CA2246427A1 - Methods for treatment of allergic diseases with ige - Google Patents

Abstract

Methods of treatment of allergic diseases, e.g. allergic rhinitis and allergic asthma, with anti-IgE antibodies and other IgE antagonists are described.

Description

W o 97/33616 PCTrUS97/03443 METHODS FOR TREATMENT OF ALLERGIC DISEASES WITH IG~

This invention relates to methods of treatment of allergic diseases with IgE antagonists, including anti-lgE antibodies.
BACKGROUND OF THE INVENTION
Allergic rhinitis is the most common form of atopic disease, affecting dlJI.)lO~ y 25%-30% of the U.S. population. Ragweed is the most common cause of seasonal allergic rhinitis (hay fever) in North America and is widespread throughout the warmer portions of the Western Hemisphere.
Current therapies for the treatment of allergic rhinitis symptoms include allergen avoidance, pharmacological interventions (e.g. ~ntihict~rnines, sympathomimetics, topical and systemic corticosteroids and chromones) and immunotherapy . Although helpful, many of these pharrnacological interventions provide only moderate or partial relief of symptoms and may be associated with cignifir~nt side effects. Traditional allergy immunotherapycan be employed alone or in conJunction with these phannacological interventions to provide relief of symptoms, but is only effective in a narrow, antigen-specific fashion.
Asthma is a common condition characterized by reversible airflow obstruction and airway hyperresponsiveness, attributed to bronchial infl~mm~tion. It is not understood precisely how airway h~Jc--ca~ /ity is produced in pre~licposed individuals but allergen-induced infl~ vly ~~ ullses appear to be pivotal in many forms of clinical asthma. In this regard, allergen exposure can increase airway responsiveness in s~ d subjects and, in the allergic subject with asthma, can lead to worsening and 20 p~ ,ce of asthma symptoms.
IgE plays a central role in the cascade of events leading to the early and late airway responses t allergen exposure in allergic asthma. The interaction of allergen with mast cell bound IgE triggers a~,~,- c~,dLion and crossl inking of IgE receptors, in turn provoking mast cell degranulation with the release of preformed mediators, such as hict~minc and tryptase, and the synthesis and release of p~ ,.gl"..riinc, leukotrienes, and 25 cytokines. These ~ . "" .~ . ymediators are thought to cause the airway mucosal edema and contraction of smooth muscle responsible for the imme~ t~ bronchoconstriction that cha~ the early asthmatic response to allergen ch~ ng e Airwayhy~c..ca-,liviLyreactionscan be inducedin the lal~ol~Lolyby exposing subjects with allergic asthma to nebulized solutions of allergen extract, the conc~,..ll ~Lion of which can be determined by airway 30 hyperreactivity to methacholine and skin test reactivity to the same allergen. This p~vcedu-c is known as experimentalaerosolizedallergen rh~lllPnec or bronchial provocation. Bronchial provocation is a useful and relevantmodelforthestudyofanti-~cthm~meriir~tionc(cockcroftetal. J. AllergyClin. ImmunoL 79:734-40 (1987);CresciollietaL Ann. Aller~66:245-51 (1991);Wardetal. Am. Rev. Respir. Dis. 147:518-23 (1993)).
For example, it is known that beta agonists inhibit the early s~ethm~tic response (EAR) but not the late 35 ~cthm~tic response (LAR) to allergen, and that a single dose of inhaled steroid inhibits the LAR but not thc ~ EAR (Cockcroft et aL, J. Allergy Clin. Immunol. 79:734-40 (1987)). Theophylline and ~lico~
cromoglycate attenuate both the EAR and LAR ~ onses to allergen (Cresciolli et aL, supra; Ward et al, W O 97133616 PCTrUSg7/03443 supra). Most drugs with proven efficacy in asthma m~n~gement have been shown to attenuate airway responses to inhaled antigens ~minic~Pred in bronchial provocation.
Other allergic diseases, such as allergic rhinitis, result when IgE antibodies bind to Fc~RI receptors located on circulating basophils. Like the functioning of mast cells in allergic asthma, basophils mediate S allergic disease by release of hict~mine and other mediators upon allergen crosclinking of allergen-specific IgEantibodiesoccupying Fc~ cel lu-~ on the basophil cell surface. Malveaux etal., J. Clin. Invest., 62:
176-181 (1978) reported a study of human basophil receptor density and noted an excellent correlation between the plasma total IgE level and the number of Fc~ ece~Lo.~ per basophil. This and subscquent studies noted that FccRI densities in allergic and non-allergic individuals range from 104 to I o6 1 e~e~Lu. ~ per 10 basophil.
Allergic rhinitis symptoms can be induced by exposing subjects to nasal challenge with allergen.
There is a highly ~iEnific~nt correlation between the results of nasal challenge and nasal symptoms among rhiniticpatients (Bousquetetal., J. Aller. Clin. Immunol., 85: 490-497(1990)). In addition, many drugs with proven efficacy in rhrnitis management have been shown to attenuate symptoms induced by nasal challenge 15 with allergen. Nasal challenge is a useful model for demonstrating the efficacy of anti-allergy medications (see e.g.,Naclerio et al., Arch. OtolaryngoL, 110: 25-27 (1984); Bascom et aL, J. Aller. Clin. Immunol., 81:
580-589 (1988)).
The concept of using anti-lgE antibodies as a ll c;allllcul for allergy has been widely disclosed in the scientificliterature. A few lep..s~ eexamples are as follows. Baniyash and Eshhar (European Journal 20 of Immunology 14:799-807 (1984)) demonstrated that an anti-lgE monoclonal antibody could specifically block passive eL~ eousanaphylaxisreaction when injected intradermallybefore rh~llengingwith the antigen;
U.S. 4,714,759 discloses a product and process for treating allergy, using an antibody specific for IgE; and Rup and Kahn (International Archives Allergy and Applied Immunology, 89:387-393 ~1989) discuss the prevention of the development of allergic responses with monoclonal antibodies which block mast cell-lgE
25 S~
Anti-IgEantiho~ sthat block the binding of IgE to its receptor on bsl~ophilc yet fail to bind to IgE
bound to the receptor, thereby avoiding hict~nninl~ release are ~licclocerl for example, by Rup and Kahn (supra), by Baniyash et aL (Molecular Immunology 25:705-711, 1988), and by Hook et al. (Federation of American Societies for E~ i,..c,.lal Biology, 7 I st Annual Meeting, Abstract #6008, 1987).
Antagonists of IgE in the form of receptors, anti-lgE antibodies, binding factors, or fragments thereof have been disclosed in the art. For example, U.S.4,962,035 disclosesDNA f-nc o-1ingthe alpha-subunit of the mast cell IgE receptoror an IgE binding fragmentthereof. Hook et aL (FederationProceedings Vol. 40, No.
3, Abstract #4177) disclose monoclonal antibodies, of which one type is anti-idiotypic, a second type binds to common IgE determinants, and a third type is directed towards determinants hidden when IgE is on the 35 basophil surface.
U.S. 4,940,782 discloses monoclonal antibodies which react with free IgE and thereby inhibit IgE
binding to mast cells, and react with IgE when it is bound to the B-cell membrane, but do not bind with IgE
when it is bound to the mast cell Fc~ receptor, nor block the binding of IgE to the B-cell receptor.

WO 97133616 PCT/US97/034'13 U.S. 4,946,788 discloses a purified IgE binding factor and ~ragments thereof, and monoclonal antibodies which react with IgE binding factor and Iymphocyte cellular receptors for IgE, and derivatives thereof.
U.S.5,091,313 discloses antigenic epitopes associated with the extracellular segment of the domain which anchors immunoglobulinsto the E cell membrane. The epitopes recogni7~d are present on IgE-bearing B cells but not basophils or in the secreted, soluble form of IgE. U.S. 5,252,467 discloses a method for producingantibodiesspecific for such antigenic epitopes. U.S. 5,231,026 discloses DNA encoding murine-human antibodies specific for such antigenic epitopes.
U.S.4,714,759 discloses an immunotoxin in the form of an antibody or an antibody fragment coupled 10 to a toxin to treat allergy.
Presta et aL (J. ImmunoL 151:2623-2632 ~1993)) disclose a hllm~ni7ed anti-lgE antibody that preventsthe binding of free IgE to Fc~RI but does not bind to Fc~RI-bound IgE. Copending WO 93/04173 discloses polypeptides which bind dil~l ~ ulially to the high- and low-affinity IgE receptors.
U.S. 5,428,133 discloses anti-IgE antibodies as a therapy for allergy, especially antibodies which 15 bind to IgE on B cells, but not IgE on basophils. This publication mentions the possibility of treating asthma with such antibodies. U.S. 5,422,258 discloses a method for making such antibodies.
Tepper et al. ("The Role of Mast cells and IgE in Murine Asthma", presented at "Asthma Theoly to Treatment", July 15-17, 1995) disclose that neither mast cells nor IgE greatly inflnt~nre the anaphylaxis, airway hyperreactivity, or airway i . . n ~ . " ~ . . ~1 ;VII in a murine model of asthma.
SUMMARY OF THE INVENTION
The invention provides for a method for treating an allergic disease, including allergic rhinitis and allergic asthma, CO~ illg ~rlminietering a loading dose of an IgE antagonist for a period of at least about 14dayssufficienttoreducethepatient'saverageserumfreelgEieveltoalevelno~c;al~, Llla,labout50ng/ml at the end of the loading period, followed by a ", ~ lce dose of the IgE antagonist averaging about 25 0.00008 to 0.0024 mg/kg/week IgE antagonist for every lU/ml baseline free IgE in the patient's serum, wherein the m lint-?n~n ce dose is at least about three fold lower than the loading dose in units of mg/kg/week IgE
antagonist for every IU/ml baseline free IgE in the patient's serum.
The invention further provides for a method for treating allergic disease, in~ ling allergic rhinitis and allergic asthma, Cvll~ u~g ~nninict~ring a loading dose of an IgE antagonist averaging at least about 30 0.003 mg/kg/week IgE ~nt~gf.nict for every lU/ml baseline free IgE in the patient's serum for a period of at least about 14 days, followed by a ~ r~ edose of the IgE antagonistaveraging about 0.00008 to 0.0024 mg/kg/weekIgEantagonistforeverylU/mlbaselinefreeIgEinthepatient'sserum,whereinthem~ r~n~ne dose is less than about one third of the loading dose.
Also provided is a method for treating allergic disease, including allergic rhinitis and allergic asthma, 35 cv",~ i"g~-lmini~tPringa loading dose of an IgE antagonist for a period of at least about 14 days, followed by a ms~int~n~nce dose of the IgE antagonistthat m~int:~in~ the patient~s serum free IgE conc~ inn at a level no greater than about 600 ng/ml, wherein the loading dose exceeds the m~int,-n~n-~e dose by at least about three fold in units of mg/kg/week IgE antagonist for every lU/ml baseline free IgE in the patient's serum.

W O 97/33616 PCTrUS97/03443 BRIEF DESCRIPTION OF THE FIGURES
Fig. I is a graph depicting serum concentrationsof free IgE in patients undergoing anti-lgE antibody therapy. The values shown are mean l standard deviation (SD) free IgE concentrations predose and two hours post-dose on days 0, 7, 14, 28, and 42 of the study for the twelve patients for whom free IgE data was S available. The means and standard deviations were ç~ fed on the log-transformedfree IgE concentrations.
Figs. 2A-C are graphs depicting expression of endogenous IgE and Fc~RI on basophils of donors undergoing anti-IgE antibody therapy. Figs. 2A-C show the results for 12 treated patients and 2 controls and the data were generated using the acetate buffer "stripping" terhni~ f Fig. 2A shows the results for endogenous IgE antibody. Fig. 2B displays the results of Ig~ measurements for cells fully s~nciti7Pd and 10 therefore plots total receptor fl~nci~i~c Fig. 2C displays the BPO-specific IgE densities and therefore plots unoccupiedreceptord~ncitiec AsexplainedingreaterdetailinExamplel below,thestars~ .c;~t:..tcalculated maximums for measurementswhere the IgE level was not detectable by the total IgE radioimm--no~cc~y. The open symbols indicate the controls and closed symbols indicate the treated patients.
Figs. 3A-B are graphs depicting the levels of basophil IgE with and without se ~C~ ion as 15 determ ined by flow cytometry. Fig. 3A shows the results for endogenous IgE e~ iOll (without sensitization) for 12 patients on therapy at three time points and Fig. 3B shows similar data for cells first s~ncifi7Pd with IgE myeloma antibody.
Figs. 4A-B are graphs depicting flow cytometric measurements of endogenous IgE and Fc~RI
expression during the first 2 weeks of therapy for 3 patients with the highest serum levels of IgE at enrollment.
20 Fig. 4A shows the results for IgE ~ e~iu.. without prior s~ n (open symbols) and with prior S~ ,.l ;nn (closed symbols) for three different subjects. Fig. 4B shows the results for Fc~ a~;vn as dct~,....i-.cd by the binding of the anti-Fc~Rla antibody, 29C6. Values of zero in Fig. 4A ~ t;~ etect~ble Fc~RI expression levels as compared to background signal produced by FITC-conjugated normal goat IgG.
Values for irrelevant IgG controls in Fig. 4B were 3-5. The dosing regimen indicated by the arrows is 25 described in Example I below.
Fig. 5 is a graph depicting the histamine release response of basophils challenged with 3 sc. .t?t~ gogues before therapy and 3 months into therapy. The left portion of the figure shows the anti-IgE
antibody dose response curves at the two time points; pre-therapy (~ .cs~ ed as open circles) and the 3 month time point (-~ s~ das closed circles). The right side histograms show the -~ ,onses to FMLP and 30 dust mite antigen (D. farinae at 10 PNlJ/ml). The box plots show the median ~25% of the median at the two time points.
DETAILED DESCRIPTION OF THE INVENTION
A. DEFINITIONS
The terms "allergy" and "atopy" and all their ~ ", l l~ l variants are used synonymously herein to 35 refertoanydiseaseme~ tf~dbyaTypeI(Gell&coombsc!~ccifi~ition)hy~tl~ ilh/ityreaction~including allergic rhinitis, atopic dermatitis~ anaphylaxis, allergic asthma.
The term "asthma" as used herein refers to a lung disease chald~ d by airway obstruction that is reversible (although not entirely in some patients) either sp~nlancollsly or with treatment, airway W O 97/33616 PCTrUS97/03443 infl~mm~tion,and increasedairwayresponsiveness to a variety of stimuli. "Allergic asthma" as used herein refers to an ~cthm:ltic response to inhalation of an antigen to which the patient is sensitive.
The term "early s~cthm~tic response" (EAR) as used herein refers to an asthmatic response to an antigen within about two hours of exposure. The terrn "late ~c~hm~ticresponse" (LAR) as used herein refers to an acfhm~tic response to an antigen within about two to eight hours after exposure.
The term "allergic rhinitis" as used herein refers to any allergen-induced nasal symptoms, including itching, cnep7ing nasal congestion, nasal di~chal~,e, and symptoms associated with nasal mucosal infl~mm~ti~n.
The term "IgE antagonist"as used herein refers to a sllhsf~nce which inhibits the biological activity of IgE. Such antagonists include but are not limited to anti-lgE antibodies, IgE I ~c~ ~,lu, " anti-lgE receptor antibodies, variants of IgE antibodies, ligands for the IgE receptors, and fragments thereof. Antibody antagonists may be of the IgA, IgD, IgG, or IgM class. Variant IgE antibodies typically have amino acid cllhctitlltinnc or deletions at one or more amino acid residues. Ligands for IgE rccel,Lc"~ include but are not limited to IgE and anti-receptor antibodies, and fragments thereof capable of binding to the receptors, including amino acid 5nh5ti~1tion and deletion variants, and cyclized variants.
In general, in some embodiments of the invention, IgE antagonists act by blocking the binding of IgE
to its l~ce~lola on B cells, mast cells, or basophils, either by blocking the receptor binding site on the IgE
molecule or blocking its l~c~ a. ~ liti~n~lly, in some embodiments of the invention, IgE antagonists act by binding soluble IgE and thereby removing it from circulation. The IgE antagonists of the invention can alsoactbybindingtomembrane-boundIgEonBcells,therebyelimins~tingclonalpopulationsofBcells. The IgE antagonists of the instant invention can also act by inhibiting IgE production. Preferably, the IgE
antagonists of the instant invention do not result in hict~mine release from mast cells or basophils.
The term llciallllc.l~" as used herein denotes therapy or prophylaxis that prevents or ameliorates symptoms of a disorder or responsive pathologic physiological condition.
The term "mg/kg/week IgE antagonist for every lU/ml baseline free IgE in the patient's serum" as used herein is defined as a drug dosage unit expressed in terms of an average amount of IgE antagonist in milligrams~minictPredperkilogramofpatientbodyweightperweekforeveryInternationalUnitofbaseline free IgE per milliliter of patient serum. An l-,t~ )io"~l Unit of IgE is defined as 2.4 ng/ml. The per week term does not necessarily indicate weekly ~lminictration, but rather the average weekly amount of drug received ~ tl~d by dividing the amount of drug received over a given treatment period by the number of weeks in the Ll~:dlll.e.ll period.
"Polypeptide" asusedhereinrefersgenerallytopeptidesandproteinshavingatleastabouttwoamino acids.
The term "free IgE" as used herein refers to IgE not complexed to a binding partner, such an anti-lgE
antibody. The terrn "total IgE" as used herein refers to the measurement of free IgE and IgE comptexed to ~ a binding partner, such as an anti-lgE antibody. The terrns "baseline IgE" aDd "baseline free IgE" as used herein refer to the level of free IgE in a patient's serum before treatment with an IgE antagonist.

W O97/33616 PCTrUS97/03443 The term "polyol" as used herein denotes a hydrocarbon including at least two hydroxyls bonded to carbon atoms, such as polyethers(e.g. polyethylenegiycol3,trehalose, and sugar alcohols (such as mannitol).
The term "polyether" as used herein denotes a hydrocarbon containing at least three ether bonds.
Polyethers can include other functional groups. Polyethers useful for practicing the invention include polyethylene glycol (PEG).
B. GENERAL MET~IODS
The present invention arises from the inventors' discovery that treatment of atopic patients with IgE
antagonistresults in the down regulation of the high affinity IgE receptor (Fc~RI) on the basophils from the treated patients. Based on these surprising and unexpected results, the inventors determined that the down 10 regulation of the Fc~RI produced by an unusually high loading dose of IgE antagonist significantly reduces thelevelofIgEantagonistthatwouldneedtobemaintainedinordertoblockIgE-mP~ t~dallergicresponses in atopic patients. Accordingly, the inventors designed and developed methods for treating allergic disease with IgE antagonistswherein a loading dose of IgE antagonist that is ~igni~c~ntly higher than that ordinarily designed to reduce the time to attain a target serum drug concentration is used to down-regulate IgE receptor 15 onbasophils(basophilstripping),followedbyam~ cedoseregimenprovidingsignific~ntlylowerdrug amounts and/or fewer drug at~minic~ations than would a typical Il~ lCt: regimen that is designed to maintain equilibrium at the target serum drug conce~ a~ioll attained with the ordinary loading dose. The methods of the invention advantageouslyreduce the cost and other burdens affecting the treatment of allergy with IgE antagonists by eignific~ntly reducing the amount of drug and/or frequency of treatment required.
l . IgE Antagonists IgE antagonists suitable for use in the methods of the invention include anti-IgE antibodies, IgE-binding proteins, soluble IgE receptors, anti-lgE receptor antibodies, IgE variants and other IgE receptor binding competitors.
Polyclonal antibodies to IgE generally are raised in animals by multiple sub~ ous (sc) or 25 h.L.~,t.iloneal (ip) injections of IgE and an adjuvant. It can be useful to conjugate IgE or a fragment contS~iningthe target amino acid sequence from the Fc region of IgE to a protein that is illllllUllOg~:lliC in the speciestobeimmnni7~l,e.g.,keyholelimpethemocyanin,serumalbumin,bovinethyroglobulin,0rsoybean trypsin inhibitorusing a bifunctionalor derivatizing agent, for example, m:~leimi-lQbenzoyl sulfosuccinimide ester(conjugationthroughcysteineresidues),N-hydroxysuccinimide(throughlysineresidues),gll~t~r~ ehyde, 30 succinic anhydride, SOCI2, or RIN = C = NR, where R and Rl are different alkyl groups.
Animals ordinarily are immnni7.od against the cells or immunogenic conjugates or derivatives by combining I mg or I ~g of IgE with Freund's complete adjuvant and injecting the solution intradermally at multiple sites. One month later the animals are boosted with 1/5 to 1/10 the original amount of conjugate in Freund's incomplete adjuvant by sllhcl-t inPous injection at multiple sites. Seven to 14 days later, animals are 35 bled and the serum is assayed for anti-IgE titer. Animals are boosted until the titer plateaus. Preferably, the animal is boosted with a conjugate of the same IgE, but conjugated to a different protein and/or through a different cross-linking agent. Conjugates also can be made in recombinant cell culture as protein fusions.
Also, a~ L~.Ig agents such as alum can be used to enhance the immune response.

W O 97/33616 PCTrUS97/03443 Monoclonal antibodies are prepared by recovering spleen cells from immllni7f d animals and immortalizingthe cells in conventional fashion, e.g. by fusion with myeloma cells or by Epstein-Barr (EB)-virus transformation and screening for clones ~ IC;5~illg the desired antibody. The hybridoma tP~hniqll~
described originally by Koehler and Milstein, ~ur. J. ImmunoL, 6: 511 (1976) and also described by Hammerling et al., in: Monoclonal Antibodies and T-Cell Hybridomas, Elsevier, N.Y., pp. 563-681 ( 1981 ) has been widely applied to produce hybrid cell lines that secrete high levels of monoclonal antibodies against many specific antigens.
The hybrid cell lines can be maintained in vitro in cell culture media. The cell lines producing the antibodies can be selected andJor m~int~in--d in a composition C~ lisi,lg the continuous cell line in 10 hypoxanthine-amino~,L~lhllllymidine(HAT) medium. In fact, once the hybridoma cell line is established, it can be maintainedon a variety of nu~ ollallyadequate media. Moreover, the hybrid cell lines can be stored and preserved in any number of conventional ways, in~ ing freezing and storage under liquid nitrogen.
Frozen cell lines can be revived and cultured in-lefinitf?lywith resumed synthesis and secretion of monoclonal antibody.
The secreted antibody is recovered from tissue culture si ~ I by conventional methods such as precipitation,ion-exchangechromatography, affinity cl~l~,nlalography, or the like. The antibodies described herein are also recovered ~om hybridoma cell cultures by conventional methods for purification of IgG or IgM, as the case may be, that heretoforehave been used to purify these immunoglobulinsfrom pooled plasma, e.g., ethanol or polyethylene glycol ~l~cilJiL~lion IJIoCedult;S. The purified antibodies are sterile-filtered.
While mouse monoclonal antibodies are routinely used, the invention is not so limited; in fact, human antibodies can be used. Such antibodies can be obtained, for example, by using human hybridomas (Cote et aL, Monoclonal Antibodies and Cancer Therapy, Alan R. Liss, p. 77 (1985)). In fact, according to the invention, t~rhniqllee developed for the production of chimeric antibodies (Cabilly et aL, U.S. 4,816,567, Morrison et al., Proc. NatL A.cad. Sci. 81: 6851 (1984); Boulianne et aL, Natz~re 312: 643-646 (1984);
25 Neubergeretal.,Nature,312:604(1984);N~ t;letal.,Nattlre314:268-270(1985);TakedaetaL,Nature 314: 452 (1985);EP 184,187; EP 171,496; EP 173,494; PCT WO 86/01533; Shaw etaL, J. Nat. ~anc. Inst.
80: 1553-1559 (1988); Morrison, Science 229: 120Z-1207 (1985); Oi et aL, BioTechniques, 4: 214 (1986)) by coupling an animal antigen-binding variable domain to a human constant domain can be used; such antibodies are within the scope of this invention. The term "chimeric" antibody is used herein to describe a 30 polypeptideco~ hlgatleasttheantigenbindingportionofanantibodymoleculelinkedtoa~leastpartof another protein (typically an immunoglobulin constant domain).
In one embodiment, such chimeric antibodies contain about one third rodent (or other non-human species) sequence and thus are capable of eliciting a ~ignifi~nt anti-globulin response in humans. For example, in the case of the murine anti-CD3 antibody OKT3, much of the resulting anti-globulin response is 35 directedagainstthevariableregionratherthantheconstantregion(Jaffersetal., Transplantation41: 572-578 (1986)).
Hnm~n i7Pd antibodies are used to reduce or elim inate any anti-globulin immune response in humans.
In practice, hnm~ni~Pd antibodies are typically human antibodies in which some amino acid residues from the CA 02246427 l998-08-l2 WO 97/33616 PCTrUS97/03443 complementarity det~rrninin~; regions (CDRs), the hypervariable regions in the variable domains which are directly involved with formation of the antigen-binding site, and possibly some amino acids from the framework regions (FRs), the regions of sequence that are somewhat ~;OIISt~l .rcd within the variable domains, are ~nlJ~ d by residues from analogous sites in rodent antibodies. The construction of hllm~ni7~d S antibodies is described in Riechm~nn et al., Nature 332: 323-327 (19883, Queen et al., Proc. Natl. ~cad. Sci.
USA 86: 10029-10033 (1989), Co et al., Prof~. Natl. Acad Sci. USA 88: 2869-2873 (1991), Gorman et al., Proc. Natl. Acad. Sci. 88: 4181-4185 (1991), Daugherty et al., Nucleic Acids Re~. 19: 2471-2476 (1991), Brown et al., Proc. Natl. Acad Sci. USA 88: 2663-2667 (1991), Junghans et al., Cancer Res. 50: 1495-1502 (1990), Fendly et al., Cancer Res. 50: 1550-1558 (1990) and in PCT applications WO 89/06692 and WO
1 0 92/22653 .
In some cases, ~-lb~ h~gCDRs from rodent antibodies for the human CDRs in human frameworks is sufficient to transfer high antigen binding affinity (Jones et al., Nature 321: 522-525 (1986); Verhoeyen et al., Science 239: 1534-1536 (1988)) whereas in other cases it is necessary to ~Miticm~lly replace one (Riechm~nn et al., supra) or several (Queen et al., supra) FR residues. See also Co et aL, supra.
Theinventionalsoenc~ p~c~stheuseofhumanantibodiesproducedintransgenicanimals. Inthis system, DNA ~ncofling the antibody of interest is isolated and stably incorporated into the germ line of an animal host. The antibody is produced by the animal and harvested from the animal's blood or other body fluid. Altematively, a cell line that G~ ses the desired antibody can be isolated from the animal host and used to produce the antibody in vitro, and the antibody can be harvested from the cell culture by standard methods.
Anti-lgE antibody r. ~ .IL~ can also be used in the methods of the invention. Any fragment of an anti-lgE antibody capable of blocking or di~- u~Li~g IgE interaction with its receptor is suitable for use herein.
Suitable anti-lgE antibody fragments can be obtained by screening combinatorial variable domain libraries for DNA capable of ~ln~;,ail~g the desired antibody fragments. These te~hni~ es for creating recombinant DNA versions of the antigen-bindingregions of antibody molecules which bypass the generation of monoclonal antibodies, are ~nc~",~ ced within the practice of this invention. One typically extracts antibody-specific m~cc~ng~r RNA molecules from immune system cells taken from an immllni7f-d animal, ~l dns~ .sthese into compl~ lL~ y DNA (cDNA), and clones the cDNA into a bacterial expression system.
"Phage display" libraries are an example of such t~ hni~ln~c One can rapidly generate and screen great numbers of cznAillAtl?s for those that bind the antigen of interest. Such IgE-bindingmolecules are specifically encompassed within the term "antibody" as it is defined, AiccllcceA and claimed herein.
In some embodiments, the IgE allLzlgoni~Lbinds to the IgE receptor binding site on the IgE molecule, thereby preventing the IgE molecule from binding to the IgE receptor. In other embodiments, the IgE
antagonistis an anti-lgE antibody that binds to the IgE receptor binding site on the IgE molecule. Preferred embodimentsofthe invention include methods using the monoclonal antibodies MAEI 1, MAE13, MAE15, and MAE 17 disclosed in copending application WO 93/04173 . Particularlypreferred are embodiments using hllmzlni7~d versions of MAEI 1, including variant 8b disclosed in Table 9 on page 68 of WO 93/04173.
Variant8b is also describedas hums-ni7lod variant 12 of MAEI I in Presta et al., supra, and is identical to the rhuMAb-E25 antibody described in Example I below. Other preferred embodiments of the invention use antibodies capable of binding to (B cell) membrane-boundIgE, such as rhuMAb-E25. In some embodiments, the methods of the invention use antibodies that suppress serum IgE levels by clonal deletion of IgE-producing B cells or inhibit IgE production by other mefh-nicmc In a further embodiment of the invention, soluble IgE receptor can be used as the IgE antagonist.
Soluble I ecc~Lol ~ suitable for use herein include, for example, molecules comprising the IgE binding site in the extracellulardomain (exodomain3Of the Fc~ -chain. The Q-chain of Fc~RI can be geneticallymodified such that the exodomain is secreted as a soluble protein in a recombinant expression system according to the method of Blank et aL, J. Biol. Chem., 266: 2639-2646 (1991) or Qu et al., J. Exp. Med.,167:1195.
The invention also enco~,-passes the use of IgE-binding peptides in addition to anti-IgE antibodies and soluble receptor. Any IgE-bindingpeptide capable of disrupting or blocking the interaction between IgE
and its receptors is suitable for use herein.
In addition to Ig~ antagonists which interfere with IgE/receptor interaction by binding to IgE, such as anti-IgE antibodies, rla~ thereof, soluble IgE receptor and other IgE-binding peptides described above, the invention encompasses the use of IgE antagonists which disrupt IgE/receptor interaction by Li~.g with IgE for binding to its receptor, thereby lowering the available IgE receptor.
IgE variants are an example of a receptor-binding culll~lilol that is suitable ~or use in the methods of the invention. IgE variants are forms of IgE po~ie~;..g an alteration, such as an amino acid cnhctitll~ion or suhstitlltions and/or an amino acid deletion or deletions, wherein the altered IgE molecule is capable of c~nlllJ~.li-lg with IgE for binding to its l~ce~lul~.
~ragments of IgE variants are also suitable for use herein. Any fragment of an IgE variant capable of CIJIIIP~ with IgE for binding to its I~C~ ~JL~)Ia can be used in the methods of the invention.
l~e inventionalso enco.npass~the use of IgE receptor-binding peptides in addition to IgE variants and fragments thereof. Any IgE receptor-binding peptide capable of disrupting or blocking the interac~ion between IgE and its l-,C~ , is suitable for use herein.

2. Th~la~ lic Compositions Cnnt~ining IgE Ant~gonict In general, the formulations of the subject invention can contain other components in amounts not detracting from the preparation of stable forms and in amounts suitable for effective, safe ph~ f e--ti~
~-lmini~tration. Forexample,other~ e~1;call~acc~,Lnl,let;,.ci~ well known to those skilled in the art can form a part of the subject compositions. These include, for example, salts, various bulking agents, additionalbufferingagents, chelatingagents, antioxidants,cosolventsand the like; specific examples of these include tris-(hydroxymethyl)aminomethane salts ("Tris buffer"), and disodium edetate.
In one embodimentofthe invention,IgE antagonistformulationscomprisea buffer, a salt, optionally, ~ a polyol, and optionally, a preservative.
One exemplary formulation of the invention is a liquid formulation of about 1-100 mg/ml IgE
~ antagonist in 10 mM acetate buffer, pH 5.0-6.5, 100-200 mM sodium chloride, and about 0.01% polysorbate 20, more preferablyabout 5 mg/ml IgE antagonist in 10 mM acetate buffer, pH 5.2, 142 mM sodium chloride, and 0.01% polysorbate20. In other embodiments of the invention, the formulation may be freeze-dried and W O 97/33616 PCTrUS97/03443 recc-nctih~t~d for a~minictration For example, anti-lgE antibody can be f~rmnl~tPd at about 25 mg/ml in 5 mM histidine, pH 6.0, and 88 mM sucrose, free~-dried, and reconstitutedin water to 100 mg/ml antibody for su1minictration. Mixed sugars can also be used, such as a combination of sucrose and mannitol, etc.
In one embodiment,the invention provides for the treatment of allergic diseases, including allergic 5 asthma diseases by ~flminictration of IgE antagonist to the ~ aLoly tract. The invention contemplates formulations coll.prish.g an IgE antagonist for use in a wide variety of devices that are designed for the delivery of pharmaceutical compositions and therapeutic formulations to the respiratory tract. In one aspect ofthepresentinvention,anlgEantagonistisadministeredinaerosolizedorinhaledform. ThelgEantagonist, combined with a dispersingagent, or di~ d,~L,can be ~-~minicteredin an aerosol formulationas a dry powder 10 or in a solution or suspension with a diluent.
Suitabledispersingagentsarewellknownintheart,andincludebutarenotlimitedto~ulra.,l~.lL~and the like. Surfactantsare generallyused in the art to reduce surface induced aggregation of protein caused by z-tnmi7Ationofthe solutionformingthe liquidaerosol. Exarnplesofsuch :,ulra~l~lL:,include polyoxyethylene fatty acid esters and alcohols, and polyoxyethylenesorbitan fatty acid esters. Amounts of surfactants used will 15 vary,beinggenerallywithintherangeofabout0.001to4%byweightoftheform~ tion. Inaspecificaspect, the ~ulra-;Lanl is polyoxyethylene sorbitan monooleate or sorbitan trioleate.
The liquid aerosol formulationscontainthe IgE antagonistand a dispersingagent in a physiologically acceptable diluent. The dry powder aerosol formulations of the present invention consist of a finely divided solid form of the IgE antagonist and a dia~J~.aillg agent, and optionally a buL~cing agent, such as lactose, 20 sorbitol, sucrose, or mannitol, and the like, to facilitate dispersal of the powder. With either the liquid or dry powderaerosol formulation,the formulationmust be aerosolized. That is, it must be broken down into liquid or solid particles in order to ensure that the aerosolized dose actually reaches the bronchii and/or alveoli, as desired. For example, in the methods for treatment of asthma provided herein, it is preferable to deliver aerosol ized IgE antagonist to the bronchii. In other embodiments, such as the present methods for treating 25 adult respiratory distress syndrome, it is preferably to deliver aerosolized IgE auLagOlli~L to the alveoli. In general the mass median dynamic diameter will be 5 mi~ - u---e~ (,um) or less in order to ensure that the drug particles reach the lung bronchii or alveoli (Wearley, L.L., Crit. Rev. in Ther. Drug Carrier Systems 8:333 (1991)).
With regard to construction of the delivery device, any form of aerosolization known in the art, 30 including but not limited to nebulization, atomization or pump aerosolization of a liquid formulation, and aerosolization of a dry powder formlll~tion, can be used in the practice of the invention. A delivery device that is uniquelydesignedfor ~lminictration of solid formulations is envisioned. Often, the aerosolization of a liquid or a dry powder formulation will require a propellent. The propellent can be any propellant generally used in the art. Examples of useful propellants include chlorofluo.~ca~ s, hydrofluo,u~a-l/olls~
35 hydochlorofluorocarbons, and hydrocarbons, including triflouromethane, dichlorodifluoromethane, dichlorotetrafluoroethanol, and I, I, I ,2-tetrafluoroethane, and combinations thereof.
In a preferred aspect of the invention, the device for aerosolization is a metered dose inhaler. A
metereddose inhalerprovidesa specificdosagewhen a-~minict~red, rather than a variable dose depending on W O 97/33616 P~TnJS97/03443 ~Aminictration. Such a metered dose inhaler can be used with either a liquid or a dry powder aerosol formulation.
Systems of aerosol delivery, such as the pressurized metered dose inhaler and the dry powder inhaler are disclosed in Newman, S.P., ~erosols and the Lung, Clarke, S.W. and Davia, D. editors, pp. 197-22 and 5 can be used in connection with the present invention.
Additional pharm~e~ methods may be employed to control the duration of action of the antagonists of this invention. The antagonists also may be entrapped in microcapsules prepared. for example, by coacervation techniques or by interfacial polymerization (for example, hydroxymethylcellulose or gelatin -microcapsulesand poly-(methylmethacylate)microc~rs~ s,respectively), in colloidal drug delivery systems 10 (for example, liposomes, albumin microspheres, microemulsions, nano-particles and n~nocslrslll~c), or in macroemulsions. Such terhniqu~ sare disclosedin Remington'sPha~ r~ ic,7/Sciences, 16th editionj Osol, A., ed., 19~.0).

3. Therapeutic Administration of IgE Antagonists In general, the invention provides for methods for treatment or prophylaxis of allergic disease in a 15 patientby~lminict~oringaloadingdoseofanIgEantagonistthatissufficienttoreducethepatient'sserumlgE
level for a period of time that is suffcient to ci~nific~ntly suppress the expression of Fc~RI receptor on the basophil cell surface, such that a ,..~ t ;~llyreduced ~ r~ e regimen of lgE antagonist is sufficient to prevent the reoccurrence of allergic symptoms. The basophil IgE receptor ~.u~ t;..ion resulting from the unusually high loading dose reduces the receptor density available to trigger an allergic cascade by interaction 20 with allergen-specific IgE, and enables the treating physician to use a 5ignifics~ntly lower m~int~n~n~e dose to prevent basophils from reaching the threshold level of allergen-specific serum IgE s ..~ l ion required for degranulation.
In some embodiments, the loading regimen and/or ll~ilUr~l~lllre regimen dosing amounts are determined according to the baseline serum IgE level of the patient. Patient serum IgE levels are typically 25 assayed by standard ELISA ~erhnirl~les well known in the art. Total serum IgE can be measured by commerciallyavailable assays, such as Abbott Laboratories' Total IgE assay. Free IgE, e.g., IgE not bound to antibody can be measured by a capture type assay in which, for example, IgE receptor is bound to a solid support. IgE complexed to an anti-IgE antibody which binds at or near the site on IgE which binds to the receptor will be blocked from binding the receptor, and thus only free or unbound IgE can react with the 30 receptor bound to the solid support in this assay. An anti-lgE antibody which recognizes IgE even when the IgE is bound to its receptorcan be used to detect the IgE captured by the receptor on the solid support. This anti-lgE antibody can be labeled with any of a variety of reporter systems, such as alkaline phocrh~tslce, etc.
In some embo-limPntc the methods of the invention provide for treatment of allergic disease by ~ ~lminiclering a loading dose of an IgE antagonist for a period of at least about 14 days, or at least about 28 35 days, or at least about 42 days, sufficient to reduce the patient's average serum free IgE level to a level no greaterthan 50 ng/ml, or no greater than 30 ng/ml, or no greater than 16 ng/ml, or no greater than 10 ng/ml, or no greater than 6 ng/ml, at the end of the loading period, followed by a ~ r~ lce dose of the IgE
antagonist averaging about 0.00008 to 0.0024 mg/kg/week, or about 0.00025 to 0.0024 mg/kg/week, or about W O97/~3616 PCT~US97/034430.0008 to 0.0024 mg/kg/week, of IgE antagonist for every IU/ml baseline free IgE in the patient's serum. In otheremborlim~?ntc,the ~"~.;"1~ ..re dose averages about 0.0007S to 0.0024 mg/kg/week, or about 0.00125 to O.OOZ4 mg/kg/week, or about 0.00175 to 0.0024 mg/kg/week, of IgE antagonist for every lU/ml baseline free IgE in the patient's serum. Preferably, the m~int~n~nce dose is at least about three fold lower, or at least aboutsix fold lower, or at least about 12 fold lower, or at least about 25 fold lower, or at least about 50 fold lower, than the loading dose in units of mg/kg/week for every IU/ml baseline free IgE in the patient's serum.
In these embodiments, the patient's serum free IgE response to the loading regimen can be determined by assaying the patient's serum free IgE according to the methods for baseline serum free IgE assay described above.
In the practice of the foregoing methods, the patient's serum IgE level can be reduced to the ~, ese. il,ed level at the end of the loading period by a~lminictPring for the prescribed loading period a loading dose of at least about 0.003 mg/kg/week of the IgE antagonist, or at least about 0.007 mg/kg/week of the IgE
antagonist, or at least about 0.021 mg/kg/week of the IgE antagonist, or about 0.003 to 0.030 mg/kg/week of the IgE antagonist, or about 0.007 to 0.021 mg/kg/week of the IgE antagonist, for every IU/ml baseline free 15 IgE in the patient's serum. However, the invention also ~illC~ aSSeS the use of other loading doses that are sufficient to strip basophils of IgE receptor such that m~intPn~nce dose levels provided in the methods are sufficientto substantially maintain IgE receptor aul.p.c~ion and avoid .Goe~iu-.~..ce of symptoms. In some embodiments, the foregoing methods are used to treat allergic rhinitis or allergic asthma.
In other embodiments,the methods of the invention provide for the treatment of allergic disease by 20 arlm in ictf~ringa loading dose of an IgE ~nt~gon ict averaging at least about 0.003 mg/kg/week IgE ~nt Ig(-ni ct, oratleastaboutO.007mg/kg/weeklgEant:~gonict,oratleastaboutO.021 mg/kg/weeklgEantagonist,orabout 0.003 to 0.030 mg/kg/week IgE antagonist, or about 0.007 to 0.021 mg/kg/week IgE ~nt;-gonict, for every lU/ml baseline free IgE in the patient's serum for a period of at least about 14 days, or about 14 days to 56 days, or about 21 days to 56 days, or about 28 days to 56 days, or about 35 days to 56 days, or about 42 days 25 to S6 days, or about 49 days to 56 days, followed by a ~ e dose of the lgE antagonist averaging about 0.00008 to 0.0024 mg/kg/week IgE antagonist, or about 0.00025 to 0.0024 mg/kg/week IgE ant:l~onict or about 0.0008 to 0.0024 mg/kg/week IgE antagonist, for every lU/ml baseline free IgE in the patient's serum.
In other embodiments, the ~ .t~ e dose averages about O.00075 to O.0024 mg/kg/week, or about O.00 125 to 0.0024 mg/kg/week, or about 0.00175 to 0.0024 mg/kg/week, of IgE ~ntS~g()nict for every lU/ml baseline 30 free IgE in the patient'sserum. Preferably,the ~ e dose is at least about three fold lower, or at least about six fold lower, or at least about 12 fold lower, or at least about 25 fold lower, or at least about 50 fold lower, than the loading dose in units of mg/kg/week for every lU/ml baseline free IgE in the patient's serum.
In some embodiments, these methods are used to treat allergic rhinitis or allergic asthma.
The invention additionally encol..,oa~l s methods for treating allergic disease Cu~ hlg 35 ~iminict~ringaloadingdoseofanlgEantagonistforaperiodofatleastaboutl4days~orabout28to56days~
orabout42toS6days,followedbyam~int~n~n~edoseofthelgEantagonistthatm~int~incthepatient'sserum free IgE concentration at a level no greater than about 600 ng/ml, or no greater than about 300 ng/ml, or no greaterthan about 150 ng/ml, or no greater than about 75 ng/ml, or no greater than about 50 ng/ml, wherein the loadingdose exceedsthe m~inten~ncPdose by at leastaboutthree fold, or by at least about six fold, or by at least about ~2 fold, or by at least about 25 fold, or by at least about 50 fold, in units of mg/kg/week IgE
~nt~gon ict for every lU/ml baseline free IgE in the patient's serum. In some embodiments, these methods are used to treat allergic rhinitis or allergic asthma.
Intheseembodiments,atypicalloadingdoseusedisatleastaboutO.003mgtkg/weeklgE~nt~nict, oratleastaboutO.007mg/kg/weeklgEantagonist,oratleastaboutO.021 mg/kg/weeklgE~nt~gonict,orabout 0.003 to 0.030 mg/kg/week IgE antagonist, or about 0.007 to 0.021 mglkg/week IgE :,nS~gr.nict, for every lU/ml baseline free IgE in the patient's serum. However, the invention also cl,c-""~ çs the use of other loading doses that are sufficient to strip basophils of IgE receptor such that m~inten~nce dose levels which providethe serum free IgE levels recited in the methods are sufficient to 7~ 1y maintain IgE receptor bu~lc~.~.ionand avoid reoc- ull~llccof symptoms. Suitable m~inten mcedosing for practice of these methods includems~int~n~ncedosesofaboutO.00008toO.0024mglkg/weekIgEantagonist,oraboutO.00025toO.0024 mg/kg/week IgE antagonist, or about 0.0008 to 0.0024 mg/kg/week IgE :~nt~gonict, for every lU/ml baseline freelgEinthepatient'sserum. Inotherembodiments,them~int~n~ncedoseaveragesaboutO.00075toO.0024 mg/kg/week, or about 0.00125 to 0.0024 mg/kg/week, or about 0.00175 to 0.0024 mg/kg/week, of IgE
antagonist for every IWml baseline free IgE in the patient's serum. The patient's serum free IgE response to thel"~;"~ eregimencanbedeterminedbyassayingserumfreelgEaccordingtothemethodsforbaseline serum free IgE assay described above.
The invention further provides methods for reducing reactivity to intrabronchial allergen ~h~llPng~
20 inapatientc~....l.,;~;~.g~mini~t~ringaloadingdoseofanlgE~nt~g~ni~tforapeFiodofatleastaboutl4days~
or at least about 28 days, or at least about 42 days, ~llffi~i( nt to reduce the patient's average serum free IgE
level to a level no ~"- c.lt~ h~l. 50 ng/ml, or no ~" calcl lhdll 30 ng/ml, or no greater than 16 ng/ml, or no greater than 10 ng/ml, or no greater than 6 ng/ml, at the end of the loading period, followed by a m~ r..,...cc dose of the IgE al,l~,onL.I averaging about 0.00008 to 0.0024 mg/kg/week, or about 0.00025 to 0.0024 mg/kg/week, or about 0.0008 to 0.0024 mg/kg/week, of IgE antagonist for every lU/ml IgE in the patient's serum. Inotherembodiments,ther.~ a~ .fedoseaveragesaboutO.00075toO.0024mg/kg/week,orabout 0.00125 to 0.0024 mg/kg/week, or about 0.00175 to 0.0024 mglkg/week, of IgE antagonist for every IU/ml baseline free IgE in the patient's serum. Preferably, the m~intç~nce dose is at least about three fold lower, or at least about six fold lower, or at least about 12 fold lower, or at least about 25 fold lower, or at least about 50 fold lower, than the loading dose in units of mg/kg/week for every IU/ml baseline free IgE in the patient's serum . In these embodiments, the patient's serum free IgE response to the loading regimen can be determined by assayrng the patient's serum free IgE according to the methods for baseline serum free IgE assay described above. The patient's reactivity to intrabronchial allergen challenge can be ~et~?rrnin~od according to the methods of Naclerio et al., "IN VIVO METHODS FOR THE STUDY OF ALLERGY: Mucosal tests, tçchni~l~lf sand hllcl~lcl~lionsllAller~ Principles & Practice, Middleton et al., cds, pps. 613-627 (1992), or - as described in Example 2 below.
In the practice of the foregoing methods for reducing bronchial h~l,cl-~a.~ ity, the patient's serum IgE level can be reduced to the prescribed level at the end of the loading period by ~-lmini~tf -ing for the CA 02246427 l998-08-l2 W O 97/33616 PCTrUS97/03443 prescribed loading period a loading dose of at least about 0.003 mg/kg/week of the IgE antagonist, or at least about 0.007 mg/kg/week of the IgE antagonist, or at least about 0.021 mg/kg/week of the IgE antagonist, or about 0.003 to 0.030 mg/kg/week of the IgE antagonist, or about 0.007 to 0.021 mg/kg/weelc of the IgE
antagonist, for every IU/ml baseline free IgE in the patient's serum. However, the invention also encompasses 5 the use of other loading doses that are sufficientto strip basophils of IgE receptor such that maintenance dose Ievels provided in the methods are sufficient to ~cuhst~nti:~lly maintain IgE receptor suppression and avoid reo~:u, . .,.I~,e of symptoms.
The invention a~ itionzllly provides methods for reducing reactivity to intrabronchial allergen challengein a patient CUUl~Uliaillg ~-lminict~ring a loading dose of an IgE antagonist averaging at least about 10 0.003 mg/kg/week IgE antagonist, or at least about 0.007 mg/kg/week IgE antagonist, or at least about 0.02 l mg/kg/week IgE antagonist, or about 0.003 to 0.030 mg/kg/week IgE antagonist, or about 0.007 to 0.021 mg/kg/week IgE antagonist, for every lU/ml baseline free IgE in the patient's serum for a period of at least about 14 days, or about 14 days to 56 days, or about 21 days to 56 days, or about 28 days to 56 days, or about 35 days to 56 days, or about 42 days to 56 days, or about 49 days. to 56 days, followed by a ~ e dose of the IgE antagonist averaging about 0.00008 to 0.0024 mg/kg/week IgE antagonist, or about 0.00025 to 0.0024 mg/kg/week IgE antagonist, or about 0.0008 to 0.0024 mg/kg/week IgE antagonist, for every lU/ml baseline free IgE in the patient's serum. In other embodiments, the m~inten~nce dose averages about 0.00075 toO.0024mg/kg/week,oraboutO.00125toO.0024mg/kg/week,oraboutO.00175toO.0024mg/kg/week,of IgE ~ hg~ i .I for every lU/ml baseline free IgE in the patient's serum. Preferably, the m~inlPnz~nre dose is 20 at least about three fold lower, or at least about six fold lower, or at least about 12 fold lower, or at least about 25 fold lower, or at least about 50 fold lower, than the loading dose in units of mg/kg/week for every IU/ml baseline free IgE in the patient's serum. The patient's reactivity to intrabronchial allergen rh~llPng~ can be ~l~tPrmined as described above.
The invention also provides methods for reducing reactivity to hlll~blullchial allergen challenge in 25 apatientco~ Jl;aillg~minicteringaloadingdoseofanIgEantagonistforaperiodofatleastaboutl4days~
or about 28 to 56 days, or about 42 to 56 days, followed by a ~I,.;lln ,~ e dose of the IgE antagonist that m~int~incthe patient'sserum free IgE collc~ alion at a level no greater than about 600 ng/ml, or no greater than about 300 ng/ml, or no greater than about 15~) ng/ml, or no greater than about 75 ng/ml, or no ~ a~ hàll about 50 ng/ml, wherein the loading dose exceeds the m lint~Pn~nre dose by at least about three fold, or by at 30 least about six fold, or by at least about 12 fold, or by at least about 25 fold, or by at least about 50 fold, in units of mg/kg/week IgE antagonist for every lU/ml baseline free IgE in the patient's serum. The patient's reactivityto hlllàlJlvllcl~ialallergenrh~llçngecan bedeterrninedas describedabove. The patient's serum free IgE response to the ." ~ r..~nce regimen can be determined by assaying serum free IgE according to the methods for baseline serum free IgE assay described above.
In these embodiments, a typical loading dose used is at least about 0.003 mg/kg/week IgE antagonist, or at least about 0.007 mg/kg/week IgE antagonist, or at least about 0.021 mglkg/week IgE antagonist, or about 0.003 to 0.030 mg/kg/week IgE antagonist, or about 0.007 to 0.021 mg/kg/week IgE antagonist, for every lU/ml baseline free IgE in the patient's serum. However, the invention also encolll,oaaa~ s the use of other W O 97133616 PCT~US97/03443 loading doses that are sufficient to strip basophils of IgE receptor such that m~interl~n~e dose levels which provide the serum free IgE levels recited in the methods are suf~cient to ~ul .,~ lly maintain IgE receptor SU~pl c~ion and avoid reoccurrence of symptoms. Suitable m linten~nce dosing for practice of these methods includem~intt~n~n~edosesofaboutO.00008toO.0024mg/kg/weeklgEantagonist,oraboutO.00025toO.0024 S mg/kg/week IgE antagonist, or about 0.0008 to 0.0024 mg/kg/week IgE antagonist, for every IU/ml baseline free IgE in the patient's serum. In other embodiments, the maintenance dose averages about 0.00075 to 0.0024 mg/kg/week, or about 0.00125 to 0.0024 mg/kg/week, or about 0.00175 to 0.0024 mg/kg/week, of IgE
antagonist for every lU/ml baseline free IgE in the patient's serum.
The invention further provides methods for reducing reactivity to intranasal allergen challenge in a 10 patient COl~ illg ~rlminict~ring a loading dose of an IgE antagonist for a period of at least about 14 days, or at least about 28 days, or at least about 42 days, sufficient to reduce the patient's average serum free IgE
leveltoalevelnogreaterthanSOng/ml,ornogreaterthan30ng/ml,ornogreaterthan 16ng/ml,ornogreater than 10 ng/ml, or no greater than 6 ng/ml, at the end of the loading period, followed by a ~--ai.lltlla-.ce dose of the IgE alllagoni~l averaging about 0.00008 to 0.0024 mg/kg/week, or about 0.00025 to 0.0024 15 mg/kg/week, or about 0.0008 to 0.0024 mg/kg/week, of IgE antagonist for every lU/ml IgE in the patient's serum. In other embodiments, the m ~ u~ e dose averages about O.00075 to O .0024 mg/kg/week, or about 0.00125toO.0024mg/kg/week,oraboutO.00175toO.0024mg/kg/week,oflgEa-,l~r...;~l foreveryIU/ml baseline free IgE in the patient's serum. Preferably, the m~intl~n~nce dose is at least about three fold lower, or at least about six fold lower, or at least about 12 fold lower, or at least about 25 fold lower, or at least about 50 fold lower, than the loading dose in units of mg/kg/week for every IU/ml baseline free IgE in the patient's serum. Intheseembodiments~thepatient~sserumfreelgEresponsetotheloadingregimencanberlet~rmin~cl byassayingthepatient'sserumfreeIgEacc~ gtothemethodsforbaselineserum freelgEassaydescribed above. The patient's reactivity to intranasal allergen challenge can be (1et~nnin~d according to the methods of Naclerio et al., "IN VIVO METHODS FOR THE STUDY OF ALLER/~Y: Mucosal tests, t~rhni~ c and ;IIICIIJI c~ ons~ Allergy Principles & Practice, Middleton et al., eds, pps. 595-613 (1992) or as described in Example 2 below.
In the practice of the foregoing methods for reducing reactivity to intranasal allergen ch~l konge the patient's serum IgE level can be reduced to the ~lcsclibed level at the end of the loading period by ~r1minictloringforthe~le;tclil)edloadingperiodaloadingdoseofatleastabouto.oo3mglkg/weekofthelgE
antagonist, or at least about 0.007 mg/kg/week of the IgE antagonist, or at least about 0.021 mg/kg/week of the IgE antagonist, or about 0.003 to 0.030 mg/lcg/week of the IgE ~ntslgonict, or about 0.007 to 0.021 mg/kg/week of the IgE antagonist, for every lU/ml baseline free IgE in the patient's serum. However, the invention also c.lc.),.ll,a~:,. s the use of other loading doses that are sufficient to strip basophils of IgE receptor such that m~ - .imce dose levels provided in the methods are sufficientto ~ul.~l n ~ ~l inlly Illailll~ IgE receptor ju~vp,c~ion and avoid reoc,ùll~ .lce of symptoms.
~ The invention additionallyprovides methods for reducing reactivity to intranasal allergen challenge in a patient C~,lll,ulisillg ~-lminictl~ring a loading dose of an IgE antagonist averaging at least about 0.003 mg/kg/week IgE ~ntlgr~nict or at least about 0.007 mg/kg/week IgE antagonist, or at least about 0.021 mg/kg/week IgE antagonist, or about 0.003 to 0.030 mg/kg/week IgE antagonist, or about 0.007 to 0.021 mg/kg/week 3gE ~nt~gnnict, for every IU/ml baseline free IgE in the patient's serum for a period of at least about 14 days, or about 14 days to 56 days, or about 21 days to 56 days, or about 28 days to 56 days, or about 35 days to 56 days, or about 42 days to 56 days, or about 49 days to 56 days, followed by a m~inten~nce dose S of the IgE antagonist averaging about 0.00008 to 0.0024 mg/kg/week IgE ant~gl-rlict, or about 0.00025 to 0.0024 mg/kg/week IgE ~nt~gf nict, or about 0.0008 to 0.0024 mg/kg/week IgE antagonist, for every l~/ml baselinefree IgE in thepatient'sserum. In otherembodiments,the l"~;lllr~ .cedose averages about 0.00075 to 0.0024 mg/kg/week, or about 0.00125 to 0.0024 mg/kg/week, or about 0.00175 to 0.0024 mg/kg/week, of IgE antagonist for every lU/ml baseline free IgE in the patient's serum. Preferably, the m~int~n~nce dose is 10 at least about three fold lower, or at least about six fold lower, or at least about 12 fold lower, or at least about 25 fold lower, or at least about 50 fold lower, than the loading dose in units of mg/kg/week for every lU/ml baseline free IgE in the patient's serum. The patient's reactivity to intranasal allergen challenge can be determined as described above.
The invention also provides methods for reducing reactivity to intranasal allergen challenge in a 15 patientconlpl;~illg~lmincteringaloadingdoseofanlgEantagonistforaperiodofatleastaboutl4days,or about 28 to 56 days, or about 42 to 56 days, followed by a ..l~i"~ re dose of the IgE antagonist that m~int~incthe patient'sserum free IgE ~;onc~ ldLion at a level no greater than about 600 ng/ml, or no greater thanabout300ng/ml,ornogl~dL~,l llldnabout 150ng/ml,ornogreaterthanabout75ng/ml,ornogreaterthan about 50 ng/ml, wherein the loading dose exceeds the m~inten~nce dose by at least three fold, or by at least 20 about six fold, or by at least about 12 fold, or by at least about 25 fold, or by at least about 50 fold, in units of mg/kg/weeklgE dllLdgu-li~Lfor every lU/ml baseline free IgE in the patient's serum. The patient'sreactivity to intranasalallergen ch~ ngecan be ~eterminedas des~ il,ed above. The patient's serum free IgE response to the maintenance regimen can be determined by assaying serum free IgE accul.li,.g to the methods for baseline serum free IgE assay described above.
In these embodiments, a typical loading dose used is at least about 0.003 mg/kg/week IgE antagonist, or at least about 0.007 mg/kg/week IgE antagonist, or at least about 0.021 mg/kg/week IgE antagonist, or about 0.003 to 0.030 mg/kg/week IgE antagonist, or about 0.007 to 0.021 mg/kg/week IgE ~ntZ~gonict for every lU/ml baseline free IgE in the patient's serum. However, the invention also ~ ,""~ cc~c the use of other loading doses that are sufficient to strip b~Cophilc of IgE receptor such that ~ e dose levels which 30 providethe serum free IgE levels recited in the methods are sufficient to ~ lly maintain IgE receptor ~u~ s~ionand avoid reoccurrenceof symptoms. Suitablem~ r~ edosing forpractice of these methods include-..~ r~ edosesofaboutO.00008toO.0024mg/kg/weeklgEantagonist,oraboutO.00025toO.0024 mg/kg/week IgE antagonist, or about 0.0008 to 0.0024 mg/kg/week IgE antagonist, for every IU/ml baseline freelgEinthepatient'sserum. Inotherembodiments,the",~i.-lP~ .,cedoseaveragesaboutO.00075toO.0024 35 mg/kg/week, or about 0.00125 to 0.0024 mg/kg/week, or about 0.00175 to 0.0024 mg/kg/week, of IgE
antagonist for every lU/ml baseline free IgE in the patient's serum.
The invention furtherprovides methods for reducing reactivity to skin prick allergen challenge in a patient Culllplisillg ~-~minictering a loading dose of an IgE antagonist for a period of at least about 14 days, CA 02246427 l998-08-l2 or at least about 28 days, or at least about 42 days, ~u~cicllL to reduce the patient's average serum free IgE
leveltoalevelnogreaterthan50ng/ml,ornogreaterthan30ng/ml,ornogreaterthan 16ng/ml,ornogreater than 10 ng/ml, or no greater than 6 ng/ml, at the end of the loading period, followed by a m~int~ n~nce dose of lhe IgE antagonist averaging about 0.00008 to 0.0024 mg/kg/week, or about 0.00025 to 0.0024 mg/kg/week, or about 0.0008 to 0.0024 mg/kg/week, of IgE antagonist for every IU/ml IgE in the patient's serum. In other embnfiimPntc the m~ "~ dose averages about O.00075 to O.0024 mg/kg/week, or about 0.00125 to 0.0024 mg/lcg/week, or about 0.00175 to 0.0024 mg/kg/week, of IgE antagonist for every lU/ml baseline free IgE in the patient's serum. Preferably, the m~intPn~nc. dose is at least about three fold lower, or at least about six fold lower, or at least about 12 fold lower, or at least about 25 fold lower, or at least about 50 fold lower, than the loading dose in units of mg/kg/week for every lU/ml baseline free IgE in the patient's serum. In these embodiments,the patient's serum free IgE response to the loading regimen can be determined by assaying the patient's serum free IgE according to the methods for baseline serum free IgE assay described above. The patient's reactivity to skin prick allergen challenge can be determined according to the methods of Bousquet and Michel, "IN VIVO METHODS FOR STUDY OF ALLERGY: Skin tests, techniques and interpretation" Allergy Principles & Practice, Middleton et al., eds, pps. 573-594 (1992).
In the practice of the foregoing methods for reducing reactivity to skin prick allergen challenge, the patient's serum IgE level can be reduced to the prescribed level at the end of the loading period by afiminictt-ringfor the prescribed loading period a loading dose of at least about 0.003 mglkg/week of the IgE
antagonist, or at least about 0.007 mg/kg/week of the IgE antagonist, or at least about 0.021 mg/kg/week of the IgE antagonist, or about 0.003 to 0.030 mg/lcg/week of the IgE dlltaguni~L, or about 0.007 to 0.021 mglkg/week of the IgE ~nt~gonict, for every IU/ml baseline free IgE in the patient's serum. However, the invention also e.~co~ sthe use of other loading doses that are s-.ffiei~nt to strip basophils of IgE receptor suchthatm;~;"l~ ,cedoselevelsprovidedinthemethodsaresufficienttos~kst:~nti~11ymaintainlgEreceptor lc:,~ion and avoid reoccurrence of symptoms.
The invention additionallyprovides methods for reducing reactivity to skin prick allergen challenge in a patient cu~ ing ~tlnninict~ring a loading dose of an IgE antagonist averaging at least about 0.003 mg/kg/week IgE ?nt~gl~nict or at least about 0.007 mg/kg/week IgE ~IlLagulli~L, or at least about û.0?1 mg/kg/week IgE antagonist, or about 0.003 to 0.030 mg/kg/week IgE ~nt~g.-nict or about 0.007 to 0.021 mglkglweek IgE all~gOl~ , for every IU/ml baseline free IgE in the patient's serum for a period of at least about 14 days, or about 14 days to 56 days, or about 21 days to 56 days, or about 28 days to 56 days, or about 35daystoS6days,orabout42daysto56days,orabout49daystoS6days,followedbyamaint~n~ncedose of the IgE antagonist averaging about 0.00008 to 0.0024 mg/kg/week IgE antagonist, or about 0.00025 to O.Oû24 mg/kg/week IgE antagonist, or about 0.0008 to 0.0024 mg/kg/week IgE antagonist, for every IU/ml baselinefreelgEinthepatient'sserum. Inotherembodiments,them~int~n~n~edoseaveragesaboutO.00075 toO.0024mg/kg/week,oraboutO.00125toO.0024mg/kg/week,oraboutO.00175toO.0024mg/kg/week,of - IgE antagonist for every lU/ml baseline free Ip,E in the patient's serum. Preferably, the m~intPn:~nce dose is at least about three fold lower, or at least about six fold lower, or at least about 12 fold lower, or at least about 25 fold lower, or at least about SO fold lower, than the loading dose in units of mglkg/week for every IWml CA 02246427 l998-08-l2 W O 97/33616 PCT~US97/03443 baseline free IgE in the patient's serum. The patient's reactivity to skin prick allergen challenge can be determined as des~ . ii,ed above.
The invention also provides methods for reducing reactivity to skin prick allergen ~ hAllengP in a patient comprising a~lminict~ring a loading dose of an IgE antagonist for a period of at least about 14 days, or about 28 to 56 days, or about 42 to 56 days, followed by a mAint~nAnce dose of the IgE antagonist that mAintz~inc the patient's serum free IgE concentration at a level no greater than about 600 ng/ml, or no greater thanabout3oong/mltorno~lea~lLlldllaboutlsong/ml~ornogreaterthanabout7sng/ml~orno~l~;aLc;lLll~l aboutSOng/ml,whereintheloading doseexceedsthe,.,A;~ A..eedosebyatleastaboutthreefold,orbyat least about six fold, or by at least about 12 fold, or by at least about 25 fold, or by at least about 50 fold, in 10 units of mg/kg/week IgE antagonist for every lU/ml baseline free IgE in the patient's serum. The patient's reactivity to skin prick allergen challenge can be determined as described above. The patient's serum free IgE
response to the . . .Ai. -~ e~ cc regimen can be determined by assaying serum free IgE according to the methods for baseline serum free IgE assay described above.
In these embodiments, a typical loading dose used is at least about 0.003 mg/kg/week IgE AntAg~n ict, 15 oratleastaboutO.007mg/kg/weeklgEantagonist,oratleastaboutO.021 mg/kg/weeklgEantagonist,orabout 0.003 to 0.030 mg/kg/week IgE antagonist, or about 0.007 to 0.021 mg/kg/week IgE antagonist, for every lU/ml baseline free IgE in the patient's serum. However, the invention also enc~....p l~PC the use of other loading doses that are sufficient to strip basophils of IgE receptor such that m~ lrl~ e dose levels which provide the serum free IgE levels recited in the methods are ~ r~i -1 to s~ lly maintain IgE receptor 20 su~ s~ionandavoidreoc~ Ull~ .lceofsymptoms. Suitablem iintPnAncedosingforpractice ofthese methods includemAi..lr~- ~cedosesofaboutO.00008toO.0024mg/kg/weekIgEantagonist,oraboutO.00025toO.0024 mg/kg/weekIgE antagonist, or about 0.0008 to 0.0024 mg/kg/week IgE antagonist, for every lU/ml baseline free lgE in the patient's serum. In other embodiments, the m ~ .. e dose averages about O.00075 to O.0024 mg/kg/week, or about 0.00125 to 0.0024 mg/kg/week, or about 0.00175 to 0.0024 mg/kg/week, of IgE
25 antagonist for every lU/ml baseline free IgE in the patient's serum.
The invention further enc-- --~ c~ methods for reducing the average number of Fc~RI l~:;C~ Lol:~
expressed per basophil in an atopic patient's serum Culll~ illg A.lminictf~ring to an atopic patient a loading dose of an IgE antagonist over a period of at least about 14 days, or at least about 28 days, or at least about 42 days, ~nffici~nt to reduce the average number of Fc~RI receptors ~ ed per basophil in the patient's 30 serum at the end of the loading period to a number that is ~ Ally lower~ or at least about 9o% lower~ or at least about 95% lower, or least about 99% lower, than the baseline average number of Fc~RI ,ec~
expressed per basophil in the patient's serum, followed by a mAintensln-~e dose of the IgE antagonist sufficient to maintain the average number of Fc~RI receptors expressed per basophil in the patient's serum at a number that is subst~ntiAliy lower, or at least about 90% lower, or at least about 95% lower, or at least about 99%
35 lower, than the baseline average number of FccRI receptors expressed per basophil in the patient's serum, wherein the loading dose exceeds the mAintenAnce dose by at least about three fold, or by at least about six fold, or by at least about 12 fold, or by at least about 25 fold, or at least about 50 fold, in units of mg/kg/week for every lU/ml baseline free IgE in the patient's serum. In these embodiments, the patient's Fc~RI expression CA 02246427 l998-08-l2 W O 97/33616 PCTrUS97/034~3 levels at baseline and at various points du}ing the loading and ~ ;..f~-~,.,.ce regimens can be determined as described in Malveaux et al., s7~pra, or as described in Example I below.
In these embodiments, a typical loading dose used is at least about 0.003 mg/kg/week IgE antagonist, oratleastaboutO.007mg/kg/weeklgE~nt~gl)nict,oratleastaboutO.021 mglkg/weeklgE~nt~gQnict,orabout 0.003 to 0.030 mg/kg/week IgE antagonist, or about 0.007 to 0.021 mg/kg/week IgE zlnt Igonict, for every lU/ml baseline free IgE in the patient's serum. Suitable m~int~n:ln~e dosing for practice of these methods include ~ ( e doses of about O.00008 to O.0024 mg/kg/week IgE antagonist, or about O.00025 to O.0024 mglkg/week IgE antagonist, or about 0.0008 to 0.0024 mg/kg/week IgE antagonist, for every IU/ml baseline freelgEinthepatient'sserum. Inotherembodiments,them~inten~ncedoseaveragesaboutO.00075toO.0024 10 mg~cg/week, or about 0.00125 to 0.0024 mg/kg/week, or about 0.00175 to 0.0024 mg/kg/week, of IgE
antagonist for every lU/ml baseline free IgE in the patient's serum. However, it will be ~ lcd that the invention also ~ncomr~ss~s the use of other loading doses and/or m~int~n~nce doses that are capable of effecting and mzlin~ining the ~u,u~cs~ion of basophil Fc~RI expression prescribed in the present methods.
Typically, IgE antagonists are ~minict~-red by intravenous, ~ .e~)u5~ intramuscular, or 15 hlllal~cliLoneal in~ection, or by other pa.~ ldl routes, or by i~ nullcl~ial or intranasal inhalation. Other suitableroutesof~lminictrationarealsoellcu~p~ dwithinthescopeoftheinvention. Itisenvisionedthat injections(intravenous,subc-lt~neo~eorilll-~u--us-,ular)willbetheprimaryrouteforthcla~,~ulic~iminic~Tation of the IgE antagonistof the invention, although delivery through catheter or other surgical tubing can also be used. Alternative routes include s~ .t .~:-...c, tablets, capsules and the like for oral ~lminictration7 20 commercially available nebulizers for liquid formnl~tionc and inhalation of Iyophilized or aerosolized mi-,lu.,al~ules,andsuppositoriesforrectalorvaginal~lminictration. Liquidf-rml-l~tionccanbeutilizedafter reconstitution from powder formulations. The IgE all~agOllial~ can be ~lminict(~red before and/or after the onset of symptoms.
As provided herein, the particular loading dose, loading dose period, ~ lr .~.ce dose, and 25 . . .~ r~ cc dose period selected for an individual patient within the methods of the invention are determined accordingtogoodmedicalpracticetakingintoaccountthel,lu~lLi~softheIgEàllla o..i,Lemployed,e.g.its in vivo plasma half-life, the formulation of the IgE antagonist employed, the disorder to be treated, the condition of the individual patient, the clinical tolerance of the patient involved, and the like, as is well within the skill of the physician. It will be understood that individual patients require different levels of free IgE
30 clearance to produce the desired level of basophil stripping (down-regulation of IgE receptor). Also, individualpatients may respond di~ llly to the IgE antagonist used. Thus, individual patients may require different levels of drug in order to achieve the desired effect. Such variations are well within the skill of the treating physician, and are ~.lcc.lllpas~ed by the scope of the invention as described and claimed herein. Aside from any variation in absolute dosing levels that may be required among individual patients, the underlying 35 biology remains the same for each patient such that the basophil stripping caused and maintained by the methods of the invention will result in improvement in clinical outcome while using cignifir~nf1y less drug than would be indicated under an ordinary treatment regimen.

W O 97/33616 PCTrUS97/03443 Although the invention enc~"-,passcsthe treatment of any allergic disease according to the methods described herein, the preferred indications include allergic asthma and allergic rhinitis. The present methods for therapy of allergic rhinitis, allergic asthma and other allergic diseases can be combined with known therapiesforallergy,asthmaorotherallergicdiseases,includingtreatmentswithanti-hict~minec,theophylline, S salbut~rnol, beclomethasone dipropionate, sodium cromoglycate, corticosteroids, anti-in 11 ~ c.~ y agenes, and the like.
Further details of the invention can be found in the following examples, which further define the scope of the invention . All references cited throughout the specification, and the references cited therein, are hereby expressly incorporated by ~rclellce in their entirety.
EXAMPLES
EXAMPLE I
Materials and Methods Buffer: PIPES (Pi,~.~i"e-N,N-bis-2-eth~nPcl-lfonic acid) (Sigma Chemical Co., St. Louis, MO) stockbuffer,25mMPIPEScont~iningllOmMNaCl,SmMKCl,and40mMNaOH,adjustedtopH7.3and 15 stored at 10 times the above concentration. PAG: PIPES (IX) cont~ining 0.003% human serum albumin (HSA) (Miles Laboratorieslnc., Elkhart, IN) and 0.1% glucose. PAGCM: PAG with I .0 mM CaCI2 and I .0 mM MgCI2. PAG-EDTA: PAG with I .0 mM (ethylent?diS~rnin~tetra~ceti~cid (EDTA). Acetate elution buffer contained 0.05 M sodium acetate, 0.085 M NaCI, 10 mM EDTA and 0.03% HSA at pH 7.3.
Reagents: Polyclonalgoatanti-humanlgEwaspreparedasdescribedby~kincon,in"~5e~u.~ ent 20 of total serum immunoglobulin E and allergen-specific immunoglobulin E antibody" (2nd ed), W~chingtnn, D.C.: American Society of Microbiolo~ (1980), p. 800; the antibody used for these studies ~ ,s. .ll~d the IgG fractionofgoatserumpreparedbyDE-$2chrom~t-Jgla~Jlly.Dustmiteantigen(5000proteinnitrogenunits (PNU)/ml, containing 0.4% phenol), D. farinae (Miles, Inc.) was obtained as the skin test reagent used to screen the donors used in the study. This antigen was therefore dialyzed against 3 changes of saline and finally 25 IX PlPESbufferusingSpectroPorlO,OOOMWCOcutofftubing(Spectrum Medical Industries, Los Angeles, CA). The resulting antigen was tested on three types of donors, a non-releaser (no IgE-mediated histamine release with anti-lgE antibody), a highly sensitive non-atopic donor and an atopic donor without sensitivity to dust mite antigens. The dialyzed antigen did not induce release in these three types of donors while it was an effective stimulus for b~Cophils from dust mite allergic donors. The optimal Col~ct;llllalion on the dose 30 response curve was determinedto be a~ ,ly 10 PNU/ml for dust mite allergic donors. FMLP (formyl-methionyl-leucyl-phenylalanine) was obtained from Sigma. Benzylpenicilloyl~BPO)-specificlgE was partially purified from the serum of a penicillin allergic patient by affinity methods describcd by MacGlashan, J.
Immunol., 130: 2337-2342(1983). It is a combination of both IgG and IgE specific for penicillin and >95%
ofthe IgE is specificforthe penicillin hapten. BPO(I I)-~SA was prepared as described by MacGlashan, J.
35 Immunol., 127: 2410-2414 (1981). Purified IgE - PS myeloma was obtained as described in Ishizaka, Immunoch~".i~l~y, 7: 687-702 (1970).
Subjects: Fifteensubjectswererecruitedthroughpublicadvertisementforparticipation in the open-labeled, Phase I trial of intravenous recombinant human monoclonal antibody E25 (rhuMAb-E25). They all W O 97/33616 PCTrUS97/03443 c~ mpl~; ed of perennial nasal symptoms and fulfilled the following entry criteria: age range of 18-65, negative serumpregnancytest(~HCG)'atentryandusageofanacceptablemethodofcollLIac~;~"ionforfemales,weight within the 70 to 130% range of the ideal value for their height using the 1983 Metropolitan Height and Weight Table, positive (~5 mm wheal) epicutaneous skin test to either Dermatophagoides farinae or S Dermatophagoide;,~ )llnysinus,serum total IgE ranging between 85 and 550 lU/ml and a stable therapeutic regimen. Eleven subjects were also affected by asthma. Subjects were excluded if they had ever experienced anaphylaxis, if their rhinitis included marked seasonal exacerbations, if they had to be hospitalized or visit the emergency department for asthma Ll ~allll~lll within 6 weeks prior to the initiation of the study or if they had required merh~nic~l ventilation for status ~cthm~ticus in the year prior to the initiation of the study. In 10 addition, individuals were excluded whose baseline forced expired volume in one second (FEVI ) was <50%
of the predicted value, or who had been on allergy immunotherapy in the year prior to the study, or who llad ahistoryofamedicalillnessthatraisedreasonablesuspiciononpossibleinterferencewiththesafetyoutcomes of the study. Pregnancy or lactation were also exclusion criteria.
Nine patients received a loading dose of rhuMAb-E25, followed by ms~inten~nne dosing equivalent 15 to7.511g/kg/weekperlU/mLbaselineIgE. Threepatientsreceived.. ,.;.. l~.,.. cedosesofl5~g/lcg/weekper IU/mL baseline Igl~. Two controls were also enrolled, however, these donors were only monitored for receptor density changes, i.e., they did not receive any form of placebo therapy.
Cell preparation: Two types of basophil preparation were employed. For the receptor mea~ul Clllelll:~
by acid elution, venous blood was drawn by v~n ;~ and the cells were separated on a single-step Percoll 20 gradient as describedby MacGlashan, J. Immunol., 124: 2519-2521 (1980). Briefly, blood was centrifuged overPercoll(specificgravity=1,080xg),thenthemononuclearcelllayerwascollectedandwashedwithEDlA
saline, PAG-EDTA, and twice with PAG, and then used as described below. For the flow cytometric studies, the blood was diluted with Percoll to a final density of 1.065 g/ml and layered over Percoll at a density of I .079 g/ml as described by Warner, J. Immunol. Methods, 105: 107- 110 (1987). After centrifugation at 450xg 25 for 15 minutes the interface between the 1.065 Percol]/plasma upper layer and the I .079 lower Percoll layer was harvested and washed as above.
General Protocol: For receptor ~ a~ul~ ents by acid elution, washed mononllcle~r cells were divided into 3 portions, 2/9 for h ;~ ..i.-e release, 2/9 for endogenous lgE density measurements, and S/9 for s~ n i~n(see below) with ~ gE density mea~ulc~ The cells destined for hict:~mine release 30 werechallengedassoonasthecellswerefinishedbeingpreparedinordertominimi~-theeffectsofhandling on intrinsic functional l~ullses of the basophils. The cells destined for determination of endogenous IgE
densities were 1~ ed in 5 ml of PAG buffer and incubated at 37~C for 30 minutes, after which they were centrifugedandr~osllcpPndcclin I ml saline.FurtherprocessingforlgEdensities is described below. The cells destined for se~ lion were prepared as described below.
Cell challenge: Dose response curves for polyclonal anti-lgE antibody were eY~rn in~d to obtain the optimal or m~ximnm release value. The response to two doses of D. farinae previously shown to be optimal and suboptimal for release (10 and 0.5 PNU/ml, respectively)was included. To determine the response of the basophils to a non-lgE mediated stimulus and to determine if this response remained constant throughout W O 97/33616 PCTrUS97103443 therapy, cells were ~h~ n~cl with I ,uM FMLP. Cells were r~c~lcp~n~d in PAGCM, then challenged with stimulus for 45 minutes at 37~C at a final volume of 1.0 ml. All reactions, performed in duplicate, were stopped by centrifugationafter 45 minutes and the ~uiJ~Illa~ L was removed for histamine analysis. In each experiment, perchloric acid at a 1.6% final concentration was added to duplicate tubes to determine total 5 histamine content. Histamine was assayed by the ~--tc-msltcd fluorometric technique of Siraganian and Brodsky,J. Clin. Immll~ol, 57: 525-540 (1976) and the percentage of histamine release was calculated From the ratio of sample to total hict~mine after spontaneous release was subtracted from both.
S~ u~ion: For cells used to determine both the total and unoccupied receptor densities, the cells were first sensitizedwith penicillin-(BPO) specific IgE. The cells were incubated with IgE antibody at S llglml 10 for20minutesat37~CinRPMI-1640media(LifeTechnologies,G~ithercbPrg~MD)c~nt~ining I mMEDTA
and 10 llg/ml heparin (250 111 final volume). Previous studies demonstrated that senciti~tion with this concentration of BPO-specific IgE, for this length of time, effectively saturates unoccupied It:c~ ul~ and thereforeallowed a 1l.~ a~ul~ lllent of total i ecel,L~ (by measuring the total IgE from saturated cells) as well as measurementof the unoccupiedreceptordensity (MacGlashan, J. ImmunoL, 130: 2330-2336 (1983)). After 15 washing once, the cells were layered over I ml of chelated FCS (5 mM EDTA in heat inactivated fetal calf serum) and centrifuged for 5 minutes to rapidly separate the cells from their diluted se~c.l ;~ ion buffer. The cells were further washed twice in PAG and then l~ y. .~ded in 5 ml of PAG buffer and incubated for 60 minutesat37~C.Thisincubationsteportheoneusedfom~ncl~nciti7~dcells(seeabove)wereincol~oldL~dto facilitate the elution of IgE from low affinity receptor binding, whether it be to FccRII or Fc~RII, as 20 determined in previous studies. After centrifi-g?til-n the pellets were r.~s~cpf nflPd in I ml of saline.
Acetate elution: Prior to a final c~..LI;rugaLion of the cells sucpended in saline (saline is used to eliminate the buffering capacity of the PIPES, reducing its influence on the s--hse(luent acetate elution), duplicate samples were taken for cell counts. For the cells that were s~nciti7~d and after centrifugation, a sample of the saline s~ t was kept for analysis of lgE to ensure that there was no carry over from the 25 sPnciti7~tion step. None of these samples c~nt~in~d measurable BPO-specific IgE. The pellets were rçs--cpPnded in ice-cold acetate buffer, pH 3.7, and inr~llhat~d in an ice bath for 10 minutes. For the cells destined for a determinationof endogenous IgE, the elution volume was 1).35 ml and for the cells used for total and unoccupied I ect~ L~ the final volume of elution was 0.7 ml. After a brief C~.IL. h~u~aLion (15,000 Xg), the ~u,u~,.l.aLail~as removed, and neutralizedwith I N NaOH. These samples were frozen immc~ tely for later 30 mea~ul~li.c.lt of total and specific IgE by radioimmunosorbent test (RIST) or radioallersorbent test (RAST) as described by A~1kincorl, in "Measurement of total serum immunoglobulin E and allergen-specific immunoglobulinE antibody",2nd ed., Washington, D.C.: American Sociely of Microbiology (1980), p. 800.
Acid elution was done in duplicate. In addition to these samples, each day of testing also included a sample of IgE treated with the acetate buffer (or not treated as a control) and these samples were also frozen with the 35 cell elution samples. These later samples were used to determine if acid Li~aLIllent and storage affected the measurement of IgE, however, no effect was observed. Basophil counts (determined by alcian biue staining according to the method of Gilbert, Blood, 46: 279-286 (1975), made before elution, were also done in duplicate. This method of acetate "stripping" determines the amount of IgE bound to mononuclear cells but W O 97133616 PCT~US97/03443 previous studies have shown that it is the basophil that is the primary, if not sole, contributor to "stripped" IgE
antibody.
lg~ mec~surement: The eluted IgE was measured in either a total IgE RIST (endogenous IgE or total receptormeasurement) or BPO-RAST (unoccupied l~c~ tOl~, see below). Cells counts (alcian blue positive S cells) obtained prior to elution allowed a c~ tinn of the receptor density to be made (the amount of IgE
measured by RIST or RAST divided by cell count with the result expressed as IgE molecules per basophil).
This ~eneral procedure and the acetate elution techni~ f? are described in detail by MacGlashan, J. Im~nunol., 127: 2410-2414(1981);Conroy,J. Immunol., 118: 1317-1321 (1977);MacGlashan,J. Immunol., 136: 2231-2239 (1986).
~low Cytometry: At various timepoints throughout the study, human basophils were isolated from 10 ml of venous blood by Percoll density gradient cellLliru~;dlion as described by Warner, J. Immunol.
MeJhoc~, 105: 107-110(1987). Basophilpurityrangedbetweenl and9%(basedonlightmicroscopiccounts after alcian blue staining). A flow cytometric technique inc~ ,laLillg light scatter characteristics was used to 4uall1ilal~ cell surface IgE and Fc~ chain expression on basophils as des.,l ibcd by Bochner, J. Immunol.
Methods, 125: 265-271 (1989). Cell surface IgE was detected using fluorescein isothiocyanate ~FITC)-conjugatedpolyclonalgoat IgG anti-human IgE (Kirkegaardand Perry, Gaithersberg, MD); FITC-conjugated polyclonal normal goat IgG (Kirkegaard and Perry) was used as a control. To fully saturate FccRI, separate ali~uots of cells were passively s~nciti~Pd with PS IgE myeloma (10 llg/ml, 37~C, 15 minutes in PAG/EDTA/heparin buffer, see above) prior to immunofluorescent labeling. Cell surface expression of 20 Fc~Rla chain was detected using a mouse IgG1 anti-human Fc~ chain monoclonal antibody (29C6) obtainedas describedin Riske,J. Biol. Chem., 266: 11245-11251 (1991) and was compared to labeling with an irrelevantmouse IgGl (Coulter, Hialeah, FL). The 29C6 antibodyhas been shown to recognize an epitope that is unaffected by Fc~RI occupancy (Riske, J. Biol. Chem., 266: 11245- 11251 (1991)). Aliquots of cells were labeled in pho5rh~te buffered saline containingO.2% bovine serum albumin (BSA) with 4 mg/ml human 25 IgG to minimi7t? nonspecific binding to FcyR as dcs.,.;bcd by Bochner, J. Immunol. Methods, 125: 265-271 (1989). Binding of monoclonals was detected using saturating concentrations of phycoerythrin-conjugated polyclonal goat anti-mouse IgG (Tago, Bullill~allle~ CA). An EPICS Profile flow cyfometer was used to analyzefluor~cc~ntcign~lcafterexcitationat488 nM. The use of "bitmap" gates to select fora population of cellswhichareprednmin~ntlybasophilsispossiblebecausebasophilshavereasonablydistinctiveforwardand 30 side scatter~l~ala~ ;aliCs. Since the cells were also enriched using a double Percoll gradient, these bitmaps can select a population of cells that is generally greater than 80% basophils, with the primary cnnt~rnin~nts being Iymphocytes. Monocytes were also gated for study by their distinctive forward/side scatter characteristics. Data is expressed as the mean fluorescence in labeled cells minus the mean fluorescence of - IgG 1 controls. Of note, incubation of fresh h~corh ilc from naive donors with up to 200 ~lg/ml of E25, in vitro, 35 failed to alter labeling of basophils with FITC-anti-IgE or 29C6 antibodies, suggesting that E25 does not interfere with immune fluorescent labeling. For some samples, 100 111 of whole blood was treated with perchoric acid and hict~mine content analyzed as above. These samples were used to determine if histamine content was measurable even if IgE fluorescence became nndet~çf~hle - =
CA 02246427 l998-08-l2 W O 97/33616 PCT~US97/03443 ,~t~,t;5tjCS~ A non-parametric Mann Whitney U rank order statistic was generally examined for each of the data sets although in some instances simple paired t-tests were also applied.
Results Twelve atopic subjects with baseline serum IgE concentrationsbetween 85 lU/mL and 550 lU/mL
5 were ~ln~inici~redrhuMAb-E25 intravenously. Baseline cha~ L~ Lics of the study participants are noted in Table I . All subjects received regular administration of rhuMAb-E25 during the course of this study.
Table 1: Baseline charn- .;,li~, of study subjects Age range 29.8 ~ 6.3 (mean + SD) 10 Sex male 7 female 5 Weight(lcg) 77.2 1 16.3 (mean + SD) 15 Baseline IgE (lU/mL) 192 + 79 (mean + SD) ~i.,l~.. ~e dose of rhuMAb-E25 57.1 ~ 20.2 mg/wk (mean ~ SD) The data in Figure I show serum titers of free IgE pre-dose and two hours post-dose on the first day of 20 therapy and on 7, 14, 28, and 42 days after the start of therapy for the 12 atopic patients. Free IgE titers dropped imme~ tPlyafter the first dose and were ~ccPnti~lly constant thereafter, averaging 1% of the pre-therapy titers.
Theprotocolforreceptordensitymeasurementswascarriedoutjustpriortothestartoftherapyand 3 months after the start of therapy. Figure 2 shows the results for the three palalllcte.a measured. Fig. 2A
25 plots the basophil surface IgE densities for each patient. As noted in the Methods section above, it was previously establishedthat little, if any, of the IgE llleaa~ ,d by this te~hniqlle comes from cells other than basophils. The pre-therapy average was approximateiy 250,000 IgE molecules per basophil. At the three month time point, the amount of IgE "stripped" from the cells and measured in the total IgE '*IST was not detectable for most of the treated patients. Since b~sophiic were counted, a maximum value for the IgE
30 density could be ac~ign~fl The 0.512 ng/ml standard on the standard curve for the total IgE '~IST ~ .L~d t'ne least detectable concel.l, dtion ( 2 2 standard deviations from blanks). Therefore this value was used in the numeratorof the expression to calculate IgE density (amount of IgE/basophil count). In Fig. 2, the "stars"
represent data calculated in this manner, and it applies to all but one treated donor at the three month time point for the data in Fig.2A. While a true average can not be c~lc~ t~-d since most of the three month data 35 represents only calculated maximums for each patient, the average of the data c~lrul~rd in this manner was a,u~"~,..i",ately2200 IgE molecules per basophil, a greater than 99% decrease (p=.0001 by Mann-Whitney U test).

Fig. 2B shows a similar set of data for sensitized cells, i.e., a ll,ca~u.~,.nent of IgE density after saturating the unoccupied I ccclJlol ~ and therefore a measurement of total receptor density. It can be seen that all samples had measurable IgE after "stripping," indicating a detectable number of total Icccpl~. The average receptor number before s~ ion was again approximately250,000 IgE receptors per cell. This 5 was reduced to approximately 8600 IgE t eccluLol :j per basophii at the three month mea~ul clllcnt. Thus, after therapy there were greaterthan three fold more unoccupiedthan occupied receptors, but like the plasma IgE
levels and the endogenous basophil surface IgE~ densities, total receptor density had markedly decreased, by approximately 97% (Mann-Whitney U, p=.0001).
Fig. 2C shows data for the unoccupied receptors before and during treatment, i.e., the "stripped"
10 IgEfrom~ dcellsmeasuredbytheBPO-RAST. SincethelgEusedfors~ ,".~ionwasnearly 100%
specific for penicillin, this measurement determines the unoccupied receptor density. Prior to therapy this density averaged approximately2700 IgE receptors per basophil while after therapy, the average increased to 7100/basophil. This increase was st~tict;c~lly cignifif~nt (Mann-Whitney U, p=.0001). The difference between total leccl~Lol a (Fig. 2B) and unoccupied receptors (Fig. 2C) for the three month time poinr~ using 15 the average values, suggests a value of d~ I UXillldL~ly 1500 endogenous IgE molecules which is just below the m~imllm estimate obtained in Fig. 2A. However, it should also be pointed out that this acetate "stripping"method of measuring-cc~Jt~n~hasanaverageerrorof~ ,?si..-alcly25%,sothattheaverage total receptor density at three months of 8600, if calculated from an individual experiment, would have a one standard deviation error of ~2200. Therefore, the di~,~ ~,ce of 1500 should be i.~L~ .I,. cled cautiously. At 20 the pre-therapy and three month time points, basophil numbers were not signifi- s~ntly different, with an average ratio 1.06~.1 I counts at 3 months counts pre-therapy.
At three times during therapy, the leukocytes were also analyzed for IgE by flow cytometry. As noted in Figure 3, cells were studied just prior to therapy, 5 weeks, and 10 weeks after the start of therapy.
Flow cytometry, as described in the Methods section above, provided an important control to rule out the 25 possibilitythat the absolute receptormeasurementswere also ~ccPccing high affinity binding of IgE on cells other than b~cophilc, i.e., on monocytes. The enriched cells were eY~mined with or without prior s~ l ic n with IgE myeloma to determine the endogenous cx~, c~sio.. of IgE or total receptor expression.
As can be seen in Figure 3A, by S weeks, and p~ li-.gat 10 weeks~ the cells of most donors expressed very little endogenouslgE, mostpositivelystained flow cytometric distributions becarne inriictingllich~kle from 30 controlstainingdi~LIil~ulious (Mann-Whitney U, p=.0001). These points are plotted as a zero in the figure.
IuLc.c:,Li..gly,the one subjectwith persistently deLe-,lable levels of IgE (Figure 3A) was the same subject in which a definitely detectable level of IgE was found by RIST (Figure 2A). Total receptor expression ~Figure 3B, after passive sc~ l ion) also decreased rapidly but was detectable at five weeks for many patients while it became generally lm-1~tert~hle by 10 weeks. It should be pointed out that in previous calibrations 35 of the sensitivityof the flow cytometerfor cl~tecting basophil IgE, the minimnnn detectable e~ es~ioll was ~ found to be 8000-10000 IgE receptors per basophil. Therefore these undetectable levels of expression are compatiblewith the data shown in Figure 2. Indeed, the sporadic net positive results (among patients) after W O 97/33616 PCT~US97/03443 sensili~lion are also compatible with the unoccupied receptor data in Figure 2 since the average total receptor density borders on the threshold of detection for the flow cytometer.
For the last three patients enrolled in the study, flow cytometric measurements were made several times during the first two weeks of therapy in order to more carefully examine the kinetics of IgE and Fc~RI
5 down-regulation. The dosing regimen was slightly different for these three patients and the timing of the doses is indicated in Figure 4. Figure 4 demonstrates that the decrease in both endogenous IgE density and total receptors occurred with a half life of approximately three days for all three patients. In these experiments,the total receptorexpression was also ~lPtl~rmined more directly using an anti-Fc~ subunit antibody, 29C6, which binds to both occupied and unoccupied receptors (Fig. 4B). Reduction in Fc~RI
10 paralleled the total receptor data obtained by first 5~lcil;,,..g the cells with IgE myeloma (Fig. 4A). Both endogenous IgE and Fc~RI decreased at similar rates. ~low cytometric distributions remained unimodal throughout the two week time course.
Figure5 illustratesthefunctionalconce~ ncPsofthesechangesonbasophilsasmeasuredbytheir ability to secrete histamine in response to several physiologic stimuli. The basophils were assessed before 15 and during therapy by challenge with I ) dust mite antigen, D. farinae, at two doses, optimal and suboptimal, 2) a full dose response to polyclonal goat anti-lgE antibody and 3) a single C- IlC~ alion of FMLP. Since FMLP acts through a distinct receptor that has a markedly different signal transduction pathway from IgE-mediated release, the response to this stimulus was not expected to change during therapy and, indeed, as can be seen in the right side histogram in Fig. 5, it did not. In contrast,the response to dust mite antigen (the 20 response to an optimal c~n~ C.-tl ~Lion is shown) dcclLased by approximately 90% (median ratio of post/pre was 0.09, p=.0002 by Mann-WhitneyU). In fact, in halfthe patientsthere was çccPnt~ y no response. The response to the 20 fold lower ~ub~ ti-l~al concentration of dust mite antigen was proportionally lower or zero. The response of the two controls used in these studies was çccçnti ~1 Iy unchanged (ratio of post/pre was 0.83~.25 (range, n=2)3. The response to anti-lgE antibody was also decreased in the treated patients but not 25 to same extent as antigen, with the peak response de~ a~ing a~lJI u~illlately4oo/o (p=.046 by Mann Whitney U) and no meaningful change in the clla~a~ of the dose response curve. There was signifirS~nt variability among donor basophil responses, with nearly no change in the response to anti-lgE antibody in some donors, while in several cases the response to anti-IgE was nearly ablated. The response of the basophils that had been sPnciti7pdwithanti-BpolgE(forthereceptorstudies)tobothanoptimalc~"lc~,ild~ionofanti-lgE(o.2 30 ,ug/ml) and to an optimal col~c~;~lLI~lion of BPO(I l)-HSA was dPtPrminPd for seven of the patients at the three month time point. On average, the histamine release to BPO-HSA was 70~7%, ç5cenfi~11y the same as the pre-therapy I ~ se to the natural antigenic sensitivity, D. farinae (78~7%). The response to anti-lgE
antibody also increased to 52 t8%, which is also the same as the pre-therapy response to an optimal concentration of anti-lgE antibody (53~7%). These studies indicate that the number of unoccupied receptors 35 remz~iningon the treated patients' basophils were sufficient to drive a normal response to both antigen and anti-lgE when sPnciti7Pd (see Discussion below). It was not possible to properly correlate the responses of the cells to rpnn~ining (endogenous) IgE densities because, as noted above, the three month data generally only n ~ ;sellL~d the maximum possible IgE densities. However~ there were two anecdotal observations.

W O 97/33616 PCTrUS97/03443 First, the only patient whose basophils had a measurable endogenous IgE density (the only circle point in Figure lA at "90" days3 was also the only patientwhose response to dust mite antigen was only marginally reduced. Second, the two patients whose Pctims-ted "maximum" endogenous IgE densities were the lowest (1400-1500/basophil) also showed strong responses to anti-lgE antibody but poor responses to dust mite antigen.
Discussi~n These studies demonstratea remarkabledown-regulationof Fc~RI on basophils during a period of time when the free circu1atingtiters of IgE reached a small fraction of the patients' pre-therapy levels. Both measuresofFc~ ,ressi()llon basophils, absolute ~ I;nn by acetate stripping and flow cytometry, 10 resulted in the same conclusion. Although the number of controls used was small, many previous studies have shown that IgE and Fc~RI densities are relatively stable over long periods of time, varying at most 2-3 fold but usually varying no more than the error of this measurement technique (MacGlashan, J. All. Clin.
Immunol., 91: 605-615 (1993).
The kinetics of the decrease in IgE expression, under conditions where the ii ee IgE cvllc~ Lion 15 was less than I ng/ml, indicate that the ~Jieeoci~tion constant for Fc~RI was on an scale similar to that found for the interaction of rat IgE antibody and rat Fc~RI (T1~2 a~ hlla~cly 50-70 hrs) (Kulczycki, ,~. Exp.
Me~.,140: 1676-1695(1974);Metzger,lmmunochemisty,13:417-423(1976)).Thedatadoesnotsupport the possibilitythat this loss l l -~sP--L~ the replacement of an original pool of basophils with new basophils thatdidnotexperienceup-regulationofFc~RlbecauseofthelowplasmalgEcollc~llL-~.livn.Theobservation 20 that the flow cytometric distributions for samples taken within the first several days of therapy were unimodal suggests that there were not two populations of basophils, but rather that the existing population was uniformly losing IgE.
These observations have several implications with respect to the observed changes in receptor density as they relate to the changes in function. Previous studies have noted that b~cophil~ have many 25 "spare"lt~ JL(J.~.R~cophilcfromatypicalpatientwillgiveahalfmaximalresponseto~ntigenicctim~ ti~n with a cell surface density of 2000 antigen-specific IgE molecules (MacGlashan, J. Immunol., 91: 605-615 (1993)). Asnotedinthisstudy,thestartingdensityofIgEwasal,l,lv~i---a~ely250,000moleculesandsince the ratio of antigen-specificlgE to total IgE can be as high as 50% (in ragweed allergic patients for example), there will clearly be many "spare" IgE molecules. In order to reduce the presence of antigen-specific IgE on 30 the basophil surface to levels signifi~ ~ntlybelow this ECso threshold, in the absence of a change in receptor density, total IgE titer would have to decrease at least 100 fold. Factoring in the down-regulation of the receptor, however, suggests that the decrease in IgE titer need be much less profound. As an illustration, given the following conditions: I ) a patient naturally c~ s~ing 250,000 Fc~RI molecules,2) occupied with IgE which was 25% specific for ragweed antigens, 3) a typical basophil sensitivity of 2000 molecules for 35 a half maximal response,4) using a log-linear extrapolation to a minimum of 8300 Fc~RI molecules as the IgE titer decreased,5) a starting total IgE of 100 ng/ml and 6) an equilibrium constant for IgE:Fc~RI of 101 ~
to decrease the antigen specific IgE to the EC50, or 2000 molecules per cell, it would require a decrease in plasmalgE concentration to 0.63 ng/ml (1/160) without a change in IGC~ OIa and a decrease to 6.7 ng/ml W O 97133616 PCT~US97/03443 (1/15)withthechangein~ ,c~)1ul~,i.e.an~pl~lv~Lh--ately99%decreasevsanapproximately90%decrease.
Therefore,the responsivenessofFccRI e,~ s~.ion to IgE antibody conc. .,ll~,Lion ~cc~ lrs the ability of the therapy to down-regulate the overall responsiveness of the basophil.

A placebo-controlled,double-blindstudy is performed to determine tne safety, pharmacokinetics, pharrnacodynamics and efficacy of long-term dosing of anti-lgE recombinant h~m:~ni7ed monocIonal antibody E25 (rhuMAb-E25) (described as h--m~ni7Pd variant 12 of monoclonal antibody MAE 11 in Presta et al., and alsodescribedas variant 8b in Table 9 on page 68OfSer.No.08/405,617) following short term high-dose loading in patients with allergic rhinitis or allergic asthma. In particular, the objectives are to evaluate the safety of rhuMAb-E25 a~lTninictered by high-dose loading in patients with perennial allergic rhinitis or asthma, and to determine if a significant reduction in dose of rhuMAb-E25 can maintain suppression of basophil Fc~RI expression and clinical outcomes.
The study can include appl ~J~hl.ately 150 to 200 patients with history of allergic rhinitis or allergic asthma, and who are positive skin prick test-positive to dust mite allergens (greater than 5 mm wheal and erythema). Exclusion criteria are FEV I less than or equal to 50% of the predicted FEV I at screening, use of topical nasal cromolyn or ~ o 7Lrl~ids for 14 days before, or during the course of the study, pregnant women or women of child-bearing potential not on an accepted forrn of cOllL.aC~,I"iull, or chronic disease ~e.g.diabetesmellitus,hypertension,chronicobstructivepulmonarydisease(COPD),coronaryarterydisease (CAD).
Serum conc~ .~lla~ions of total serum IgE and free serum IgE can be l-l~ asnl~d at baseline and at various time points during the study. Total IgE can be measured using a collllll~ ,ially available kit (IMx TotalIgE,AbbotLaboli~L~ s,AbbottPark,IL). SerumfreelgEcanbemeasuredusingsolidphaseELISA.
High binding flat bottom polystyrene plates (Costar, Cambridge, MA) are coated overnight at 2-8~C with 100 ng of human IgE receptor a-chain IgG chimera (FccRI-IgG) (obtained as described in Haak-Frendscho 25 etal.,J.Tmmunol.. 151:351(1993))inl00~LlofpH9.6calboll~L~buffer. Theplatesarethenwashedwith 0.05% Tween 20 in ph~ cph~te-buffered saline (PBS) and incubated with 200 1ll of assay diluent (0.5%
bovine serum albumin (BSA) ~Intergen Co., Purchase, NY]/0.05% Tween 20/0.01 % thimerosal in PBS) for 1 to two hours. The plates are sealed and frozen until use. All subseguent assay steps are performed at room t~ llly~ . For the ELISA, plates are thawed and washed, and 50 111 of sample or standard are added in 30 duplicate to 50 ,ul of assay diluent in the wells and incubated for one hour. Fc~RI-lgG captures only free IgE (IgE that is not complexed with E25). The plates are washed, and 100 ng of biotinylated monoclonal anti-human IgE in 100 ~LI of assay diluent is added to the wells and in~ llb~ted for one hour. The plates are washedand 100,ul of avidin-horseradishperoxidase(HRP)(VectorLaboratories, Burlingame, CA~ diluted 1/2000 in assay diluent is added to the wells and inrllb~t~d for 30 minutes. The plates are washed and then 35 developedwith 0.4 mg/ml o-phenylPne~ mineand 4 mM H2~2 in PBS. The colorreaction is stopped with 4.5 N H;!SO4. The absorbance at 492 nm is measured with an SLT EAR 340AT (Research Triangle Park, NC). The concentrationof free IgE is ~lr--l~t~d for the standard curve, using a 4-parameter logistic fit for the standards.

W O 97/33616 PCT~US97/03443 In the ~lh"~,.l ;~ arm of the study, the primary efficacy variable is the change in FEVI measured within one hour of allergen provocative bronchial challenge (EAR) between day -I and after final administration of the study drug. The baseline is defined as the observed dirr. .en. c in the percent change from prechallenge levels in FEV I response between an allergen diluent challenge and an allergen challenge.
S The follow-up is defined as the difference in the percent change from prerhAll~nge levels in FEV I response betweenanallergendiluentchallengeandanallergen~hAIIengeafterfLnaladministrationofthestudydrug.
In each case, two variables are derived: maximal observed decrease and area under the curve (AUC) as approximated by the trapezoidal rule. Treatment efficacy is based on the between L~ ~dLIl.cll~ comparison of the baseline and follow-up of AUC and maximal increase. Between group dirr~,c,.ces for the change 10 between day - I and post-final drug AAmini~tration are assessed by the Wilcoxon Rank sum test.
LAR is measured in a similar fashion as the primary efficacy variable of change in the FEV I (AUC
and maximal decrease).
The bronchoprovocation tests can be cor -ln~ted as follows. The initial dosing of allergen for inhalation is four doubling doses below that cAl~ulAted from the prediction forrnula: y = 0.69% + 0.1 1, where 15 y=log~OallergenPD20FEVI (thedoseofallergenthatcausesa20%fallinFEVl~andx=loglOmethcholine PDIo (the dose of methocholinethat cases a 10% fall in FEV I ) multiplied by skin allergen sensitivity (skin sensitivity to allergen is defined as the smallest allergen dilution that gives a wheal 2 mm in diameter).
When the dose causes a fall in FEV I of 20% or more, no further allergen is delivered. When the dose causes a fall in FEVI of less than 10%, then the chAllPn~e is advanced to the next doubling step, etc. The FEVI
20 is measured at 20, 30, 45, 60, 90 and 120 minutes and at hourly intervals up to seven hours after inhAlAtic)n Dosing of allergen for the follow-up bronchial challenge c~ mmt9nr~s at an allergen cunc~ ..Lion of four doubling doses more dilute than the dose which caused a 20% fall in FEV I during the first challenge.
The dosing then proceeds in two-fold more concentrated steps until the FEV I falls by 20% or until allergen is delivered at a co ,.,.,.,11 dliull one doubling dose higher than that delivered on day -I .
In the rhinitis arrn of the study, the primary efficacy variable is the change in clinical symptom score for nasal response to allergen provocative intranasal challenge from day -I to various times during treatment and after final A-lminidTation of the study drug. The nasal rhAll~nge tests can be con~ t.od as follows. An atomized allergen solution, beginning with the lowest concentration (highest dilution), is applied to each nasal mucosa only, with no portion of the dose delivered beyond the posterior nasal mucosa.
30 Based on the patient's tolerance, At~ mi7ed allergen solution is reapplied using the next most concentrated (10 fold base increase from the lowest concentration) until the onset of symptoms listed in Table 2 below are exhibited. Usually, the first symptom is marked by nasal congestion or di~cul,lru, L

W O 97/33616 PCT~US97/~3443 Table 2: Clinical Symptom Score for Nasal Response Symptom Response Points S Sneezes o o >3 Z
Rhinorrhea 0 o mild abundant 2 Congestion o 0 mild nasal block 2 Itching o 0 l 5 itchy eyes and/or throat watery and itchy eyes, and 2 itchy palate and throat Upon determination of a positive clinical eh~llPn~e score of greater than or equal to four points, the rh~ nge procedure is complete. A positive clinical challenge score is defined as greater than four points (mz-ximnm possible score is eight). Treatment efficacy is assessed by C~ ,(Jal isol) of pre~treatment, during-ll~a~ ll and post-treatment nasal symptom scores.
In both arms of the study (~cthm~tic and rhinitic), treatment efficacy is assessed by allergen-titrated skin prick tests con~lllctpd at various times before, during and after ~I.,aLll~clll with the study drug.
In both arms of the study, basophils are harvested from the patients at various times before, during and after treatment and assayed for Fc~RI expression as described in Example I above.
In both arms of the study, patients are r~n-1omi7Pd to receive one of the following two loading regimens or placebo as set for~ in Table 3 below. Loading doses are expressed in units of mg/kg/week E25 ~miniqtPredfor every IU/ml baseline free IgE in the patient's serum. Patients can receive the loading dose in one or more ~rlminictrations by intravenous or sub.,ul~c~us injection during the loading period.

Table 3: rhuMAb-E25 Loading R~ ~ -Loading Dose Duration of Load High: 0.021 4-6 weeks Low: 0.007 4-6 weeks After completing the loading regimen, patients in each loading group receive one of the m~ r~ . ce regimens (or placebo) shown in Table 4 below. Loading doses are w~Jlc~acd in units of mg/kg/week E25 ~riminicfPredfor every lU/ml baseline free IgE in the patient's serum. Patients can receive W O 97133616 PCTrUS97/03443 the ".~;..1. ..~..cedosebyintravenousorsnbrnt~neousinjectiongivenatanyconvenientcombinationofdose amount and time interval, e.g., ~Am in jct~ring fiour times the weekly dose once every four weeks.

Table 4: rhuMAb-E25 M~in~en~nce Rl~o S High Loading Group Low Loading Group 0-007 0.0024 0.0024 0.0008 0.0008 0.0002 0.00025 Placebo Placebo The above-described therapeutic treatment of atopic patients with E25 is expected to reduce reactivity to bronchoprovocation, nasal challenge, and skin test with allergen, maintain su~ sion of 15 basophil FccRI expression, and improve clinical outcomes.

Claims (33)

WE CLAIM:

1. A method of treating an allergic disease selected from the group consisting of allergic rhinitis and allergic asthma in a patient comprising administering;
a) a loading dose of an IgE antagonist for a period of at least about 14 days sufficient to reduce the patient's average serum free IgE level to a level no greater than about 50 ng/ml at the end of the loading period; followed by, b) a maintenance dose of the IgE antagonist averaging about 8 x 10 -5 to 2.4 x 10 -3 mg/kg/week IgE antagonist for every IU/ml baseline free IgE in the patient's serum;
wherein the maintenance dose is at least about three fold lower than the loading dose in units of mg/kg/week IgE antagonist for every IU/ml baseline free IgE in the patient's serum.

2. The method of claim 1 wherein the loading dose is sufficient to reduce the patient's serum IgE level to an average level no greater than about 30 ng/ml at the end of the loading period.

3. The method of claim 2 wherein the loading dose is sufficient to reduce the patient's serum IgE level to an average level no greater than about 16 ng/ml.

4. The method of claim 3 wherein the loading dose is sufficient to reduce the patient's serum IgE level to an average level no greater than about 10 ng/ml.

5. The method of claim 4 wherein the loading dose is sufficient to reduce the patient's serum IgE level to an average level no greater than about 6 ng/ml.

6. A method of treating an allergic disease selected from the group consisting of allergic rhinitis and allergic asthma in a patient comprising administering;
a) a loading dose of an IgE antagonist averaging at least about 3 x 10 -3 mg/kg/week IgE
antagonist for every IU/ml/ baseline free IgE in the patient's serum for a period of at least about 14 days; followed by, b) a maintenance dose of the IgE antagonist averaging about 8 x 10 -5 to 2.4 x 10 -3 mg/kg/week IgE antagonist for every IU/ml baseline free IgE in the patient's serum;
wherein the maintenance dose is less than about one third of the loading dose.

7. The method of claims 1 & 6 wherein the maintenance dose of the IgE antagonist averages about 0.00025 to 0 .0024 mg/kg/week IgE antagonist for every IU/ml baseline free IgE in the patient' s serum.

8. The method of claims 1 & 6 wherein the maintenance dose of the IgE antagonist averages averages about 0.0008 to 0.0024 mg/kg/week IgE antagonist for every IU/ml baseline free IgE in the patient's serum.

9. The method of claim 6 wherein the loading dose of the IgE antagonist averages at least about 0.007 mg/kg/week IgE antagonist for every IU/ml baseline free IgE in the patient's serum.

10. The method of claim 9 wherein the loading dose of the IgE antagonist averages at least about 0.021 mg/kg/week.

11. A method of treating an allergic disease selected from the group consisting of allergic rhinitis and allergic asthma in a patient; comprising administering, (a) a loading dose of an IgE antagonist for a period of at least about 14 days, followed by;

(b) a maintenance dose of the IgE antagonist that maintains the patient's serum free IgE
concentration at a level no greater than about 600 mg/ml;
wherein the loading dose exceeds the maintenance does by at least about three fold in units of mg/kg/week IgE antagonist for every IU/ml baseline free IgE in the patient's serum.

12. The method of claim 11 wherein the loading dose exceeds the maintenance dose by at least about six fold in units of mg/kg/week IgE antagonist for every IU/ml baseline free IgE in the patient's serum.

13. The method of claim 12 wherein the loading dose exceeds the maintenance dose by at least twelve fold.

14. The method of claim 13 wherein the loading dose exceeds the maintenance dose by at least twenty-five fold.

15. The method of claim 14 wherein the loading dose exceeds the maintenance dose by at least fifty fold.

16. The method of claim 11 wherein the maintenance dose of the IgE antagonist maintains the patient's serum free IgE concentration at a level no greater than about 300 ng/ml.

17. The method of claim 16 wherein the patient's free IgE concentration is maintained at a level no greater than about 150 ng/ml.

18. The method of claim 17 wherein the patient's free IgE concentration is maintained at a level no greater than about 75 ng/ ml.

19. The method of claim 18 wherein the patient's free IgE concentration is maintained at a level no greater than about 50 ng/ml.

20. The method of claims 1, 6 and 11 wherein the loading dose is administered for a period of 14-56 days.

21. The method of claim 20 wherein the period is 21-56 days.

22. The method of claim 21 wherein the period is 28-56 days.

23. The method of claim 22 wherein the period is 35-56 days.

24. The method of claim 23 wherein the period is 42-56 days.

25. The method of claim 24 wherein the period is 49-56 days.

26. The method of claims 1, 6 and 11 wherein the IgE antagonist is an anti-IgE antibody.

27. The method of claim 26 wherein the antibody is chimeric.

28. The method of claim 27 wherein the antibody is humanized.

29. The method of claim 26 wherein the antibody is a human antibody.

30. The method of claim 28 wherein the antibody is rhuMAb-E25.

31. The method of claim 26 wherein the antibody binds to soluble IgE and blocks the binding of IgE to the IgE receptor on basophils.

32. The method of claim 26 wherein the antibody binds to membrane bound IgE.

33. An IgE antagonist for use in the preparation of a medicament for treating an allergic disease selected from the group consisting of allergic rhinitis and allergic asthma in a patient wherein said medicament is administered as;
a) a loading dose of the IgE antagonist for a period of at least about 14 days sufficient to reduce the patient's average serum free IgE level to a level no greater than about 50ng/ml at the end of the loading period; followed by, b) a maintenance dose of the IgE antagonist averaging about 8 x 10 -5 to 2.4 x 10 -3 mg/kg/week IgE antagonist for ever IU/ml baseline free IgE in the patient's serum; wherein the maintenance dose is at least about three fold lower than the loading dose in units of mg/kg/week IgE antagonist for every IU/ml baseline free IgE
in the patient's serum.

-33a-