CA2092801A1 - Method for treating inflammation using anti-idiotypic antibodies - Google Patents

Abstract

A method for inhibiting ICAM-1 dependent inflammatory responses by immunizing a patient with anti-ICAM-1 anti-idiotype antibodies or fragments thereof. Anti-ICAM-1 anti-idiotypic antibodies are also disclosed whose antigen combining site binds to an idiotype of an anti-ICAM-1 antibody that inhibits binding of ICAM-1 to an ICAM-1 leukocyte receptor.

Description

2~9 ~8~ ~ :
os2~6lls PCT/US91/~7365 METHOD FOR TREATIN~ INFLAMMATION USING
ANTI-IDIOTYPIC ANTIBODIES

FIELD OF THE INvE2~EEQ~

The present invention relates to a method for inhibiting ICAM-l-dependent inflammatory responses utilizing anti-ICAM-l anti-idiotype antibodies or fragments thereo~. In particular, this invention relates to a method for treating ICAM-l-dependent in1ammation in a pati~nt by immunizing the patient with a sufficient amount of an anti-ICAM-l anti-idotype antibody to induce an immune response against ;
ICAM-l-bearing cells.

BACKGROUND OF THE INVEN~

Cellular adhesion is a process through which leukocytes attach to cellular substrates, such as end~thelial cells, in order to migrate from circulation to sites of ongoing inflammation, and properly defend the host against foreign invaders such as bacteria or viruses.
An excellent review of ~he defense system is provided by Eisen, H.W., (~: Macrobioloqv, 3rd Ed.j Harper &
Row, Philadelphia, PA ~1930), pp. 290-295 and 381-413).

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One o~ th~ molecules on the surface of endothelial cells which participates in the adhesion process is the intercellular adhesion molecule ICAM-l. See Rothlein et al, J. Immunol. 137:1270 (1986), thereinafter referred to as ("Rothlein et al"), herein incorporated by reference. This molecule has been shown to mediate adhesion by binding to molecules of the CD18 family of glycoproteins which are present on the cell sufaces of leukocytes (Sanchez-Madrid, F. et ~., J. Ex~er. Med 1785-1803 (1983); Keizer, G.D. et al., ~. J.
Immunol. 15:1142-1147 (1985)). This glycoprotein family is composed of heterodimers having one alpha chain (also rPferred to as "CDll") and one beta chain (also referred to as "CD18") There are three major members of the CD18 family: p.l50,95, Mac-l and LFA-l. Mac-l is a heterodimer found primarily on macrophages, granulocytes and large granular lymphocytes. LFA-l is a heterodimer found on most lymphocytes (Springer, T.A.
et al. Immunol. Rev. 68~ 135 (1982)). plS0,95 has a tissue distribution similar to Mac-l, and also plays a role in cellular adhesion (Keizer, G. ç~ al., Eur. J.
Immunol. 1~:1142-1147 (1985)).

Frequently, however, the adhesion mediated by ICAM-l results in undesirable inflammation, as, for e~ample, in arthritis, in asthma, in rejection and destruction of a transplanted organ or in the reperfusion injury which occurs when blood is abla to re-entar pre~iously ~locken (occluded) arteries. Accordingly, in appropriate circumstances, it is desirable to inhibit or eliminate ICAM-l mediated adhesion by inhibiting or preventing ~CAM-l from binding to its CD18 receptor molecules. One method to inhibit or prevent IC~M-l from binding to its CD18 receptors is to utilize a molecule which competes with the CD18 receptors for binding to ICAM-l. One such competing molecule is an anti-ICAM-l antibod~.

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' ' 1 ' WO92/061l9 2 0 9 ~ ~ O PCT/US91/0~36 Mouse monoclonal antibodi~s to ICAM-l have been shown to inhibit lymphocyte pr~iiferdtive response requiring cell/cell interactions as well as inhibiting granulocyte attachment and subsequent migration thraugh endothelial cell monolayers i~ vitro. Mouse anti-ICAM-l antibodies are also known to inhibit leukocyte migration to inflamed lungs in rabbits, kidney allograft rejection and antigen-induced airway hyperreactivity in primates. See, e.g., Dustin et al, J. Immunol. 137:245 (1986~ and Wegner et al Science 247:456 (1990). The use of mouse monoclonal anti-ICAM-l -antibodies has therapeutic potential because the mouse anti-ICAM-l can attenuate an in1ammatory response by binding to ICAM-l and interfering with leukocyte adhesion. However, treatme~t must be of short duration due to the innate immunogenicity of mouse immunoglobulin in primates.

According to the ~network~ theory ~Jerne, N.K. Ann.
Immunol. (Inst. Pasteur) 125C:373, (1974)], a network .of idiotypes and anti-idiotypic antibodies regulate the expression of immune responses. Anti-idiotypic antibodies (or anti-idiotypes) are antibodies raised against another (primary) antibody, which recognize unique epitopes on the primary antibody. Some anti-idiotypes actually mimic the epitope that the primary antibody is directed against. For example, certain anti-insulin anti-idiotypes act as agonists o the insulin receptor tShechter et al, Anti-IdiotYPes, RecePtor5 and MQlecular MimicrY, p. 73 (D.S. Linthicum and N.R. Farid, eds., New York, 1988)]. Certain anti-morphine anti-idiotypes will hind to opiate receptors (Ng et al, Eur. J. Pharmacol. 10~:187, lg84).
Certain anti-viral anti-idiotypes have elicited an im~une response to the virus where the host has been Y~92/06119 ~ CT/US91/07365 ^

immunized with the anti-idiotype. (See, e.g., Gaulton et al. J. Immunol. 137:2930, 1986). U.S. Patent No.
4,918,164 describes anti-tumor anti-idiotypes and their use in immunotherapy and immunoprophyla~is. Monoclonal anti-CD18 anti-idiotypic antibodies have been described in Hildreth et al., Mol. Immunol. 26:1155 (1989).

Accordin~ly, it is a purpose of the present invention to provide a method for treating ICAM-1 dependent inflammation using anti-ICAM-1 anti-idiotypic antibodies. It is also a purpose o the present invention to provide an anti-ICAM-l anti-idiotype.

DESCRIPTION OF THE FIGURES

Fiqure 1 SPecifiç bindinq of CA3 to R6.5 F(ab) fraqments.
8inding of control mouse Ig (solid bars) or CA3 (hashed bars) to R6.5 and RRl F(ab) fragments was detected by pero2idase-conjugated anti-mouse IgG Fc in an ELISA
assay.

Fiaure 2 Titration of CA3 bindin~ to R6.5. In an ELISA assay the binding of varying concentrations of purified CA3 to F(ab) fragments of R6.5 (solid line); RRl (dotted line); and normal mouse IgG (broken line~, was quantitated.

Fiqure 3 The effe~t of sICAM-l on CA3 and anti-kaPPa liaht chain bindinq to R6.5. C~3 alone (solid bar); CA3 + sICAM-l (~/////); anti-kappa alone (cross hatched bar);
anti-kappa + sICAM-l ( ).

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20~2~
~v092/06119 _ 5 _ PCr/US91/07365 ~re 4 Anti-CA3 blndinq to sICAM-l. In an ELISA assay the binding of varying concentrations of rabbit anti-CA3 and normal rabbit IgG to solid phase sICAM-l was ~
quantitated. ~solid bars), anti-CA3: (cross hatched ~ .
bars), normal rabbit IgG.

Fiqure 5 ;~:

Immunoblot comparison of the binding of normal rabbit IgG ~lanes l-S), rabbit anti-CA3 (lanes 6-lO), and R6.5 (lane ll), to native and reduced sICAM-l.

Fiqure 6 Western blot analysis of anti-CA3 binding to reduced ;~
s-ICAM-l (left hand blot) and R6.5 binding to native sICAM-l (right hand blot3. The lanes to the right of each blot are molecular weight standards as identiied.

E~g~ .

FACS analysis of rab~it anti-CA3 and normal rabbit IgG ~-~binding to JY cells. .. ~..... , anti-CA3 l:lO; ;
---------, anti-CA3 l:20; . . . . ., normal rabbit IgG ;~
l:lO; , normal rabbit 1:20. ~ :

DESCRIPTION OF THE INVENTION

This invention relates to a method for treating ICAM-l dependent inflammation in a patient, which comprises adminstering to the patient a therapeutically effective dosage of an anti-ICAM-l anti-idiotype (Ab2B) or a ;.

~092/06l19 ~ 0~ - 6 - PCT/US91/0736 fragment thereo~, wherein the Ab2~ or the fragment thereof, specifically recognizes an anti-ICAM-l antibody (Abl) idiotype that inhibits the binding of ICAM-l to the ICAM-l receptor participating in the inflammation. The Ab2B has conformational homology with the antigenic epitope of ICAM-l. Because of this conformational homology (or internal image), administration of the Ab2B to the patient results in the elicitation of anti-Ah2B antibodies (Ab3) in the patient. At least one Ab3 (Ao3') mimics the Abl. The Ab3~ will bind to the ICAM-l and inhibit ICAM-1 function in adhesion, i.e., the Ab3' will inhibit IC~M-l binding to the leukocyte receptor participating in the inflammation. As a result, the inflammation is reduced or eliminated.

The Abl und Ab2B antibodies can be prepared using procedures known in the art for producing antibodies, e.g. by immunopurification of polyclonal serum, by hybridoma technology or by recombinant cell culture technology. See, e.g. Kohler et al, P.N.A.S. USA
77(4):2197 ~1980) and U.S. Patent No. 4,816,567.

To prepare a monoclonal Abl, e.g., mice are initially immunized with ICAM-l. The mice are later sacrificed and their spleens removed. Spleen cells are fused with myeloma cells to produce hybridomas. The hybridomas are cultivated and their supernatants screened for anti-ICAM-l activity, e.g. by radioimmunoassay ~RIA), enzyme-linked immunoassary (ELISA), or, preferably, by an aggregation assay as described in Rothlein et al. `
Hybridomas secreting monoclonal antibody (mAb) with the desired anti-ICAM-l activity are cloned and cultivated to produce the Abl.
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W092/Ohll9 _ 7 PCT/US91/0736 Preferably~ the Ab2B is a monoclonal antibody.
Monoclonal Ab2B can be prepared in a manner similar to the preparation of monoclonal Abl. Mice are initially immunized with Abl. The mice are later sacrificed and their spleens removed. Spleen cells are fused with myeloma cells to produce hybridomas. The hybridomas are cultivated and their supernatants screened for anti-Abl activity, e.g., by ELISA or RIA. Hybridomas secreting Mab with the desired anti-Abl activity are cloned and cultivated to produce the Ab2~.
'~ , Many methods for administering the Ab2B to the patient can be used including, for eYample, intradermal, intramuscular, intraperitoneal, intravenous, subcutaneous and intranasal routes. Preferably, administration of Ab2~ utilizes subcutaneous or `
intramuscular injection of the Ab2B in the presence of various adjuvants. The amount of Ab2B to be administered will vary depending on the route of administration, type of inflammation, patient response, etc. but in general 0.1 mg to 100 mg of Ab2B, preferably about 1 mg of Ab2~, can be administered to the patient. The Ab2~ can be formulated with a suitable ad~uvant in order to enhance the immunological response, including e.g., mineral gels such as aluminium hydroxide; surface active substances such as lysolecithin; pluronic polyols; polyanions; peptides and oil emulsions.

It is to be understood that the Ab2~ useful in this invention includes whole antibodies and ~ragments, or any chemical modiications thereo, which mimic the desired ICAM-l binding site that binds to the idiotype of Abl. Ab2B fragments can be generated using various techniques known in the art, e.g., by proteolytic clcavage of the ~b2B, or by rncombinant construction.

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As an illustration of the present invention, the following e~amples are set forth in detail below:
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XAMPLE

A. PREPARATION OF CA3 lnAb Monoclonal anti-ICA~-l antibody R6.5 ~hereinafter referred to as "R6.5"), produced by hybridoma cell line R6~506'Eg~B2 (ATCC HB 9580), at a concentration of l mg/ml~ was incubated with keyhole limpet hemocyanin (KLH~ (Sigma Chemical, St. Louis, MO.) also at l mg/ml, with 0.05 % glutaraldehyde (Sigma) for two hours at room temperature and dialysed against phosphate buffered saline (PBS), to produce a KLH-R6.5 conjugate.
Si~ to eight week old femable Balb/c mice (Charles River Laboratories, Cambridge, mA.~ were injected, intraperitoneally (i.p.), with lOO ~g of the KLH-R6.5 conjugate in Complete Freunds Adjuvant (CFA) (Dico Labs) in a total of 0.4 ml per mouse. After three weeks mice were boosted with lOO ~g of KLH-R6.5 conjugate with Incomplete Freunds Adjuvant (IFA) (Difco Labs).
The last boost (3 weeks later) was an i.p. injection of 5~lO6 R6.5 hybridoma cells (ATCC HB 9580) per mouse.
Cells were irradiated, (lOOOR) before injection.
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Three days after the last boost, mice were sacrificed and spleens removed for fusion. Spleen cells were fused with P3x63Ag8.653 myeloma cells at a ratio o~ 4:l with PEG 4000 (VWR, Philadelphia, PA). The fusion mi~ture was aliquoted into 96-well microtiter plates ~Costar, Cambridge, M~). The resulting hybridoma supernatants were screened for anti-R6.5 activity by ELISA. Briefly, 2 ~} (J~
~VO92/o~lls PCT/USgl/0736 E.I.A. plates (Flow labs, '~clean, VA) were coated with Mab R6.5 F(ab')2 Jackso; labs, West Grove, PA), or the appropriate control a.itibody, l ~g/well, overnight in Dubleccos Phosphate Buffered Saline ~DPBS) at 4C.
The plates were washed 2x with DPBS and blocked with a 2 % BSA solution or one hour at 37C, washed 3x with DPBS and then rabbit anti-mouse IgG-Fc-specific ;
peroxidase conjugated antibody was added (~ccurate, Westbury, NY) and allowed to incubate for one hour at 37C. The plates were than washed 4~ with DPBS and the su~strate, 2,2~Azino-di(3-ethylbenzthiozoline Sulonic Acid) (ABTS) dissolved in 0.1 M citrate buffer, pH 4.2 and 0.03 % hydroyen peroxide (ZYMED, San Franzisco, CA) was added. Optical density was read at 410 nM (Dynatech plate reader MR600~. One hybridoma of interest was cloned and subcloned by limiting dilution. The clone,, CA3/Hl0, secreted an IgG, class mAb (hereinafter referred to as ~CA3"), as determined by Ouchterlony and ELISA.

Pristine-primed Balb/c mice were injected i.p. with 5x 106 CA3/H10 cells in 0.5 ml of DPBS. Ascited tumor development was pronounced by day 14 after injection and mice were sacrificed by cervical dislocation.
CA3-rich ascites fluid was collected and centrifuged to remove cells.

B. AGGREGATION ASSA~

A qualitative aggregation was carried out as described in Rothlein et al. Brie1y, 100 ~1 of culture supernatant from hybridoma cell line CA3/H10, was preincubated with 50 ~1 containing 0.1 ~g R6.5 Ig solution in complete media for 30 minutes at 37C in a , ... ..... .
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~ 092/06119 2 ~ 9 2 ~ ~ i 1 o PCT/ US91/0736~

microtiter plate. At this time 1~105 JY cells in 50 ~1 of media containing 10 ng of phorbol 12-myristate 13-acetate (PMA) (SIGMA Chemical Co., St.
Louis, M~) were added. Cells were incubated for 0.5 to 20 hours at 37~C and viewed with an inverted microscope for scoring aggregation.

C. COMPETITIVE BINDING STUDIES

An E. I .A. microtiter plate was coated with R6.5 or normal mouse IgG F(ab~)2, 1 ~g/well, overnight at 4C in DPBS. The plates were washed 3x and blocked at 37C for 1 hour with a 2 % bovine s0rum albumin (BSA) solution. The BSA was then flicked out and replaced with 50 ~1 of soluble ICAM~l (sICAM-l) (prepared as described in Marlin, S., et al, Nature 344, 70-72 (1990)) or control solution (25 ~g/ml final concentration) and incubated for 30 minutes at 37C.
Without removing the solution, 50 ~1 of the appropriate mAb solution was added (10 ~l~ml final concentration) and allowed to incubate for 1 hour at 37C. The plates were washed 4~ and the appropriate dilution of peroxidase-conjugated rabbit anti-mouse IgG-Fc specific antibody was added, incubated and washed again. ABTS substrate was added and color de~elopment was read at 410 nM.

D. ANTI-CA3 IMMUNI2ATION

White (NZW) rabbits (Hare Marland, Hewitt, N.J.) weighing between 2.5 and 3.5 kg were immunized with subcutaneous injections of CA3, 0.~ mg in CFA, and boosted with 0.5 mg of CA3 in IFA at three week intervals. Blood and serum samples were taken after the second boost.
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WO 92/061 1~ PCI`/US91/07365 E . B I ND I NG OF ANT I - CA3 ANT I BODY

E.I.A. plates were coated with 1 ~g/well of native or reduced sICAM-l overnight at 4C. Reduced sICAM-l was prepared by incubating native sICAM-l prepared as described in Marlin, et al (supra), in lM
2-mercaptoethanol (final concentration) at room temperatur for 30 minutes. A 2 % ovalbumin-coated plate (OA) was prepa~ed as a negative control. The plates were washed 3~ with DPBS and blocked with 2 % OA for 1 hour at 37~C. Blocking solution was replaced with 50 ~l/well of either 2 % OA, R3.1 (100 ~g/ml, control IgGl~ mAb (prepared as described in Smith et al, J. Clin Investigation 82: 1746-1756 (1988)) or CA3, 100 ~g/ml, prior to adding the anti-CA3 antibody or normal rabbit IgG (control) to the assay plate. The microtiter plate was then incubated for 1.5 hours at 37C, washed 3~ and incubated with goat anti-rabbit IgG-pero~idase enzyme conjugate for 1 hour at 37C
followed by 4 washes and incubated with 100 ~l/well ABTS substrate. Optical density was evaluated at 410 nM.

F. WESTERN BLOT ANALYSIS

Soluble ICAM-l was run on an 8 % Laemmli SDS-polyacrylamide gel in a pH 6.9 sample buffer containing 6 % SDS, 4mM urea, 125 mM Tris, 4 mM EDTA, O.25 % bromphenyl blue and 10 ~ glycerol. sICAM-l was loaded at a concentration of 5 ~g per lane in a inal volume of 150 ~1 and run ~or 16 hrs at 45 volts.
Transfer to nitrocellulose membranes was perormed at 4C for 2 hrs. at 50 volts in a Trans Blot ~Bio-Rad, Richmond, CA.) in a solution containing 25 mM Tris, 192mM glycine, 0.1 SO SDS and 20 % v/v methanol.
Nitrocellulose membranes were then washed in a 10 mM
Tris-buffered 150 mM NaCl solution containing 0.05 %
Tween-20 (TBST) and blocked for 2 hrs at 37C in 5 %
bovine serum albumin ~BSA) ~Sigma).

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~vos2/06119 2 0 ~ L - 12 - PCr/US91/0736 Individual lanes were then cut and incubated for l hour at room temperature with either normal rabbit IgG, polyclonal anti~ody anti-CA3, R6.5 or no anti~ody at a concentration of 20 ~g/ml. Membranes were then washed 3x in TBST and incubated for l hour at room temperature with the appropriate anti-IgG alkaline phosphatase conjugated secondary antibody (Promega, Madison, WI.).
Nitrocellulose membranes were again washed 3~ in TBST
and visualized with a solution containing 0.33 mg/ml nitroblue tetrazolium (NBT), 0.16 mg/ml ;
5-bromo-4-chloro-3-indolyl phosphate (BCIP), lO0 mM
Tris-HCL pH 9.5~ lO0 mM NaCl and 5 mM MgC12.
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G . DOT BLOT ANALYS I S

Using a Bio-Dot microfiltration apparatus (Bio Rad~
either native sICAM-l, reduced sICAM-l, or TBS was spotted on nitrocellulose membranes at a concentration of l mg per well in a volume of lO0 ml. The samples were allowed to filter through the membrane by gravity flow, blocked by the addition of 400 ~l of 5 ~
ovalbumin (Sigma) and washed with 400 ~l of TBST.
Primary antibody solutions of either normal rabbit IgG, polyclonal antibody CA3, or no mAb were at dilutions of 1:20, l:S0, 1:250, l:lO00 and l:lO,000 were then added.
R6.5 was used at a concentration of 20 ~l/ml as a positive control because it did not bind to reduced sICAM-l. After primary antibodies were allowed to filter through, the membrane was washed wi~h 400 ~l of TBST and the appropriately diluted secondary anti-IqG
alkaline phosphatase-conjugated antihody was added. The membrane was then washed 3x with TBS to remove excess Tween-20 and color development was achieved by the addition of substra~e containing NBT and BCIP. Color development was stopped by adding deionized water.

- ' w0~2/06ll9 2~h ~ ~ ~ PCT/US91/0736 H. RESULTS

1. Immunization, production and sele~ion o~
anti-ICAM-l anti-idiotY~e mAb. As described above, Balb/c mice were immunized with a RLH-R6.5 conjugate and with irradiated R6.5 hybridoma cells. The sera ~rom immunized mice showed detectable binding to R6.5 F(ab) but not to a control mouse Ig. Hybridomas resulting from the fusion of immunized Balb/c mouse spleen cells with P3~63A~8.653 myeloma cells werP screened for the production of antibodies binding to R6.5 F(ab) fragments by ELISA using a peroxidase-conjugated anti-mouse IgG Fc for detection. The supernatants of all positive hybridomas were tested for binding to RRl F(ab) fragments, a second anti-ICAM-1 mAb ~Rothlein et al.). One hybridoma, CA3~H10, was selected and, following cloning by limiting dilution, was shown to produce an IgGl mAb, CA3.

2. Chara~erization_of the anti-ICAM-l anti-idiotYPe mAb, ~3. The results in Figure 1 show the binding of CA3 to F(ab) fragments of the two anti-ICAM-l mAbs. CA3 bound to R6.5 F(ab~
fragments but no binding to RRl F(ab) fragments was detectable. Titration of CA3 showed detectable binding to ~6.5 at a concentration of 15 ng/ml (Fig. 2~. The binding of CA3 to control mouse IgG F(ab) fragments was also negative.

The anti-ICAM-l mAbs, R6.5 and RRl, have been shown to e~hibit functional anti-adhesion properties. ~he aggregation of cells from the human lymphoid cell line, JY, is dependent upon the interaction of ICAM-l and LFA-l leukocyte adhesion molecules and antibodies to both LFA-1 and ICAM-l inhibit aggregation (Rothlein et al.).
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0~2/061~9 '~ ~ 9 2 ~ G 1 PCT/US91/07365 As shown in Table l below, both R6 . 5 and RRl inhibited JY cell aggregation by 60 %; C~3 blocked the ability of R6.5 but not of RRl to inhibit JY cell agyregation. CA3 alone had no demonstrable effect on the aggregation. Titration of CA3 showed that a concentration as low as 0.78 ~g/ml will completely block R6.5 inhibi~ion of JY cell aggregation. Soluble ICAM-l has been shown to bind specifically to the anti-ICAM-~
mAb's R6.5 and RRl. The effect of sICAM-l on CA3 binding to R6.5 F(ab) fragments is shown in Figure 3. Both CA3 and control anti-kappa light chain antibbdies bound to the R6.5 fragments. The binding of CA3 was inhibited by approximately 70 % by sICAM-l whereas no significant inhibition of anti-kappa binding was detected. "

TAB~E l CA3 Inhibition of R6.5 Activity in JY Aaqreaation SamPle Agqreaation MedialO0 %
R6.5 RRl40 %
CA3lO0 ~ `
R6.5 & CA3 lO0 %
RRl & CA3 40 % `~
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3. CA3 as an immunoqe~. The characterization of CA3 suggested that it shared conformation homology wi~h the epitope bound by R6.5. Rabbits were immunized with CA3 to induce a heterologous anti-antiidiotype (Ab3) response. An IgG ~ `
preparation of rabbit anti-CA3 sera was tested ~

2~2~0~
092/06119 PCT/~S91/0736 or binding to sIC~-l. As shown in Figure 4, in an ELISA assay with solid phase sICAM-l, significant binding to ICAM-l was detected at 200 ~g/ml. Comparison of binding to native and to reduced sICAM-l in an immunoblot showed significant binding to reduced sICAM-l at lO-fold lower IgG concentrations than was detected with native sICAM-l (Fig. 5). R6.5 bound to native sICA~-l but no binding to reduced sICAM-l was detected. Western blot analysis supported the binding o~ anti-CA3 to reduced ICAM-l which migrates, as expected, at an apparent higher molecular weight (Fig. 6).

Rabbit anti-CA3 was also tested for its ability to bind to cells expressing ICAM-l. As shown in Figure 7, rabbit anti-CA3 showed significant binding to JY cells which decreased with decreasing antibody concentration. That the binding of anti-CA3, like that of R6.5, was specific not only for ICAM-l but also for the do~ains of the molecule that are functional in agyregation is indicated by the fact that the rabbit anti-CA3 inhibited JY cell aggregation.

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Claims (12)

WHAT IS CLAIMED IS:

1. A method for treating ICAM-1 dependent inflammation in a patient, which comprises administering to the patient a therapeutically effective dosage of an anti-ICAM-1 anti-idiotype (Ab2B) or a fragment thereof, wherein the Ab2B or the fragment thereof, specifically recognizes an anti-ICAM-1 antibody (Ab1) that inhibits ICAM-1 function in inflammation.

2. A method as recited in claim 1 wherein the Ab2B
is a monoclonal antibody.

3. A method as recited in claim 2 wherein the monoclonal Ab2B is CA3.

4. A method as recited in claim 1 wherein the fragment of the Ab2B comprises an Fab, F(ab')2 or Fv fragment of the Ab2B.

5. A method as recited in claim 2 wherein the Ab2B
binds to the idiotype of the monoclonal antibody designated as R6.5, which idiotype is directed against an antigenic epitope of ICAM-1.

6. An anti-ICAM-1 anti-idiotypic antibody, the antigen combining site of which binds to an idiotype of an anti-ICAM-1 antibody that inhibits binding of ICAM-1 to an ICAM-1 leukocyte receptor.

7. An anti-ICAM-1 anti-idiotypic antibody as recited in claim 6 which is a monoclonal antibody.

8. An anti-ICAM-1 anti-idiotypic antibody as recited in claim 7 which is monoclonal antibody CA3.

9. An Fab, F(ab')2 or Fv fragment of the anti-ICAM
anti-idiotypic antibody of claim 6.

10. An anti-ICAM-1 anti-idiotypic antibody produced by hybridoma cell line CA3/H10.

11. An anti-ICAM-1 anti-idiotypic antibody as recited in claim 6 wherein the ICAM-1 leukocyte receptor is LFA-1.

12. An anti-ICAM-1 anti-idiotypic antibody as recited in claim 6 wherein the ICAM-1 leukocyte receptor is Mac-1.