This glyprotein family is composed of heterodimers having one alpha chain and one beta chain. Although the alpha chain of each of the proteins differes from one another, the beta chain was found to be highly conserved (Sanchez-Madrid et al, supra) . The beta chain of the glycoprotein family (sometimes referred to as "CD18") was found to have a molecular weight of 95 kd whereas the alpha chains were found to vary from 150 kd to 180 kd [Springer, T. , Fed. Proc. 4 : 2660 (1985)]. Although the alpha subunits of the membrane proteins do not share the extensive homology shared by the beta subunits, close analysis of the alpha subunits of the glycoproteins has revealed that there are substantial similarites between them. There are three major members of the CD18 family: Mac-1, LFA-1 and pl50,95. Mac-1 is a heterodimer found on macrophages, granulocytes and large granular lymphocytes. LFA-1 is a heterodimer found on most lymphocytes [Springer et al, Immunol. Rev. 68.: Ill (1982)]. pl50,95 has a tissue distribution similar to Mac-1, and also plays a role in cellular adhesion [Keiser et al, Eur. J. Immunol. 35: 1142 (1985) ] . Reviews of the similarities between the alpha and beta subunits of the LFA-1-related glycoproteins are provided by Sanchez-Madrid et al (supra) and Sanchez-Madrid et al, J. Exper. Med. 158: 586 (1983). Anti-CD18 antibodies have been described as useful in ' treating hemorrhagic shock in rabbits. Vedder et al, J. Clin. Invest. JJl: 939 (1988) . Anti-CD18 antibodies have been shown not to increase susceptibility to sepsis when used to inhibit neutrophil adherence in rabbits. Mileski et al, Surgical Forum. Infection and its Mediators, p. 107 (1989) . U.S. Patent no. 4,388,318 describes a method for treating endotoxin shock using a pyrimido-pyrimidine derivative. U.S. Patents Nos. 4,472,420 and 4,472,431 describe a method for treating shock using benoxaprofen and fenoprofen, respectively. Prior to present invention, however, no method was known for treating endotoxin shock using anti-ICAM-1 antibodies. Accordingly, it is the purpose of this invention to provide a novel method for treating endotoxin shock using anti-CD18 and anti-ICAM-l antibodies. BRIEF DESCRIPTION OF FIGURE 1 J Figure 1 is a graphic representation of the survival of rabbits suffering from septic (endotoxin) shock, treated with saline or monoclonal anti-ICAM-l antibody R6.5. DESCRIPTION OF THE INVENTION This invention relates to a method for treating endotoxin shock in a patient which comprises administering to the patient a therapeutically effective amount of an anti-CD18 antibody or an anti-ICAM-l antibody, or fragment thereof. The anti-CD18 antibody and anti-ICAM-l antibody useful in the method of this invention, can be prepared using known procedures, e.g., by immunopurification of polyclonal serum, by hybridoma technology or by recombinant DNA techniques. See, e.g., Kohler et al, PNAS USA 22(4): 2197 (1980) and U.S. Patent No. 4,816,397. Preferably, the anti-CD18 antibody and anti-ICAM-l antibody is a monoclonal antibody. Monoclonal anti-CD18 antibody can be prepared in the following manner. Mice are immunized with the CD18-containing cells. The mice are later sacrificed and their spleens removed. Spleen cells are fused with myeloma cells to produce hybridomas. The hybridomas are cultivated and their εupernatants screened for anti-CD18 activity, e.g., by ELISA or RIA, or by an aggregation assay as described in Rothlein et al (supra) . Hybridomas secreting monoclonal antibodies with desired anti-CD18 activity are cloned and cultivated. Monoclonal anti-ICAM-l antibody can be prepared in a similar manner by initially immunizing the mice with ICAM-l or ICAM-1-containing cells. Several methods for administering the anti-CD18 antibody or anti-ICAM-l antibody to the patient can be used including, for example, intradermal, subcutaneous, intramuscular, intraperitoneal, and intravenous routes. Preferably, administration of the anti-CD18 or anti-ICAM-l antibody utilizes subcutaneous or intramuscular injection of the anti- CD18 or anti-ICAM-l antibody in the presence of various adjuvants. The amount of anti-CD18 or anti-ICAM-l antibody to be admimistered will vary depending on the severity of the shock, route of administration, etc., but, in general, 0.1 mg/kg to 10 mg/kg or higher, of antibody can be administered to the patient. The anti-CD18 or anti-ICAM-l antibody can be formulated with a suitable adjuvant, including, e.g., mineral gels such as aluminum hydroxide; surface active substances such lysolecithin; pluronic polyols; polyanions; peptides; or oil emulsions. The anti-CD18 or anti-ICAM-l antibodies can be utilized as whole antibodies in the method of this invention or as fragments. The anti-CD18 or anti-ICAM-l antibody fragments must comprise at least the binding site of the anti-CD18 or anti-ICAM-l antibody. Useful fragments of the anti-CD18 or anti-ICAM-l antibody include the Fab and F(ab) 2 fragments. Fragments of the anti-CD18 or anti-ICAM-l antibody can be prepared using known procedures, e.g., by digestion of the antibody with proteolytic enzymes or by recombinant DNA techniques. The following examples illustrate the present invention. EXAMPLE 1 A. PREPARATION OF MONOCLONAL ANTI-CD18 ANTIBODY CR15.7) The monoclonal anti-CD18 antibody, R15.7, was prepared as described in Ent an et al, J. Clin. Invest. 85_: 1497 (1990) , herein incorporated by reference. Briefly, BALB/c mice were immunized with canine peritoneal macrophages. Spleen cells from the immunized mice were fused with P3X63Ag8.653 myeloma cells to create hybridomas. Supernatants of the hybridomas were screened for inhibition of JY cell and SK 3 aggregation. Clones positive in both assays were then screened for binding to mouse/human hybrid cells expressing mouse CDlla and human CD18. One clone, R15.7, was selected. Ascites fluid was prepared in pristane-primed BALB/c mice for the R15.7 clone and the R15.7 antibody was isolated on protein A. R15.7 is a murine IgG*, ^ antibody to canine CD18. Although directed against canine CD18, R15.7 cross reacts with human CD18 and rabbit CD18. B. PREPARATION OF ANIMALS White (NZW) rabbits (Hare Marland, Hewith, NJ) weighing between 2.5 and 4.0 kg were primed with lOμg/kg E. Coli lipopolysaccharide (LPS) by intravenous injection into the right marginal ear vein (priming dose) . The rabbits were anesthetized by i.m. injection of ketamine-HCl and xylazine, 50 mg/kg and 5.7 mg/kg, respectively. Systemic blood pressure was monitored via the right carotid artery. 17.5 hours after the priming dose, the rabbits were given intravenous injections into the right marginal ear vein with one of the following test compounds: i) Sterile saline ii) Mouse IgG (l mg/kg) iii) R15.7 (l mg/kg) 0.5 hours after injection of the test compounds, the rabbits were then given a second LPS intravenous injection (100 Mg/kg) . Blood samples were taken from the rabbits after the priming dose; after injection of the sterile saline, mouse IgG or R15.7; after the second LPS injection; 1 hour after the second LPS injection; and 2 hours after the second LPS injection. C. RESULTS df All 5 saline-treated rabbits developed peripheral leukopenia with marked neutropenia and thrombocytopenia; four experienced severe hypotension, expiring within 30 minutes (27±3.6 min.) after the second LPS injection. All 3 rabbits treated with mouse IgG experience severe fall in systemic blood pressure, hematologic parameters similar to the saline-treated rabbits, and all 3 died after the second LPS injection (18, 44 and 114 minutes, respectively) . All rabbits treated with R15.7 survived 2 hours after the second LPS injection. Mean arterial pressure decreased only slightly (15.5-19.3 mm Hg by 2 hours). Peripheral neutrophil counts remained significantly higher (p<.05) in R15.7 rabbits than in the saline or mouse IgG treated rabbits. EXAMPLE 2 A. PREPARATION OF MONOCLONAL ANTI-ICAM-1 ANTIBODY (R6.5) The monoclonal anti-ICAM-l antibody R6.5 was prepared as described in European Patent Application No. 289,949, from hybridoma cell line R6 I 506'E9 » B2 (ATCC HB 9580). B. PREPARATION OF ANIMALS Male white New Zealand rabbits (Hazelton Research, Denver, PA), weighing between 2.0 and 4.0 kg, were given an intravenous injection with one of the following: i) Sterile saline (0.2 ml/kg); or ii) R6.5 (2.0 mg/kg). The R6.5 was in a solution of sterile saline. Following a period of 0.5 hours, the rabbits were primed with 20 μg/kg lipopolysaccharide (LPS) (Sigma E. Coli. LPS, serotype 0111:B4, lot 39F4030) , by intravenous injection into the right marginal ear vein. After 18 hours the rabbits were anesthetized by intramuscular ketamine (50 mg/kg)/xylazine (6 mg/kg) followed by a constant xylazine (0.32 mg/kg/min) infusion. Systemic blood pressure was monitored via the right carotid artery by a P23 10 Gould/Statham pressure transducer. Rabbits initially receiving sterile saline were then given a second intravenous injection of sterile saline (0.2 ml/kg) • into the right marginal ear vein. Animals receiving R6.5 as a priming dose were given a second intravenous injection of R6.5 (2.0 mg/kg) delivered in a solution of sterile saline. 0.5 hours later, the rabbits were given an additional intravenous injection of LPS (400 μg/kg) and monitored for 2 hours. Blood samples of 1.0 ml were taken from the rabbits via the carotid artery catheter before the initial injection of sterile saline or R6.5; before the second injection of either sterile saline or R6.5; before the second (additional) injection of LPS; and 30 minutes, 1 hour and 2 hours after the second injection of LPS. C. RESULTS Only 1 of the 4 saline-treated rabbits survived the entire protocol (Figure 1) . All of the saline-treated rabbits developed an inflammatory response (conjunctival discharge, rhinorrhea) following the initial LPS injection. Two the these saline-treated rabbits died prior to the second LPS injection. The other 2 rabbits experienced severe hypotension with 1 expiring within 30 minutes after the second LPS injection. All 5 rabbits treated with R6.5 survived until the second LPS injection. There was noticeably less of an inflammatory 9 response following the initial LPS injection in the R6.5- treated rabbits. Four out of the 5 rabbits treated with R6.5 survived the entire protocol (Figure 1). One of the R6.5- treated rabbits expired 30 minutes following the second LPS injection. In the 4 R6.5-treated rabbits which survived, mean arterial pressure decreased only slightly (16±5 mmHg) by the end of the experiment.

Claims: WHAT IS CLAIMED IS: 1. A method for treating endotoxin shock in a patient which comprises administering to the patient a therapeutically effective amount of an anti-ICAM-l antibody or fragment thereof. 2. A method as recited in claim 1 wherein the entire anti- ICAM-l antibody is administrated. 3. A method as recited in claim 1 wherein a fragment of the anti-ICAM-l antibody is administered. 4. A method as recited in claim 3 wherein the fragment is an Fab or F(ab) 2 fragment. 5. A method as recited in claim 1 wherein the anti-ICAM-l antibody or fragment thereof, is administered intravenously. 6. A method as recited in claim 1 wherein the anti-ICAM-l antibody is R6.5.

Inventors: LETTS LINDSAY GORDON, POSSANZA GENUS J, WELDON STEVEN M, LETTS, LINDSAY, GORDON, POSSANZA, GENUS, J., WELDON, STEVEN, M.
Applicants: BOEHRINGER INGELHEIM PHARMA, BOEHRINGER INGELHEIM PHARMACEUTICALS, INC.
Application year: 1991
Main classification: A61K35
IPC codes: A61K35, A61K38, A61K39, A61P31, C07K16, C12N15, C12P21