Patent Application: US-31030402-A

Abstract:
the present invention discloses a method to induce the cd4 + th1 immune response , combined with a repression of the th2 mediated activities . the method comprises administering an igg isotype antibody , which is not an igg1 isotype antibody . the antibody is an igg2a and / or igg2b isotype anti - allergen antibody . the shift from a th2 response towards a mixed th1 / th2 response is useful in the treatment of diseases , such as asthma . since the disclosed method corrects the immuno - pathological cause of the disease , the polarized th2 response against allergen , a sustained cure from asthma may be achieved .

Description:
definitions . the following definitions are set forth to illustrate and define the meaning and scope of various terms used to describe the invention herein . “ igg2 isotype antibody ” as used herein refers to an isotype antibody derived from a polyclonal or a monoclonal preparation . the isotype antibody may be purified to a degree such that it is free from immunological active amounts of antibodies of other isotypes or of other immunological active compounds . “ substantially free of allergen ” refers to the ratio of the number of antibodies to the number of antibody - binding epitopes which bind to the antibodies as measured in vitro before administration . the ratio may be 10 / 1 , 100 / 1 , or even 1 , 000 / 1 . one or more igg isotype antibodies “ substantially free of other isotype antibodies ” means that the ratio of the total number of igg isotype antibodies to the total number of non - igg isotype antibodies , as determined in vitro , before administration is at least 10 / 1 , but maybe 100 / 1 or even 1 , 000 / 1 . “ environmental allergen ” as used herein refers to allergen to which an animal subject , including a human , is exposed to by external contact , such as inhalation . “ aeroallergens ” include , without limitation , pollen . the pollen may originate from gymnosperms , dicotyledonous angiosperms , monocotyledonous angiosperms , dust mite antigens and mold antigens , such as alternaria antigens . “ a persistent reduction ” of aeroallergen - induced inflammatory response is a reduction wherein , after contact with the allergen , a significant decrease in inflammatory response is noticed . the reduction may occur even after stopping the treatment for at least four days and even after stopping the treatment for at least six days . the significance of the decrease may be evaluated by comparing the treatment to a result obtained in a persistent reduction of aeroallergen - induced inflammatory response with a placebo treatment . mouse strains : in all experiments , unless otherwise indicated , balb / c mice were used . induction protocol : sensitization by repeated injection of ova . alternatively , mice were sensitized by injection of 10 μg ova adsorbed with 1 mg al ( oh ) 3 ( ova - alum ). dependent on the experiment and degree of sensitization desired , mice were injected with ova - alum once on day 0 or received an additional injection on day 7 and day 14 . challenge was by 8 consecutive exposures to nebulized ova over 8 days , unless otherwise indicated . parameters monitored : number of bal cells in individual animals ; composition of bal regarding numbers of eosinophils , macrophages , cd4 + t cells and cd8 + t cells ; number of cytokine - positive cd4 + t cells from bal following in vitro activation with anti - cd3 and anti - cd28 monoclonal antibody ; cytokine concentration in the supernatant of the above described t cell cultures collected after 24 hrs ; serum titers of ova - specific ige , igg1 , igg2a and igg2b antibodies ( ova - specific elisa ). anti - ova antibodies : mouse monoclonal anti - ova antibodies of the isotypes ige , igg1 , igg2a and igg2b were isolated in the laboratory . anti - ova ige - containing crude hybridoma culture supernatant was used as internal standard for ova - specific ige elisa . the various anti - ova igg monoclonal antibodies were similarly used as internal standard for specific elisa . in addition , cultures of the corresponding hybridomas were expanded for large - scale antibody production followed by purification of the monoclonal antibody . all preparations were found to be free of endotoxin . route of administration of anti - ova antibody : antibodies were administered either by intravenous ( i . v .) injection or by intranasal instillation . a well - established experimental model for allergic asthma includes sensitization of balb / c mice to the protein antigen ova , followed by challenging the sensitized mice by repeated exposure to nebulized ova ( hofstra et al ., 1998 ). sensitization was achieved by 7 intraperitoneal injections of 10 μg ova in pbs given on alternate days . exposure of treated mice , 3 weeks after the last injection , to inhaled ova resulted in induction of atopy which was apparent from strongly increased serum titers of anti - ova ige ( fig1 ). this ige response was accompanied by a strong increment of cellular infiltration in the lungs . the cell infiltrate included mainly eosinophils , cd4 + and cd8 + t lymphocytes , and macrophages ( fig2 ). both responses to inhaled allergen , namely induction of atopy and eosinophilic airway inflammation , are characteristic of allergen - induced asthma and as a consequence represent a valid experimental model for the human disease . an essential feature of the approach described herein relates to the spontaneous formation of antibody - allergen immune complexes formed as inhaled allergen reaches the airways . therefore , administration of antibody specifically to the airways is important . the feasibility of introducing antibodies to the lungs by aerosol or by intranasal instillation was investigated . the presence of functional anti - human catalase igg antibody ( anti - hcat ) in the bal was measured by specific elisa after administration of the antibody by aerosol or intranasal instillation . as shown in fig3 administration by aerosol allowed recovery of functional antibody and intranasal instillation allowed nearly 40 % recovery of functional antibody . control experiments showed the dramatic loss of functional antibody in the bal after aerosol administration which reflected loss of function of the antibody rather than inadequate inhalation . altering the pressure used for aerosol and / or the concentration of the antibody did not lead to significant gains in antibody stability . accordingly , intranasal instillation was chosen as an administration method for delivery of antibody to the upper airways . a second parameter that was established dealt with the time of retention of antibody in the lung which is used to define the time range wherein the administered antibody may exert its presumed effects . ova - specific elisa on bal fluid of mice that received anti - ova igg by the intranasal route showed a slow clearance of free antibody while significant titers were still detectable after 24 h ( fig4 ). after 48 h , most of the antibody appeared cleared from the lungs . to determine whether cell - bound antibody exhibited a similar clearance rate , fluorescent - labelled antibody was administered and the presence of cell - bound fluorescence was measured on bal cells using flow cytometry . in c57bl / 6 mice , cell - bound antibody was detectable within 1 h after intranasal instillation and reached maximal intensity after 6 h ( fig5 ). unlike free antibody , cell - bound antibody remained detectable 48 h after administration . a similar result was obtained with balb / c mice . from these results , it may be concluded that intranasal administered antibody may exert its local effects in the airways within a time span of 24 h to 48 h . in another set of experiments , it was verified whether administration of anti - allergen igg antibodies to sensitized mice 2 hrs before challenge with aerosol affects the airway inflammatory response . preliminary experiments indicated that an antibody dose range of about 50 to 200 μg igg antibody was effective ( fig6 ). the following experiments were used for an antibody dose of 100 μg and the following experimental parameters were varied : the number of administrations was once ( 2 h before the first exposure to aerosol ) or twice ( an additional administration of antibody 2 h before the 5 th aerosol exposure ); and the extent of eosinophilia in the bal was analyzed . since eosinophilia is the major indicator of allergen - induced airway inflammation , the extent of eosinophilia revealed a pronounced reduction in the conditions where igg2 antibodies were administered to the lungs by intranasal instillation ( fig7 upper panel ). igg2a appears to be a potent igg2 isotype in generating this protective effect . in contrast , intranasal administration of igg1 appeared to have no protective effect . administration of the same antibodies by the intravenous route had no , or only marginal , effects on the degree of eosinophilia ( fig7 lower panel ). a comparison in two separate experiments between the same igg2a antibody dose ( 100 μg ) given in a single administration or divided over two separate administrations of 50 μg each revealed a diminished eosinophilia and diminished cell infiltration in the airways with both treatment schedules ( fig8 ). however , two separate administrations of 50 μg igg2a each produced a more pronounced reduction in both independent experiments of allergen - induced airway inflammation as compared to a single administration of 100 μg igg2a antibody . analysis of the serum titers induced by a first round of aerosol challenge analysis of the serum titers of ova - specific ige , igg1 , igg2a and igg2b induced by challenge with ova aerosol revealed no significant changes between the various experimental groups ( table i ). thus , despite the presence of ova - specific igg antibodies in the airways , a challenge with inhaled antigen induced a secondary antibody response similar to the antibody response induced in placebo - treated mice . this result indicates that the reduced airway inflammation observed in the igg2 - treated mice did not result from molecular avoidance or immune exclusion of the acroallergen by the administered allergen - specific antibodies as was previously reported for allergen - specific iga ( schwarze et al ., 1998 ). table i anti - ova ig - serum titers induced by ova aerosol challenge of treated and untreated sensitized mice . nd = not determined . i . n . = intranasal administration . in the columns labeled ige , igg1 , igg2a , and igg2b , the anti - ova ig - serum titer of the indicated antibody type is presented . mouse treatment ( i . n .) number ige igg1 igg2a igg2b 100 μg igg1 ab 1 66667 1000 nd nd 100 μg igg1 ab 2 66667 1000 nd nd 100 μg igg1 ab 3 50000 1000 nd nd no antibody 10 66667 1000 nd nd no antibody 11 50000 600 nd nd no antibody 12 50000 700 nd nd 100 μg igg2a ab 1 44444 2778 1375 20 100 μg igg2a ab 3 94444 4444 2500 71 100 μg igg2a ab 4 44444 2778 1375 36 no antibody 18 87500 5600 3333 160 no antibody 19 75000 6400 1667 50 no antibody 20 75000 7200 3333 60 100 μg igg2b ab 1 33333 3750 275 120 100 μg igg2b ab 2 83333 10000 775 280 100 μg igg2b ab 3 46667 6500 350 140 no antibody 19 33333 4000 225 119 no antibody 20 17667 2750 25 35 no antibody 21 28333 4000 250 143 based on the data in table i , an active process involving a modulation of the allergen - induced immune response by the administered igg2 antibodies appears to be responsible for the attenuation of the airway inflammatory response to allergen . also , the recurrent response pattern observed in example 3 with various administration schedules of allergen - specific igg antibodies indicates an alternative modulation of the anti - allergen immune response . thus , all treatments involving intranasal instillation of igg2 , but not igg1 , antibodies consistently resulted in a diminished airway inflammatory response to inhaled allergen whereas the intravenous route of administration did not produce this consistent response pattern . the discrepancy between administration routes indicates that the protective effect of the allergen - specific igg2 antibodies requires interaction of the antibody with the allergen at the site of allergen entry . as the protection is not the result of shielding of the immune system from the allergen by the administered antibody , an active , not passive , mechanism must be responsible for the observed reduction in inflammation . the observations , specifically the dependence of the protective effect on intranasal instillation and its occurrence despite contact of the immune system with the allergen , indicate that the disclosed method modifies the nature of the anti - allergen immune response and is valid for obtaining a sustained cure for asthma , rather than a symptomatic treatment . analysis of the serum titers induced by a second round of aerosol challenge . the absence of decreased ige and igg antibody responses in the treated animals , despite a marked reduction of the inflammatory airway response , can be explained as follows . the observed antibody titers reflect the activation of antibody - producing memory b lymphocytes by allergen , wherein the b lymphocytes are generated during the preceding sensitization . antibodies derived from newly generated antibody - producing b cells marginally contribute to the antibody response due to the short period ( 7 days ) between the aerosol challenge and the serum collection . however , upon renewed challenge with allergen , memory b cells derived from the newly generated antibody - producing b cells will significantly contribute to the antibody response . to verify whether the treatment with antibody affected the generation of new antibody - producing b cells and subsequently of new memory b cells , igg2a treated mice were exposed to a second round of aerosol after a 2 week rest period . a marked increase of the th1 - dependent igg2a and igg2b isotypes was observed in the treated mice ( fig9 ). the th2 - dependent isotypes remained at the same level ( igg1 ) or showed a slight decrease ( ige ). thus , although the mice did not receive an intermittent treatment with antibody , the memory ige response ( th2 dependent ) of the mice was reduced , whereas the memory igg2 response ( th1 dependent ) of the mice was enhanced . accordingly , the treatment with anti - allergen igg2 at the time of the first challenge not only reduced the airway inflammatory response to aeroallergen , but also selectively affected the formation of th1 versus th2 - dependent memory b cells . persistence of the reduced airway inflammatory response to aeroallergen during a second round of aerosol challenge the previous observations that locally administered anti - allergen igg2 protects against allergen - induced airway inflammation through an active instead of passive mechanism ( see examples 3 - 5 ) implies that a modification of the nature of the anti - allergen immune response drives the airway eosinophilic inflammation . if this implication is true , a likely consequence would be that the immune response retains a memory of the altered nature and may cause a persistence of the therapeutical effect . to verify this possibility , sensitized mice received a first challenge with ova by exposure to aerosol during two consecutive days along with two separate administrations of 50 μg anti - ova igg2a given 2 h before each aerosol ( fig1 ). this treatment with antibody resulted in a pronounced reduction of bronchial alveolar cell infiltration and eosinophilia measured 48 h after the last ova aerosol ( fig1 ). the persistence of the protective effect was verified by exposing the treated mice to a second round of aerosol challenge 6 days after the first round ( fig1 ). in this case , the mice did not receive an additional treatment with anti - ova igg2a which allowed analysis of the endurance of the protective effect during a new allergen exposure . as shown in fig1 , the mice retained a memory of the first treatment as apparent from the significantly lower airway inflammation and eosinophilia induced by the second round of allergen challenge . this result confirms the active nature of the treatment method and the capacity of the method to generate a sustained cure for asthma rather than a symptomatic treatment . conversion of the anti - allergen cd4 + t cell response from a th2 polarized response to a th1 and th2 mixed response the reduced airway inflammatory response , the persistent nature of the reduction and the increased formation of th1 - dependent memory b - cells , but not of th2 - dependent memory b - cells after intranasal administration of anti - allergen igg2 , indicate that an increased participation of th1 cells represents the actual modification of the anti - allergen immune response that is responsible for the reduced asthmatic phenotype . to verify this possibility , the number of ova - responsive th1 and th2 cells in the bal was determined . bal cells were stimulated in vitro with anti - cd3 antibody in the presence of anti - cd28 antibody ( maximization of t cell costimulation ) and the number of ifn - γ , il - 4 and il - 5 - secreting cd4 + t cells were determined by cytoplasmic cytokine staining and 2 - color flow cytometry ( table 2 ). cd4 + t cells from the bal of sensitized mice challenged with ova and treated with placebo produced predominantly the th2 cytokine il - 4 , whereas a smaller fraction of the cells produced the th1 cytokine ifn - γ . the prevailing th2 nature of the bronchial alveolar cd4 + t cells is in agreement with the well - established th2 nature of the airway inflammatory response . treatment with anti - ova igg2a reversed the immune response to a prevailing th1 response as apparent from the reduced number of il - 4 - secreting th2 cells , the doubling of ifn - γ - secreting th1 cells and the resulting shift in the th1 / th2 ratio from 0 . 5 to 1 . 9 . from this result , it can be concluded that the anti - ova igg2a exerts its protective and sustained effect on allergen - induced airway eosinophilia by altering the th1 / th2 ratio of the immune response and shifts the response pattern from a pathological th2 response towards a benign th1 response . intranasal administration , prior to aeroallergen exposure , of a compound that binds inhaled allergen and allows the inhaled allergen to be directed to antigen - presenting cells that induce and / or support th1 cell responses and counteract th2 responses , in casu igg2 and macrophages ( such as ifn - γactivated macrophages ), respectively , has an inhibitory effect on aeroallergen - induced eosinophilic airway inflammation in sensitized mice by modifying the cd4 + t cell response from a predominant th2 response to a predominant th1 response . to verify whether the generation of a prevailing th1 environment by local treatment with anti - allergen igg2 promotes the generation of a prevailing th1 response against unrelated aeroallergens , mice were simultaneously rendered sensitive to two inhaled antigens , ova and human catalase ( hcat ). the occurrence of cross - protection was analyzed by intranasal administration of igg2a antibodies against allergen , followed 2 h and 26 h later by intratracheal instillation of both antigens . analysis of the bal 2 days later reveals a clear reduction in airway inflammation as apparent from the reduced cell infiltration and degree of eosinophilia . this reduction was not observed in mice treated with the mismatched antibody which confirms the requirement for a high - affinity interaction between the administered igg2a antibody and the allergen . to verify the occurrence of cross - protection , the mice were exposed to aeroallergen 6 days after the last challenge . however , the aeroallergen was mismatched with respect to the specificity of the antibody instilled during the first round of allergen challenge . in this manner , mice treated with anti - hcat igg2a and challenged with hcat and ova were rechallenged with ova without further treatment with antibody . inversely , mice treated with anti - ova igg2a and challenged with hcat and ova were rechallenged with hcat . in both instances , a reduction of the bal cell infiltration and airway eosinophilia was observed despite the mismatch between the treating antibody given during the first challenge and the allergen instilled during the second challenge . these results demonstrate that an increase of th1 reactivity against a single allergen exerts a bystander activity on the immune response against a second allergen and promotes the induction of a th1 response against the second allergen . as a consequence , treatment concomitantly suppresses airway hyperreactivity to unrelated inhaled allergens through this bystander activity although the treatment specifically targeted a single allergen . azuma , i ., k . sugimora , t . taniyama , m . yawakawi , y . yamamura , s . kusumoto , s . okada , and t . shiba . 1976 . adjuvant activity of mycobacterial fractions : immunological properties of synthetic n - 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