AU673508B2 - Directed human immune globulin for the prevention and treatment of staphylococcal infections - Google Patents

Description

OPI DATE 13/09/93 AOJP DATE 25/11/93 APPLN. ID .32718/93 Ilflll111111ii PCT NUMBER PCT/US92/09830 IIIIII ILliIII I AU9332718

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(51) International Patent Classification 5 (U1) International Publication Number: WO 93/17044 C07K 15/06, A61IK 35/16, 39/40 Al (3 nentoa ulcto ae etme 93(20.3 A61K 39/085, 39/395, 49/00 (3 nentoa ulcto ae etme 93(20.3 C12Q_1/00,_GOIN_33/536 (21) International Application Number: (22) International Filing Date: 9 IN Priority data: 804,317 25 Febru PCT/US92/09830 Jovember 1992 (09.11.92) ary 1992 (25.02.92) US 4W~72)&p :ln~dIventor: FISCHER, Gerald, W. [US/ US]; 6417 Lybrook Drive, Bethesda, MD 20817 (US).

(74) Agent: BELLAMY, Werten, Intellectual Property Law Division, Office of The Judge Advocate General, DA, 901 N. Stuart Street, Suite 400, Arlington, VA 22203-1837 (US).

(81) Designated States: AT, A U, BB, BG. BR, CA, CH. *CS.

DE, DK, ES, Fl, GB, HU, JP, KP, KR, LK, LU, MG, MN, MW, NL, NO, PL, RO, RU, SD, SE, US. European patent (AT, BE, CH, DE, DK, ES, FR, GB, GR, IE, IT, LU, MC, NL, SE), OAPI patent (BF. 13J. CF.

CG, CI, CM, GA, GN, ML, MR, SN, TD, TG).

Published Wi~th international search report.

4~ uJo Pc 0 0+.

\AT O/ -1 .1 1 C k! C -r (54)Title: DIRECTED HUMAN IMMUNE GLOBULIN FOR THE PREVENTION AND TREATMENT OF STAPHYLO- COCCAL INFECTIONS (57) Abstract This invention is directkd to a Directed Human Immunoglobulin and~ compositions thereof for preventing or treating staphylococcal infections such as S. epidermidis.

6 738 WO 93/17044 PCT/US92/09830 -1- 1 DIRECTED HUMAN IMMUNE GLOBULIN FOR THE PREVENTION 2 AND TREATMENT OF STAPHYLOCOCCAL INFECTIONS 3 I. GOVERNMENT INTEREST 4 The invention described herein may be manufactured, licensed uQriec) S Otes qo\emnr.4O\\ and used by or for/gees metal purposes without the payment of any royalties 6 to us thereon.

7 II. FIELD OF THE INVENTION 8 This invention relates to Directed Human Immune Globulin for 9 the prevention and treatment of staphylococcal infections.

1II. BACKGROUND OF THE INVENTION 11 Over the last two decades, staphylococci have become important 12 causes of infection in hospitalized patients. Because of their high prevalence 13 on the skin, staphylococci are ideally situated to cause serious infections in 14 debilitated or immunosuppressed patients. The st ,)hylococcal species most frequently pathogenic in humans are Staphylococcus aureus (SA) and 16 Staphylococcus epidermidis Both groups have developed resistance to 17 multiple antibiotics making antimicrobial therapy difficult. In recent years SE 18 has become a major cause of nosocomial infection in patients whose treatments 19 include the placement of foreign materials such as cerebrospinal fluid shunts, WO 93/17044 PCT/US92/09830 -2- 1 vascular catheters or joint prostheses. SE is a common cause of post operative 2 wound infections peritonitis in patients with continuous ambulatory peritoneal 3 dialysis. Patients with impaired immunity (malignancy, bone marrow 4 transplant) or those receiving parenteral nutrition through central venous catheter are also at high risk for developing SE sepsis (Patrick, J. Pediat., 6 1990).

7 SE has emerged as a common cause of neonatal nosocomial 8 sepsis in premature infants. As shown by Fleer and colleagues, (Pediatr Infect 9 Dis, 1983) SE infections frequently occur in immature babies that have received parenteral nutrition. Premature babies have impaired immunity with 11 deficiencies in antibodies, complement and neutrophil function. Lipid infusion 12 is now a standard ingredient of parenteral nutrition therapy in many nurseries 13 and may further impair immunity to bacterial infection as disclosed by Fischer 14 and colleagues (Lancet, 1980; 2:819-20). Recent studies have associated coagulase negative staphylococcal bacteria in neonates with lipid emulsion 16 infusion (Freeman and colleagues, N. Engl. J. Med, 1990). Further studies by 17 Fleer and colleagues (J Inf Dis, 1985) showed that neonates had low levels of 18 opsonic antibody to SE despite the fact that the sera had clearly detectable 19 levels of IgG antibodies to SE peptidoglycan (opsonic antibodies for staphylococcus have been considered to be directed to the peptidoglycan 21 antigens). While these studies suggested that neonatal susceptibility to SE 22 might be related to impaired oposonic activity, it is not clear if antibodies WO 93/17044 PCr/US92/09830 -3- 1 directed against SE are opsonic or would be capable of providing protection 2 when given passively to neonates. Further, it is unknown whether the 3 presence of intralipid, which further impairs phagocytosis and killing of 4 bacteria by phagocytes, would inhibit the activity of antibody.

The opsonic activity of pooled human immunoglobulin for SE 6 was studied by Clark and colleagues (J Med Microbiol, 1986), and showed that 7 complement and IgG were both critical for efficient opsonization of SE. They 8 noted, however, that in some studies complement was not required and that 9 contrary to the report of Fleer (1985), absorption of serum with peptidoglycan may remove the opsonic activity for SE. Further studies by Clark and Easmon 11 (1986) showed that several lots of standard intravenous immune globulin 12 (VIG) had variable opsonic activity for SE. One third of the IVIG lots had 13 poor opsonization with complement and only 2 of 14 were opsonic without 14 complement. Despite the fact that the IVIG lots are made from large plasma donor pools good opsonic antibody to SE was not uniformly present. Their 16 studies focused on potential use of immunoglobulin to boost peritoneal defenses 17 in patients receiving continuous ambulatory peritoneal dialysis and did not 18 examine whether IVIG could be utilized for the prevention or treatment of 19 bacterial sepsis, or the use of antibody to prevent or treat sepsis and lethal infection in immature or immunosuppressed patients and Specifically, no in 21 vivo studies were done to test antibody to prevent or treat WO 93/17044 PCT/US92/09830 1 SE. There is no evidence therefore that the antibody would provide beneficial 2 therapy in a setting of immaturity or impaired immunity.

3 The opsonic assays, that are currently used are slow and 4 cumbersome for screening blood, plasma or immune globulin for antibodies to SE. It would be important to have a rapid antigen binding assay to screen for 6 SE antibody, if that assay further correlated with opsonic activity in vitro and 7 protection in viyo.

8 In order to determine if IgG is capable of enhancing protection 9 against SE, a suitable animal model that is comparable to patients with SE infections is reauired. This is critical since neonates have low levels of 11 complement and impaired neutrophil and macrophage function. While opsonic 12 activity of immune globulin may be adequate under optimal conditions in vitro, 13 protection may not occur in patients with immature or impaired immune 14 systems. As has been demonstrated by Clark and colleagues (J Clin Pathol, 1986), most IVIG preparations were not opsonic when complement was 16 removed. However, since SE has low virulence, suitable animal models of SE 17 sepsis have not been available.

18 Yoshida and collegues, (J Microbiol, 1976) reported on a 19 virulent strain of SE that infected mature mice with 90 100% of mice dying within 24 48 hours. This model is very different from that seen in patients 21 and may represent an unusual type of SE infection. When they analyzed 22 fresh isolates of SE from humans, they were not able to kill mice. Non-human WO 93/17044 PCT/US92/09830 1 antibody to a new SE surface polysaccharide protected the mice from the 2 virulent SE strain. A later report by Yoshida and colleagues (J Med 3 Microbiol, 1977) confirmed their previous observations. Passive prophylaxis 4 with immunization induced non-human antibody showed that the IgG fraction did not protect while the IgM fraction did provide protection. Thus 6 demonstrating in this model that IgG antibody was not protective. As noted 7 previously herein neonates had good levels of IgG to SE, but had low levels of 8 opsonic antibody (Fleer and colleagues, J. Infect. Dis, 1985), consistent with 9 the findings in this study and showing that the role of IgG in protection against SE is unclear. In 1987 the report by Ichiman and colleagues (J Appl Bacteriol, 11 1987) extended their animal studies to include analysis of protective antibodies 12 in human serum against their selected virulent strains of SE. Protective 13 antibody was found in the IgA, IgM and IgG immunoglobulin fractions. These 14 studies are in conflict with their, previous data showing that IgG was not protective and fails to establish a definitive role for any of the immunoglobulin 16 classes (IgG, IgM or IgA).

17 In the animal model described by Yoshida, Ichiman and 18 colleagues mature, non-immunosuppressed mice were used and death was 19 considered to be related to toxins not sepsis (Yoshida and colleagues, J.

Microbiol, 1976). Most clinical isolates did not cause lethal infections in their 21 model. Since quantitative blood cultures were not done, it is not known 22 whether antibody would prevent or treat SE sepsis in immature WO 93/17044 PCT/US92/09830 -6- 1 immunosupressed patients or specifically in the presence of intralipid.

2 Antibody provides protection in humans against certain 3 encapsulated bacteria such as Hemophilus influenzae and Streptococus 4 pneumoniae. Individuals such as young infants who are deficient in antibody are susceptible to infections with these bacteria and bacteremia and sepsis are 6 common. When antibody to these bacteria is presenit it provides protection by 7 promoting clearance of the bacteria from the blood. Immunoglobulin with 8 antibody to influenzae and S pneumoniae protects infants from sepsis with 9 these bacteria. The article by Espersen and colleagues, (Arch Intern Med, 1987) discloses the use of an antigen binding RIA assay to analyze IgG 11 antibody to SE in patients with uncomplicated bacteremia and those with 12 bacteremia and endocarditis. This assay used an ultrasonic extract of SE to 13 identify SE specific IgG (the surface antigen in this study differs from the 14 antigen used by Yoshida and colleagues which was obtained by a different method; gentle sonic oscillation). None of the patients with uncomplicated 16 bactermia had IgG antibodies to SE. These data would suggest that IgG is 17 unnecessary for effective eradication of SE from the blood. In addition, 89% 18 of bacteremic patients with endocarditis developed high levels of IgG to SE.

19 In these patients, IgG was not protective since high levels of IgG antibody (which may have developed late) were associated with serious bacteremia and 21 endocarditis. Based on these studies the protective role of IgG in SE sepsis 22 and indocarditis is not established, especially in the presence of immaturity, WO 93/17044 PCT/US92/09830 -7- 1 debilitation, intralipid infusion, or immunosuppresion. In addition, the 2 extensive review of Patrick et al. Pediat., 1990) does not include 3 immunoglobulin as a potential prophylactic or therapeutic afent for SE 4 infections.

It has been recognized by the medical community that SE is an 6 important pathogen in certain high risk individuals, such as patients with 7 foreign body implants, premature neonates and immunosuppressed patients.

8 Accordingly there is a need for a human immune globulin that would prevent 9 or treat SE infections such as, sepsis or endocarditis and promote clearance of SE from the blood of such high risk people.

11 IV. SUMMARY OF THE INVENTION 12 It is therefore an object of the present invention to provide a 13 novel Directed Human Immune Globulin for preventing or treating 14 staphylococcal infections. We have found that it is useful to screen serum (plasma) or pooled immunoglobulin for specific antibody to S. epidermidis to 16 produce Directed Human Immune Globulin to this pathogen. This Directed 17 Human Immune Globulin is different from standard human immune globulin 18 preparations in that it has high levels of human anti-staphylococcal antibodies 19 that react with surface antigens of S. epidermidis and enhance phagocytosis and killing of epidermidis in vitro, (opsonophagocytic bactericidal activity 21 greater than In addition, Directed Human Immune Globulin for S.

I WO 93/17044 PCT/US92/09830 -8- 1 epidermidis enhances immunity in vivo and prevents lethal infection as well as 2 enhancing clearance of S epidermidis from the blood in conditions of 3 immaturity and impaired immunity. This is surprising since 4 immunosuppression or immaturity would be expected to render the antibody ineffective by impairing the ability of phagocytic cells to engulf and kill the S.

6 epidermidis.

7 It is also another advantageous object of the present invention 8 that while standard immunoglobulin pools or normal donors do not have 9 reliable levels of opsonic antibody for S. epidermidis, Directed Human Immune Globulin when given intravenously immediately provides specific 11 antibodies to promote phagocytosis and killing of S. epidermidis by 12 phagocytes. A further advantages of the present invention is that by providing 13 opsonic antibody to immature or immunosuppressed patients infected with SE, 14 antibiotic therapy may be enhanced by improved epidermidis clearance from the blood or site of infection. Another advantage is that since Directed Human 16 Immune Globulin given intravenously or intramuscularly can raise the level of 17 antibodies in the blood of patients, Directed Human Immune Globolin could 18 prevent epidermidis from causing bacteremia and local infections.

19 The method of producing the Directed Human Immune Globulin for idermidis involves: 21 a) screening plasma (pools of immunoglobulin or plasma; 22 immunoglobulin or immunoglobulin preparations) for antibodies to S WO 93/17044 PCT/US92/09830 -9- 1 epidermidis using an in vitr antigen-binding assay: (ELISA), followed by 2 confirmation of functional activity using an in vitro opsonophagocytic 3 bactericidal assay (bactericidal activity greater than 4 b) Protective efficacy can be documented in vivo by analyzing protective activity of the Directed Human Immune Globulin using a 6 suckling rat model of neonatal S. epidermidis sepsis (mortality and bacterial 7 clearance). We believe that this is the first in vivo model to test antibody 8 effectiveness in the presence of immaturity and/or intralipid induced immune 9 suppression.

These methods could be repeated using other staphylococci such as SA 11 instead of SE to produce Directed Human Immune Globulin for S. aureus.

12 This novel Directed Human Immune Globulin for SE could be used to 13 prevent lethal SE infections in high risk patients such as neonates and adults in 14 intensive care units or patients with in-dwelling foreign bodies such as venous and arterial catheters or ventricular shunts. Directed Human Immune Globulin 16 could also be used in addition to antibiotics as adjunctive therapy to enhance 17 bacterial clearance in patients treated for SE infections.

18 Other objects, features and advantages of the present invention will 19 become apparent from the following detailed description. It should be understood, however, that the detailed description and specific examples, while 21 indicating preferred embodiments of the invention, are given by way of 22 illustration only, since various changes and modifications within the spirit and WO 93/17044 PCT/US92/09830 1 scope of the invention will become apparent to those skilled in the art from 2 this detailed description.

3 The terms Standard Human Immunoglobulin and Directed 4 Human Immune Globulin for &S epidermidis as used in this application are defined as follows: Standard Human Immunoglobulin immune human 6 globulin that was prepared by pooling immunoglobulin from many donors, 7 without selecting donors or screening the immunoglobulin to ensure antibody 8 acitivity for S. Epidermidis.

9 Directed Human Immune Globulin for epidermidis Immune globulin prepared by screening for antibody to S, epidermidis (Bactericidal 11 Activity thereby providing a human immune globulin with protective 12 levels of antibody to S. epidermidis and suitable for preventing or treating S.

13 epidermidis infections. Bactericidal Activity-The percentage of bacteria killed 14 with the addition of antibody, using a neutrophil mediated opsonophagocytic bactericidal assay after 2 hours of incubation at 37 0

C.

16 V, BRIEF DESCRIPTION OF THE DRAWINGS 17 Figure 1 18 Figure 1 shows that when several pools of human standard intravenous 19 immunglobulin were analyzed, there was a marked difference in the antibody activity to I epidermidis as measured by an antigen binding assay (ELISA, WO 93/17044 PCT/ US92/09830 -11- 1 highest 0.0. reading at 1 1/2 hrs using 1: 00 Dil). These were large pools of 2 IgG, purified by several companies using various techniques. Of three pools 3 with the highest titers, two were from Cutter Laboratories, Berkeley 4 California, (40P07, 40R09) and one was from Sandoz, East Hanover, N.J.

(069). One preparation from Cutter also had next to the lowest activity 6 (2801). These data show that standard unscreened human immunoglobulin has 7 variable levels of antibody to S, epidermidis and that no single method used to 8 prepare the immunoglobulin or utilizing a large donor pool size will ensure 9 good antibody activity to S. epidermidis. In addition, a donor was shown to have high antibody activity (Sam) to S. epidermidis demonstrating the 11 feasibility of identifying units of plasma or, plasma donors with high levels of 12 antibodies to staphylococcus.

13 Figure 2 14 Figure 2 shows that using an in vitro functional (opsonic) assay that measures the ability of immunoglobulin to promote phagocytosis and killing of 16 2 epidermidis by neutrophils in the presence of complement, that opsonic 17 activity is also variable in various lots and preparations of standard human 18 immunoglobulin. The figure also shows that the immunoglobulins identified 19 by ELISA as having high levels of antibody to S. epidermidis also had high levels of functional antibody in vitro. This is critical since this study shows 21 that IgG that binds to TCA extracted S. epidermidis antigen will promote WO 93/17044 PCT/US92/09830 -12- 1 phagocytosis and killing of S. epidermidis. Therefore, using i vitro screening 2 assays, one could select a Directed Human Immune Globulin for 1 3 epidermidis that would have reliable levels of antibody to prevent or treat S.

4 epidermidis infections.

It also shows that unscreened immune globulin would not 6 provide reliable protection, since many standard human immunoglobulin lots 7 have little or no opsonic activity for S epidermidis. Hence, standard human 8 immune globulin would not ensure uniformly high levels of antibody to SE and 9 would not be uniformly protective despite the fact that !Lrge numbers of donors might be expected to provide good levels of antibody to a common bacteria 11 such as S epidermidis.

12 Figure 3 13 Figure 3 shows that Directed Immune Globulin protects animals from 14 developing prolonged epidermidis bacteremia while standard immune globulin did not. Animals treated with Directed Immune Globulin had lower 16 peak bacteremia levels (9.2 x 102 vs. 6.5 x 103) and cleared the bacteremia 17 more efficiently (at 72 hours, 5 bact. per ml vs. 380 bact. per ml; geometric 18 mean level). In addition 72 hours after infection, 18/24 animals given 19 Directed Immune Globulin had cleared their bacteremia and 100% survived, while only 4/20 animals given standard immune globulin died and only 21 1/16 cleared their bacteremia during that 72 hour period. In addition to WO 93/17044 PCT/US92/09830 -13- 1 prevention, since Directed Immune Globulin enhanced S. epidermidis 2 clearance, it would be a valuable adjunct to antibiotic therapy for people 3 infected with S epidermidis, since many of these patients hve imparied 4 immunity and may not cleor the bacteria efficiently.

VI. DETAILED DESCRIPTION OF PREFFERED EMBODIMENTS 6 EXAMPLES 7 The herein offered examples provide methods for illustrating, without any 8 implied limitation, the practice of this invention in the production of Directed 9 Human Immune Globulin for Staphylococcus epidermidis and the use of said Immune Globulin for the prevention or treatment of infections caused by 11 Staphloccus epidermidis.

12 The profile of the representative experiments have been chosen 13 to illustrate methods for producing Directed Human Immune Globulin to S.

14 idpid.idis and to demonstrate its usefulness to prevent or treat 5 epidermidis infections.

16 Materials and Methods 17 taph.yloccal Strins: Although any R epidermidis strains 18 could be used, in these experiments we used two strains from the American WO 93/17044 PCT/US92/09830 -14- 1 Type Culture Collection, Rockville, MD (ATCC #31432 and ATCC #35984).

2 A clinical isolate (Hay) from the blood of a child with epidermidis sepsis 3 was also used and is also on deposit at the American Type Culture Collection.

4 Materials and Method; Immunoglobulin: Standard Intravenous Immunoglobulin was 6 used in these experiments to represent large immunoglobulin pools.

7 Preparations from several companies were analyzed for comparison, to include 8 Gamimmune, Cutter Laboratories Inc. Berkeley, California; Sandoglobulin, 9 Sandoz, East Hanover, Gammagard, Hyland, Los Angeles, California.

Serum from individual donors were also analyzed for antibody activity to S.

11 epidermidis.

12 Trichloroacetic Acid (TCA) Antien Extraction 13 Staphylococcus epidermidis strains (ATCC #35984, ATCC 14 #31432 and Hay) were grown to log phase at 37°C in 1000 ml of Tryptic Soy Broth (Difco). The bacteria were then centrifuged at 2500 RPM for 16 minutes and the supernatant was aspirated and discarded. The bacterial button 17 was resuspended in 200 ml of 2% trichloroacetic acid (TCA) and stirred 18 overnight at 4°C. The mixture was then centrifuged at 2500 RPM for 19 minutes and the supernatant aspirated. To the supernatant, 4 volumes of absolute ethanol were added and refrigerated overnight at 4°C. After 21 centrifugation at 2500 RPM for 10 minutes, the supernatant was removed and WO 93/17044 PCT/US92/09830 1 discarded. Then, five milliliters of normal saline was added to the antigen 2 precipitate, it was cultured to ensure sterility and then lyophilized for storage.

3 Antigen Binding Studies Using Enzyme-Linked 4 Immunoabsorbent Assay ELISA) L epidermidis Antigen was dissolved in carbonate buffer at a 6 concentration of 25 micrograms/ml. To each well of A 96-well flat-bottomed 7 microtiter plate (NUNC, Roskilide, Denmark) 100 microliters were added and 8 stored at 4°C until used. Immunoglobulin was diluted to 1% and 2-fold 9 dilutions prepared in phosphate-buffered saline-Tween To each well was added 100 microliters of the serial dilutions and the plates were incubated for 1 11 hour at 4 0 C. The plates were washed four times with H 2 0-Tween Alkaline 12 phosphatase linked goat anti-Human IgG (100 microliters; 1:250) was added, 13 the plates were incubated for 1 hour at 4°C and then washed H 2 0-Tween and 14 100 microliters of P-nitrophenyl phosphate substrate in diethanolamine buffer were added. After 90 minutes of incubation at room temperature, the color 16 development was determined by absorbance at 405 nm.

17 Opsonic Assay: 18 To determine the functional antibody to S epidermidis in the 19 immune globulin pools and sera, a neutrophil mediated bactericidal assay was WO 93/170444 PCT/US92/09830 -16- 1 used. Neutrophils were isolated from adult venous blood by dextran 2 sedimentation and ficall-hypaque density centrifugation. Utilizing a microtiter 3 plate assay that requires a total volume of 0.1 ml/well, washed neutrophils 4 (approximately 10 6 cells) were added to round-bottomed microtiter wells along with 3x10 approximately mid-log phase bacteria. Newborn rabbit serum 6 microliters; screened to assure absence of antibody to S. epidermidis) was 7 used as a source of active complement. Forty microliters of 5% standard 8 immune globulin (or serum) was.added and the microtiter plates were 9 incubated at 37"C with constant, vigorous shaking. Samples (10 microliters) were taken from each well at zero time and after 2 hours of incubation, 11 diluted, vigorously voiiexed to disperse the bacteria and cultured on blood agar 12 plates overnight at 37 0 C to quantitate the number of viable bacteria. C .oh 13 consisted of neutrophils alone, complement alone and neutrophils plus 14 complement.

Staphylocccal Sepsis Model: 16 A suckling rat model was used to determine the in vivo activity 17 of antibody to S. epidermidis. Wistar rats (2 days old) were given 0.2 ml of 18 20% Intralipid (Cutter, Berkeley California,) intraperitoneally at 0800 and 19 1400. At three days of age each animal was again given, 0.2 ml of intralipid at 0800 and 1400 and 0.2 ml of 5% immunoglobulin or serum was 21 given IP. Shortly after the last dose of intralipid, 0.05ml (approx. 5x107) WO 93/17044 PCT/US92/09830 -17- 1 mid log phase S. epidermidis were injected subcutaneously just cephalad to the 2 tail. Suckling rats less than 24 hours old also develop lethal S. epidermidis 3 sepsis when infected with 10 7 l10 S epidermidis subcutaneously. To analyze 4 bacteremia levels in selected animals, 0.01 ml of blood was obtained from the tails of the suckling rats, 24, 48, and 72 hours after infection. The blood was 6 collected under sterile conditions in micropipettes and serially diluted in 7 Tryptic Soy Broth (Difco). Bacteria were subcultured onto plates to ensure S 8 epidermidis bacteremia and all animals were followed five days to determine 9 survival.

Results 11 Antigen Binding Activity of Human Immunoglobulin for S.

12 epidermidis.

13 The results of the ELISA testing of several standard 14 immunoglobulin preparations for antibody to S. epidermidis are presented in Figure 1. Most standard immune globulins contained low levels of antibody to 16 S epidermidis. However, by screening for antibody to TCA extracted antigens 17 of S. epidermidis, some immunoglobulin lots and serum from one volunteer 18 donor were found to have increased levels of antibody to epidermidis (O.D.

19 readings 1.014, 1.026, and 1.002). Variations in antibody to S. epidermidis occurred between preparations prepared by different techniques and lot to lot 21 variation in a single preparation was seen as well, indicating that all 22 immunoglobulin pools were not the same.

WO 93/17044 PCT/US92/09830 -18- 1 Opsonic Activity of Human Immunoglobulins for S. epidermidis.

2 All antibody directed against a given organism may not enhance 3 immunity and provide enhanced protection from infection. Stated differently, 4 antibodies can bind to bacteria and yet not enhance opsonization in vitro or clearance from the blood of an infected host. Therefore a functional assay was 6 also utilized to determine if the antibody to S. epidermidis detected by ELISA 7 was also capable of promoting phagocytosis and killing of the organism by 8 neutrophils (Figure Opsonic antibody activity ranged from low 9 bactericidal activity), to moderate activity (25-80%) and a few had high bactericidal activity Therefore two standard human immune globulin 11 preparations with high bactericidal activity were selected as Directed Human 12 Immune Globulin for S. epidermidis based on in vitro assays that measured 13 antibody binding to TCA epidermidis antigens and opsonic antibody activity 14 determined by in vitro testing. Serum from a single donor also had good opsonic activity for S, epidermidis 80% opsonophagocytic bactericidal 16 activity). While serum and plasma from several individuals have been studied 17 only this donor had high opsonic activity. Therefore donor screening could 18 detect individual blood or plasma donors that could contribute immunoglobulin 19 that could be pooled as an alternate method to produce a WO 93/17044 PCT/US92/09830 -19- 1 Directed Human Immune Globulin for S, epidermidis. In addition blood or 2 plasma units could be screened for pooling as well.

3 Animal Protection Studies 4 Discription of Tables Table 1 6 Table 1 shows the effect of Directed Human Immunoglobulin for 7 epidermidis (40R09) (which was selected by ELISA and opsonic assay 8 screening) compared to standard human immunoglobulin (that had moderate 9 activity for SI epidermidis) and saline control. Table 1 shows that untreated control animals had about a 50% mortality while animals given Directed 11 Immune Globulin for S. epidermidis were fully protected (NO mortality).

12 Standard immune globulin gave only partial protection. Other standard 13 immune globulin lots with lower levels of antibody to S epidermidis would be 14 even less effective, since mortality was much higher with saline. However, one would not expect that Directed Immune Globulin would be always 100% 16 effective, but that it would consistently improve survival over standard immune 17 globulin or untreated animals.

18 Tabl 2 19 Table 2 demonstrates that Directed Immune Globulin produced in rabbits by immunization (S epidermidis vaccine) produced survival similar WO 93/17044 PC/US92/09830 1 to Directed Human Immune Globulin produced by screening immunoglobulin 2 for antibody to epidermidis. Immunization of individuals with S 3 epidermidis vaccine and collecting plasma for immunoglobulin extraction 4 would be another method for producing Directed Human Immune Globulin for preventing or treating epidermidis infections.

6 Table 3 7 Table 3 shows that intralipid causes a dose related increased 8 mortality in suckling rats infected with S. epidermidis. Control animals 9 receiving Intralipid alone had 100% survival (43/43) while immature rats given 16 gm/kg of Intralipid had only 46% survival The high dose of 11 Intralipid appears to impair the immune system sufficiently to allow the 12 normally avirulent epidermidis to overwhelm the baby animals.

13 Table 4 14 Table 4 shows that normal 3 day old suckling rats not given Intralipid, but infected with epidermidis develop bacteremia. However, 16 over 72 hrs their immune system is able to clear the organisms from the blood 17 and all of the baby rats survive.

WO 93/17044 PCT/US92/09830 -21- 1 Table 1 shows the Directed Human Immune Globulin for S.

2 epidermidis (selected by screening standard immunoglobulin for opsonic or 3 antigen binding activity for &S epidermidis) provides complete protection from 4 lethal infection in the setting of impaired immunity with Intralipid while standard immune globulin (with moderate antibody levels) had only partial 6 protection (1 out of 5 aminals died compared to about 50% with saline).

7 Additional studies with another immu.noglobulin preparation, (Alpha 8 Pharmaceuticals; Directed Human Immune Globulin 8016A >90% opsonic 9 activity, versus standard human immune globulin, 8007A 50% opsonic activity) showed that the Directed Human Immune globulin also provided 11 enhanced survival (8016A-64/95 vs. 8007A-39/90 over standard 12 human immune globulin. Even more striking was the fact that the Directed 13 Human Immune Globulin decreased the peak level of S. epidermidis 14 bacteremia and promoted rapid clearance of the bacteria (Figure These studies showed that antibody was important for protection against S.

16 epidermidis enhanced bacterial clearance from the blood and could be an 17 effective prophylactic or therapeutic modality even in the immature host with 18 impaired immunity. Many of the animals treated with standard human immune 19 globulin remained bacteremic 72 hours after infection while only 1/20 animals was still bacteremic at 72 hours after receiving the Directed Human Immune 21 Globulin. In addition the mean bacteremia level at 72 hours was WO 93/17044 PCT/US92/09830 -22- 1 markedly different (bacteremia with Directed Human Immune Globulin 0.5 x 2 10' vs. bacteremia with standard human immune globulin 3.8 xl0 2 3 In further studies, rabbit Directed Immune Globulin for S 4 epidermidis was produced by immunizing rabbits with S. epidermidis vaccine.

The vaccine induced Directed Immune Globulin was compared with Directed 6 Human Immune Globulin produced by screening immunoglobulin for antibody 7 to S epidermidis (Table Vaccine induced Directed Immune Globulin had 8 similar protective activity to Directed Human Immune Globulin produced by 9 screening (9/11 vs. 12/13 survived) and each was better than controls (11/19 survived). These data show that S. epidermidis vaccine induced antibody 11 could be used for prevention and treatment of S. epidermidis infections and 12 that vaccine could be used to produce a Directed Human Immune Globulin.

13 TABLE 3 14 Many bacteria such as S. epidermidis are not pathogenic in normal people. However, in babies with an immature immune system or 16 impaired immunity as is seen with intralipid, j epidermidis may cause sepsis 17 and death. It is critical therefore, that any animal model to test antibody 18 effectiveness should include these factors. To our knowledge this is the first 19 time that antibody to Staphylococcus epidermis has been shown to provide protection and enhance bacterial clearance in an immature and/or WO 93/17044 WCr/ US92/09830 -23- 1 immunosuppressed host. Intralipid given in dosage up to 16 gm/kg did not 2 cause death in any baby animals (controls, table In the absence of 3 Intralipid, the 3 day old animals will become bacteremic with S. epidermidis 4 after infection, but will clear the infection over 72 hours and survive (Table 4).

However, Intralipid did impair immunity in a dose related fashion and when 6 the 3 day old animals were infected with S. epidermidis lethal sepsis occurred 7 in up to 67% of the animals. Baby rats in the first day of life also do not clear 8 bacteriemia well (due to immature immunity) and develop lethal sepsis. In 9 these models baby rats were unable to clear the S. epidermidis bacteremia and developed lethal sepsis. Directed Human Immune Globulin was able to 11 enhance survival and promote bacterial clearance while standard human 12 immune globulin did not enhance clearance (Fig 3).

13 TABLE 4 14 When SE is injected into normal baby rats, they become bacteremic in 2 hours and then begin to slowly clear the bacteria from the 16 blood. All of the animals cleared the bacteremia 72 hours after the infection.

17 thus suggesting that under normal circumstances neonatal immunity while 18 impaired can eventually control SE. However, studies in rats infectedd with S 19 eidermidis shortly after birth have demonstrated that they can also develop a lethal infection.

I TABLE 1 2 E-ffectiveness of Standard Immune (ilubul in, 3 and Directed Immune Globulin to 4 Staphylococcus epidermidis in Providing Protection from lethal S. epidermidis Infection 6 in a Suckling Rat Model 7 Iminunoglobulin Treated Died Mortality 8 Type 9 Directed Immune I I Globulin *(40R09) 24 12 Standard 13 Immune Globulin 20 4 14 Control Untreated"* 13 7 54% 16 lininfectcd** I 1 0 0 17 #20-23 -3/25/90 19 2/11l/90, #4 1/29/90) 2 Comparision of Therapeutic Efficacy of 3 Vaccine Induced Anti-staphylococcal 4 Directed Immune Globulin with Screened Directed Immune Goobulin ;n a S. epideridis Sepsis Model* 6 Treatment Exp. Treated Survived Survived 7 8 Vaccine Induced 16,19 11 9 82% 9 Directed Immune Globulin I I Screened Directed 17,18 13 12 *92% 12 Immune Globulin 13 (40R09) 14 Saline 16,17 Control 18,19 19 If 58% 16 17 *1990 Studies Animal Model: Effect of Intralipid Dosage on~ Staphylococcus epidermidis mortality in suckling rats Intralipid Dose 4 gm/kg 8 gm/kg 12 gm/kg 16 gm/kg *16 gm/kg Survival Infected 10/10 (100%) 10/13 (76%) 7/12 (58%) 6/13 (46%) 2/6(33%) Control 7/7 (100%) 9/9 (100%) 11/11 (100%) 11/11 (100%) 5/50(00%) I I Infection with S. epidermidis (Ilaywood); approximately 10' bacteria SQ.

12 Standard model starts 11- on day 2 of life with infection after last IL dose on day 3 if full 4 doses given.

13 *11, started on day I of life with Infection after the 4th dose on day 2.

TABLh 4 Staphylococcus epiderffidis Bacteremia Levels in Normal Suckling Rats Given Normal Saline Instead of Intralipid Time Post Number Per Cent Bacteremia Infection liacicremic Bacteremic Level 2 hours 8/8 100 3.8 x 4 hours 7/8 87.5 1.3 x 6 hours 8/8 100 7.5 x 24 hours 6/8 75 8.8 x 48 hours 3/8 37.5 0.5 x 72 hours 0/8 0 0 Exp. 93 +94: 818 survived *MeaIn number of bacterial per ml of blood

Claims (13)

1. Substantially pure Directed Human Immune Globulin having a bactericidal activity of greater than 80% which comprises a measured level of anti-staphylococcal IgG antibodies that react with surface antigens of Staphylococcus epidermidis, promote phagocytosis and killing of Staphylococcus epidermidis in vitro and/or protection against Staphylococcus epidermidis in vivo.

2. A pharmaceutical composition comprising an amount of Directed Human Immune Globulin of Claim 1 sufficient to prevent or treat infections by S. epidermidis, together with a pharmaceutically acceptable carrier therefor.

3. A method of preparing the Directed Human Immune Globulin of Claim 1 said method comprising 15 a) screening serum, plasma, or an .immunoglobulin pool for antibodies to S. epidermidis by an in vitro antigen-binding assay and S* b) confirming the functional activity of said antibodies by an in vitro opsonophagocytic bactericidal 20 assay wherein said bactericidal activity is greater than

4. The method of Claim 3 wherein the serum is screened by S. epidermidis ELISA or Opsonic Assays.

5. The method of Claim 3 wherein the plasma is screened by S. epidermidis ELISA or Opsonic Assays.

6. The method of Claim 3 wherein the immunoglobulin is screened by S. epidermidis ELISA or Opsonic Assays.

7. The method of any one of Claims 4 to 6 wherein said screening is by ELISA.

8. The method of any one of Claims 4 to 6 wherein said screening is by Opsonic Assays.

9. A method of preparing the Directed Human Immune globulin of Claim 1 comprising the steps of (a) immunising plasma donors and removing plasma from said donors for Directed Immune Globulin preparation. staffhronrkeop/sped32718.93 5.9 29 A method of assessing the protective level of Direct Human Immune Globulin by using an immature or intralipid induced lethal model to provide minimum protective standard comprising the steps of screening with in vitro assays and using animal lethality tests to ensure that the immunoglobulin preparation provided protective antibody to S. epidermidis.

11. A method of prevention or treatment of Staphylococcus epidermidis infections in a patient comprising administering a therapeutically-effective amount of the Directed Human Immune Globulin of claim 1 to said patient.

12. The method of claim 11 wherein said patient is administered the amount of Directed Human Immune Globulin S 15 intravenously.

13. The method of claim 11 wherein said patient is administered the therapeutically effective amount of Directed Human Immune Globulin intramuscularly.

14. The method of Claim 12 wherein the patient is treated prior to infection with S. epidermidis. The method of claim 13 wherein the patient is treated after infection with S. epidermidis. DATED THIS 5TH DAY OF SEPTEMBER 1996 HENRY M JACKSON FOUNDATION FOR THE ADVANCEMENT OF MILITARY MEDICINE By Its Patent Attorneys: GRIFFITH HACK CO., Fellows Institute of Patent Attorneys of Australia staff/hronkeep/specd32718.93 5.9