IMPROVED IMMUNOTHERAPEUTIC METHOD OF PREVENTING OR TREATING VIRAL RESPIRATORY TRACT DISEASE

Abstract: An improved immunotherapeutic method of preventing or treating viral respiratory tract disease is described. The method comprises administering into the lower respiratory tract of a susceptible host a small particle (< 2 mu m) aerosol of a mixture of specific monoclonal antibodies directed against the various protective antigenic sites present on a major protective viral surface antigen(s).

Description: IMPROVED IMMUNOTHERAPEUTIC METHOD OF PREVENTING OR TREATING VIRAL RESPIRATORY TRACT DISEASE The present invention is related generally to immunotherapy of respiratory disease caused by viral infection. More particularly, the present invention is related to an improved immunological method of preventing or treating viral respiratory tract disease by administering into the lower respiratory tract of a susceptible host a small particle (<2μm) aerosol of a mixture of specific monoclonal antibodies directed against the various protective antigenic sites present on a major protective viral surface antigen (or antigens) . An immunotherapeutic method of treating pulmonary disease due to respiratory syncytial virus (RSV) by intranasal administration of a purified human immunoglobulin preparation (designated IVIG because it is prepared as a monomeric suspension of human IgG that is suitable for administration by the intravenous route) containing a high titer of neutralizing antibodies ("1:1000 to 1:2000) has been described in U.S. patent 4,800,078. In this instance IVIG was administered into the nose of anesthetized cotton rats which resulted in quantitative delivery of IVIG into the lungs, thereby simulating the aerosol mode of administration that is used in humans for pulmonary therapy. Also described in this patent was the important discovery that topically administered human immunoglobulins (IVIG) with a high titer of RSV neutralizing antibodies were retained in the lungs for up to 7 days and provided a significant prophylactic effect against RSV infection throughout this interval. However, with the presently available technology, direct introduction of the requisite amount of these antibodies into the lungs of infants and young children, who are most susceptible to RSV or other respiratory viral disease, can be achieved effectively only for those patients who are intubated in order to be connected to a mechanical ventilator which assists the respiratory function of the patient. It is clear that it would be difficult as well as unsafe to deliver the requisite amount of an IVIG suspension of 24 mg per kg, i.e., 125 mg to 250 mg of human immunoglobulin IgG for a 5 to 10 kg infant or young child, into the lungs of a patient who was not intubated. An improved therapeutic modality is, therefore, needed. SUMMARY OF THE INVENTION It is, therefore, an object of the present invention to provide an improved method of treating or preventing lower respiratory tract viral disease through the delivery of protective and therapeutic antibodies in a small particle aerosol (<2μm) , which overcomes the difficulties and problems associated with heretofore known methodology. Pursuant to this goal it is a further object of the present invention to utilize a mixture of neutralizing and/or therapeutic and/or prophylactic monoclonal antibodies, each of which has a different antigenic specificity that is directed to a specific highly conserved epitope on the surface of a major protective viral protein. Also, these epitopes against which the monoclonal antibodies are directed collectively represent the known repertoire of the major protective viral surface antigenic sites, i.e., monoclonal antibody to at least one epitope in each major protective antigenic site is included in the monoclonal antibody mixture. The mixture contains monoclonal antibodies suitable for human use and directed to highly conserved epitopes that are: (1) shared by the known viral antigenic subgroups, such as RSV subgroup A and subgroup B, or (ii) unique to a viral antigenic subgroup, such as RSV subgroup A or subgroup B. This strategy is dictated by the need to: (i) protect against infection of (ii) treat infection by a large variety or virus strains that exhibit antigenic variation in some of the protective antigenic sites on viral surface proteins. Other objects and advantages will become evident from the following detailed description of the invention. BRIEF DESCRIPTION OF THE DRAWINGS These and other objects, features, and many of the attendant advantages of the invention will be better understood upon a reading of the following detailed description when considered in connection with the accompanying drawings wherein: Figure 1 shows the therapeutic efficacy of a mixture of 16 neutralizing monoclonal antibodies directed against antigenic sites A, AB, B, or C of the RSV fusion (F) surface glycoprotein with neutralizing antibody titers ranging from 1:320 to 1:5120, and one monoclonal antibody without neutralizing activity, directed against site B (Beeler et al, J. Virol, £1:2941-2950, 1989). The RSV F monoclonal antibodies were combined to form an antibody mixture in which the geometric mean titer of the neutralizing antibodies was 1:1100. 0.1 ml of the antibody suspension was administered by the intranasal route to anesthetized cotton rats (average weight 100 grams) 3 days after infection with RSV, and the quantity of the RSV present in the lungs was titrated 24 hours later. Figure 2 shows the results of aerosol immunotherapy with RSV neutralizing antibodies. Three days after infection with RSV a 5% suspension of purified pooled human immunoglobulin G (IVIG) , with a RSV neutralizing antibody titer of 1:800, was administered by: (i) small particle (<2μm) aerosol or (ii) intranasal drops (25 mg/kg) to anesthetized cotton rats (average weight 100 grams) and the lungs were titrated for RSV 24 hours later. Figure 3 shows the effectiveness of topical immunotherapy of human parainfluenza virus type 3 (HPIV3) . A pool of cotton rat human parainfluenza type 3 virus (HPIV3) convalescent sera, with an HPIV3 neutralizing antibody titer of 1:10,000, was administered by the intranasal route to anesthetized cotton rats (average weight 100 grams) 3 days after infection with HPIV3 and the lungs were titrated for the amount of virus present 24 hours later. Figure 4 shows the effectiveness of a mixture of neutralizing HPIV3 monoclonal antibodies in systemic passive immunoprophylaxis of HPIV3 infection. Monoclonal antibody (MAb) pool #1 contained 4 neutralizing monoclonal antibodies directed against antigenic site A or the viral surface hemagglutinin-neuraminidase (HN) glycoprotein of HPIV3 and 1 neutralizing monoclonal antibody directed against site B (Coelingh et al. Virology 141: j569-582, 1985). MAb pool #2 contained the aforementioned 5 MAbs plus an additional neutralizing MAb directed against antigen site A of the HPIV3 HN. Infant cotton rats (average weight 10 grams) were inoculated intraperitoneally with 0.4 ml of the mixture of monoclonal antibodies (derived from ascites fluid) per 10 grams body weight. 24 hours later anesthetized infants were challenged intranasally with HPIV3, and the lungs were titrated for the amount of virus present 4 days later. The mean HPIV3 serum neutralizing antibody titer of cotton rats at the time of virus challenge was 1:6400 in Experiment #1 and 1:3200 in Experiment #2. DETAILED DESCRIPTION OF THE INVENTION The above and various other objects and advantages of the present invention are achieved by a method of preventing or treating viral respiratory tract disease, comprising the step of administering directly into the lower respiratory tract of a host susceptible to lower respiratory tract viral disease, an effective amount of a small particle (equal to or less than 2μm in diameter) aerosol of a mixture of distinct monoclonal antibodies directed against one or more specific protective epitopes on each of the major protective antigenic sites of a major protective viral surface protein (or proteins) for the purpose of treating or preventing said disease. It should be noted that there are several factors leading to the evolution of the inventive methodology described herein. First, the use of human immunoglobulin IgG suitable for administration by the intravenous route (IVIG) described in previous reports requires a preparation of purified, monodispersed IgG derived from a pool of human plasma. This is problematic from the point of view of available amounts of human IgG with high titer of virus neutralizing antibodies. There is also a possible risk of infectious contaminants and the like which may be present in the plasma. However, it was found that the combination of the appropriate monoclonal antibodies provided an inexhaustible supply of efficacious reagents for the prophylactic and therapeutic usage described herein. Also, the use of monoclonal antibodies decreases the amount of immunoglobulin suspension required for a topical therapeutic effect by a factor of 10 to 50 or more. RSV or HPIV3 antibodies (or other viral antibodies) constitute only a very small proportion of the immunoglobulin molecules in murine or human immunoglobulin IgG. In contrast, a suspension of (i) purified murine monoclonal antibodies, (ii) purified human neutralizing monoclonal antibodies derived from lambda phage recombinant-infected E. coli expressing the Fab fragment of such antibodies (as described recently by Huse et al., Science 246:1275-1281,. 1989) or (iii) purified "humanized" mouse monoclonal antibodies produced by recombinant DNA technology to contain the variable region antigen combining sites of murine monoclonal antibodies with the remaining regions of the molecules being of human immunoglobulin origin (as described by Reichman et al., Nature 332:323-327, 1988), would contain essentially only the relevant virus neutralizing and/or prophylactic and/or therapeutic antibodies. This would decrease the dose of IgG required for a therapeutic effect by a factor of 10 to 50 or more. Second, the therapeutic efficacy of the RSV neutralizing antibodies delivered directly into the lungs in a small particle aerosol (<2.0μm), which is specifically targeted to the smallest air passages and alveoli, has been demonstrated in cotton rats and constitutes an important feature of the current invention. In a similar manner 2.5 to 25 mg of RSV neutralizing antibodies could be delivered directly into the lungs of young patients by aerosol during a short interval and the ■ therapeutic effect of these antibodies would be experienced soon after administration. This represents a major advantage of topical therapy in addition to its greater efficiency compared to parenterally administered antibodies. For example, in infants and young children with severe RSV lower respiratory tract disease the time required to safely attain a therapeutic level of serum RSV neutralizing antibodies by intravenous administration of purified human immunoglobulin IgG (IVIG) ranges from 2 to 4 hours. It should be noted that parenterally administered antibodies exert their therapeutic effect in the lungs following transudation onto the lumenal mucosal or epithelial surface of the lungs and the concentration of such antibodies on the lumenal surface appears to be at i ast two orders of magnitude less than in serum. In contrast, a therapeutic level of these antibodies in the lungs can be attained with considerably greater efficiency ("100 times greater efficiency) within a few minutes following aerosol administration of: (i) immunoglobulin IgG (IVIG) with very high RSV, HPIV3 or other viral neutralizing activity or (ii) a purified suspension of neutralizing and/or protective and/or therapeutic monoclonal antibodies with even higher specific activity which would decrease the amount of immunoglobulin required for therapy of prophylaxis to a level which could be delivered by current aerosol technology to unintubated patients. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, the preferred methods and materials are now described. All publications mentioned hereunder are incorporated herein by reference. Unless mentioned otherwise, the techniques employed or contemplated herein are standard methodologies well known to one or ordinary skill in the art. The materials, methods and examples are illustrative only and not limiting. MATERIALS AND METHODS RSV Monoclonal Antibodies Murine monoclonal antibodies directed against 16 distinct epitopes present on the 4 known neutralization antigenic sites of the F glycoprotein of RSV and conserved among subgroup A and subgroup B strains of RSV were prepared by standard methodologies well known to one of ordinary skill in the art and pooled for treating cotton rats previously infected with RSV. Cotton rats were inoculated intranasally with 10 5 plaque-forming units (PFU) of RSV. Three days later, at the peak of viral replication, the animals were anesthetized and inoculated intranasally with 0.1 ml of a dilution of the pooled monoclonal antibody suspension. One day after treatment the animals were sacrificed, and pulmonary viral titers determined by plaque assay. As shown in Fig. 1, at a 1:4 dilution (i.e., an antibody suspension with a neutralizing titer of 1:275) the pooled monoclonal antibody preparation of the present invention effected a 100-fold reduction in pulmonary viral titer. It should be noted that while the percentage of antibody molecules in human plasma directed against RSV is small (about 0.1% or less), purified "humanized" (murine-human recombinant monoclonal antibodies) or purified human monoclonal antibodies derived from Fab fragments of such antibodies produced in Fj« coli. by definition, have 100% specificity. Thus, a preparation of pooled monoclonal antibodies which can be prepared by hybridoma and recombinant DNA techniques (Riechmann et al.. Nature 332:323-327. 1988) or by direct cloning and expression of Fab fragments in E. coli using recombinant DNA technology (Huse et al. , Science 246:1275-1281. 1989) and which have activity against both A and B subgroups or RSV should yield a suspension as much as 50-fold or more potent than purified IgG (IVIG) derived from human plasma. It may be noted that a mixture of several MAbs directed against different antigenic sites of the viral protein(s) would be required for maximal therapeutic or prophylactic efficacy. The use of a single MAb should not be as effective as a combination of MAbs directed against different antigenic sites because of the high frequency of mutants that acquire resistance to neutralization by a specific monoclonal antibody directed against a single epitope. The frequency of such a "neutralization escape mutation" affecting a single epitope is " 1 mutant for every 10 to 10 5 virus particles produced during virus replication. Hence, the use of monoclonal antibodies representing different antigenic sites reduces the frequency of neutralization escape mutants as a function of the product of the frequency of such mutants for each site represented in the monoclonal antibody mixture. For example, when monoclonal antibodies for 2 antigenic sites are used in prophylaxis or therapy the frequency of "neutralization escape mutants" would be 10 ~ * to 10 "5 X 10 " * to 10 "5 (i.e., 10 "8 to 10 10 ) , for 3 antigenic sites 10 "12 to 10 "16 and for 4 antigenic sites 10 ~16 to 10 "20 . F(ab) 2 fragments of human or murine neutralizing antibodies can also be used for topical therapy of RSV or other respiratory viral infection. Recent studies summarized in Table 1 demoristrate that F(ab) 2 fragments of human RSV neutralizing antibodies are as effective in topical therapy of RSV infection of cotton rats as the full immunoglobulin molecules from which the F(ab) 2 fragments were derived (Prince et al., J. Virol. In press 1990) . Cotton rats were infected intranasally with 10 50 PFU of RSV (strain A2) on day 0, anesthetized animals were treated with topical IgG or F(ab') 2 by direct instillation into the respiratory tracts on day 3, and the animals were sacrificed on day 4 and the lungs were titrated for the quantity of RSV present; days 3 and 4 represent the time of peak viral titer in the lungs. Concurrently infected animals which were not treated with IgG of F ab'Jj served as the basis for determining the extent of viral clearance effected by topical immunotherapy. When used at an equimolar concentration, IgG (group 2) and F(ab*) 2 (group 5) were equally effective, each bringing about a 100-fold reduction in pulmonary virus. The possibility that the small amount of Fc remaining in the F(ab') 2 preparation (<0.2%) contributed to viral clearance was examined by using 10- fold (group 3) and 100-fold (group 4) dilutions of IgG. The IgG administered to group 4 contained a 10-fold higher amount of Fc than that found in the F(ab') 2 preparation but had no effect on viral titer, indicating that the antiviral effect seen in group 5 was due exclusively to F(ab') 2 . The role of complement was studied by comparing the efficacy of antibody in clearing virus from complement-depleted and-sufficient animals (Table 1) . Cobra venom factor (Naja Naja; Cordis Laboratories, Inc., Miami, Fla.) was administered intracardially, at a dose of 200 U per kg of body weight, to cotton rats challenged 2 days earlier with 10 50 PFU of RSV. These animals (group 7) were treated with topically administered IgG 24 h after administration of Cobra venom factor (day 3) . On day 4 the animals were sacrificed, and viral titers in lung homogenates were determined. Serum 50% hemolytic complement titers at the time of IgG administration (guinea pig C test kit, catalog No. 737- 303; Cordis) were greater than 1:5,600 in complement- sufficient animals, whereas a 1:50 dilution of serum of Cobra venom factor-treated animals did not contain detectable complement activity. Complement-depleted animals treated with IgG (group 7) showed a greater than 100-fold decrease in virus titer compared with the untreated, complement-sufficient (group 1) and untreated, complemented-depleted (group 6) animals. Complement- sufficient animals treated with IgG (group 2) showed a similar level of viral clearance as that seen in group 7, indicating that antibody-mediated clearance of RSV is not dependent on complement. Additionally, complement- depleted cotton rats were treated with F(ab') 2 (group 8) and were found to experience a similar degree of viral clearance as that in groups 2, 5 and 7. Comparison of Efficacy of Aerosol and Dropwise Intranasal Administration of IσG A small-particle ultrasonic nebulizer (Portasonic 8500 D. DeVilbiss Co., Somerset, PA) was used to generate aerosol particles (<2 μm) and to deliver the antibodies into the lungs of anesthetized cotton rats (Fig. 2) . Optimal results were obtained using a 5% suspension of purified human IgG (IVIG) because more concentrated suspensions became frothy when used for production of an aerosol. Animals were inoculated intranasally with 10 5 PFU of RSV. Three days later they were anesthetized and administered IVIG by: (i) small particle (<2.0 μm) aerosol during a 10 or 15 minute exposure, or (ii) by intranasal drops (Fig. 2) . In the former instance, cotton rats were allowed to breath an IgG aerosol as it emerged from the exit orifice of the chamber lid of a Porta-Sonic Nebulizer Model 8500D (DeVilbiss) . A 15- minute exposure to aerosolized IgG effected a 30-fold reduction in pulmonary virus which was slightly more effective than a dose of 25 mg IgG per kg delivered dropwise into the respiratory tract of anesthetized cotton rats. The results obtained are important for two reasons: (1) They show that IgG directed against RSV can be delivered topically with an aerosol nebulizer already in widespread clinical use. (2) The exposure time required for significant reduction in pulmonary virus (i.e., 15 minutes) compares very favorably with ribavirin, the only antiviral drug currently approved for treatment of RSV disease in human infants, which requires daily aerosol treatment of up to 18 hours for 3 to 5 days. Parainfluenza Virus a) Treatment. Preparations of purified human IgG (IVIG) which were effective in treating RSV infection in cotton rats had a neutralizing antibody titer against RSV approximately 50-fold higher than against human parainfluenza virus type 3 (HPIV3) . Whereas these preparations effected a 100-fold or greater reduction in pulmonary titer or RSV in infected cotton rats, the same preparations reduced pulmonary HPIV3 titers only 3-fold (data not shown) . Therefore, a pool of HPIV3 convalescent cotton rat serum with a neutralizing antibody titer against HPIV3 of 1:10,000 was prepared by standard methodology well known to one of ordinary skill in the art and used for investigating the feasibility of topical immunotherapy of HPIV3 (Fig. 3) . Cotton rats were inoculated intranasally with 10 55 PFU of HPIV3. Three days later anesthetized animals were treated intranasally with 0.1 ml of a dilution of the convalescent cotton rat serum pool (Fig. 3) . One day after treatment the animals were sacrificed and pulmonary viral titers determined. An HPIV3 convalescent serum dose of 10 mg/kg effected a 100-fold reduction in pulmonary virus titer. The fact that the amount of neutralizing antibodies required for a 100-fold reduction in titer of HPIV3 was several times higher than required for RSV, probably reflects the greater permissiveness of cotton rats for HPIV3 infection. HPIV3 replicates in the cotton rat lung to a titer 10- to 50-fold higher than RSV. Therapeutic efficacy of HPIV3 antibodies in this experiment demonstrates the effectiveness of topical immunotherapy for another important human viral respiratory pathogen and clearly indicates that (i) the usefulness of topical immunotherapy is not limited to RSV infection and (ii) topical immunotherapy should prove useful for other respiratory viral pathogens, such as parainfluenza virus types 1, 2 and 4, and influenza A and influenza B viruses whose pathogenic effects are also limited to the cells which line the lumen of the respiratory tract. b) Prophylaxis. Monoclonal antibodies directed against the A and B neutralization sites on the HN glycoprotein of HPIV3 were pooled and administered intraperitoneally to infant cotton rats. One day later the animals were challenged intranasally with 10 50 PFU of HPIV3. Four days thereafter the animals were sacrificed and pulmonary viral titers determined (Fig. 4) . At the time of challenge, the HPIV3 neutralizing antibody titer in the serum of monoclonal antibody recipients ranged from 1:3200 to 1:6400. Passive immunoglobulin prophylaxis resulted in a reduction in pulmonary virus titer of 300- to 1000-fold, thus demonstrating that pooled monoclonal antibodies directed against major neutralization sites on a major protective viral envelope glycoprotein are effective in vivo against at least two major human viral respiratory tract pathogens, viz., RSV and HPIV3. It is understood that the examples and embodiments described herein are for illustrative purposes only and that various modifications or changes in light thereof will be suggested to persons skilled in the art and are to be included within the spirit and purview of this application and scope of the appended claims. a All animals were challenged intranasally on day 0 with 10 5 PFU of RSV (strain A2) . Abbreviations: CH JQ , 50% hemolytic complement; i.n., intranasally; NT, not tested. b P < 0.001, compared with group 1. c P < 0.001, compared with group 6.

Claims: WHAT IS CLAIMED IS: 1. A method for preventing or treating viral respiratory tract disease, comprising the step of administering by small particle aerosol of a size equal to or less than about 2 μm, directly into the lower respiratory tract of a subject at risk of severe viral lower respiratory tract disease, an effective amount of the aerosol containing a pooled suspension of different, purified, monoclonal antibodies or F(ab') 2 fragments thereof, directed against conserved epitopes located on major protective antigenic sites of protective antigens of the disease causing virus, to prevent or treat said disease. 2. The method of claim 1 wherein the virus is respiratory syncytial virus (RSV) . 3. The method of claim 1 wherein the virus is human parainfluenza virus type 3 (HPIV3) . 4. The method of claim 2 wherein each of the RSV monoclonal antibodies constituting the pooled suspension of monoclonal antibodies, is directed at a unique, highly conserved, protective epitope located on a major protective antigenic site of a major protective viral surface protein and one or more of the MAbs in the pooled suspension are directed at each of the major protective antigenic sites in order to provide effective immunologic prophylactic or therapeutic effect and to prevent the emergence of neutralization-resistant virus mutants during prophylaxis or therapy. 5. The method of claim 3 wherein each of the HPIV3 monoclonal antibodies constituting the pooled suspension of monoclonal antibodies, is directed at a unique, highly conserved, protective epitope located on a major protective antigenic site of a major protective viral surface protein and one or more of the monoclonal antibodies in the pooled suspension are directed at each of the major protective antigenic sites in order to provide effective immunlogic prophylactic or therapeutic effect and to prevent the emergence of neutralization-resistant virus mutants during prophylaxis or therapy. 6. The method of claim 1 wherein said monoclonal antibodies are humanized murine-human monoclonal antibodies or human monoclonal antibodies derived from Fab fragments and cloned and expressed in £. coli. 7. The method of claim 6 wherein F(ab')2 fragments of said monoclonal antibodies are employed for therapy or prophylaxis. 8. The method according to claim 1 of administering said small particle aerosol to a subject without using intubation.

Inventors: BEELER JUDY ALLYSON, CHANOCK ROBERT MERRITT, COELINGH KATHLEEN LOUISE, HEMMING VAL, MURPHY BRIAN ROBERT, PRINCE GREGORY ANTONE, BEELER, JUDY, ALLYSON, CHANOCK, ROBERT, MERRITT, COELINGH, KATHLEEN, LOUISE, HEMMING, VAL, MURPHY, BRIAN, ROBERT, PRINCE, GREGORY, ANTONE
Applicants: US COMMERCE, THE UNITED STATES OF AMERICA, AS REPRESENTED BY TH
Application year: 1991
Main classification: A61K39
IPC codes: A61K38, A61K39, A61P31, C07K16