CA2131898A1 - Methods and compositions for the modulation of host immune response to allergens - Google Patents

Abstract

The present invention provides novel compositions and methods useful in the modulation or selective suppression of host immune responses to an immunogen of interest, particularly to immunogens which are exogenous antigens or allergens. Subject compositions include antibody, antibody derived, and antibody-like molecules of primary antigen reactivity with respect to the immunogen of interest. Antibodies or antibody-like or antibody-derived molecules include antibody fragments such as Fab, and complementarily determining region peptides (CDRs) which may be grafted into a framework region of any species, particularly human. They also include human antibodies, derived from sensitized human lymphocytes produced by cell fusion with heterohybridomas, or by DNA cloning and expression. Other compositions include T cell receptor (TCR) molecules, obtained either from T cell clones or hybridomas or as purified TCR preparations. Immunoreactive peptides corresponding to some or all of the complementarily determining regions or hypervariable regions of the TCR are also employed.

Description

WO 93/17703 PCI'/US93/02082 21 31 ~9$

~B9r~1OD8 AND CO~I~?OS:CTIO~8 FOR THE ~ODIlJa~TION
OF ~{O~q~ T~1~7E ~E8PO~8E TO AI.I-ERGE:~8 loFXEI,D OF THE ~ ENTION
5The in~ention relates generally to the field of immunology, and more particularly to methods and compositions for th~ modulation of the host immune response to immunog~ns, particularly for exogenous ~antigens ~uch a allergens. The invention employs :~ 10 antibodies and antibody-derived molecules, including T
c~ll receptors (TCRs), to down-regulate the immune respon~e to these antigens.

2. BaCKGROUND OF T~E INVENTION
:: 15 2.1 Allergic ~ as~
: The overall patterns of immune responses to allergens indicate that T lymphocytes are central in the generation of allergic diseases (Romagnani,1992.
: Immunol. Today. 13:379-380; O'Hehir et. al, 1991. Ann Review Immunol :67-95~. Followin:g allergen processing and presentation by antigen presenting cells (AP~:) they function as helper T cells in :cooperating with B cells to produce antibodies such as i~munoglobulin IgE. Rec~ptors on:mast cells and basophils bind all rgen specific IgE. Subsequent : exposure to allergen results in the release of inflammatory molecules which cause allergic symptoms.
: : Allergen sensi~iæed T lymphocytes also ~unction directly in initiating allergic responses. This includes the release of cytokines such as interleukin 5, so promoting eosinophil mediated allergic responses : : in asthma and:related diseases. They also initiate local inflammatory responses such as those caused by exposure to contact sensitizing a~leryens.
Environmental sources of contact~sensitizing allergens include naturally occurring compounds such as .

3 2 1 3 1 8 9 8 - 2 - PCT/US93/02~2 alkylcatechols present in poison oak and ivy as well as pollutants and industrial chemicalsO

2.I.l Ig~ m~ tea also~e~ tkma, ~llergic rhi~itis a~ atopi~
~0rmat~tis Classic immediat type allergic reactions are med~ated by the IgE subclass o~ antibodies, and are : directed against a variety of environmental allergens.
The most common of these world-wide are dust mite, pollen from grasses, tree~, broad-leaf weeds, mold spores, and animal dander. After initial sensitiz~tion, exposure results in an i~mediate reaction caused by the release of a range of inf~ammatory molecules including histamine and prostaglandins from mast cells. A second wave of ellular ac~ivation occurs several hours later, manifest in the s~in as vasculitls~with leukocyte nfiltration, and:in the lungs by eosinophil and lymphocyte in~iltration. The~se~ond phase r~action is thought to ~e responsible for promoting the chronic chang~s and~hypersensitivity which often accompanies the~allergic~diseases. :

201~1~1 I)U9~t Illite~ all~rgen~
~ Over the~l3st se~eral years~, evide~ce of the strong involvement~of dust mite antigen (DM~) allergy in~allergic rhin:itis and especially asthma ha~
~:~ strengthened. In a recen~ study (Gergen and :~' Terkel~aub~(l992). J. All. Clin. Immunol. 90:579-588) it was shown;:that allergic rhinitis without asthma was associated with:skin test reacti~ity to only three allergens, dust mite (house dust), ragweed, and rye : grass. Asthma alone was onIy associated with reactivity ~o house dust and alternaria. Additionally many reports have documented the close physical association between dust mites in the home and asthma W093/17703 . PCT/US93/02082 . ~ 3 ~ '21 31 89 ~

(Sporik Pt. al.(~992). Clin. Exp._A11.22:897-906;
Platts-Mill6.(1992). J. All. Clin. Immunol. 89:1046 1060).
There are two major species of dust mite, S Dermatophagoides pteronyssinus (Der p~ and Dermatophagoides farinae (Der p). A range of 30 pro*eins have been identified in allergenic preparations derived from each species; but only two, :~:; : group I, Mw 25,000, and group IIj Mw 14,000 are recognized as the major mite allergens. Within each : group, proteins are highly cross reactive between the two speciesO These two proteins account for 60-70~ of the~reactivity of the IgE in DMA allergic individuals.

~ 2.1.1~2 Rye gras~ allerge~
Grass and weed allergens are important causes of allergic rhinitis and asthma. Most of the grasses are cultiva~ed agriculturally or ornamentally and are prevalen~ in:residentia;l:areas. Only about a dozen of more:~han 5000 species of grasses~are important ~ a:llergens since~many of the grasses do not produce }~ abundan~pollén:~. Import~nt allergenic grasses include Rye,~Timothy, Kentucky blue and June. Many of the :allergens of~dif~erent~;species~;cross react antigenically, and show amino acid sequence homology and may share~:~T and B lymphocyte epitopes.
e grass~ (Lol~ium perenne) which is widely distributed~in~te~perate zones~induces allergi:c reactions in pollen sensitive individuals. Five groups of allergens; ILol p I, II,:~Ill, IV, and V), some in mult:iple~forms, have been identified (Marsh et. al., 1987. J:~. All. Clin. Immunol.80:63~6~5;
Thomas, 1991. Clin. Exp. All. 2I:255-257; Mathiesen et 1., 1991. Clin. ExP. AlI. 21:309-320~. Lol p I is a ;~ 35 major allergic~species. Lol p I is an acidic :;glycoprotein of~molecular weight~ 34 kD, located in the cytosol of the pollen. levated levels of Lol p I

specific IgE have been found in sera of up to 95% of grass poll~n-allergic subjects (Freidhoff ~t al., 1986. J. All. Clin. Immunol. 78:~190-1201).

2~1.2 T c~ aiata~ ~110rgic ~
~i~e88~: Poiso~ o~ ivy !ltiti~l Poison oak and ivy dermatitis is one example of a direct tr cell mediated allergic reaction. This ::~ reaction occurs against the hapten urushiol when it : 10 contacts the skin, bronchial epithelium or the like.
The hapten is a mixture of 3-n-alkylcatechols found in the oil of the plant, which coniugates to body ~: proteins~ The conjugate stimulates T cells to directly attack the skin or Qther cells to which the hapten is bound.
The T cell e~iology of the disease has been confirmed by studies in animal models of poison oak dermatitis which:~include the guinea~pig and mouse.
he allergic reaction:can be;transferred ~rom :0::sensitized:~o naive animals by T lymphocytes. Human and:animal~stud~ies:~ave identified::the specificity of the T cell~r~ea~tion against the al~lergen. Both the speoificity~and:~antigenicity of~the compounds reside prima~rily:~in the~common catechol structure. The irm~unologic reacti~ons:~seen with~urushiol:are typical :o~ those caused~:by various identified ~ontact sensitiz rs,~such~as~chlorinated hydrocarbons and epoxy resins. :~

30~ ~ 2.2. IgG~ aiated~re~poDse to protel~ a~tige~:

There are other cases in which undesirable immune responses occur~;~o protein antigens, such as ricin A
chain (RTA) a protein which, when coupled to a targeting agent such~as a lymphokine or monoclonal antibody such as MAb 791T/36 (immunotoxin), directed ~ against colorectal cancer, can kill the targeted cell~

:;~:: :: :

W093/17703 _ 5 _ 2131 8 9 8 Pcr/us93/o2o82 RTA is a very potent immunogen, and a single injection ~ of immunotoxin in animals or humans produces a :~ vigorous IgG immune response against all parts of the :~ molecule.
~; . 5 2.3 ~mu~otherapy with al1erg~ns ~:~ : lmmunotherapy of allergic diseases including ; asthma, allergic rhinitis, as well as urushiol dermatitis ~has been undertaken for many years using ;hyposensiti;zation with~crude~allergen extracts.
Immunotherapy is~effective in many~patients and can provide lasting benefit even after:immunotherapy has : : been discontinued~(Busse,~ 1988. J. All. Clin. Immunol.
82:8gO-9~0).Immunotherapy with grass extracts has pro~en:useful~in treating allergic~rhinitis and asthma (Creticos and~Norman,1987.~ AMA. 258: 2874-2880;
C~eticos. l989.:J.~Al1.~ Clin~. Immunol. 83:554-562;
Re~id~et.:~al.~, 1986.~J. All~Clin. Immunol. 7&:590-60:0~. In like~faæh:ion~,~ patients with:severe asthma 2Q~ a~h:ieved~significant~improvement:fo11Owing treatmeht with~a~DMA-a~tibody~complex (Machiels:et. a:l:., l99~.
3.~ Clin.~ Invest~.8~5:~1024-1035), also clinical :impr~vement~and~dec~ eased bronchial sensitivity on bronchial~provocation~in~ch~i1dren immunized for 3 2~5 y~ars~with~à~hous:e~dust~extract;~Creticos nd~Nor an.
l987~ ;3AMA~2~58~::2874-2880,~Creticos:.lggO. J. All.~Clin.
Immuno~ 3~ 5S4-562;: Bousquet et. al. t 1985.~J. All.
C1~__ Immuno}.~7~6:734-744)~,~ and~in~patients with a1lergic; rhinitis~desensitized:with either purified :30 ~:AgE from ra~gweed,~ or~with the;~who1e extract ~Norman et.~ a~ 1968.~:J~ A11. 42:93-108):~ Similarly, with - poison oak/ivy~urushiol, oral:hyposensitization is quite:effective~ a~1though~cumbersome~;since it requires :: 3:-6 months of:daily~administrati:on of the oil.
35;~Indirect:evid~ence~for a regulatory~role for anti-idiotypic ant~ibodies and/or~regulatory T cells is provided by several:experimental;and clinical studies.

WO93/17703 PCT/U~g3/02082 21 31 89 8 - 6 ~

SubJects hyposensitized with urushiol generate immunoglobulins which can transfer tolerance to mice ~Stampf et. al., 1990. J. Invest. Derm.). In immediate type hypersen~itivity, a general finding : 5 during hyposensitization is an initial incxease in I~E
antibodiesO followed by a decrease. Concomitantly, : ther~ is an increase in the allergen specific IgG
(Creticos, 1987 . JAMA ~58: 2834-2839) . When the ;~, specificity of the response is investigated carefully, ~0 it is found that auto-anti-idiotypic antibodies : develop (Gurka et al. (1988). Ann. Aller~. 61:239-243). This has been shown to be the case in rye grass hyposensitization:as well. Clinically, anti-idiotypic ~: antibodies are elevated in human subjects following hyposensitization with rye grass extracts concomitant ; with reduction in allergic rhinitis (Hebert et al., 1990. Clin. Ex~ Immunol. 80:413-419).

2.4 Dows~R-gul~tio~ of Im~u~ ~8po~ by 20: ~re~*~e~t ~it~ A~tiboai~
2.~ : A~ti-idiotypi¢ antibodio~
Transfer:of anti-idiotypic antibodies results in suppression of both T and B lymphocyte initiated responses~ Treatmen~: of mice with polyclonal anti-2S idiotypic antibod~ies generated against a mouse monoclonal antibo~y~:reacting with a tobacco mosaic virus~protein:~polypeptide suppxessed the ability of mice to generate:~antibody res~onses to the decapep~ide (:Norton and Benjamini, ~1987. Cell~ nol ~09:419-~18). Polyclonal anti-idiotypic antibodies produced by ; immunization of guinea pigs with polyclonal an~i-benzylpenicilloyl antibody wer shown to down regulate the anti-benzylpenicilloyl IgE antibody responses when :~ given either pre or~post sensitization (Wetterwald et alO,1986O Mol. Immunol. 23:347-356). Transfer of serum from bee keepers who had tolerated multiple bee stings into individuals who previously developed anaphylaxis ' r .... WO 93/17703 PCT/US93/02082 213183''~
to desensitizatlon decreased skin test reactivity and subsequently allowed the rec~ipt~ to undergo successful desensitization (Bousquet, ~987~Jo All.
Clin. Immunol. 79:947-954), and it was shown that the : 5 transferred whole sera ~ontained anti-idiotypic antibodies against bee venomu Boutin et. al.,1993. JO
Allo Clin Immunol. in press~.
In general, however, treatment with monoclonal Ab2 antibodies has been undertaken to up-regulate responses to specific antigens. These include cancer : and viral antigens (Fung et al.1990. J. Immunol.
145:~199-2206; Raychaudhuri et al.,1990. J Immunol.
145:760-767; reviewed in Kohler, et al. 1989. lin.
Immunol. and Immunopath $2:104-116~. Several of these are in clinical trials, for example against cancer : ~ antigens (Robins, et al.1991. Can~ Res. 51:5425-5429).
Consistent with these findings, vaccination with a .
monoclonal Ab2 directed against Lol p I allergen produced up-regulation of the IgE and IgG anti-Lol p I
antibodies ~Boutin et al.,l991. J._Al1. Clin.
~3~9ng~ 197 (abstrac~)). In all cases the up-regulation has been explained b~ the induction of ~B3 anti~odies.

2.~.2~ Immu~oth~r~py with ~ ~ 3~tibodie~
Monoclonal antibodies against bovine serum albumin, given to ~aive mice in the absence of adjuvant, could down regulate the IgG response to .~, later immunization with that antigen (Eddy et.
al.,1987. J. Immunol. 138:1693 1698~. Polyclonal antibodies against sheep red cells administered pre-sensitization could also nonspecifically down-regulate the IgG response to that antigen (Heyman and WigzellO
(1984). J. Immunol. 132:1136-1143).
Saint-Xemy et al. have shown in several ~: publications ~Machiels et al. (1990). J. Clin Invest.
85: 1024-1035; Saint-Remy et al. (1988) Eur. J.

WO93/17703 PCT/~S93/020~2 2131898 - 8 ~

lmmunol. 18:1009-1014) that autologous antigen-antibody complexes, ma~e by adding antigen to sera from patients who were refractory to hyposensitization, could down-regulate the IgE
response to a variety of allergic diseases including asthma and atopic dermatitis. After admini~tration of antigen-antibody complexes, the level of anti-idiotypic antibodies was increased; this was associ~ted with clinical improvement.
Thus, polyclonal antisera can down-regulate the IgG response. The use of monoclonal antibodies to down-regulate the immune response is considerably more complex, in par~ because of the dif f iculty in selecting the specificity of the monoclonal antibody.
: 15 : ~: : 3~ B~IEF DE8CRIPTION OF ~HB FIGUR~S
igure 1 represents the suppression in ear thickness of mice immunized with Der p I, then ~reated with t~ree different doses of anti-Der p I MAb H11, at doses of 3 x 5 ug, 3: x 10 ug, or 3 x 20 ug, in alum, at days 4,7,: ~nd:14:after sensitization. Animals are then~challenged by~ear injection with 10 ug DMA or ` with saline, on day 28.
Figure~2 represents an immunoblot profile of : 25 three monoclonal antibodies, all directed against rye roup I:.~ The f:irst,~:shown in immunoblot A i~ Mab 290;
: the second, shown in immuno~lot B is Mab 348; and the ` third, shown in~immunoblot C is Mab 539. Three antigen mix~ures were used; rye group I ~rom NIH (lane 1 in each blot), and were crude rye grass extracts from Omega ~lane 2~ and Pharmacia (lane 3).
; Figure 3 shows~Silver nitrate-stained SDS slab gel (7.5-15% run under reducing conditions) demonstrating the molecular weights of the rye Group I
antigens. ~ane 1 is molecular weight markers, and lanes 2 and 3 are rye Group I antigen purified by ;: :

~ W~93/~7703 PCT/US93/~2082 2t~18~

affinity chromatography with either 5 ~g/well (lane 2) or lO ~g/well (3) r~cpectively.
Figure 4 shows inhibition by unlabeled MAbs ~ _, 290A-167; ~ , , 348A-6; and 539A-6) of the binding of labeled 290A-167 (a), 34BA-6 (b) and 53~A-6 (c) M~bs to their homologous polystyrene-bound anti-ID Abs. 13A-12 (x-----x); 3E12 : : (+-~ , and 3E-3 ( ~ ) MAbs, were used as negativ~ controlsO Each point represents the mean value of the three axperimental determinations.
Figure 5 shows inhibition by anti-ID Abs (~
, anti-ID of 290A-167; ~ , anti-ID of 348A-6;
and ~ , anti-ID of 539A-6 MAbs) of the binding of labeled 290A-167 (a), 348A-6 (b), and 539A-6 (c) MAbs : 15 to polystyrene ound:rye I. Normal rabbit IgG was , ~
used as negatîve~control (x~ -x)O Each point ; represents the mean value;of three experimental determinations~
Figure 6 shows inhibition by anti-ID Abs ( 2~ ~anti-ID:of 290A;~ anti-ID of 348A, and , anti ID
of~539A) of~the~:b~nding of labelled rye I to polystyrene-bound:29~0A (a); 348A:(b), and 539 (c~
: :mAbs~ Normal~rabbit IgG:was used as negative control ). Each point:~repr~sents the mean value of three 25~ e~perimental~determinations.: :
: Figure 7 æhows~inhibition by anti-rye I MAbs ( [290A-167 (a~, 34~8A-6;tb) and 539~-6 (c)3 or their homologous:anti-idiotypic Abs~(_); of the ~inding of human IgE to polystyrene-bound rye I. The con~entration of~ e~ch Mab was I ~g/ml and ~or anti-idiotypic antibody, the concentration was 250 ~g/ml.
: Figure~8 shows inhibition of binding of labeled anti-rye I MAbs (2:9;0A-167 ~ , 348A-6 ~
539A-6 ~ ) to~solid phase~fixed rye I antigen by 35~ human auto anti-ID antibodies from three (A, B, C) sera depleted of anti-rye I antibodies. The inhibited bindings cf Mabs were as follows: 290A-167 ~ 34,400 ~: :

W093/17~03 2 1 3 1 8 9 8 lo - P~T/US93/020,~2 c.p.m.~ 348A-6 - 78,526 c.p.m. and 539A-6 = ~7,006 c.p.m. ~The binding of anti-Hbs Mab (3E~) as control to its antigen was 43,988 c.p.m. and was not inhibited by any autoanti-ID antibodies.]
Figure 9 shows specific suppression of anti-allergen antibodies by administration of idiotypic antibody. Groups of mice were immunized by i.p.
;~ injection of various amounts (O.Ol-lO ~g3 of 2gO~-l67 (Id) or lO ~g of unrelated Mab (5 mice/group). Blood samples wexe~regularly drawn and ~ested for specific : Ab in:ELISA or PCA assays.
Figure lO shows~change in specific antibody levels over time. Animals were injected with Mab 290, or an anti-transferrin Mab of the same subclass as Mab ~: 15 290, which does~ot cross react with that antigen; and Lol p I antig~en~ using the scheme shown in Fig ll. Sera were drawn at 2 week intervals, and binding to Mab Z90 was measured at a:dilution of l:lO0 by an ELIZA assay as described. ~: ~
Figure ll shows anti-idiotypic antibody l~vels as a function of Mab 290 dose. Groups:of five mice were injected with:Lol p I antigen, and concentrations of Mab:290 or with ~10~ g of the anti-transferrin Mab.
ranging from O~.Ol to lO ~g using ~he Scheme pictured 25~ in Fig 10; ~he~levels of anti-idiotypic antibodies ~eactive~with Mab~290~were assayed at a dilution of ;lOO~by; an ELISA assay as described in the text.
Figures 12 and l3 depict specific inhibition of IgE and IgG's antibodies by Mab 29~. Groups of five mice were inje¢ted~with~lO ~g of Mab 290 (termed ID in the figurè legend) or unrelat~d Mab (anti-transferrin) : :of the same subclass. After depletion of anti-~: idlotypic antibody activity, sera were either diluted l/50 and assayed for an i Lol p I: antibody of the IgE
class, or diluted ~ljlO0 and assayed for anti-Lol p I
~:~: antibody of the IgGl class. The mice had last been immunized with Lol p I at week 9.
:~: :

~ WO93/17703 ~ PCT/US93/02082 21318~8 Figure 14 depicts the structure of urushiol, the ~eries of alkylcatechols in poison oak/ivy, with a C15 ; or C17 side chain.
Figure 15 summarizes studies in which binding of radiolabelled MAb 991 to pentadecylcatechol ~PDC) was measured in the presence of a variety of ~elated ~: compounds. Maximal inhibition of binding was noted with PDC and the catechols; minimal with pentadecanoic : :acid, an analog of ~h~ side chain, or with ~e~orcinol,an an~log of the~aatechol ring but with hydroxyl groups in different positions.
Figure l~ depicts results of experiments in which ~ mice were pre-treated with one or more doses of M~b : 991, then sensitized~to PDC, and challenged (by ear painting~) with~the-same compound.
: Figure 17 depicts~results of experimen s in which mice are initially~:~ensitized with~PDC or urushiol, then injected with MAb:991, 21~days later, after which they are challenged on day 42 by ear painting.
~ Figure 18~depicts results~of experiments in which sensitized mice~:are;inje-ted~with~a variety of different doses:~:of:MAb 991 in alum prior to hallenge.
In~this~:case~ the;perc:ent reduction in ear thickness : :o~er~controls~is~:plotted, with:the background ~non-2~5;~specific) swelling subtracted.~
Figure 19~ Binding of three~anti RTA MA~s to either~;native~RT,~heat treated~:~TA (56C for 30 minutes (H RTA)~recombinant RTA which is not glycosylated, .~rRA)~ or deglyrosylated RTA produced by :30 ~periodate treatment of the polypepti~e to oxidize the terminal mannose:residues (d~
Figure 20::Anti-RTA antibody~titers resulting : from treating BALB/c mice with a single dose of loo ~g ~ . ~ . , :
; ~Ab delivered intravenously 24 hours after initial : 35 sensitizatioD with;50~g of~immunotoxin. Anti-RTA
titers were~measured by an ELISA assay, and results are expressed~as percent reductlon compared to the ... .... , .. ,.. .. ~ .. ... .

W093/17703 - 12 - PCTJUS93/020~82 213:~898 controls, mice sensitized with immunotoxin and injected with comparable amounts of an irrelevant Mab, 365, which is directed against carcinoembrionic 4 antig~n.
: 5 Figure 21: Dose response effect of Mab 608/7/
given at doses ranging from 25-200 ~g 24 hours after immunization with Immuno~oxin.
~ Figure 22, 23 Effec~ of treatment at various :: times after sensitization, with 100 ~g of Mab ffO8/7 ~ 10 upon the anti-RTA IgG response of BALB/c mice :~ immunized with immunotoxin, and treated with either Mab 608/7 (solid barsj or an equivalent amount of -irrelevant Nab 365~(anti-CEA).

4. 8UMN~RY OY ~B INV~NTION
: The present invention pro~ides novel compositions and methods~useful in:the modulation or selective suppression~of~:host immune responses;to an immunogen of~interest, particularly to immunogens which are :20~: exogenous antigens such as allergensO Subject : ; compositions~include;antibody, antibody derived, and a~tibody-like~molecules:of primary~antigen reactivity with~respect~to:~the immunogen~of interest. Antibodies or:antibody-like~:~or antibody-derived molecules include 25~ ::antib~dy~fragments~such~as Fab,:~and complementarily : determining~region peptides (CDRs) which may be grafted into~a~:~fràmework region of~any spe~ies, ; particularly human~. They also include human antibodies, derived~from sensitized human lymphocytes produced:by~cell~;fusion with he~erohybridomas, or by DNA cloning and expressio~. Other:compositions ;: include T cell receptor (TCR) molecules, obtained : either from T cell;clones or hybridomas or as purified : TCR preparations. Immunoreactive peptides corresponding to some or all of the complementarily determining regions: or hypervariable regions of the ~ ~ TCR are also employed. In preferred embodiments, :: :

,.~ WO93/17703 2 1 3 1 8 9 ~ PcT/~s93to2ox2 antibody and antibody-derived molecules and TCRs are directed against d~minant epitopes of the antigen.
Allergens to be down regulated may be from environmental sources, from food, from cosmetics, or from drugs and th~rapeutics. Immune responses to be down regulated include, but are not limited to, T cell ~ reactions such as those causing poison oak and ivy :~ dermatitis, as well as those initiating IgE mediated :: diseases:such as asthma, allergic rhinitis and atopic :dermatitis. IgE-mediated allergic responses such as asthma, allergic rhinitis and atopic dermatitis, are regulated:by the~proposed~methodst as~well as those mediated by:IgG responses.:IgG-mediated allergic reactions are especially important in mediating l5~ sensitivity to:drugs~and to injected allergens, as :exemplified~by ricin A chain.~
Compositions: of the present invention are bel~ieved to suppress~host;~:immuneiresponse to the immunogen of interest,~ at least in part by stimulation :20 of antiidiotypic:antibodies and by interactions with ce~lls:involved~in antigen processing and stimulation of~the:immune network.
The presént;:~invention also provides methods for selective suppression::~of host~i~mune response to an 25~ ~immunogen of interest by administering a compound of the invention.~ n a preferred embodiment, subiect compositions~:may-~be administered to the host after the host has been :sen~itized to the immunogen, thereby ~, ~ downregulating the:host sensitivity. Composition also ~: 30:: may be administered~o the host::prior to : sensitization:. :

~; 5. DETAILED DESCRIP~ION OF T~E:INV~TION
~-; : As used herein, an Abl or idiotypic mole~ule is an antibody, TCR, or antibody-:or TCR-derived molecule which binds to or reacts with a primary immunogen or allergen. An Ab2 or anti-idiotypic molecule is an W093/17703 2 1 ~ 1 8 9 8 - 14 - PCT/US93/02Q~2 antibody, TCR, or antibody- or TCR- derived molecule which binds to or reacts with the ~ombining ~ite of the Abl or idiotypic molerule against the primary immunogen. Thus the Abl or idiotypic molecu~e itself functions as a secondary ~ntigen, in that it can produce an anti-idiotypic response.
As used herein, the term "antibody derived"
molecule refers to an antibody, an antibody fragment, - a hybridized antibody or antibody fragment, or a lo chimeric molecule:comprising an antibody binding domain and a heterologous polypeptide. Antibody-derived molecules of the invention contain at least a blnding domain of an~Ab1 against the allergen of ~` interest. The binding domain încludes thP
15: complementarily determining regions (CDRs) of the Abl, joined to a~framawork region (FR). The FR may be a : sequenoe from an animal or a human; replacement of animal FRs with huma~ FRs produces a humanized binding domain.
The ¢onstant domains of an antibody from an ' animal~may be:~replaced~by~:~orrespon~ing domains from a human`to produce:humanized antibodies. Antibody-derived molecules;~of the invention: comprise both~
:human~zed:binding:domains and:humanized antibodies.
~ A~TCR may~be employed~analogously to an antibody-deri~ed~Abl.: ~A TCR which contains a binding site for an epitope~of~an~al~lergen of interest is capable of unctioning in a~manner similar to an antibody-deri~ed Abl~ A~"TCR-derived~' molecule refers to a TCR, a TCR
fragment, a hybridized TCR or TCR fra~ment or a chimeric molecule.~ Part:icular embodiments of TCR
deri~ed moleGules incorporate the:binding domain of a TCR, but may have other domains of the TCR deleted, or may~b formed into~a chimeric molecule with soluble : 35 domai:ns from other proteins, such as serum albumin.
~; For convenience, references hereln to 'IAbls" includes TCR-derived mole ules which bind to an epitope of the : :

.~ WO93/17703 - 15 - - 21 31 8 9 ~
., allergen of inter~ t, unless the context precludes this meaning.
A preferred embodiment of the invention comprises antibody or TCR-derived molecules which have been isolated and purified. As used herein, the term "isolated" means a composition which is free of substantial or immunologically active amounts of serum c~mponents other than antibody - or TCR-derived molecules. In particular, isolated antibody-derived molecules are substantially depleted in major serum proteins and cytokines or lymphokines. "Purified"
antibody~- or TCR-derived~molecules are depleted of molecules which~do not bind to the allergen of interest; in particular, purified Abls are depleted of 15 ~Ab2æ and of Abls~directed to other antigens.
However,~compositions of the;~present invention ;may~comprise~Abls to wh~ich non-immunologically active molecules have~been added, e.g.~, serum albumin from the~species~to wh~1ch the~compos~it~ion is to be administered.~
A-~parti~cul~ar~preferred embodiment comprises Abl antibody~ or~TCR-d~rived molecules which;are substantially~free~of the;allergen;to which the Abl molecule~bin~s~ "Substantially~free'~ means that the 2~5~ ratio of~the~numbér~of ant~ibody~binding épitopes is at least~l0~ prèferably~100:1, more preferably l000:l.
; One~categ~ry~of~exogenous~;anti~ens of~interest is referréd to as~environmental~ allergens. ~As used . , herein,! the term~"environmental allergen" refers to an 30~ allergen to which~an~;animal, including a human, is exposed by external cont`act~, and includes dermal or conjunctival;c~ntact and inhalation of airborne allergens. Environmental al~lergens include both immunogenic macromolecules and;haptens.
3S ~ ~ Particular~environmental allergens of interest include aeroallergens such as pollens, which are wind-dispersed~by many~types of higher plants, 1ncluding W093/17703 PCT/US~3/02082 2 13 18 ~ 8 ~ 16 -gymnosperms (e.g., pines and cypresses), dicotyledonous angiosperms (including broadleaf trees such as elms and many common broadleaf weeds such as ragweed and plantain) and monocotyledonous ~ ~ S angiosperms (e.g., the grasses), or dust ~ite antigens ; consisting of proteins rom the body and feces of the dust mite, found in house dust,~ mattresses and carpet, but capable of becoming air-borne. They also include molds such as al ternaria . The methods preqented here 0 are applicable to any o f these allergen.
A second category of exogenous allergens of interest are those~derived from foods and food products. Numerous~food components, particularly , proteins, are know~n to cause food allergies. Examples are proteins of wheat and; related cereal grains, and of legumes such~as peanuts.~ The allergenic proteins of;wheat~and~peanuts h~ve been isolated. ~Abls against allergenic~wheat~and~ pe~nut~proteins are particular embodiments~of~one~aspect of the~invention, e.g., 20~ ~compositions comprising Abls against food allergens.
Anothér~example~is~provided~by wheat gluten and related~protelns.~ These~are~potent allergens in some individuals~ and~provoke IgG mediated reactions to wheat products~in~the diet.~ Administration of 2~5~ ompounds;compris;ing Abl-derived molecules against food~allergens~are~useful for~suppression of food allergies.~Other~food allergens for~whi h particular proteins have~bèén~implicated~are summarized in Sampson~et al.,l992.~JAMA 268:2840-2844).
~A third category~of exogenous~aller~ens of interest is;~drug~and~therapeutic~allergens. Prominent examples of drug~allergens are the various penicillins and~ related ~-lactam~antibiotics. Allergic reaction to penicillin~can~be ~life-threatening. Administration ; 35 of a composition comprising~an Abl-derived molecule ::
against a ~-lactam antibiotic is useful for suppression of antibiotic allergy. Compounds and ~, : ~ :

, WO93/17703 2 ~ 318 9 8 PCT/US93/~2082 methods of the present invention provide for down regulation of Ig~ reactions, as seen with ricin A
chain which can be useful as a component of cytotoxic drugs. Another example of an immunogenic protein drug is provided by insulin. Diabetics receiving either animal or recombinant human insulin often develop anti-insulin antibodies.
A fourth category o~ interest are those haptens :~ and low molecular weight chemicals such as urushiol (poison ivy/oak) or industrial and environmental chemicals causing either contact dermatitis or ~: pneumonitis.
The responses to be down-regulated can be those mediated by T cells, as exemplified by poison oak : lS dermatitis , or IgE, exemp~ified by Dust Mite Antigen, or Rye Grass, or by IgG, exemplified by ricin A chain or Rye grass.
Abls useful in down regulation of an allergic r~sponse may be of any antibody class and subclass, e.g., IgM, IgE, IgG~j:IgG2, IqG4, etc., and may include TCRs. Class identity~is of~secondary importance compared to the characteristics:of the binding domain.
; Ab1s~ are believed to act in part by stimulating the formation of AB2s:directed against the binding domain ;2:5~ of the Abl. ~Although:the speci~ic binding domain of the Abl there~ore:is critical, other portions of the Abl molecule may~be modified appreciably, including those por~ions of the antibody which determine class.
For similar reasons, fragments of the Abl are ~ 30 ac~ive, provided that the desired allergen epitope : ~inding domain is present. Any of the common antibody fragments which retain binding specificity may be ~: used. Als3, cloned "fragments," or partial antibody sequences ubtained by expression of abbreviated genes encoding the:~inding domain, are within the scope of ~ the invention.

:~ :

Furthermore, chimeric molecules in which the ~bl binding domain is joined to other proteins or protein domains also are active immunoregulators. The chimeric prote~n "partner" to which the allergen binding domain is joined may be selected from a great variety of sequences. In some cases the binding domain from an antibody of one class may be joined to the constant regions of another ~lass to produce a class-switched antibody. When the constant region is lQ from a different species from that of the binding : domain, the resulting chimeric antibody is partially : species switched. This procedure may be used to create partially humanized antibodies which have lessened ~ntigenicity when injected into humans.
In particular instances, a chimeric partner may : be chosen because it imparts desirable solubility or locaIization characteristics. For example, an : : :
allergen binding domain may be joined to a collagen domain ~f the target species. The resulting chimera ` 20: remains localized at the injection site due to binding of the collagen domain to extracellular matrix. In other:instances a chimeric partner is chosen because ` it is itself immunologically active and acts as an adjuvant. Examples:~include antigenic domains from protein~ of~common bacterial pathogens.
Since immunoregulatory mole~ules of the inven~ion do not require constant domains for activity, the Fc portion of the IgG constant domain is not necessary for immunosuppression. ~This re`sult is counter to the suggestion in the work by Heyman et al., that the Fc ~ domain is critical for immunosuppression.

; 5.1 Production of ~urin~ monoclo~al a~tibodie Murine monoclonal antibodies to the antigen of interest are prepared by methods well known to those skilled in the art ~Goding, supra.) BALB/c mice are immunized with specific antigen preparations using a WO93/17703 2 P~T/US93/0208~
131 ~y8 variety of protocols including use of immunological adjuvants ~uch as complete an~ incomplete Freund's adjuvant. Spleen cells are prepared from mice shown to be producing antibodies of khe proper specificity, and fused with cells from murine myeloma cell lines such as PS-NS1, Sp 2J0, X63-Ag8~654, NSO/l or the like ~y establish~d procedures ~Kohler and Nilstein,1975.
Nature 256:495-4g7; US Paten~ No 4,376,110). ClonPs are : ~ selected by two rounds of limiting dilution.
lo Hybridoma cells are then either maintained in culture ; or propagated ~n the peritoneal cavity of BALB/c ~ female mice after prior injection of prîstane (0.5 : ml)~ Ascitlc fluid is harvested from mice lo to 15 days after hybridoma cell injection and antibodies purified by passing the fluid through a protein ~ -Sepharose 4B column.

5.2 Pro~u~tio~ of hu~izQd ~o~oclo~al antibodie~
:: :
: 2~ Humanized monoclonal ~ntibodies containing only murine variable regions may be constructed by CDR
: grafting/reshaping using various techniques. The ~ complementarity~determining regions (CDRs) of a murine : ~ ~ MAb are:transplanted onto the corresponding regions-in 25~ a human antibody. The CDRs :tthree in antibody hea~y chains and three in light chains) are the regions of the murine MAb which bind to the antigen epitope.
ransplantation of the CDRs is achieved by manipulation whereby~CDR DNA sequences are determined by cloning of~murine heavy and light chain variable V~ region gene segments and transfer to corresponding : human V regions by site-directed mutagenesis. Human ~ ~:
constant region gene segments of the desired isotype are ad*ed and the~humanized heavy and light chain genes are co-expressed in mammalian cells to produce ~humanized antibodies.

W093/17703 PCT/US93/OX~082 - 20 - ~

A general procedure has been developed to graft CDRs from rodent monoclonal antibodies onto human FRs to make fully humanized antibodies or antibody fragments using PCR methodology (Lewis and Crowe 1991.
Gene xx 297-302). PCR primers which bind to the FR
regions bracketing the CDRs have been developed; these permit amplification of the CDR from hybridoma cells ~ecreting the Mab of interest. The PCR product is cloned a~d sequenced, and joined to the framework and c~nstant regio~s of choice. These methods have been extensively described;(Winters and Milstein.1991.
Nature 349:293-299; Riechmann et al.1988. Nature 332:32~3-327; Hird et al., l991 Br. J. Cancer 64:911-914;~Gussow and Seemann, 1991. Methods in Enzymoloay,203:99-121; ~ones~ et al.,1986. Nature 321:522-525;~Verhoeyen, et~ al;.,1988. Science 239:1534-1536; Kamman et.~ al. ,1989. Nucl. Acids Res 17:5404;
Maeda, ee~ ~ al., 1991~ Nu~.~ Ant;ibod. Hybridomas 2:124-5.3 Chimeriz~tion-hum~ni~tio~ o~ murin~
monoc~on~l ~tibodi~ ~
Monoclonal antibodies of murine origin are humanized~by~various~methods so as~to reduce the immunogenicity~ of~the~proteins with respect to the murine immunoglobulin~ component. ~One way;involves producti~on of~chimèric;antibody by;linking cDNA
molecule enc;oding~the V~ region o~the murine Mab to DNA encoding the~human constant region. This can be effected using;several approaches described in issue - patents~. ~The~chimerization~procedure in~olves:
Isolation~of messenger RNA (~mRN~) from mouse hybridoma~cél1s,~then cloning~and cDNA
production.
(2) ~Preparation of a ~full length cDNA
library from~purified~mRNA fr~m which appropriate variàble (V)~reglon gene fragments, of the light (L) -~:
,:

W093/17703 - 21 - 2 1 3 1 8 9 ~ PCT/V~93/02Q82 a~d heavy (H~ chain genes can be identified, se~uenced and made comp~tible with a constant (C) region gene segment.
(3) Preparation of C region gene segment modules by cDNA preparation and cloning.
(4) Co~struction of complete H or L chain coding sequences by linkage of the cloned specific i~munoglobulin V region gene segments to cloned human C region segment modules.
~0 (5) Expression and production of chimeric L
and H chains in prokaryotic and eukaryotic cells.

Detailed procedures are provided in Cabilly et al., US Patent 4,816,567 (28 March, 1989); Taniguchi et al., Eur. Patent EP 171496 (19 February, 1986) and Kudo et ~l., EP 184187 (11 June, 1986) and ;~; pub~ications including Cabilly et. al. (19B4) Proc.
: Natl. Acad. Sci., USA, 81: 3273-3277; Morrison et al. tl984) Proc. Natl. Acad! Sci., USA, 81: t 6851-6855; Neuberger et al. ~1985) Nature. Lond., 314: 268-270; Tan et al. (1985). J.l~nl3~ a~ 3564-3567;
Sun et al. (1987) Proc. Natl. Acad. Sci. L USA, 84:
~ 21~-218, Lui et al. (1987) J. Immunol., 139: 3521-:~ 3526; and Horowitz (1988) Proc. Natl. Acad. Sci. USA, 95: 86~6~8682).

: s~ RepertoirQ Clo~i~g Antibodies reacting with Der:p and Der f allergens were produced by procedures based upon antigen selection from a combinatorial library and cloned ~O~landi et al.,l989. Proc. Nat. Acad. Sci.o ~: 8S: 3833-3837; Persson et al., l991.Proc. Nat. Acad.
Sci~ U SoA~ 88 2432-2436; Kang et. al.,l991, Proc.
Nat. Acad. Sci., U.S.A., 88 4363-4366; Duchosal et al.,l992. atur~355: 258-262; Clackson et al.,1991.
: Nature~_352: 624-628; Marks et al.,l991. J. Mol.
Biol. 222: 581-597). The general approaches well 21~1898 ;`' known to those versed in the art include extraction of messen~er RNA (mRNA) from human lymphocytes derived from various tissues including peripheral blood (PBL) tonsillar tissue (Ton-Ly) and spleen (Spl Ly). The lymphocytes may be derived from human allergic subjects and in addition may be initially stimulated by e~posure to allergens and immunological adjuvants as already desoribed. Another procedure for stimulating lymphocytes involved injecting lymphocytes from sources such~as PBL or tonsils, into severe combined immune deficiency mice (SCID) and stimulation of injected mice with allergen preparations surh as ~ the whole dust mite extracts (DMA) or purified ;; ~ proteins such as Der p I and Der p II. Mice may also be treated with i~munological adjuvants such as bacillus Calmette Gurin (BCG) or by combining ;immunogen preparations IDer pI/ll etc) with aluminum hydroxide gel ~alum), variously described as adjuvant.
` The polymerase~chain reaction (PCR) together with appropriate~primers~is~used to amplify the variable heavy (VH)~ and~variable light (VL)~genes from the m~NA
preparations~us~ing~well established procedures (Orlandi~et al.~,~l989. Proc.~Nat. Acad._Sci.. U.S.A., 86:~3833-3837~as~an~example). The mRNA preparations 25 ~are~used~to~c~nstruct Fav cmbinatoriaI libraries in phage (Persson~et al.,l99l. Proc. Nat. Acad. Sci.. 88:
24~32-2436~ Nuse~et~al;.,~1989. Science. 246: 1275-1281).
Clones~selected by~screening are~plaque-purified and the phagemids~excised and used to transform Escherichia c~ Fa~s from culture tranformants were then produced~by~procedures well known to those versed in the art (Gusow~and Seemann,l991. Methods in Enzvmoloqv, 99: ~121,~ Pluckthun.1991. Biotechnoloqy, 9:
545-551, and 1991. Nature 347: 497-498.

~: :
::

W093/17703 2 ~ 3 1 8 9 ~ PCT/U~93/020~2 5.5 Bel~c~io~ of all~rge~ sp~cifia ~b18 S~veral criteria may be used to select a preferred monoclonal antibody against an antigen to be down regulated. These include:
5.5010 D~o~str~tion th~t th~ ~ele~t~
mo~oclonal antibo~y re~ogniæe~ a hu~an IgE or IyG im~u~odomi~ant : prctein i~ the all~rga~ ~i~ture i~
tho~e C~3~ in which the ~ rg~
i~ ~ mixtur~ r~ther th~ i~glo compon~t Many native allergens are composed of mixtures of proteins, which may number more than 20.
Functionally, the majority of the human immune response which is clinically significant (IgE in the case of most aeroaller~ens) is directed against 1-4 of these proteins.~ An example is short ragweed, in which over 50% of the IgE response is directed against Amb v (known in the ear~lier literature as AgE3. Another example is the Lol p 1 protein in Rye grass, in which up to 80% of the IgE rea~ts against th~ s protein. A
20~ third is Der~p~l prstein an~ Der p 2 protein in dust mi~e allergerls:, in which these ~wo prot~ins account for well o~er 50~ of the IyE response.
To identify :such proteins, i~nunoblotting may pe used. In this procedure the crude antigen extract is subjected to SDS-PAGE to separate the proteins by molecular~weight. ~Then, sera from patients clinically r~active to the given allergen mixture are overlaid onto the separated proteins, which have been electrotransferred:onto nitrocellulose paper. After 30~ incubation, the strips are developed by addition of : reagents such as peroxidase-labeled anti-human-IgE
sera. This identifies the pro~ein(s~, and their :~ ~ relative contributlon can be demonstrated by isolation of the proteins, and absorption of the IgE from patient's sera. Identification of an Ig~

: ~ :

WO93/17703 PCT/US93/~2082 - 24 - ~
2131~3~8 immunodominant epitope may be performed using anti-human-IgG ~erum; see 5ection ~.

5.5.2. D~mon~tr~tion th~ th~ ~le~tQd ~o~o~lo~l a~t~bo~y r~ogni~e~
h ~ a~ Ig~ or Ig~ iæ~u~o~o~nant apitcp~ o~ tho prot~i~
A ra~ge of murine or human ~onoclonal IgG or IgM
antibodies are made by standard methods defined elsewhere in this dorument and well known to those 0 skilled in the art. This panel of antibodies is used to identify the immunodominant epitope on the molecule by checking the ability of each antibody to substantially inhibit the binding of the human IgE or IgG containing sera to the protein. Substantial inhibition is at least 20%, preferably by 40~, more : preferably hy 50%, 60%, 75%, or even 90%.
Alt~rnatively, the ability of the human IgE or IgG
cont~ining sera to inhibit the binding of each ~onoclonal may be tested. In~the latter case, the :2~ monoclonal antibody may be radiolabeled with 1~I, mixed w~th the human serum, and added to polystyrene remoYawell strips coated with the protein.
Determination of radioactivity remaining after incubation and washing of the wells will ~uantitate : 25 the amount of b1ocking, and identify immunodominant epitopes.~ Usually such a protein~contains 2-6 : immunodominant epitop~s, and the monoclonal an~ibodies ; reacting with these epitopes are selected for further study.
: 30 ~: : 5~5~3O Demon~tratiQ~ th~t the ~lected ~onoclo~a~l a~tibody ~imulates a~
anti-i~iotype ~peCif iGity ~imilar to that induced i~ h~ma~s, ~- includi~g tho~e receivi~g alI~rgen : ~pecific i~u~other~py:
: 35:
: In the case of allergens, humans subjected to hyposensitization and enjoying clinical improvement of .

, 2131~
WO93/17703 PCT/~S93/02082 their symptoms are available. The identification of the specificity of the auto-anti-idiotypic antibodies in the sera of these patients also aids in identification of Abs likely to be e*fective in down-regulation of the lgE response, since it isanticipated they will be directed against the : naturally occurring Abl whi~h in turn identifies the appropriate epitope fcr down regulation. Therefore, ~; one approach to identify the specificity of the 13 candidate monoclonal Abls is to measure their ability to inhibit binding of ~I labeled "candidate"
: : monoclonal antibody to the protein. In one such procedure naturally occurr~ing polyclonal human Abls ~: are remo~ed from the sera from hyposensitized humans : l5 ~y a~sorption with ~he antigen, so they will not interfere~witb the assay. They are also absorbed with normal murine IgG coupled to Sepharose C~4B, if a ` murine monoclonal antibody is to be tested. The sera, now containing only AB2 antibodies against the protein in question,:are then tested for the inhibition of inding of murine monoclonal anti-rye Lol p I
: : monoclonal antibody ~to the antigen. The antigen is coated onto polystyrene removawell ~trips, and the : can~idate monoclonal~antibody, now radiolabeled, is Z5 ~mixed with the absorbed human sera, or incubated alone a~ the positive con~rol). Wells arP washed and measured~ Reduction of radioactivity confirms the percent of inhibition, and identif:ies the~monoclonal ~, antibody which recognizes the specifi~ity of the auto-an~i-idiotypic antibody present in highest amount.
In general, an antibody binds at least 20%, more prefably 40%, 50%, 60%, 75% or greater of the auto : anti-idiotypic antibody induced in a hyposensitization ~ subject.
: 35 .
:

5.5.4. De~o~tratio~ th~t tr~at~e~t of ~ with tho ~ab ~igni~i~a~tly aown-regulat~ the ~u~e r~po~e agai~t th~ ~llergen or a~tige~
If an animal model exists, the ability of the selec$ed monoclonal antibodyts) which may be used as a cocktail, to down-regulate the immune response may be tested. In this aase, the animal may be sensitized eith~r by injection of the allergen in adjuvant, without adjuvant, or in the aerosolized form. The candidate monoclonal antibodie(s) are then injected before or at various periods after sensitization, and their ability to down-regulate either the primary response or the response to antigen challenge is measured. In most cases tested, the response is down~
regulated by àt least 50%.

6. ~PLE~ 8 ~0 DlJ8~r IIITE ~LBRG~
Dust mite aIlergy is caused by exposure to mites Qf the genus Dermatophagoi~es ~ particularly Dérma~ophayoides pteronyssinus (Der p) and Dérmatophagoides farinae (Der f~.~ A range~ of at~least 24 different proteins has been identified:aIthough ~er I; (molecu1ar:weight~24,000) and ~Der II (molecular weight lS,OOO)~ are generally recogniz~d as the major mite alIergens.

Pr:~par~tio~ of Du~t ~it~ allerg~
6.1~ ; E~tr~tio~ o~ khe ~tigen Whole dust mite preparation (~MA) from Dermatophagoi~es ~teronyssinus or Dérmatophagoides farinas including;mite bodies, feces and culture medium are extracted with phosphate buffered saline pH

7.3, 10 ml/gm, and the extract~is centrifuged, and the supernatant precipitated with 50~ saturated ammonium sulfate. The sediment is re-dissolved in phosphate ~uffered saline pH 7.3 (PBS~, dialyzed against PBS (4 changes) , and stored at 20 C. The concqntrations of WO93/177~3 ~ P~T/US93/~2082 - 27 - ~1 3~ 8g~

Der p I prot2in i~ determined by immunoassay, compared to standards provided by Dr. Martin Ch~pman, University of Virginia, Charlottesville, US~
tLuczinska et. al.,1989~ J. Immunol. Methods 118:227-~359).

6.1~2 Purif~catio~ of Group IIII Mite AIl~rg~ns Der p I/II and Der f I/II preparations are isolated from DMA extracts of the appropriate mites by affinity chromatography on in~olubilized:anti-Der I or anti-Der II monoclonal antibody respectively tfor example, 4C1:, anti-Der I, 7Al anti-Der II; Lombardero et al., 1990. J. Immunol~ 144:1353-1360).
~S ~
: 6.2 Proauc~io~ of muri~e a~ti D~r p ~d anti : D~r f ~onoclo~al ~tibodies 6.2~1 Immunizatio~ protocol :: :
:~ ~ : BALB/c mice are immunized ~ccording to the following schedule: 25 ug DMA in Freund's complete adjuvant (FCA),~delivered intraperitoneaIly, as the initial immunlæation (Day 0) then, at various intervals, be~ween days s;and 56, they are again inj`ect~d~with 25~ug DMA in incompl~ete Freund's ad~uvant (IFA~,: intraperitonealIy, two to four times.
;25~ Usually~:3 days pr:ior to sacrifice 25 ug is injected DMA:in PBS,~intravenously. Mice are killed on days 45 to 60~, and single~cell:suspenæions of the spleen are prepared and~fused with:mouse myeloma NSO cells by established p~ocedures ~Kohler and Milstein, 1975.
~:~ 3 Nature. 256: 495-497; US Patent No. 4,376/110).

:: : :
6.2.2 Hybridoma ~election protocol - Hybridomas are selected by screening of culture : : :
~ supernatants for antibody reacting with DMA by an ., ELISA test (ELISA and Other Solid Phase Immunoassays ds., D.M. Kemeny and S.J. Challacombe 1988, Wiley).

: :

WO93/17703 - 2 1 3 1 8 9 8 28 - PCT/US93/0 0~82 Brîefly polyvinyl microliter plates (Falcon Microtest II or the likej are coated with the equivalent of 0.5 ng/well Der p I by overnight incubation of a dust mite preparation tDMA~. The mi~roliter plates are blocked S with casein in tris-bu~fer pH 7.3. Tissue culture supernatant is added to the microliter plate wells,for 45 minutes, then the excess is removed by washing in PBS. Peroxidase conjugated rabbit antibody to mouse immunoglobulin i6 added and bound antibody detected by color formation~following addition of substrate 2,2'-azino-di-(3 et~ylbenzthiazoline sulfonic acid) (ABTS).
Hybridomas selected~in the primary~screen~are cloned two times and~hybridoma lin~es established.
Immunoblotting with~MAb purified from hybridoma 15~ sùpernatents~;~is carried~out in~which ~r p extract is subjeoted to SD5 PAGE~fo;llowed by transfer to ' A'`,~ nitrocellulose'and subsequent~staining with Mab ; (Thompson et al.,; 1988~ J. Immunol.~64:311-314. By aomparison with~5tandard (e.g.~, Der~p~ this allows ~components of~the~Dér~p extract reacting with the Nab to be id'entified'.~'The characteristics~of ~elected MAhs~in~comparison~wlth~other~anti-dust mite antïbodies'is~fur~her~èstablished using a bl,ocking assay~ In~this~-~procedure the Mab under investigation 2~5';~i~s'~added to'~micxoliter~plates~coatéd with~dust mite ex~ract~(DMA),~so blocking antigen sites. Inhibition of the~binding~of defined antibodies such as anti Der p~I~antibod~SH8~(Ch~apman~et al.;,1987. J.~Immunol.
139:~1479-1484,)~indicates that~the Mab under test 30~ ~recognizes the,~same~or closely~related epitope~ on DMA
to~the~standard~anti~body. ~ To~measure~ inhibition of Mab binding,~,biotinylated~Mab is~added to the blocked microliter plates'~and bound~antibody is detected by addition of streptavidin-peroxidase conjugates, 3S~ fol'lowed after~washing by the subs~rate (ABTS).
The ability of murine MAbs~t~o inhibit binding of 'antibodies ln~serum of patients~sensitized to dust WO93~17703 21 31 89~ P~T/vs93/o2o82 mite allergens is similarly detected by a bloc~ing a~ay. In theæe tests, the ~ab under investigation is added to microliter plates coated with dust mite extract. Inhibition of the binding of patients serum i~ then detected by first adding patients serum to microliter plates, washing and detecting bound human :: Ig with goat-antihuman Ig peroxidase conjugate followed by substrate ~ABTS).

6.2~3 Anti-iaiOtypic a~tiboay e~po~se to muri~e ~ti-Der p ~d ~ti Der f m~oclo~al a~tibodies ~:~ Murine anti-idiotypic antibodies to monoclonal antibodies are produced by immunizing inbred BALB/C
~: 15 mice with monoclonal antibodies coupled to keyhole limpet hemocyanin together with Freund's complete, then incompl~te, adjuvant. In an example, BALB/C mice are immunize~ first with Mab-KLH (50~g) incomplete Freund's adjuvant~and then twice at two week interval with Mab-KLH~(50~g) in incomplete~adjuvant. Antisera are then obtained 7 to 14 days later as a source of ~ ~-anti-idiotypic antibody~ These anti-idiotypic antibodies are:used~to define the diotypes of anti-DPr : : p I monoclonal antibodies 2C7 ~Table 2~ and 4Cl 25~ (Chapman et.~al., 1987 J. Immunol. 13g, 1479-1484).
: Binding~o~ Mab 2C7 to Der p I is detected by radio-; ; immunoassay using~12~5 I labelled Mab 2C7. Inhibition of this binding by a range of anti-idiotypic antibodies is detected by mixing 125 I labelled Mab 2C7 with antise~um before addition to microliter plates coated with Der p allergen. Binding of 2C7 is inhibited only by anti-idiotypic antibody to 2C7.
; :Binding of 4C1 to Der p I is only inhibited by anti-idiotypic antibody to 4Cl, these findings shGw that ~: 35 although Mabs 2C7 and 4Cl recognize closely related eptiopes on Der p I they possess separate and distinct idiotypes.

WO~3/17703 PCT/US93/02082 - 30 - ~
2 i~l898 6.2.3O1 i~uri~ ~nti-Der p hybridom~
As an ~xample, Table 1 summarizes murine ; monoclonal antibodies which react with Der p I or Der p ~I. Mab 1102/Hll and 1114/2C7 react with Der p I
(24 kD) as defined by immunoblotting after SDS PAGE.
Blocking assays with MAb 2C7 and MAb 4C1 (Chapman et.
al. ,1987. ~L_JI~Y~L~ , 1479-1484) indicated tha~
these MAbs react with Der p I epitope that are identical or closely related. Ta~le 1 also summarizes other MAb to Der p I and Der p II produced by these methods.

T~BLE 1 Mab Isotype Allergen Mw 1102/Hll IgG1 Der pI 24000 1107~2~Bll~ ~ IgG1 Der pI 27000 1111/A2~ ~;IgGl ~Der pI 24000 20 ~ 1114/lF7 IgG2a Der pI 24000 1114J2F10~: IgG2a Der~pII 15000 1119/I~12 I ~ ::ND 27000 ~;6~.3 Productio~ o~:~u~ Anti-der p ~onoclo~l Anti~odi~s ~
;Human monocl~onal~antibodies~ rearting with Der p antigens:are~produced:by various procedures. These : include immortalization:of antibody-producing cells from human donors sensitive to DM~ by construction of human hybridomas~or human-mouse he~erohybridomas using techniques well known to those versed in the art Austin et al. 1989j Immunoloqv, 67: 525-530) or by Epstein Barr virus transformation:(Roder et al.l986t Meth. in Enzymoloqy, lal: 140-167).~ Human antibodies are also obtained by repertoire cloning (Marks et al.,l991. J. Mol. BioI. 222: 581-5g7; Huse et ::::: :

:: :

~:: :

WO93/17703 - 31 - 2 1 ~ ~ 8 ~ ~cr/~S93/02082 al~l989, Science, 246: 1275-1281; Kang et al., Proc.
~a~l~ ~aad. Sci, USA~ 80: 4363-4366; Duchosal et al.,1992, Nature. 355: 258-262).

6.3~1 ~ybridoma Proauc~io~
: Lymphocytes are obtained from various tissues including peripheral blood, tonsils and spleen from human subjects sensitized to dust mite. Cell preparations:are obtained by Ficoll/Hypaque density céntrifugation and fused i D ediately after separation or following i~ vitro stimulation. Various in vitro stimulation methodæ are used including treatment.of lymphocytes cultured in medium RPMI 1~40 with dust mite extracts. In some cases in vitro culture co~ditions include~immunological adjuvants such as ::: adjuvant peptide:(N-acetylmuramyl-L-aIanyl-D-oglutamine, Sigma).

S.3~.2~ Fu~ion:Proto~ol :20 ::: Mouse/human:heteromyeloma EL41~cells (deposited on~Nay~16, 1990 with~:the European~Collection of Animal ell:~ultures::~;and~having Accession:No. 9~051602) as :; described by Aùstin~et al., Immunology, (1;989) 67: 525:-530,~are;~: f used~ with human~lymphocytes~reactive 25~ with dust~mite~;antigen using polyethy~lene glycol:
aG~Gording~ to the~method of Galfre~et al~1977. Nature 26~: 55~0~ ybridomas producing Ig~of interest are immediately cloned.~ Established~hybridoma~s~:are frozen down; in aliquot ~:of~2-5 x 106 cells in 1 ml o~ 95% FCS
and 5~ Dimethyl~sulp~oxide ~DMSO) and stored in liquid n1trogen.

6.3.3 :~ ~ybridoma 8electis~
Hybridomas are~selected by screening of culture supernatan s for~antibody reacting with dust mite antigen (DMA) preparations using ELISA assays using peroxidase conjugated rabbit antibody to human : ~ :
: `

immunoglobulin tc detect human monoclonal antibody.
Hybridomas selected in the primary screen are cloned two times and hybridoma lines established using culture procedures for stable growth.
~onoclonal antibodies are purified by various procedures and are characterized. Immunoblotting is carried out in which Der p and Der f extracts are sub~ected to SDS PAG~ followed by transfer to : nitrocellulose and subsequent staining with Mab ~ 10 (Thompson et al. 1988, Immunol.. __4: 311-314,; Chapman : et al.1987, J. Immunol., 139: 1479-1484, ; Platts-Mills and Chapman,1987 J. All. Clin. Immunol..
80:755-775) so as to d:efine the molecular weights of ~: the dust mite allergens each Mab.
The relationship~of human Mab to antibodies produced in human subjects sensitized to dust mite allergens is det cted using several immunoassays well knawn to those versed in the art (as an example :Didierlaurcnt and~Garcelon:. 1991. J. Immunol. Meth., ~ 22-41). In~:one approach microliter plates (Chapman et al.,~1987. J.:Immunol.. 139: 1479-1484) or cyanogen-bromide; activated:paper discs (Didlexlaurent and~arcelon, l`991. J. Immunol. Meth., 145: 33 41) are coated with dust mite ex~ract. Serum from allergic^
25 :donors:is~incubated~ in the~microliter wells or discs and~after washing inhibition of the reactîvîty with the~human Mab:detected.~ For thîs~purpose, ~ab îs conjugated to:biotin~by well establîshed methods. The : : înhîbîtîon of binding o~ bîotînylated Mab in mîcrolîter wells or dîscs îs then determined by încubatîon fîrst with s reptavidin-biotin~peroxidase : conjugate (Amersham, UK) and then with o-pheneylenediamin. Table 2 lists human monoclonal ; : antibodies produced by hybridomas;formed by fusion of : 35 human lymphocytes from:tonsils or peripheral blood wîth a human-mouse heteromyeloma EL 41.

W~ 93/~7703 PCI'/VS93~02082 - 33 ~ 213~98 q!ABLE 2 iElu~a~ a~ti-du3t ~ito ~lono~:loaal ntibodios ISI,ISA OD O~ 'latas (:oatod wi~h:
:
Hybridoma Lymphocyte DNa2 Caqein Re~ctivi~y with:
S Donc:~r~
Der p I Der p II3 DM27 lEll 0 .196 0 . 049 +
~ ~ DM27 lAll Ton~ 0.065 0.000 -:~ : DM27 lFlQ 0 . 094 0. 000 - +
: D2~27 2G9 0 .111 0 . 000 : DM28 lD11 ~ 0.160 0 . 019 +
D~28 2A2 PBL ~ 0 .172 0 . 04~ - +
DM28 22:9 0 . 391 0.176 ~ -`DM31~ lH2 : ~: 0.138 0 . 072 -- ~
:
DM31 lG7 ~TonsiI 0 .179 0 . 096 ~ -: D~131 2B2 ~ 0.191 ~ 0.000 ~ -31~ 2C10 ~ 0.202 ~ 0.000 +

6 . 4 . ~imal ~ ~od~ls ~ :
6.4.;1~ M~a~ureme~t ~of~ T cell~ Respo~qes 20~ 4.:1.~ uction ~o DT~ in ~ic~ to .~ ; Du~t~ M~it~ Extr~cts~
Dust mite~ extr~act ~(DMA)~ 10 to~200g mixed with 1:1 Y/V with ~comp~lete;~:Freundis ~adjuvan t ~CFA~ is injected sub~cutaneously into ~two sites in;~BALB/c mice. Con~rol mice~are~:~;injected~:~with FCA mixed~with Pss~;Or an irre~levan~p~o~ein.~ :Mice are:~hallenged S to 28 days later~ in~éction;o~DMA~(lOg) or:`purified~Der p I/II
preparations~gi~ven intradermally~into ~he~left: ear pinna using:~:the~procedures::described by Aus~in et al., :1991 (Austin ~et al., J. Natl. Cancer Inst. . 83 :1245-1248)~. T~e ~uninjected right ear ~serves as the control.
Twenty four ~and~: 48 hours after cha~llenge, the used with human-mouse heteromyeloma : : 3 5 Dust mite extract 3 ~ ELISA test:

:: :

: ~: :
: :

W093/l7703 PCT/US93/02082 thickness of the ears is measured. The differences between challenged and non-challenged ears is calculated and the measurements expressed as increment in ear swelling as a measure of the DTH response.
(Austin et al.1991, J. Nat.Cancer Inst 83:1245-1248, Kuriboyashi et al. l98?, Cell Immunol., 108: 366-377, , ; Morikawa et al.199l., Immunoloqy, 74: 146-152, To measure the effect of anti-dust mite Mab treatment, mice receive antibody at various times ~ lO~ before or after sensitization with DNA. Mab may be ; ~ administered by various routes and immunological ; adjuvants may be included in the preparation.
Suppression of~DTH responses against dust mites may be improved~by combination treatment with MAbs ~15 reacting with ~different~epitopes on the major allergic proteins such;as Der p I and Der p II. The general approach then~has been to identify MAbs singly or~in combination~which are effective for suppressing DTH~responses to individual Der p (~and Der f) 20~ proteins. Further~selection of~NUB~com~inations may also be~employed~for~suppression of DTH responses to mixed dust~mite~allergens such as those contained in arude extracts~-(DMA)~
A DTH response is~eli ited~in BALBJc m~ice 25;~sensitized~ with;`~DMA~(lO~g)~;and chal~lenged in the ear with~DMA ~5 days~ later.~ There is a significant (p<O.~Ql);~increase~in ear~swelling in DMA-sensitized mice eompared to~non sensitized~controls DTH responses are be similarly detected in BALB/c mice sensitized with DMA followed~ by ear challenge with Der p I
protein preparations~

6.4.1.2 8uppre~;~io~ of Der p I
induced DT~ Re~ponse in Mice Tr*ated with Anti-Der p Mab Mice sensltized with DMA~extract ~10 ~g) and challenged in the ear 28 days later elicit a delayed ~ ' .

W~93/17~03 2 1 3 1 8 ~ ~ PCT/US93/02~82 ; - 35 -type hypersensitivity response as assayed by ear swelling. This responæe is ~ignificantly reduced when mice are treated after sensitization with DMA with 10 to 50 ~g anti-Der p I Mab Hll (Figure 1). Prophylactic experiments show that treatment with Mab H11 following sensitization with DMA suppresses the development of DTH responses following ear challenge with Der p I
~lO~g). When DMA sensiti2ed mice are treated with H11 (3 x 20 ~g days 4, 7 and 14) and ear challenged, on 10 day 28 signif icantly reduced ear swelling is observed.
: A signif ica~t reduction in DTH responses to Mab Hll treatment is obtained using a range of Mab doses from 1 ~g to 1 mg given up to 5 times ~Tab~e 3). This response is further enhanced when Mab H11 is administered with aluminum hydroxide as adjuvant ~:~ (Alhydrogel 85; Superphos Biosector a/s Vedbaek, Denmark).

: 6~4~3: D~cre~Y~ i~ T ¢811 r~cti~ity by ; 20 i~ ~itxo p~ra~t~rY
: In addition, lymphocytes from these sensitized animals:are~isolated from l~mph nodes or spleen, and single cell~ suspensions are placed in culture and sti~ulated with either whole DMA or Der p I and Der p : , ; 25 :II. T cell ;aGtivation is measured either by incorporation~of radiolabelPd thymidine, or by ~: production of IL20 Those animals injected with ABl have significant decrease in either of these : activation markers as compared to sensitized animals ' 30 without this treatment.

:
~:

WO93/17703 PCT/US93/020$2 - 36 - s~

21318 9 8 TA~LE 3 Suppress1on of Delayed Typa Hyper ensitivity Responses to Der p 1 in Mice by treating with Anti-Der pl ~ntibodies Treatment regimen Reagent Do~e g Schedule Day~ Increa~e in ear swelling ~) following challenge with Der p X (lO~g) 4 -- 120 ~ 11 Mab H115 20 4,7,14 250 + 9 Mab 1986 20 4,7,14 98 + 14 : Mab H11 10 4,7,14 48 + 7 : ~ Mab H11 10 4,7,14 13 + 5 in Alum Mab ~11 5 4,7,14 22 ~ 8 in Alum ~ ~ab 198 10 4,7,14 92 + 12 : : in Alum 6.~.2 I~hi~iti~ of ~g~ a~ti-DMA
acti~ity 6.4.2.1 8CID ~ou~e ~ode1 Down-regula~tion of ~ntigen processing by : injection of anti-DMA;monoc1Onal antibodies c~n be assessed by use~of mice genetica~lly bread to 1ack an :immune system tsevere com~ined immune deficiency -S~ID mice. The~ animals do not have the capacity to be sensitized, but can mount an anemnestic response~
These an~ima1s~are repopulated with human i~munP
systems usiny~ either peripheral: blood mononuclear 30 c:ells, lymphocytes from *onsil , or a mixture of T
cell clones taken from disease specif ic organs such as : ~ : the lung in asthmatics, and mixed with peripheral ~:
:
4 With respect to DMA serlsitization (Day O) S CAallenge on Day 21 6 Anti Der pl Mab W093/17703 PCT/US93~02082 _ 37 ~ 21 31 ~9 8 blood mononuclear cells. Optimally these lymphocytes should be taken from atopic patients suffering from allergic reactions to the target antigen, such as dust mite, as manifest by antigen-specific IgE, skin test positivity to the antigen, and clinical symptoms indicating ongoing exposure. Alternati~ely subjects may be deliberately exposed to the allergen prior to ~; donation of the tissues.
The SCID mice are injected intravenously with l-10 X 106 and blood ;is drawn after l week and measured for total and~antigen ~specific IgMj IgG, and IgE, to verify engrafting.~ They are then injected with either DMA specific monoclonal antibody of an irrelevant monoclonal antibody of~ the same subclass, and ;challenged with antigen, lO-IOO ug without adjuvant l-3 days later. The antigen challenge is repeated again 2 weeks later,~and blood is drawn~weekly for up to 6 weeks therea~ter. ~

6.~ oD~l IIIOU~3 mo~e1 In a second~model, BALB/c or A/j mice are exposed to~;~DMA either~as the purified~antigen, or as the sèmipurified~extract.~The exposure~ can either be as an inj~eotion~with~and~ without adjuvant, in~a dose range of~20ug to 1 mg~;given as l-3~inje~tions l week apart,~or~as~the~`~aerosolized antigen, given in a nebulized form up~to l mg/day for lO days. Following - O exposure the IgG and IgE anti-DMA antibody response is deter~ined. ~
In~order~to determine if a~candidate MAb is able to down-regulate~the~response, the MAb is either given prior to sensitlzation, at a dose~;~from O.ol ug to lOO
ug, with or without~adjuvant, as multipl~ injections ranging from l-lOj up to l week prior to : ::: :

::~ ::~ :: : :

W093/17703 PCT/US93/~2082 - 38 - . .
2l3l89~
sensitization; or after sensitiza~ion with or without :~ adjuvant at periods:ranging from immediately after the last antigen exposure~up to 1 month after exposure. In some cases the latter group of animals may be rechallenged to investigate the effect of such treatment on the::secondary response. Results are ~ ; shown in Table 4.

: : TABLE 4 : Suppression::of:Der p I-Specific IgE Responses in Mice by Treatment:with Anti-~er p I

Schedule of Mab H11Serum IgE
Hll Treatment ~ : ~g Titer l5 ~ ;~ (days)~

4,7,14 ~ :: : 3::x 1023 + 7 :: :7,14,21~ :: 3~x 5~~3 * 11 7,~14,2~ 3 x~10~+ 12 l,2~ 3~x l0~.: 21:+ 4 In~these~expériments~ mice were~immunized 3 to 5 times at~monthly interYals~with Der~p I.~: Treatment;with Mab : : Hll was: conducted~7:~days after:final:treatment with :::: 30: Der p I'extract. Serum~assays for IgE titer were performed l4~:~days~iafter~final traatment with Mab H11.

6.4~.2-.3. Aero~ol-~e~iti~zed mou~e or rat~:model~ ~:
: In a third:model to measure the abi~lity of the ': MAb to:down-regulate~the response:either BALB/c or A/j :: mice or Brown~Norway rats are treated:with nebulized ~ . :
;: - ~ ; ~:: : :

WO93/17703 39 2 1 3 1 8 9 ~ PCTtUS93/02082 allergen for 30 minutes each week for 6 weeks, with molecules in the range of less than 1 uM, and tested for production of total and antigen specific IgG and ; IgE (McMenamin et. al.1992. Immunoloqy, 77: 592-596).
To test the ability of the candidate MAb to significantly down-regulate either of these antigen-specific classes, the MAb aan be given as in the example above, either before or after antigen :~ challenge, with or without adjuvant.~
: lO In both cases the IgE and IgG response is determined by the general methods set forth in section 6 below, except that the antigens`will be Der I or Der : II instead of Lol p~:~I. In~addition, animals are injected intradermally with l-lOO ug of antigen and the cutaneous response:is determined 30 minutes later as a:measure:of IgE response.

7. ; EXAMPLE: DOWN REG~LATION OF ~NTI80DIES TO RYE
: G~A5S A~L~RGÉN~
2~0;~ 7.1. Productio~ of:Murin- Nonoolon~l A~tibodie~
to~Ry~Gr~s~ (Lolium pere~ne, ~ol p I) Pollen~Allergen~
BALB/c~female~mice~are immunized on days 1, 28, and 90-~with lOO~ g o~ rye~:grass~allergen extract (US.
National Insti~utés:~of Health,~:Bethesda ~D) emulsified ::in~comp1ete Freund'~s~adjuvant in multiple subcutaneous sites::~and~:again;,~:~5~ days~prior to fusion, with lOO ~g of rye~:grass extract~`given intra~enously. Suspensions of`cells:from~the~spleen~are prepare~ and fused with : 3~ murine Sp2/o-Ag myelomaicells ~s described by Kohler and:Milstein;~(1975)~:~Nature;~256~:~495-497, US Patent : N~.4,376,llO).~ Clon~es:~are sél cted~and hybridoma cells maintained as in~seation 4.1. ThreQ were selected for further studies lTabl~e~5) " : :
~ 35 W093/17703 . PCT/US93/020$2 Char~teri~tio~ of ~urine a~ti-Lol p I Mab8 Hea~y chain Light chain kD
., 290A 167 IgG2b K 7.4 x 10-9 348A-6 IgG1 K 15.1 x 10-9 ~0 539A-6 IgG2a K 11.3 x 10-9 7.2 Charac~erization of ~urine Anti-~ol p I
~ybrido~a Coll Lia~s 7.2.1: Charact~ri~atio~ by re~ctivity with Lol p I
~5 The reactivity of specific monoclonal antibodies ; : is defined by immunobl~tting carried out by well established procedures (Towbin et al.,1979. Proc~. Nat.
; Acad. Sci. U.S,A. 72:262-269). Lol p I protein ~:~ : dissolved in 7.5 - lS% SDS-PAGE ~Sodium 20 ~ dodecylsulphate polyacrylamide gel~electrophoxesis) are~electrotransferred onto nitrocellulose paper.
Nitrocellulose paper~is cut into strips, ~aturated with bovine~s~erum a~lbumin solution and incubated with murine monoclonàl~antibody, followed by peroxidase--lab led mouse anti-IgG serum. Antigen-antibody compl~ex~s are~detected using 4-chloro-1-naphthol and hydro~en peroxide. ~ ~
As~an example of;this approach three anti-Lol p I
monoclonal ~ntibodies were reacted~ with extracts of :30 ~rye grass (Figure 3). These were obtained from the US. National Institutes of Health ~(l), and from commercial sources: Omega Montreal, Canada (2) and : Pharmacia, Pisca~away NJ, U.S.A. (3). The three monoclonal antibodies recognize the major component of 3~ the rye grass allergens ~Lol p I) with MW 34 kD~
: ~
The molecular:characteristics of the Lol p allergen~reacting with the monoclonal antibodies is WO 93/17703 ~ 41 - 2 1 3 1 8 9 ~3 PCI'/US93/02082 further defined by u~ing these antibodies to purify allergens from whole rye grass extracts (Mourad and Hebert~1986. Immunoloaiaal Investiqa~ion$ 15:801-811, 1986; Mourad et al., 1989. Mol. Immunoloav 26: 105~-1057). To accomplish this, monoclonal antibody isconjugated to Sepharose CL-4B (Pharmacia, Piscataway NJ) using a cyanogen bromide procedure (Cuatrecasa~, 1~70. J. Biol. Chem. 245:3059-3065). The solid phase antibody Sepharose CL-4B can then be used to purify ~0 allergen binding to the i~mobilized antibody (Mourad et al., 1989. Mol. Immunol. 26:~951-1057).
: In an example of this (Fig. 4) Lol p allergen purified on an immobilized monoclonal antibody 290A-167 contained a major product Mw 34 kD and a minor component Mw 23 kD when analyzed by SDS-PAGE (Mourad et al.,1986~ J. Immunolo~ical Methods 8g: 53-59).
, ~

` 7 ~ 2 0 2 ~h~ract~rizntio~ by Compariso~ of ~o~oclo~al A~tibody a~d ~um~n IgE
boay Roa~tivity with ~ol p I
The reacti~ity of monoclonal antibodies and human IgE with Lol p I allergen is determined using competition~assays (Mourad et al.1989. Molecular qy 26:1051-1057). In one approach, serum -2S :from~rye grass sens~itive subjects mixed with monoclonal antibody ;is added to immunoassay plates coa~ed with purified Lol p I allergen. After in~ùbation, unbound protein~is removed and ~25I-labeled anti-human IgE:monoclonal antibody added to detect bound human IgE. In an example of this assay (Table 6) monoclonal antibody 290A-167 produced up to 71%, and ~ 348A-6 up to 81.9~ inhibition of human IgE hinding.

:`:~ :: ~
., ~ 35 WO 93/17703 2 1 3 1 8 9 8 PCI'/US93/02082 ~ABLE 6 i~g ~hi~itio~ ~%) by ~ of i~di~idlu~l qerum Igl3 to polystyrQns-bouna I,ol p I
Mab added in competitiorlMab f irst f ixed to with human sera polystyrene-bound Lol p I
----Patient No. 290A-167 348A-6 539A-6 290A-167 348A-6 539A-6 . O ~
7I.1 B.7 81.~ 67.2 11.5 76.3 2 56.6 6.0 63.6 54.6 10.6 61.1 3 65.7 8.1 78.7 66.6 6.2 81.1 ~: ~5 ~ 77.4 11.6 83~ 65.0 11.~ 73.4 5 ~ 61 . 2 2 . 0 72 ~ 7 56 . 7 3 O 0 64 . 7 6 ~6204 8.6 74.3 59.4 12.1 78.3 7 4703 ~ 11.5 72.3 42"4 17.0 ~2.9 2 ~

8 ~ 35.9 6.4 56~8 2~.3 15.9 43.2 9 ~ 65 . 3 12 0 3 74 . 2 ND ND ND
62 . 3 3 . 8 81.1 ND . ND ND
2~5~ 69 . 5 I1. 7 84 . 9 ND ND ND
12 ~ ~63 0 5 5, 5 86 ~ 2 ND ~D ND

In another~assay murine monoclonal antibodie~ are ;, added to Lol p ~I coated immunoassay plates. Human serum containing IgE antibodies is then added and the inhibition~of bi:ndlng determined~. In the example : shown in Table:4, monoclonal antibody 290~-167 produced up to 67% inhibition and 539A-6 76%
inhibition of IgE binding.

~ .

,~ W093/177~3 43 2 1 3 1 ~ 9 8 PCT/US93,02082 7.2.3. Char~et~ri~ti¢ of rabbit a~
hu~ nti-idiotypio a~tibo~ies 7.2.3.1 ~rep~r~tion of rab~it a~ti-idiotypic ~tiboaie~
S F(ab')2 fragmen~-s of Lol p I-specific monoclonal antibody preparations are prepared (Mourad and Hebert, (1986) Immunoloqical Investigations 15:8010811).
Rabbit~ are immunized with F(ab')2 fragments of monoclonal antibodies, the first immunization being 1 `10 mg ~(ab' )2 emulsified in an equal volume of complete Freund's adjuvant (DifCo)~ Three booster immunizations ~: ~150 ~g F~ab')2 in incomplete Freund's adjuvant (Difco)] are given at weekly intervals and rabbits are ~led 14 days af~er the last injec~ion. Rabbit anti-idiotypic antibodies are:separated by passing serum dialysed against~PBS, pH8)~through a protein A-S~pharose column and eluting IgG fraction with lOmM
gly~ine-HCl, pH 2~8. Isolated rabbit Ig fractions are purified ~y absorption with unrelated mouse monoclonal 20 ~antibodies such as mouse~anti-ferritin antibodies, with rye L~l~p I~allergen extracts~: and by passage through non-specific human Ig-Sspharose CL-4B~columns (N~our~ad et al:., 1988. Immunoloqy 63:397-401j.

2~5~ :7.2.3.~: Pro~u~tio~ of hu~a~
iot~ic~ aIlti~3ai~
Serum~samples ~rom~ry~ grass sensitive sub~ects including those:~undergoing specific immunotherapy with rye grass extra ts~are absorbed twi~e with rye Lol p I
allergen preparations~coupled t~o Sepharose CL-4B to remove any anti-allergen antibody. They are then : absorbed with norma~l murine IgG-Sepharose CL-4B to remove naturally occurring anti-mouse antibodies (Mourad et al.1~88. Mol. Immunol. 2:5:899-906). In each case, one volume of absorbent is mixed with one ~:~ volume of serum and incubated overnight at 4 C. Final products:containing human anti-idiotypic antibodies WO93/17703 PCT/US93/~2082 - 44 - .
21 ~1 8.~8 are tested by enzyme linked immunoab~orbent assay (Mourad et alO1986. J. Immunol. ~ethods 89:53-S9) to confirm absence of anti-rye Lol p I anti~ody.

7.~.3.3 I~hibitio~ of ~in~i~g of a~ti ~ol p I M~b to r~bbit ~ti-i~iotypic ~ibody The specificity of rabbit anti-idiotypic . antibodies to murine anti rye Lol p I monoclonal antibodies is determined by the capacity of unlabeled mo~oclonal antibody to inhibit the binding of l2sI
labeled murine monoclonal anti Lol p I monoclonal : antibody to rabbit idiotypic antibody coated onto polystyrene coated micro plates . Using this procedure described fully in Mourad et al.1988.
Immunol 63:397-401, unlabeled monoclonal antibody inhibi~s binding of its relevant l25I labeled monoclonal antibody. ~o inhibition is ob~ained with unrelated monoclonal antibody. This is illus rated in ~igure ~
which shows as an~example, inhi~ition of binding of l25I
; monoclonal antibodies:290A 167, 348A-6 and 539A-6 by unlabeled preparations but not by anti-ferritin monoclonal antibodies.
In a ~econd approach the ability of anti- .
idiotypic antibodies t~ inhibit binding of~2sI labeled mu~r:ine ànti-Lol~p~ monoclonal antibody to~Lol p I is deter~ined by~solid~phase radioimmunoassay (Mourad ~ :et al., (1988) Immunolo~y:63:397-401). Using ~his : assay anti idiotypic an~ibody is shown to block : binding of the homologous anti-rye Lol p I monoclonal ~ : antibody. No inhibition is obtained with unrelated :~ anti-idiotypic antibody (Figure 5:).

: ~ :
~ 3s :
: :

21~t898 WOg3~17703 PC~/US93/02082 . - 45 -7~2.3.~ A~ti-i~iotypic ~tibody i~hibitio~ of bi~di~g of ~I
~ol p I to a~ti-Lol p I
~o~o~lo~ ntibo~y In order to verify that the idiotypic determinant recognized by anti-ldiotypic antibody is associated with the combining site on the anti-rye Lol p I
monoclonal antibody, tests are carried out to show that the anti-idiotypic antibody inhibits binding to 5I labeled rye Lol p I. Polystyrene removawell strips (Dynatech, Chan~illy VI) are coated with anti-rye Lol p I monoclonal antibody followed by saturation with BSA. After:washing plates, rye Lol p I 1~I labeled i5 added alone or in the presence of anti-idiotypic antibody . After overnight incubation the wells are washed and bound l2sI determined. In an example of the approach (Fig. 6), binding of~l~I rye Lol p I to monoclonal antibodies 290A-1~7,348A-6 and 539A-6 is : ~ ~ inhibited by the~homo1Ogous anti-idiotypic antihodies.
:
7~2~3.5. I~iotypia Cro~ ~R~aeti~it~
B~eQn ~um~ a~ Muri~
: Anti-Lol p I ~tibo~
The rel~tionship between anti-idiotypic antibodies to murine monoclonal ~nti-rye Lol p I and human IgE~antibodies:in human serum from rye gra~s allergi~ individuals is determined:by asses ment of nhibit}~on of TgE:binding to rye Lol p I~(~ourad et : ~1., (19883 ~m~Yne~ggy 63:397 40l3. Polystyrene ~ removawell strips are coated with rye Lol p I (200 ng/well) and saturated with 3% BSA. Plates are incubated overnight~ with human serum 1/4 v/v3 alone or : in the presence of rabblt anti-idiotypic antibodi s to ~: murine monoclonal:antibodies (290A-167, 348A-6 and : 539A-6). After washing, human IgE bound to Lol p I is detected using 1~I labeled anti-human IgE monoclonal antibody 7H8. In an example of this assay (Figuxe 7), WO93/17703 . : PCT/US93/02082 2131898 - 46 ~

anti-idiotypic antibodies to 290A-167 and 348A-6 produc~d significant inhibition of IgE reaction with ~ol p I.

S 7~2.~ Det~ctio~ of huma~
~uto-a~ti i~iotypic ~ibodi~s by ~r~ct bi~ing to muri~e ~nti-~ol p I Hab Sera from human subjects receiving immunotherapy with rye grass extracts are absorbed (2 times) with : I normal murine IgG coupled to Sepharo~e CL4B to remove : naturally occurring anti-mouse antibodies. They are ~lso a~sorbed (2 times) with rye Lol p I (Mourad et al., ~1988) Molecular Immunoloqy 25:899-906; Hebert et al., (1990) Clin. Exp. Immunol. 80: 413-~19).
~ ~5 These serum preparations are then used to test for the : inhibition of binding of murine monoclonal anti-rye Lol p I antibody to rye Lol p I. In this assay polystyrene re~ovawall strips (Dynatech) are ~oated with rye Lol p I. ~sI labeled murine anti-rye Lol p I
: monoclonal antibody either alone or mixed with various con~entrations of human:anti-idiotypic antibody preparation are added:~and incubated for 4 hours. After incubation,~wells~are washed and radioactivity ~ aounted. In an example of this assay (Figure 8~, anti-: : : idio~ypic antibodies~from *hree:subjects are shown to nhibit murine monoclonal antibo~y 290A-167, 348A-6 and 539A-6 binding to Lol p I. Human anti-idiotypic antibody reactivity with murine anti-rye Lol p I
:~ : mono lonal antibodies i5 further confirmed by direct r~dio-immunoassay.~Polystyrene removawell strips (Dynatech) are coated with anti-rye Lol p I monoclonal ~:: antibodies (lOo ~g/well) and saturated with 3% BSA.
~: After washingr human anti-idiotypic antibody is added and incubated for 3 hours. After washing bound human : Ig is-detected by addition of 125I labeled goat anti-human Ig antibody (Table 7).
:

. WO93/17703 47 213 ~ ~ 9 ~ PCT/US93/02082 ~ABLB 7 I~iotop~s ~harea by hum~n a~d murine ~tibodies Binding of human auto anti-ID antibodies* to NAbs Mab 3E3 290Aol67 348A-6 539A-6 _, . .
Binding (~P~) 458 48,972 36,240 68,18~

* Pooled human sera from rye sensitiv~ patients and under specific immunother~py after depletion as describedO

7.3~ I~h~bi~ion of IgG and IgE r~spon~e~ in mice following ~timulation of anti-idiotypic ~nti~odis~
, 7.3.1 I~duotion of anti-iaiotypic tibodi~s to~uri~e ~nti-Lol p I
: ` mo~oclonal ~nti Oay 290 ice are injected intraperitoneally with M~b 290 (0.~01 ug to 10:ug)~ in complete Freunds adjuvant ~nd 20~ one week later with MAb 290 in incomplete adjuvant (Figure 9~. Further inj:ections of MAb 290 are then administered weekly:without adjuvant for a total of 7 : :weeks.~
In~order~to détec~anti-idiotypic antibodies ~f M~b 290:::an Fab fractions of the~MAb;is prepared by papa:in digestlon (Mecheri et. al., 1988. Immunology 64 15~. The~F~b f~raction:is coated onto microliter plate weIls ~I00 ng/well) in carbonate buffer pH 9.6.
Wells are then blocked with BSA in~ris-buffered saline and washed.~ serum samples from M~b 2~0 : immunized mice ar~e then added in dilutions ranging : from 1:50 TO 1:5000. After incubation and washing, ound anti-idiotypic antibody is detected in an ELISA
assay using anti-mouse Fc specific IgG labeled with alkaline phosphatase (Zymed San Francisco, CA).
:` : :

W093/17703 2 1 3 i 8 9 8 - 48 - PCT/US93/02,0~82 In an example of these studies ( Fig lo) anti-idiotypic antibodies to MAb 290 were detected within 4 weeks of immunization with MAb 290 in Freund's : complete/incomplete adjuvant~ In controls, mice S immunized with an unrelated MAb (Anti-ferritin~ did not produce anti-idiotypic antibodies to MAb 290.
:: Further weekly injections of MAb 290 without adjuvant : markedly increases the level of anti-idiotypic antibodies (Figure 10) Dose-response~studies~summarized in Figure 11 indicate that MAb at doses as low as 0.1 ug induced anti-idiotypic:~antibodies with 1-lO ug being an ~:: : :::appropriate dose.:

~ : 7.3.2 8erum ~nti-Lol p I IgE ~nd IgG
ntibody r~sponse Mice treated~w~th~8 doses of MAb 290 or control ~b were i~munized~with Lol p I in:alum given over 4 doses (Figure 8)~ Sera from~these~mice were then ;2~0 àssayed for the~production::~ of~: anti-Lol p I IgE and IgG
;;antibodies. ~In these:tests, it`was necessary to deplete sera:;~fr.om~any:residual anti-idiotypic antibody since~this:~interferes;with~anti-Lol~p I antibody assays.~Accord;ingly~:~sera were inltially adsorbed by ~:treatment~with~M~b~290~cross-linked to Sepharose 4B.
Th:is:~imm~noahsorbant was~prepared~by reacting MAb 290 with~cyanogen~bromide~activated~Sepharose 4B (3 mg MAb : 290~/ml gel).~ Each:serum sample~:wa~s absorbed with Mab 290-Sepharose 4B~`~(:50 ul serum/50 ul gelj. Under these conditions the~!: sera were depleted of any anti-idiotypic antibody~to MA~ 290. ~
: Anti-Lol p~ IgE antibody:in these sera was determined:by:ELISA assay.~ As shown in Figure 12 : mice treated with~:MAb~290 producing anti-idiotypic 3S antibody only produced a weak IgE:response to Lol p I.
In comparison, control mice~treated with an unrelated :

WO93/17703 21 31 8 9 ~ PCT/U~93/02082 MAb (13-A2) and immunized with ~ol p I produced a strong IgE antibody response.
Serum anti-Lol p ~ IgG antibody levels as determined by ELISA assay were also suppressed in mice S initially treated with MAb 290 so as to generate anti-idiotypic antibodies (Figure 13).

7.3.3 ~sti~tion of IgE ~tibodies by P~ive Cutan~ous A~sphylaxis : 10 IgE antibodies were also estimated using a passive cutaneous anaphylaxis test. In this test, Sprague Dawley rats are injected intradermally with serum :(100 ulj diluted from 1/40 to 1/5120. Forty eight hours later mice are injected intravenously with Lol p I (1 mg) in 1% Evans blue solution (1 ml~, and the cutaneous anaphylaxis wheal measured. As shown in Table 8, *he PCA titer from~mic~ treated with MAb 290 ; was significantly lower than that in control mice treated with MAb 13A-12.

T~BLE 8 ; ; P~A titer upon pretre~t~ent of mice with Id (2~9~0A-167) of;~o~trol NAb 25 ~ PCA titer ~ , Waek ~ ~ Id pretreated~ : Control MAb-;pret~eated : 8 ~ : ~40 : ~4~
<40 <40 12 I80. 320 ~: 30 14 32~ 512~

7 . 3 . 4 . Vse of ~n EhI8A Plaque A~3ay to D~tect 8pot FormiIlg Cell~
Spleen cells se~reting anti-Lol p I IgG
~ 3S antibodies were detected by an ELISA plaque assay : (Table 9). In this:test (laboratory Methods in ~ Immunology Volume I p. 104-108) round well tissue ~:

WO93/17703 2 i 3 1 8 ~ 8 50 PCT/US93/02~82 culture plates are coated with Lol p I in carbonate buf~er pH .6 and then blocked with BSA solution.
After washing spleen cells ranging from 20-150 nucleated cells/well were added. Plates are ~ncubated o~ernight at 37C in S% C02/95% air, washed, and then biotinylated anti-mouse Ig antibody l/2000 (Zymed) added. After incubation (2 hours) alkaline `~ phosphatase conjugated streptaviden (BRC) was added.
~ The plates are then washedj developed with 5-BCIP
;~ lO substrate and antibody forming cells (blue spots) counted. The number~of antibody forming cells in spleen cells from mice pretreated with MAb 290 was ; significantly reduced compared to spleen cells from mice treated with control MAb.

TAB~E 9 ~u~ber of ~nti-Lol p~l secreti~g cells following ; pretre~tm-nt ~itb~Id Spot-forming cells (SFC)/106 spleen cells Id-pretreated ;~; ~ control MAb-pretreated 3.75 *~l.5 ~ 2~3 + 2.~

7.4~Protocol for~de~en~itlzation of grass 2~5 ~ l}-rg~ic~ ubject~ to ~ye gr~ llergy Desensitization optimally is started before the onset of the~allergy season.~ This will vary depending ~` on the location and~climate. Thus for example, in locations in which symptoms began in May and October, desensitization would begin in the 2 month period prior to those two seasons. Subjects are those people who clinically suffer from allergy to grasses as : , demonstrated by ~symptoms, and who have a positive skin test to Rye grass ~or one of the other cross reacting grasses. They are immunized with Mab/CDR peptides, administered singly or as a cocktail of 1-20 different W~93/17703 PCT/US93/02082 Mab/CDR peptides. The vaccine is administered either as soluble protein or as protein admixed on aluminum hydroxide gel ~such as Alhydrogel 85, Vedbaek Denmark.
The protein content of ~ach injection ranges between l ~g to l0 mg of protein. Injections are repeated weekly to monthly for up to 6 months, given ~ubcutaneously (if mixed with alum) or intradermally or subcutaneously without alum. Other adjuvants besides alum also may be used.
Subjects demonstrate significant reduction of symptoms as compared to controls. Symptoms which decrease include those of allergic rhinitis such as sneezing, indicated by a decrease in frequency of sneeæes, and in requirement for medica~ions.
lS Laboratory parameters include reduction in nasal air flow resistance both spontaneously and after challenge with allergen. Asthma sufferers show a decrease in fxequency of episodes of breathlessness, decrease in reguirement for medication, decrease in number of yearly episodes~ requiring medication, and improvement : in pulmonary function testO Additionally, the amount of allergen necessary to provoke a pos}tive skin test is in~reased,~and the area of~the wheal and flare reaction at fixed~dose~ of antigen is decreased.
:25 ~ All specific:~examples and procedur~2s described herein are provided to exemplify the invention, and not ~or limit~tion:. :

8. E~CANE'LB: tJ8E OF ANTI--IIRIJ~3HIOL ~ONOC:IONAL
30 ~ ~TIBODIE8 ~ABl) TO DOWN ~E:GnLATE q!HE T CELL
RE8PON8~5 TO POI60N OAR AI~D IVY ~I,~ERGY
: Poison oak/ivy allergy is a delayed type : hypersensitivity (DTH) response to allergens ~ ~(urushiol~ in the oil of the plants. The natural ::: 35 allergen is a mixture of 3-n-alkylcatechols with a Cl5 or Cli side chain either fully saturated (e.g. 3-n-pentadecylcatechol, PDC) or having l to 3 unsaturated WO93t17703 PCT/US93/02082 2131898 .","
bonds (Figure 14) ~Symes and Daw~on, 1~54. J~_~nn Chem. Soc. 76, 295~-2963). In the initiation of an allergic response the allergen first undergoes quinone formation. The quinone then undergoes reaction with cell proteins through Sulfhydryl or amino groups and these product initiate the allergic response.

8.1 Prep~r~tio~ of 3 ~-Alkylcat~chol Conjug~t~
for U3~ a~ Im~u~ogens Urushiol and related 3-n-alkylcatechols pxoduce T
Gell responses; antibodies have never before been : demonstrated. Thus to generate anti-uru~hiol antibodies, it has been necessary to synthesize analogs capable of stimulating B cell responses.
These have been synthesized by several routes, a preferred pathway being reaction of quinones of :~ urushiol and PDC with proteins, peptides and,amino acids.

: 8.1.1 R~actio~ of auto-o~i~ uru~hio~
~; . to prot~s ~: ~ The con~ugation procedure is based upon that described by Liberato et. al.1981 ( Med Chem. 24:28-33). Urushiol, 1.56 uM dissolved in 1 ml acetone is placed into a conical flask and dried onto the inner ~: surface under a stream of nitrogen. Protein solution : ~serum albumen 100 mg in 10 phosphate buffered saline `:
pH 7, or keyhole limpet hemocyanin in sodium hydrogen carbonate buffer pH 8~4~ is added. Alternatively, urushiol may ~be added drop-wise e.g. 0.2 ml urushiol added to KLH solution 20 mg in 2 ml of buffer pH 8.4.
The heterogeneous mixture is stirred open to air at room temperature for up to 48 hours. During this time the urushiol undergoes o-quinone formation and protein coupling to yield a red-brown colored solution. The solution is chromatographed (e.g. on Sephadex G25) to sPparate the protein conjugate. In a typical example, ~r~ 93/1 7703 2 1 3 1 ~ 9 8 PCT/US93/02082 the absorbance (4B0 nM) of the albumin solution increa~ed from zero to 0.59. The final solution is sterilized and stored at 4C~
801.2 Co~jugatio~ of Alkylc~t~hol to Pxot~i~ through it~ n A~etyl Cy~tei~e I~terme~iat0 to Co~mo~
2rotei~ C~rriers Quinone derivatives of urushiol and 3-n-pentadecylcatechol (PDC) are prepared by oxidation of urushiol or PDC with silver oxi~e using procedures described by Liberato et al. ibid. to yield N-acetyl-S
(2t3)-dihydroxy-4-pentadecylphenyl cyst~ine (PDC-n-acetyl cysteine). The purity of the product was as essed ~y thin layer chromatography, elemental analysis and NMR spectroscopy to conform with published data ~ibid ) . The quinone derivative of urushiol is similarly reacted with N-acetyl cysteine.

8.1.3 ~o~jugatio~ o~ 3-~-~: p~t~cyl~t~hol (PDC) to ~:~ 20 Polya~parti~ ~cia ~ : Conjugation of PDC to polyaspartic acid i5 ; ~ effected through a carbamide complex. PDC ~10 mg) was dissolved in dioxase:(1 ml3. Dicyclohexylcarbodiimide ~DCC: 2 mg) was added~ and reacted for I8 hours at 4 C~ The omplex was then added to acid ~S mg; 41.6 uM) ~: : in phosphate buffered saline pH5 and after 30 minu~es the conjugate isolated by Sephadex G25 chromatography.
Spe~troscopy analysis (277 nm~ indicated the mean substitution ratios was 42.8 u mol/mg.

B . 2 Proauction o~ Anti~ru~hiol ~onoclo~al : ~tibGdies and TC~'~
Methods for producing murine monoclonal - antibodies have been described ~Section 5). BALBtc mice were immunized on day 0 with PDC - N
acetylcysteine (SU ug) subcutaneously in Freunds' complete adjuvant followed by an intravenous boost WO93/17703 2 ~3 1 ~9~ - 54 ~ PCT/U~g3/0~.82 dose of PDC-N acetyl cysteine on day 7. Spleen cells were harvested on day ll and fused with my~loma P3 NSO
cells. Hybridoma clones are seIected as described in Section 4.
Anti-urushiol producing monoclonal antibodies ar~
identifi~d by ELISA assays. Briefly, polyvinyl microliter plates (Falcon) are coated with urushiol -or PDC-conjugates such as PDC and urushiol - N
~: acetylcysteine and PDC~polyaspartate. Hybridoma ~ ~0 supernatants are addedj incubated 45 minutes, washed : and bound with mo~se immunoglobulin deleted with horse radish peroxidase conjugated rabbit anti-mouse immunoglobulin. This methodology yielded MAb 991/81/7, of the IgM isotype from a mouse immunized with PDC N
aaetyl cysteine.: MAb 991 is purified from hybridoma culture supernatants by ammonium sulfate precipitation and fractionation by;Sephacryl S300 gel filtration chromatography.
., .. ; ~ , 8.3. I~munor~ctivity of Mab 9gl The i~munoreactivity of ~ b 991 is initially determined by its :binding in ELISA assay to u~ushiol and PDC conjugates~but~not to carrier molecules. The spe~ificity of MAb 9gl~is~further defined b~. the 25~:inhibition of its:binding to urea-N-acetyl cysteine by compounds~rela~ed~to 3 N-àlkylcatechols. This is illustrated~in Fi~re 15 which summarizes inhibition : of:MAb:991 t~ ure~-N-acetyl cysteine. Thus compounds :
such as catechol and catechin hydrate with features of : 30 the catechol moiety inhibits MAb 991 binding. From these experiments~MAb 991 is ~escribed as reacting with thé catechol structure (Figure 15).

~ .4 Dow~-r~gulation of Delayed Type :~ 35~ypersen~iti~ity Re~pon~es to ~rushiol The use ~of anti-urushiol monoclonal antihodies to suppress allergic responses to urushiol has been ~::
, WOg3/17703 2 I 31 ~ 9 ~CT/US~3/02082 evaluated using a murine model (Dunn et~ al., 1982.
Cell Immunol. 68, 377-388~. BA~B/c mice are sen~itized by application of urushiol or PD (2 - 4 mg) to the abdomen in acetone (100 ul). Sensitization to the allergen is then detected ~rom day 4 up to day 42 by application of urushiol or PDC ~50 ug in lOul acetone) to the dorsal side of one ear. The other ear is treated with acetone only (10 ul). ~ar thickness is then det~cted using a sensitive pressure micrometer ~0 (Mitutoya, Japan) and the difference between allergen-challenged and control ears determined.
In a prophylactic approach (Figure 16) mice are : treated by a single or multiple intravenous injections of MAb using a range of doses (1 to 25 ug). Mice are ~ lS then sensitized to the allergen and challenged on the :~; ear. As an example of this approach, MAb 991 ~: treatment (3 times, lO ug) suppressed to DTH response to urushiol compared to control mice receiving an unrelated IgM MAb (B55: anti breast:cancer).
~ In therapeutic~tests MAb is given after sensitization ~o urushiol or PDC. This is illustrated in Figure:17 where urushiol sensitized mice received MAb 9:9I~as a single::dose (10 ugj intravenously 21 days after sensitization. :When ear challenged on day 42 r50~ ug urushiol~, the~DTH response was ~ignificantly redu~ed.
MAb~in adju~ant are even more effective than MAb alone. :This is~illustrated by experiments where urushiojl sensitized mice were treated on day 7 and 14 :30 with MAb 991 (1 ug) in complete and then incomplete Freunds' adjuvant. ~When ear challenged on day 21 the DTH response was reduced by almost 80 per cent ~: compared to that~in control mice receiving control MAb (Figure 18).
:~ 35 : :

WO93/17703 - 56 - PCT/US93/O~Q82 2~318~
8~5 Pro~uctio~ of Anti~Uru~iol T Cell Re~eptors ~or ~8e A~ u~oge~
Mice are vaccinated against haptens ~oxazolone, DNFB), including urushiol, by immunization with T cells derived from lymph nodes of hapten sensitiæed : donors. Mice are vaccinated by immunization with urushiol specific T cells. These may be cloned ~:T cells, T cell hybridomas, or peptides mimickiny the TCR of the specific T cells.

8 0 5 . 1 Pro~uctio~ of urushiol ~pecific T c~ll line~ from ~e~itized mi~e BAL~c mice are sensitized:following application of urushiol ~1-5 mg) in acetone onto the dorsal skin.
T cells from sPnsitized mi~e are obtained from several sources including lymph nodes or urushiol-induced cutaneous lesions~and expanded in culture by in Yitro stimulation with~urushiol~or urushiol-polymer coniugates using:stimulation procedures well known to 20~ those skilled;in the art.~ ~Urushiol specific T :cell clones~or T cell hybridomas are obtained:~y fusion of T cells wi~h murine thym~oma cells (BW 5147 cells as :described by Bi~gby, M.,~: t:al.,1989. J. Immunol.
43:3867-3872~ hese~cells~produce immune respon~es to urushiol specif~ic TCR. ~:Such cells have utility as ; T:cell vaccines~which may be one of several types including ~iable~or a~tenuated~T~:cells and T cells modified ~y cross~ linking~with agents such as glutaraldehyde ~(Cohen and Weiner,1988. Immunol. Today : 30 9:33~335). The TCR~of the urushiol responding-T cell : clone is~ identified~by reaction~:with monoclonal antibodîes which detect variable segments of the TCR
(Kappler et al.,:1988. Nature 332:35-40).
~ In another approach, messenger RNA or DNA from `~ 35 cloned T cells~reacting wîth urushîol are probed eîther directly or after amplîfication hy polymerase ~: chain reaction by specific hybridization wîth nucleic , ~
, ~ .

WO93/17703 _ 57 _ 2131~9~ PCT/US93J02082 acid probes for TCR gene families using hybridiz~tion methods well known to those skilled in the art. The TCR sequence (in part) is obtained from the amplified DNA or RNA. Regions of TCR amino acid sequences are selected on the basis of their immunogenic or antigenic properties identified using algorithms to predict ~ cell helper/cytotoxic determinants (Rothbard.and Taylor, 1988. EMBO 7:93-l00). For example, peptides containing 14 or 15 amino acids are predicted to contain specific TCR sites for urushiol.
Peptides of selected amino acid composition are then synthesized using standard procedures well known to those skilled in the art. Synthetic peptides are then used to vaccinate animals.
: : 15 : :
, ~
8.5.2 TCR pro~uced from imcune hum~n o~or~
T lymphocyte clones are established from ;
peripheral~blood mononuclear cells (PBMC) or contact d~rmatitis lesions derived from urushiol sen itized human subjects by~methods generally described by Kalish and others. (Kalish and Morimoto,l988. J.
Clin. Invest.~82~:825-~832; 1989. J.~Invest. Dermatol.
92:46-52).~ P8MC~isolated from venous blood by Ficoll-25 ~Hypaque separation are cultured in IL-2 containing média~and sensitized~or expanded by inducing prolifera~ion~in~vi~tro by the addition of urushiol in dimethylsulfoxide~or on autologous PBMC (Byers, V., et al.,1979. J._Clin. Invest.64:l437-l448). Other growth factors such as~IL-4 may be used (Paliard, et al., l989. J. Immunol. 8l:,452-457). Follow~ng culture for : ~
a period of time to establish satisfactory cell growth, cells are cloned by limiting dilution using standard cell culture techniques. Cells are then grown and positive wells expanded. This phase may include further stimulation o~ cells with urushiol and autologous irradiatéd (5000R) PBMC as feeder cells. T

.
.

WO93/17703 PCT/US93/02~2 5~
~ 31 898 cell clones are then established by standard techniques. Alternatively, T cell hybridomas may be employed as a source of TCRs. The T cell vaccine may be composed of one or more formulations including viable or attenuated T cells and T cells modified by cross linking agents, e.g. glutaraldehyde (Cohen, supra). Ideally, T cell vaccines should be of autologous origin since responses are generally restricted by the MH component of the TCR (Schwartz, R., l985. nn. Rev. Immunol. 3~237-261). Since broad NHC restriction is, however, involved in the recognition of certain haptens and antigens (Panina-Bordrgnon, et al., 1989. Eur. J. Immunol.19 2~37 2242). An alternative apprvach is to administer vaccines composed of pooled preparations of cloned T
cells derived from a~number of allogeneic donors.
In another approach,~ specific antigen binding regions of the TCR may be identified, and the peptide comprising that region~synthesized as described previously for~ use as an immunogen.

9 ,. 152~ lPLE.gtlPPRE:8SION OF; T~E IgG a~TIBODY :~E8PONSE
TO AN I~XOGENOlU8 ~IGE~o BICIN A CHAIN I~RTA~ BY
NOC!LONAI~ R~A ANT:3:BODIES
9~1 Pro~u~t~io~ of AbI to~R~
BALB/~ mice;received a single intravenous :
injection of an immunotoxin composed of the murine monoclonal antibody s791Tl36,con~ugated by a disulfide bond to RTA. Significant titers of anti-RTA
antibody were noted 7 days after injection, and continued to~ increase by day 30.
Monoclonal antibodies were generated against RTA
using splenocytes from BALB/c mice immunized with the immunotoxin 791T/36-RTA, fused with the myeloma cell line P3NS1, and cloned and characterized as previously described. Five MAbs were selected for further study.
Three (596/134; 596/192; and 608/7) reac~ed equally WO93/17703 PCT/US93/02~82 2131~98 well with native RTA, and with recombinant RTA ~rA~
and with deglycosylated RTA (dgRTA) produced by -periodate cxidation of R~A (Thorpe et. al. 1985, Eur.
J. Biochem. 14?: 197-206). These results indicate that the anti-RTA MAb react with RTA polypeptide and not oligosaccharide structures.

9.2 Down regul~tion of a~ti-RTA re~pon~e IgG
with Abl BALB/c mice were immunized with immunotoxin 791T/36-RTA, and then 24 hours later were treated either with a single~injection of 100 ~g given intravenously of the anti-RTA MAbs, or with a control Mab, Mab 365,~directed against Carcinoembryonic antigen, which does not~cross react with RTA. Animals were bled;14 days~after~initial antiqen injection and the~IgG anti-RTA serum titers were measured by ELISA, and~suppression~was~compared with tha~t of the control anima~ls treated~with the anti-CEA antibody. Three of the~five MAbs, 608/7,~596/134 and 596/192 2 ~ significantly suppréss~ed~the ant~i-RTA response (Figure 20~ Thé~effect~was~most pronounced with MAbs 608/7 and~596/134,~producing 71 and 73~ i~nhibition respectively.~ T}tration of the response with Mab 608/7~(Figure~21~)~demonstrated that~a~ single dose of ; 25 ~ antibody~ 25-50 ~g~ resulted~in~40-60% reduction~in anti-RTA antibody~ti~er.
reatment~with~anti-RTA~Mab~608/7 between 2 and 24 hours following ~immunotoxin treatment produced signifiaant inhibition (p less than 0.001) of anti-RTA
antibody;production~[Figure 22).; ~In other experiments,~(;Figure 23),~Mab 608/7~was also effective when aadministered~up to 7 days~efore immunotoxin treatme~t, and~praduced~very~effective inhibition of anti-RTA respanses.~
35~ Immunotoxlns are rapidly removed from the ~ circulation, with a T 1/2 of about 20 minutes. This :: ; : `
:

WO93Jl7703 2 1 3 1 8 9 8 - 60 - PCT/US93/020~

~hort half life is due to the mannose residues on the RTA~ which bind to receptors on the Kuppfer cells of the liver. Since antibody is ~till capable of down-regulating the IgG anti-RTA response when given as late as 3 days after antigen exposure, it cannot simply be preventing the antigen from reaching the antigen processing cells, since none remains in the clrculation. The evidence therefore is that the Mab ; is inducing tolerance by an effect on antigen lQ processing.

:: ~ 15 :

: :

: ~ :

~: 35

Claims (30)

WHAT IS CLAIMED IS:

1. A method for suppression of an allergic response to an exogenous allergen, comprising administering to an animal a noncellular immunosuppressive composition comprising an isolated and purified antibody-derived or TCR-derived molecule which is capable of binding selectively in vitro to an allergenic epitope of said allergen, said composition being substantially free of said allergen, thereby selectively suppressing the allergic response of said animal to said allergen.

2. The method of Claim 1 wherein said allergic response comprises production of IgE antibodies against said allergen.

3. The method of Claim 2 wherein said allergic response comprises asthma.

4. The method of Claim 2 wherein said allergic response comprises allergic rhinitis.

5. The method of Claim 2 wherein said allergic response comprises conjunctival inflammation.

6. The method of Claim 1 wherein said allergic response comprises atopic dermatitis.

7. The method of Claim 1 wherein said allergenic epitope comprises an immunodominant epitope.

8. The method of Claim 1 wherein said animal is sensitized to said exogenous allergen by airborne exposure.

9. The method of Claim 1 wherein said exogenous allergen is a tree or broadleaf weed pollen, a mold spore, or a dust mite antigen.

10. The method of Claim 1 wherein said exogenous allergen is a food protein or a drug.

11. The method of Claim 1 wherein said exogenous allergen is an industrial chemical which causes contact sensitization.

12. The method of Claim 1 wherein said animal has been sensitized to said exogenous allergen prior to administration of said immunosuppressive composition.

13. The method of Claim 1 wherein said immunosuppressive composition is substantially free of Ab2s to said allergen.

14. The method of Claim 1 wherein said immunosuppressive composition is substantially free of antibodies other than Ab1s against said allergen.

15. The method of Claim 1 wherein said immunosuppressive composition comprises at least two Ab1s against different epitopes of said allergen.

16. A method for obtaining an immunosuppressive composition containing an antibody-derived molecule which binds selectively to an exogenous allergen, comprising the steps of:
a) selection of a monoclonal antibody which recognizes an immunodominant epitope on said allergen; and either b) demonstration that the selected monoclonal antibody stimulates an anti-idiotypic antibody which blocks binding of said monoclonal antibody to said allergen, and demonstration that an anti-idiotype antibody which block-s binding of said monoclonal antibody to said allergen is present in a patient receiving antigen immunotherapy with said allergen; or c) demonstration that the selected antibody binds to an anti-idiotypic antibody induced in a patient receiving antigen immunotherapy with said allergen.

17. An immunosuppressive composition comprising an antibody-derived molecule obtained by the method of Claim 16 in a pharmaceutically acceptable carrier.

18. A noncellular composition for suppression of an exogenous allergic response to an allergen, comprising an isolated and purified antibody-derived or TCR-derived molecule which is capable of binding selectively in vitro to an allergenic epitope of said allergen, in a pharmaceutically acceptable injectable solution, said composition being substantially free of said allergen, said composition selectively suppressing the allergic response to said allergen when said composition is administered to said animal.

19. The composition of Claim 18 wherein said antibody-derived molecule is an antibody fragment.

20. The composition of Claim 18 wherein said antibody-derived molecule lacks an Fc domain.

21. The composition of Claim 18 wherein said composition is substantially free of antibodies other than Ab1s against said allergen.

22. The composition of Claim 18 wherein said antibody-derived molecule is a monoclonal antibody.

23. The composition of Claim 18 wherein said exogenous allergen is a food protein or a drug.

24. The composition of Claim 18 wherein said exogenous allergen is a tree or broadleaf weed pollen, a mold spore, an animal dander or a dust mite antigen.

25. The composition of Claim 24 wherein said antibody-derived or TCR-derived molecule recognizes an immunodominant epitope on cat Fel I antigen.

26. The composition of Claim 24 wherein said antibody-derived or TCR-derived molecule recognizes an immunodominant epitope on alternaria mold spore.

27. The composition of Claim 24 wherein said antibody-derived or TCR-derived molecule recognizes an immunodominant epitope on ragweed or birch pollen.

28. The composition of Claim 24 wherein said antibody-derived or TCR-derived molecule recognizes an immunodominant epitope on Der p I or Der p II.

29. The composition of Claim 28 wherein said antibody-derived molecule competes for a Der p I
binding epitope with antibody 2C7 and generates an antidiotypic antibody which binds to 2C7.

30. The composition of Claim 28 wherein said antibody-derived molecule is antibody 2C7.