METHOD FOR TREATING INFLAMMATION USING ANTI-IDIOTYPIC ANTIBODIES
FIELD OF THE INVENTION
The present invention relates to a method for inhibiting ICAM-]-dependent inflammatory responses utilizing anti-ICAM-1 anti-idiotype antibodies or fragments thereof. In particular, this invention relates to a method for treating ICAM-1-dependent inflammation in a patient by immunizing the patient with a sufficient amount of an anti-ICAM-1 anti-idotype antibody to induce an immune response against ICAM-1-bearing cells.
BACKGROUND OF THE INVENTION
Cellular adhesion is a process through which leukocytes attach to cellular substrates, such as endothelial cells, in order to migrate from circulation to sites of ongoing in lammation, and properly defend the host against foreign invaders such as bacteria or viruses. An excellent review of the defense system is provided by Eisen, H. ., (In: Macrobioloσv. 3rd Ed., Harper & Row, Philadelphia, PA (1980), pp. 290-295 and 381-418).
One of the molecules on the surface of endothelial cells which participates in the adhesion process is the intercellular adhesion molecule ICAM-1. See Rothlein et al, J. Immunol. 137:1270 (1986), (hereinafter 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. aJL. , J. Exper. Med 158:1785-1803 (1983); Keizer, G.D. et al., Eur. J. Immunol. 15.:1142-1147 (1985)). This glycoprotein family is composed of heterodimers having one alpha chain (also referred to as "CD11") and one beta chain (also referred to as "CD18") There are three major members of the CD18 family: p.150,95, Mac-1 and LFA-1. Mac-1 is a heterodimer found primarily on macrophages, granulocytes and large granular lymphocytes. LFA-1 is a heterodimer found on most lymphocytes (Springer, T.A. et aJL. Immunol. Rev. :1H-135 (1982)). pl50,95 has a tissue distribution similar to Mac-1, and also plays a role in cellular adhesion (Keizer, G. e£. a∑. , Eur. J. Immunol. 15:1142-1147 (1985)).
Frequently, however, the adhesion mediated by ICAM-1 results in undesirable inflammation, as, for example, in arthritis, in asthma, in rejection and destruction of a transplanted organ or in the reperfusion injury which occurs when blood is able to re-enter previously blocken (occluded) arteries. Accordingly, in appropriate circumstances, it is desirable to inhibit or eliminate ICAM-1 mediated adhesion by inhibiting or preventing ICAM-1 from binding to its CD18 receptor molecules. One method to inhibit or prevent ICAM-1 from binding to its CD18 receptors is to utilize a molecule which competes with the CD18 receptors for binding to ICAM-1. One such competing molecule is an anti-ICAM-1 antibody.
Mouse monoclonal antibodies to ICAM-1 have been shown to inhibit lymphocyte proliferative response requiring cell/cell interactions as well as inhibiting granulocyte attachment and subsequent migration through endothelial cell monolayers in vitro. Mouse anti-ICAM-1 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 egner et al Science 247:456 (1990). The use of mouse monoclonal anti-ICAM-1 antibodies has therapeutic potential because the mouse anti-ICAM-1 can attenuate an inflammatory response by binding to ICAM-1 and interfering with leukocyte adhesion. However, treatment 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 of the insulin receptor [Shechter et al, Anti-Idiotvpes, Receptors and Molecular Mimicry, p. 73 (D.S. Linthicum and N.R. Farid, eds.. New York, 1988)]. Certain anti-morphine anti-idiotypes will bind to opiate receptors (Ng et al, Eur. _, . Pharmacol. 102:187, 1984). Certain anti-viral anti-idiotypes have elicited an immune response to the virus where the host has been
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 immunoprophylaxis. Monoclonal anti-CD18 anti-idiotypic antibodies have been described in Hildreth et al., Mol. Immunol. 26:1155 (1989).
Accordingly, 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 of the present invention to provide an anti-ICAM-1 anti-idiotype.
DESCRIPTION OF THE FIGURES
Figure 1
Specific binding of CA3 to R6.5 F(ab) fragments. Binding of control mouse Ig (solid bars) or CA3 (hashed bars) to R6.5 and RR1 F(ab) fragments was detected by peroxidase-conjugated anti-mouse IgG Fc in an ELISA assay.
Figure 2
Titration of CA3 binding to R6.5. In an ELISA assay the binding of varying concentrations of purified CA3 to F(ab) fragments of R6.5 (solid line); RR1 (dotted line) ; and normal mouse IgG (broken line) , was quantitated.
Figure 3
The effect of sICAM-1 on CA3 and anti-kappa light chain binding to R6.5. CA3 alone (solid bar); CA3 + sICAM-1 (//////) ; anti-kappa alone (cross hatched bar) ; anti-kappa + sICAM-1 ( ) .
Figure 4
Anti-CA3 binding to sICAM-1. In an ELISA assay the binding of varying concentrations of rabbit anti-CA3 and normal rabbit IgG to solid phase sICAM-1 was quantitated. (solid bars), anti-CA3; (cross hatched bars), normal rabbit IgG.
Figure 5
Immunoblot comparison of the binding of normal rabbit IgG (lanes 1-5), rabbit anti-CA3 (lanes 6-10), and R6.5 (lane 11), to native and reduced sICAM-1.
Figure 6
Western blot analysis of anti-CA3 binding to reduced s-ICAM-1 (left hand blot) and R6.5 binding to native sICAM-1 (right hand blot). The lanes to the right of each blot are molecular weight standards as identified.
Figure 7
FACS analysis of rabbit anti-CA3 and normal rabbit IgG binding to JY cells , anti-CA3 1:10; , anti-CA3 1:20; , normal rabbit IgG
1:10; , normal rabbit 1:20.
DESCRIPTION OF THE INVENTION
This invention relates to a method for treating ICAM-1 dependent inflammation in a patient, which comprises adminstering to the patient a therapeutically effective dosage of an anti-ICAM-1 anti-idiotype (Ab2β) or a
fragment thereof, wherein the Ab2β or the fragment thereof, specifically recognizes an anti-ICAM-1 antibody (Abl) idiotype that inhibits the binding of ICAM-1 to the ICAM-1 receptor participating in the inflammation. The Ab2β has conformational homology with the antigenic epitope of ICAM-1. Because of this conformational homology (or internal image) , administration of the Ab2β to the patient results in the elicitation of anti-Ab2β antibodies (Ab3) in the patient. At least one Ab3 (Ab3') mimics the Abl. The Ab3' will bind to the ICAM-1 and inhibit ICAM-1 function in adhesion, i.e., the Ab3' will inhibit ICAM-1 binding to the leukocyte receptor participating in the inflammation. As a result, the inflammation is reduced or eliminated.
The Abl und Ab2β 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-1. 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-1 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-1 activity are cloned and cultivated to produce the Abl.
Preferably, the Ab2β is a monoclonal antibody. Monoclonal Ab2β 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 Ab2β.
Many methods for administering the Ab2β to the patient can be used including, for example, intradermal, intramuscular, intraperitoneal, intravenous, subcutaneous and intranasal routes. Preferably, administration of Ab2β utilizes subcutaneous or intramuscular injection of the Ab2β in the presence of various adjuvants. The amount of Ab2β 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 Ab2β, preferably about 1 mg of Ab2β, can be administered to the patient. The Ab2β can be formulated with a suitable adjuvant 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 Ab2B useful in this invention includes whole antibodies and fragments, or any chemical modifications thereof, which mimic the desired ICAM-1 binding site that binds to the idiotype of Abl. Ab2β fragments can be generated using various techniques known in the art, e.g., by proteolytic cleavage of the Ab2β, or by recombinant construction.
As an illustration of the present invention, the following examples are set forth in detail below:
EXAMPLE 1
A. PREPARATION OF CA3 mAb
Monoclonal anti-ICAM-1 antibody R6.5 (hereinafter referred to as "R6.5"), produced by hybridoma cell line R6'506*E9'B2 (ATCC HB 9580), at a concentration of 1 mg/ml, was incubated with keyhole limpet hemocyanin (KLH) (Sigma Chemical, St. Louis, MO.) also at 1 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. Six to eight week old femable Balb/c mice (Charles River Laboratories, Cambridge, MA.) were injected, intraperitoneally (i.p.), with 100 μg of the KLH-R6.5 conjugate in Complete Freunds Adjuvant (CFA) (Difco Labs) in a total of 0.4 ml per mouse. After three weeks mice were boosted with 100 μg of KLH-R6.5 conjugate with Incomplete Freunds Adjuvant (IFA) (Difco Labs) . The last boost (3 weeks later) was an i.p. injection of 5x10 R6.5 hybridoma cells (ATCC HB 9580) per mouse. Cells were irradiated, (1000R) before injection.
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 of 4:1 with PEG 4000 (VWR, Philadelphia, PA) . The fusion mixture was aliquoted into 96-well microtiter plates (Costar, Cambridge, MA). The resulting hybridoma supernatants were screened for anti-R6.5 activity by ELISA. Briefly,
E.I.A. plates (Flow labs, Mclean, VA) were coated with Mab R6.5 F(ab')2 (Jackson labs, West Grove, PA), or the appropriate control antibody, 1 μg/well, overnight in Dubleccos Phosphate Buffered Saline (DPBS) at 4°C. The plates were washed 2x with DPBS and blocked with a 2 % BSA solution for one hour at 37°C, washed 3x with DPBS and then rabbit anti-mouse IgG-Fc-specific peroxidase conjugated antibody was added (Accurate, Westbury, NY) and allowed to incubate for one hour at 37°C. The plates were than washed 4x with DPBS and the substrate, 2,2-Azino-di(3-ethylbenzthiozoline Sulfonic Acid) (ABTS) dissolved in 0.1 M citrate buffer, pH 4.2 and 0.03 % hydrogen 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/H10, 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 ASSAY
A qualitative aggregation was carried out as described in Rothlein et al. Briefly, 100 μl of culture supernatant from hybridoma cell line CA3/H10, was preincubated with 50 μl containing 0.1 μg R6.5 Ig solution in complete media for 30 minutes at 37°C in a
5 microtiter plate. At this time 1x10 JY cells in
50 μl of media containing 10 ng of phorbol 12- myristate 13-acetate (PMA) (SIGMA Chemical Co., St.
Louis, MO) 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 μg/well, overnight at 4°C in DPBS. The plates were washed 3x and blocked at 37°C for 1 hour with a 2 % bovine serum albumin (BSA) solution. The BSA was then flicked out and replaced with 50 μl of soluble ICAM-1 (sICAM-1) (prepared as described in Marlin, S., et al. Nature 344, 70-72 (1990)) or control solution (25 μg/ml final concentration) and incubated for 30 minutes at 37°C. Without removing the solution, 50 μl of the appropriate mAb solution was added (10 μl/ml final concentration) and allowed to incubate for 1 hour at 37°C. The plates were washed 4x 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 development was read at 410 nM.
D. ANTI-CA3 IMMUNIZATION
White (NZW) rabbits (Hare Marland, Hewitt, N.J.) weighing between 2.5 and 3.5 kg were immunized with subcutaneous injections of CA3, 0.5 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.
_
E. BINDING OF ANTI-CA3 ANTIBODY
E.I.A. plates were coated with 1 μg/well of native or reduced sICAM-1 overnight at 4°C. Reduced sICAM-1 was prepared by incubating native sICAM-1 prepared as described in Marlin, et al (supra), in 1M 2-mercaρtoethanol (final concentration) at room temperatur for 30 minutes. A 2 % ovalbumin-coated plate (OA) was prepared as a negative control. The plates were washed 3x with DPBS and blocked with 2 % OA for 1 hour at 37°C. Blocking solution was replaced with 50 μl/well of either 2 % OA, R3.1 (100 μg/ml, control IgGl) mAb (prepared as described in Smith et al, J. Clin Investigation £2: 1746-1756 (1988)) or CA3, 100 μg/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 37°C, washed 3x and incubated with goat anti-rabbit IgG-peroxidase enzyme conjugate for 1 hour at 37°C followed by 4 washes and incubated with 100 μl/well ABTS substrate. Optical density was evaluated at 410 nM.
F. WESTERN BLOT ANALYSIS
Soluble ICAM-1 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, 0.25 % bromphenyl blue and 10 % glycerol. sICAM-1 was loaded at a concentration of 5 μg per lane in a final volume of 150 μl and run for 16 hrs at 45 volts. Transfer to nitrocellulose membranes was performed at 4°C 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 % 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 37°C in 5 % bovine serum albumin (BSA) (Sigma) .
Individual lanes were then cut and incubated for 1 hour at room temperature with either normal rabbit IgG, polyclonal antibody anti-CA3, R6.5 or no antibody at a concentration of 20 μg/ml. Membranes were then washed 3x in TBST and incubated for 1 hour at room temperature with the appropriate anti-IgG alkaline phosphatase conjugated secondary antibody (Promega, Madison, WI.) . Nitrocellulose membranes were again washed 3x 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) , 100 mM Tris-HCL pH 9.5, 100 mM NaCl and 5 mM MgCl2.
G. DOT BLOT ANALYSIS
Using a Bio-Dot microfiltration apparatus (Bio Rad) either native sICAM-1, reduced sICAM-1, or TBS was spotted on nitrocellulose membranes at a concentration of 1 mg per well in a volume of 100 ml. The samples were allowed to filter through the membrane by gravity flow, blocked by the addition of 400 μl of 5 % ovalbumin (Sigma) and washed with 400 μl of TBST. Primary antibody solutions of either normal rabbit IgG, polyclonal antibody CA3, or no mAb were at dilutions of 1:20, 1:50, 1:250, 1:1000 and 1:10,000 were then added. R6.5 was used at a concentration of 20 μl/ml as a positive control because it did not bind to reduced sICAM-1. After primary antibodies were allowed to filter through, the membrane was washed with 400 μl of TBST and the appropriately diluted secondary anti-IgG alkaline phosphatase-conjugated antibody was added. The membrane was then washed 3x with TBS to remove excess Tween-20 and color development was achieved by the addition of substrate containing NBT and BCIP. Color development was stopped by adding deionized water.
H . RESULTS
1. Immunization, production and selection of anti-ICAM-1 anti-idiotype mAb. As described above, Balb/c mice were immunized with a KLH-R6.5 conjugate and with irradiated R6.5 hybridoma cells. The sera from 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 P3x63Ag8.653 myeloma cells were screened for the production of antibodies binding to R6.5 F(ab) fragments by ELISA using a peroxidase-conjugated anti-mouse IgG F V__* for detection. The supernatants of all positive hybridomas were tested for binding to RR1 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 IgG, mAb, CA3.
2. Characterization of the anti-ICAM-1 anti-idiotvpe mAb. CA3. The results in Figure 1 show the binding of CA3 to F(ab) fragments of the two anti-ICAM-1 mAbs. CA3 bound to R6.5 F(ab) fragments but no binding to RR1 F(ab) fragments was detectable. Titration of CA3 showed detectable binding to R6.5 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-1 mAbs, R6.5 and RR1, have been shown to exhibit functional anti-adhesion properties. The aggregation of cells from the human lymphoid cell line, JY, is dependent upon the interaction of ICAM-1 and LFA-1 leukocyte adhesion molecules and antibodies to both LFA-1 and ICAM-1 inhibit aggregation (Rothlein et al.).
As shown in Table 1 below, both R6.5 and RR1 inhibited JY cell aggregation by 60 %; CA3 blocked the ability of R6.5 but not of RR1 to inhibit JY cell aggregation. CA3 alone had no demonstrable effect on the aggregation. Titration of CA3 showed that a concentration as low as 0.78 μg/ml will completely block R6.5 inhibition of JY cell aggregation. Soluble ICAM-1 has been shown to bind specifically to the anti-ICAM-1 mAb's R6.5 and RR1. The effect of sICAM-1 on CA3 binding to R6.5 F(ab) fragments is shown in Figure 3. Both CA3 and control anti-kappa light chain antibodies bound to the R6.5 fragments. The binding of CA3 was inhibited by approximately 70 % by sICAM-1 whereas no significant inhibition of anti-kappa binding was detected.
TABLE 1
CA3 Inhibition of R6.5 Activity in JY Aggregation
Sample Aggregation
Media 100 %
R6.5 40 %
RR1 40 %
CA3 100 %
R6.5 & CA3 100 %
RR1 & CA3 40 %
CA3 as an immunogen. The characterization of CA3 suggested that it shared conformation homology with 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
for binding to sICAM-1. As shown in Figure 4, in an ELISA assay with solid phase sICAM-1, significant binding to ICAM-1 was detected at 200 μg/ml. Comparison of binding to native and to reduced sICAM-1 in an immunoblot showed significant binding to reduced sICAM-1 at 10-fold lower IgG concentrations than was detected with native sICAM-1 (Fig. 5). R6.5 bound to native sICAM-1 but no binding to reduced sICAM-1 was detected. Western blot analysis supported the binding of anti-CA3 to reduced ICAM-1 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-1. 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-1 but also for the domains of the molecule that are functional in aggregation is indicated by the fact that the rabbit anti-CA3 inhibited JY cell aggregation.