CA2060663A1 - Atherosclerotic plaque specific antigens, antibodies thereto, and uses thereof - Google Patents

Abstract

This invention provides purified antigens which are indicative of the presence of atherosclerotic plaque. Different concentrations of these antigens have been found to coincide with the progressiom of atherosclerosis. The subject invention also provides different hybridoma cell lines which produce monoclomal antibodies directed to antigens associated with atherosclerosis and a hybridoma cell line which produces monoclonal antibodies directed to antigen associated with normal artery and not with plaque. The atherosclerotic plaque antigen, and monoclonal antibodies made thereto, are used in various methods for detecting in a biological sample an antigen present in, and indicative of the presence of, atherosclerotic plaque.

Description

i; W091/02~52 ~ u 7~ J PCr/US90/04272 i AT~ERORCL~SRO~IC P~Q~B ~PBCI~IC ~TIG~38, A~TIBODIE8 ~ETO, AND U~ EREO~

~a~;ound of the Inven~ion This application is a continuatlon-in-part of U.S. Serial No. 388,129, filed July 3~, 1989, the text of which i~
-~ incorporated by reference into the subject application.

- Atheroscleroæis is the progressive narrowing of the lumen (inner passageway) of arterial blood vessels by layer~ of . .
pl~que (fatty and fibrous tissues~. Atherosclerosis can occur in any artery. In coronary arteries it may result in heart attacks; in cerebral arteries it ~ay result in strokes; and in peripheral arteries it may result in gangrene of ~he legs and feet.

A~herosclerosis is the single largest medical problem currently facing the United StatQs and other developed countriss. Approximately 40 million people in the United States are at risk for atherosclerosis. ~owever, only 6 million people in the United States show overt signs of the disease. The rest remain undiagnosed until the disease ~anifests itsel~ symptomatically, in the worst case as heart ; attack or stroke. Heart attack and stroke, respectively, represent the first and third leading causes of death in ,United State~. Over 500,000 people die of heart attacks ev~ry year and a signi~icant sub-group o~ these patients ; 30 expire without warning.

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The endothelium is located between the blood and arterial ~; tissue and servesi as a barrier against the accumulation of ; blood components in the vaiscular wall. ~ormation o~
atherosclerotic lesions in the sub-endothelium is associated with ma~or coronary art~ry disiease and stroke. The causes ; and detection of such lesions have been intensaly .. .
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Endothelial injury is believed to be an initial ~tep in theformation of the atherosclerotic lesions and may be caused by hemodynamic strain, hypercholesterolemia, hyperten~ion and immune complex disease. Endothelial injury leads to cholasterol accumulation and intimal thickening, cellular prolif~ration, and formation o~ connective tissue fibers.
IgG and complement ~actor C3 accumulation in injured lo endothelial cells and nonendothelialized intima has been observed. Mononuclear phagocytes derived from bloo~ are also part of the cell population in atherosclerotio lesions.
The mechanism of plaque formation is not ~ully known.
~owever, a probable mechanism is that fatty deposits lead to an influx of macrophage~, which in turn are followed by T
cells, B cells, and antibody production.

A variety of ~oluble proteins have been extracted from human atherosclerotic plaque, including IgA, IgG, IgM, 81C(C3), alpha1-antitrypsin, alpha2-macroglobulin, ~ibrinogen, albumin, L~L, HD~, alphal-acid glycoprotein, B2-glycoprotein, transferrin and ceruloplasmin. The diseased intima was also ~ound to contain a small amount o~ tissue-bound IgG, IgA and ~; ~lC EHollander, W. et al., Atherosclerosis, 3~:391-405 (1979)l. IgG has been determined in lesions and adjacent endothelial tiSSUQ tParums, D. et al., Atherosclerosis, 38:211-216 (1981), Hansson, G. et al., Experimental and Molecular Pathology, 34:2fi4-280 (1981), Hannson, G. et al., Acta Path. Nicrobiol. Immunol. Scand. Sect. A., ~:429-435 (1984)~- However, the origin, ~unction and binding propertiQ~ o~ the immunoglobulins in the atherosclerotic and associated tissue are not well characterized. Anti-low density lipoprotein tLDL) autoantibodies are reported to be higher in patients of vascular disease, suggesting that they are as~ociated in some way with atherosclerotic ; manlfestations. However, no causal relationship between .

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w09l/022s2 PCT/US90/04272 ~ ~ 5 ~

these autoantibodies and atherosclerotic plaque has been established. ~Szondy, E. et al., Mechanisms of Aging and Development, ~:117-123 (1985)].

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A wide variety o~ immunoas6ay~ have been developed for determining the presence and amount of antigenic and non-antigenic material6 in diverse body fluids and ti5SUQ8.
Total immunoglobulin and IgE immunoassays are described in ; U.S. patents 3,720,760 and 4,444,879. IgG allotype i~unoassays are described in Russian Patent 649,433. ELIS~
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immunoassays are described by ~aggio, et al.
~Enzy~e~ unoassay, Boca Raton: CRC Press pp 172-176 80)]. However, prior to this invention, no immunoassay suitable for det2rmining the presence of atherosclerotic plaque has been known.

Although atherosclerosis is qenerally a diffuse disease, human coronary atherosclerosi~ lends itself to bypass ,f~ procedures because the ma~or site of plague formation is usually proximally distributed. As a result, direct coronary artery bypass has become the most fraguently selected form o~ myocardial revascularization. The aorta-coronary artery vein gra~t or the internal m2mmary , ~rtery graft have become technically standardized and have high long-terc potency rates. These ~ong-ter~ results, however, can be co~pro~ised by progressive atherosclerosis distal to the graft anastomosis. Other cases are inoperable j~ because o~ distal disease. Previously, distal lesions have been ignored or, in selected cases, treated by endarterectomy although neither approach has proved entirely satis~actory.
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Most existing procedures for the diagnosis and treatment of atherosclerosis are invasive, costly, and of li~ited ; 35 ~ectiveneRs in a significant percentage of patient cases.
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Prior to the subiect invention, radioimaging of athero~celerotic plaque using an antibody which specifically binds to an atheroscl~rotic plaque specific ant~gen was unknown, although radioimagi~g o~ aged venous thrQmbi with fibrin-specific monoclonal antibodies label~d with a radioactive moiety has been reported [Rosebrough, S. ~t al., Radiology L~:575-577 ~February, 1987)].
: . , Radioimaging thrombi with radiolabeled ~onoelonal antibodies to platelets was first deseribed by Peter~, A., et al.
~British ~edical Journal, ~ 1525-1527 (Deee~ber, 1986)] o ~,~
~TPA-eoupl~d antibodies radiolabeled wi~h metallie radionuclides has been described by Hnatowich, D., et al.
~Journal of Immunologieal Methods, 6~:147-157 (1983)~.
NMRI, ultrasound and X-ray imaging with metal chelates are described in U.S Patent 4,647,447. In addition, antibody eoupling of metal ehelates is mentioned at eolumn 7, line 42. Monoclonal antibodias labeled wit~ polymerie paramagnetic ehelates and their use in NMRI methods have al50 been deseribed ~Shreve, P. et al., Magnetic Resonance in Medicine, 3:336-340 (1986) and Brady, T. et al. in Proeeedings of the Soeiety of Magnetie Resonanee in Medieine, Second Annual Meeting, Soe. of Magnetic Rasonanee in M~dielne, Ine., San Francisco, p. 10, (1983), refereneed by ~outcher, J. et al., J. Nuel. Med., ~:506-513 (1984)].

U.S. Patent 4,343,734 (Lian et al.) deseribes gamma-earboxyglutamie aeid (GLA) speei~le antlbedies whieh ; 30 ean be labeled with fluoreseein ~or immuno~luoreseenee staining of tissue to determine the presenee therein of GLA.
GLA speeific antibodies bind with GL~ present in advaneed atheroselerotie plaque having ealeiu~ deposits. Lian et al.
report that GLA i9 not ~ound in uncalcified plagues and that GLA is ~ound in cardiac valves and ao~tas, and in circulating proteins such as protho~bin, clotting ~actors ~.:
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wo 9 1 /022~2 PCl`/US90/04272 _5_ 2^~U~3 :

VII, IX and X, Protein C and Protein S. However, the GLA
blnding antibodies of Lian et al. do not sel~ctively bind to atherosclQrotic plaque.

The atherosclerotic plaque antibodies of the ~ubject invention bind to all stages of atherosclerotic plaque . including non-calcified stages, and do not selectively bind ~ with GLA.
'' The concept of plaque e~hancement by application of a stain . has been reported [Spears, J~ et al., J. Clin. Invest, ~: 71:395-39~ ~1983)]. These stains mark the plaque surfaces ~` wi~h a fluorescent compound. Plaque destruction by photoactivation of hematoporphyrin derivatives u~ing an intraluminal laser-transmitting optical fiber has been suggested tAb41a, G. et alO, Am. J. Cardio., 50:1199-1205 ~1982)~. ~oreov~r, tetracycline stains have also been suggested. ~Mu$phy-Chutorian, D. et al., A~. J. Cardiol., 1293-1297 (1985)].
-. 20 The above-identified stains were selected for their ability :. to bind to components of the atherosclerotic plaque. In principal, the ~tain absorbs laser light concentrating the light at the stained surface. Some staining of heal~hy tiSSU8 occurs causing stain associated damage to the ; surrounding tissue. Because laser ~avelen ~ i~ li~ited to the absorption waYelength of the stain, chro~ophores offering optimùm absorption o~ laser must be us~d to prov~de . most controllad ablation.
:. 30 .; In recent years, laserB have been used increasingly in microsurgery, both as scalpels and as coagulating instruments. Because of their ability to produce relatively ; bloodless incisions of great precision, as well as focal coagulation, they have been particularly useful in mlcrosurgical procedures in the eye, central nervous syste~, WO91/02252 PCT/USso/04272 2 ~
nasal passage~, cervix, ga~trointestinal tract, 6kin, muscle, and even in small vessels.
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L_Yi~Q experiments with heart and arterial tissue from human cadavers have demonstrat~d the feasibility of vaporizing or etching away plaque on disea~ed surfaces.
W -wavele~gths were found to offer more precision. ~aser treatment of plaque in live animals was less preci~e, causing damage and perforation of ~urrounding heal~hy tissue. [Gerrity, R. et al. J. Thorac. Cardiovasc. Surg., 85:409-421 ~1983); Lee, ~. et al , Am Heart J., lQ~:885-B89 (1983); ~ee, G. et al., Am. Heart J., pp 777-778. ~Aug.
1984); ~ee, G. et al., A~. ~eart J., lQ~:1577-1579 (1984);
Lee, G. et al., Am. J. Cardiology, S~:290-293 (1g~4~;
Linsker, R. et al., Laser~ in Surgery and ~edicine, 4:201-206 (1984); Abela, G. et al., Circulation, ~1(2):403-411 (1985); Prince, M. et al., J. Clin. Invest., 7~:295-302 tl986); and Srinivasan, R., Science, 234:559-565 ' tl986) ] .
Recent reference has been made to monoclonal antlbodies targeting differential antigQns in atherosclerotic plaque.
These antigens have included oxidized or otherwise modified ; lipoproteins t~aberland, M.E., et al., Science ~L: 215 (1988)) and glycosylated connective tissue proteins ~Curtiss, L.K. and Witztum, J.L., J. Clin. Invest, 87: 1436 tl983))o While concentrated within the plaque substance, these antigens have also been found in normal artery and/or ` other normal tissues. Some antigens and their corresponding ::;
monoclonal antibodies have shown early promise in the ~; Watanabe rabbit model but have not held up when applied to human lesions (Shih, I.L., et al., Proc. Natl. Acad, Sci., ~: 1436 (1990)), especially when di~fuse markers of extracellular plaque tissue are being sought (Rimura, J., ~t al., Virchows Arch., ~ 2L: 159 (1986)).

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w091/02252 PCT/US~0/04Z72 . -7- 2 ~

The ~u~ject inventio~ providas an ine~pensive, accuratQ
method for ~etermining the pre~ence of atherosclerotic ~' plague both in ~i~Q and i~ ~lYQ- In addition, the ~ubject inv~ntion provides methods of treating persons having ., 5 atherosclerotic plaque which include enzyme treatment, and :~. laser treat~ent. Lastly, the subject invention provides a ~. method of drug delivery to ar2as o~ athero~clerotic plaque.

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"~:' ;, w091~)2~5? pcT/usso/o4272 ~ 8-, ~n .~ The subject invention provide6 a purified antigen indicative of the presence o~ atherosclerotic plaque characterized as having a complex carbohydrate structure having a molecular ~ weight greater than 200,000 dalton~ and being present as an ,~. extracellular component of ~therosclerotic plaqu~. The .~ subject invention also provides a purified zntigen wherein : the antigen is characterized by its ~elective binding to the monoclonal antibody produced by hybridoma Q10~7. These ~:~ antigens are characterized by existing in amounts which vary ~ with the progression o~ atherosclerosis. The subject :~ invention further provides antibodies to these antigens and .: .
~: methods of detecting the presence of both the antigens and ~ 15 the antibodies thereto. ~ethods for treating ? atherosclerosis are also provided.

. The subject invention also provides a method for reducing the amount of atherosclerotic plaque in a blood ve~sel which . 20 comprises: a) contacting the atherosclerotic plague with a reagent which is capable of speci~ically binding to both the ~; plaque and to a proenzyme, the substrate o~ which enzyme i5 a connective tissue present in atherosclerotic plaque which, when clea~ed, is capable of dissolving a component of the plaque under conditions such that the reagent bind~ to the plaque so as to form a reagent-plaque complex; b) contacting s the reagent-plaque complex with the proenzyme to which the ~: reagent specifically binds under conditions such th~t the proenzyme i8 bound to th~ reagen~ forming a proenzyme-reagent-plaque complex; and c) contacting the ~:. proenzyme-reagent-plaque complex with an agent which is capable o~ specifically cleaving the proenzyme 50 that the : proenzyme is converted into the enzyme under conditions such that the enzyme digests the plaque.
;: 35 . , ~ The subject invention further provides a method for ... .
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diagno~tic analysis comprising the ~teps of: a) obtaining a value for the body ~a88 index (BMI) of a patient; b) obtaining a value for the concentration o~ an antigen or ; other ~erum or plasma analytes associated with a pathological condition or an antibody ~hich bind~ with the antigen; c) plotting the bo~y mas~ index of ~he patient :. again~t the antigen or antibody concentration oP the same patient; and d) comparing the resulting ~alue against a set of re~erence ~alue~ to determine whether the re ulting value exceeds the reference value and thereby indicates the ~ . .
presence of a pathological condition.
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wogl/02252 PCTtUS~0/04272 2~ f'l -10-f ~ çription-Qf-~he Ei~U~s.

Figure 1 - Patnways used for developing antibodies to the various stages of the atherosclerotic " 5 plaque ~peci~ic antigen and for testing the antigen and antibodies mad~ thersto.

Figure 2 - DEAE Fractionation (Preparative) Auto-Antibody Assay. The dotted line represent~ the amount of binding of CAD
serum compared to normal serum in each fraction. The solid line represents the amount of protein in each ~raction as r detected b~ absorbance at OD~. The dashed line represents a NaCl gradient ~ro~ O to ~: 1.0 M NaC~. ;
~ 1 ~:` Figure 3 - DEAE fractionation (Analytical) - Antigen capture assay. The dotted line repre~Qnts ~:. 20 the amount of auto-antigen in each fraction as detected by binding to the monoclonal antibody produced by hybrido~a 15H5. The results æhown are for peroxidase conjugated ~: antibody to the antigen and the plateæ are :
read at OD~so~ The solid line represents the ; amount o~ protein in each fraction as detected by absorbance at OD2~. The dash~d line represents a NaCl gradient fro~ O to 1.0 M NaCl.
Figure 4 - Atherosclerotic plaque 15H5-antigen sizing.
A mixture o~ the autoantigen and 4 size : ~arkers tThyroglobulin (a); IgG (b);
Ovalbumin (c); and Myoglobin ~d)] were run through a ~ioSil analytical TSX-400 column.
The auto-antigen was detected by binding ' , . .

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9l/l)t2s2 PCTtUS90/04272 with p~roxidase-labelad 17H3 and 15H5.
Binding 1~ det2rmined by measurlng , absorbance at OD~50.

~; 5 Flgure 5 - Level of IgA which specifically binds to atherosclarotic plaque antig~n ~or persons : with CAD, nor~al persons less than 35 years of age, and normal per~ons greater than 35 ~. years of agQ.
;''' 10 Figure 6 - Levels of atherosclerotic plaque antigen as . determinined by radioimmunoassay ~or person~
.`. with CAD, normal persons less than 35 years ~. of age, and normal persons greater than 35 f, 15 years of age.
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Figur~ 7 - Atherosclerotic plaque antigen level ~or ~ normal persons vs. age.
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Figure 8 - Atherosclerotic plaque antigen lev~l ~or ~ persons with severe CAD, i.e. greater than ,. 50% occlusion, vs. age.
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.~ Figuer 9 - Atherosclerotic plaque antigen levels for `~ 25 persons with ~ild CAD vs. age.
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~5 ` Figure 10 - Level of IgG which specifically binds to ~- atherosclerotic plague antigen ~or normal ~.
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~ persons vs. age. :
i~ 30 i Figure 11 - Level o~ IqG which specifically binds to atherosclerotic plague antigen for persons with severe CAD, i.e. greater than 50%
. occlusion, vs. age. :~
; 35 Figure 12 - Level of IgG which specifically binds to : -, . . .
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~vosl~022s2 ~CT/~S90/04272 athero~clerotic plaque antigen ~or personswith mild CAD v~. ~ge.

. Figure 13 - Level of IgA which specifically binds to athero~clerotic plaque antigen f or nor~al perSOn5 V8. age.

Figure 14 - Level o~ IgA which specifically binds to atherosclerot~c plaque antigen for persons with severe CAD, i.e. greater than SO%
,: oc~lusion, v~. age.
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~ Figure 15 - Level of IgA which ~pecifically ~ind~ to ,~ atherosclerotic plaque antigen ~ox per~ons with mild C~D vs. ag~.

i Figure 16 - Positive pr~valence of atherosclerotic antigen, 1.e. percent o~ persons above normal, V8. age group.
. 20 i Figure 17 - Positive prevalence o~ antibody which specifica}ly binds to atherosclerotic plaque antigen, for various age groupc. Solid bars represent IgG. Cro~s-hatched bars represent IgA.
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.;~ Figure ~8 - Positive pr~valence of either, or both, IgG
; or IgA which speci~ically binds to atherosclerotic plaqu~ antigen for variou~
age groups.
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Figure 19 - Positive prevalence of either antibody (IgG
or IgA) or antigen ~or various age groups.

Figure 20 - A chromatographi~ blank run wi ~ just distilled water u~ing a Dionex instrument J'`' ~'.
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WO9l/022~2 PCT/US90/04272 -13- 2 ~

. ~or monosaccharides analysis with a CPPA-1 col~mn.

~ Figure 21 - Chromatographic run of sevQn standard ;, 5 monosaccharide~ using a Dionex instrumen~
for ~onosaccharide~ analy~is with a CPPA-l column, 15 mM NaOH in purified water.
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~` Figure 22 -Chromatographic blank run with the auto-antigen affinity purification with the 15H5 ~` monoclonal antibody using a Dionex instrument for monosacoharides analysis wi~h a CPPA 1 colu2n, 15 ~M NaOH in purified water.
~5 Figure 23 -Graph of body ~ass index (~MI) again~t IgG+A. Valua~ in each compartment are marked at the 98th percentile, 80 that 98 percent of subjects in such compartment are below the marked threshold. I~ a ~ubj~ct is above the threshold, such sub~ect may be ~; predisposed to atherosclerosi~
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Figure 24 -Graph of body ~ass index (BMI) against antigen. Values in ea~h compart~ent are marked at the 100th percentile, ~o that 100 percent of subjects in such compart~ent are below the marked threshold. If a sub~eck is above the threshold, such 8ub~ ect may be predisposed to atherosclerosis.
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, Figure 25 -Antigen binding inhibitions percent inhibition of binding for antibodi~s produced by hybridoma 15H5 and 17H3 ~hown ; 35 after pretreatment of antigen covered ,~ ~icrobes with various dilutions of CAD
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Figure 26 - Antigen binding inhibition percent inhibition o~ binding of CAD serum after :;
pretreatment of antigen covered microwells with various ~mount~ of the a~tibodiQs produc~d by hybrido~a 15H5 and 17~3.
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~igure 27 - Schematic representation o~ ~thod No. 1 for purifying the forms of the atherosclerotic plaque antigen recognized by the antibod~es ~- produced by hybridomas Z2D3 and QlOE7.
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Figure 28 - DEAE ion ex~hange chromatography ~or CsCl . 15 fraction l. The peaX repre~enting ~he antigen form which binds to Z2D3 i8 deter~ined by the ELISA method using the antibody produced by hybridoma Z2D3.
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20Figure 29 - DEAE ion exchange chromotography ~or CsCl .. fraction 4. The peak representing the antigen ~orm which bind~ to QlOE7 i8 determined by the ELISA method usiny the :~ antibody produced by hybridoma QlOE7.
Figure 30 - Schematic of enzymatic reduction of '.............................. atheroscl~rotic plaque by proenzyme targeting with plaque speci~ic antibody fragments.
A. Representation o~ tha b~functional antibody.
. B. Representation of the bifunctional antigen binding to Z2D3 antigen.
. C. Representation of the enzyme including :., the propeptide portion which inhibits ` enzyme activity.
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D~ R e p r es e n t a t i o n o ~ t h e an~iqen-bif~nct~onal antibody proenzyme compl~x.
E. Representation of the complex agter ~-.~ .
' 5 cleavage o~ the propeptide following ::
treatment with tissue plasminoge~
activator, and the enzyme initiating plaque lysis.
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5~ 0 Figure 31 - Histological staining of rabbit aorta using ,: ths monoclonal antibody produced by ~- hybridoma Z2D3 using the ~BC
. i~munoperoxidase method, and counter stained ~:~
with hematoxylin.
,~ 15 A. Map corresponding to the rabbit ~.
'. histology photograph~
B. Photograph o~ Z2D3 monoclonal antibody l binding in rabbit aorta a~ter the aort~ :
i:: had been denuded. ~.
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, 20 :. Figure 32 - Immunostaining of an un~ixed 5 ~ thicX .
frozen tissue section of human coronary artery, from a patiQnt wi~h advanced atherosclerosis using the A~C :-:
~5 immunoperoxidase me~hod, and counter stained , with hematoxylin. Nagnification is 20x.
;.;~ A. Shows the histological staining of Z2D3 ~: ~onoclonal antibody.
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B. Shows the histological ~taining o~
non-speci~ic IgM monoclonal antibody. .
- , Figure 33 - Immunostaining of an unfixed 5~ thick frozen :~
. tissue section of human coronary artery ~rom :~:
; a patient with early atherosclerosis using ~ 35 the ABC peroxidase method, and .~ counterstained with hematoxylin.

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~'WO 91/02252 PCr/US9~ 4272

3$~3~ ~agnificatio~ is 20X.
$'. A. Show8 th~ histological ~taining of Z2D3 monoclonal antlbody.
B. Shows the histological ~tain~ng of non-speci~ic IgM monoclonal antibody.

~ Figure 34 - Immunostaining of an unf ixed 5 ~ thick .( frozen tissue section of human coronary artery ~ro~ a patient with athero clerosis, ucing the ABC peroxidase ~ethod, counterstained with hematoxylin.
.~ ~agnification is 20X.
A. Shows the histological staîning of Z2D3 .~` ` monoclonal antibody.
lS B. Shows the histological Etaining of QlOE7 monoclonal antibody.
Ca Shows! the histological staining o~
: non-specific IgM monoclonal antibody.

Figure 35 - Co~parison of a section of rab~it aorta ~rom the thorax region which was untreat~d, a, and a section of aorta from the ~ame rabbit ; which was denuded 80 as to produce athero~clerotic plaque, b.: Pr~or to ~ sacr~icing the rabbits, the rab~its were injected via the ear vein with In-DTPA-Z2D3 monoclonal antibodiQ~.
A. Photograph ~howing tho gross morphological dif~erencQs between a section o~ aorta without plaque, a, and a section of aorta with plaque, b.
; B. Photograph of an autoradiograph sho~ing - increased binding of 11~ In-DTPA-Z2D3 in .~ the region o~ the aorta containing ~ ~35 atherosclerotic plaque, b.

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WO~t/022s2 PCT/US90/~272 et~il~d ~es~riPtion o~ the Invention ~ The subject invention a purified antigen indicative of th~
,; presencP of atherosclerotic plaque characterized as having a complQx carbohydra~e structure and a molecular weight .-, greater than 200,000 daltons and as being present as an extracellular component of atherosclerotic plaqu~.

~:~ This antigen is characterized by bæinq synthesized by, or present in, smoot~ ~uscle cells. It has been purified and is characterized by lts selective binding to the monoclonal antibody produced by hybridoma 15H5 (ATCC Accession No.
,.......... HB9839). This antigen is further characterized by being '.` neutral in charge. This antigen is also characterized by haYing a carbohydrate profile depicted in Figure 22. It has been determined that the 15H5 antigen ~electively binds to lectins. ~ccordingly, this can be ~urther characterized by s` binding to the lectins CQ~avalia ens.ifor~ia, ~ri5 ~ vulaaris, ~ens culinaris, ~icinuS co~monis, and Tri~icum t .:
;,......... 20 vulqzrl~, and by no~ binding to the lectins Ara~his hvDqaea, ~` Bandeiraea sim~licitolia, Dioli~hos bifloru~, gly~ ~y~
Limulus Poly~henus~ Phaseolus ~laari3-E, ~h~as~olus .. vulqaris-L, ~isum ~atiYu~~ SophPv~ Ponica, ~lex europaç , ~: Ulex europaeus, and Vicia villosa.
~5 Another way of determining the characteristic~ o~ a molecule ;. is examine the actions of various enzymes upon the molecule.
The 15~5 antigen is ~urther characterized as being resistant to degradation by proteinases, deoxyribonuclease~, lipases, and ribonucleases, while baing partially ~uscQptible to degradation by ~-amylase, B-amylase, and glucoa~ylase.
, . , ': To determine whether the 15H5-antigen was a molecule known ; to be associated with atherosclerotic plaqu~, the antigen , 35 was evaluated for binding to antibodies which specifically bind to known components of atherosclerotic plaque. The ~:
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Other characteristics o~ the antigen include being reactive with the monoclonal antibody produced by hybridoma 15~5 (ATCC Accession No. EB98~0) after the antigen has been boiled for one hour, and being reactive wi~h ~he monoclonal antibody produced by hybridoma 15H5 (ATCC Accession No.
HB9840) after the antigen has been treated with 8 ~ urea in phosphate buf~ered saline for 24 hours at room temperatur~, 6 M suanidine HCl in phosphate bu~fered saline for 24 hours at room temperature, 2 M trifluroacetic acid for 30 minutes at raom te~perature, 3.5 ~ sodium thiocyanate ~n phosphate ~ bu~fered 6aline for 8 hours at room temperature, or 0.19 M
~odiu~ dodecyl sulfate in phosphate buffered saline for one hour at roo~ temperature.
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The 6ubject invention also provides an antigen or epitop~
~j~ associated with atherosclerosis and so~e normal tissue, ; characterized by its selective binding to the monoclonal ~ antibody produced by hybridoma 17H3 (ATCC Accession No. HB
j~ 10189).
Another antigen ifi provided by the subject invention, which antigen is characterized by being synthesised by, or present in, atherosclerotic plaque connective tissue and plaque smooth muscle cells is the antigen characterized as being a ... ..
lipid-containing molecule which selQctively binds to the monoclonal antibody produc~d by hybridoma Z2D3 (ATCC
Accession No. HB 9840) or by hybrido~a Z2D3/3E5 (ATCC
Accession No. HB 10485). This antigen may be furthar characterized by having its ability to be used for histologiaal staining destroyed upon treatment with ac~tonQ.
, . w09l/02~s2 PCT/USso/04272 . ~lg--2 ~ 5 ~
Also provid~d are antigen~ indicative of the presence of nor~al ~mooth muscle cells. One such antigen i8 characteriz~d by its selective binding to the monoclonal ; antibody produc~d by hybridoma QlOE7 ~ATCC Acc~ssion No. HB
10188). ~he molecular weiqht o~ thi5 antigen i~ gr~ater . than 150,000 daltons. This anti~en i8 further ~`
:. characterlzed by being synthesized by, or pre88~t in, normal '.............. smooth mu6cle cells and normal connective tissue ~urrounding arteries.
': 10 In one embodiment of the subject invention, an above-described antigen is labeled with a dete~table marker.
This marker may be any marker known to one skilled in the art. However, in the preferred embodiment the marker i~ an enzyme, a paramagnetic ion, biotin, a ~luorophore, a chromQphore, a heavy metal, or a radioisotope. In mo~t cases involving immunoassays, the preferred marker is an enzyme, pre~erably horseradiRh peroxidase or ~lXaline m phophatas~.
~ 20 ;~.~ The subject invention also provides puri~ied antibodies which spec~fically bind to an atheroclerotic plaque antigen or to an antigen associated with normal smooth muscle cells :~ and connective tissue. In one embodiment, the antibcdy i5 labeled with a detectable marker. The choice of marker u~ed will vary depending upon the application. However, the ;~ choice of marker is readily deter~inable to one s~illed in .~ the art. In a pre~erred embodiment of th~s invention the ~ marker is an enzyme, a para~agnetic ion, biotin, a ~.. 30 fluorophore, a chromophore, a heavy metal, or a radioisotope. These labeled antibodies may be used in . immunoassays as well as in histological applications to s detect the presence of atherosclerotic plaque. In such .. ~ applications it is preferred that the marker is an enzyme, '........ 35 and it i8 most pref~rred that the enzyme i8 horseradish ~ pQroxidase or alkaline phosphatase.

,,, :

... .

,, .

wosl/02252 ~ PCT/US90/~427 The above-identified antibodies may b~ either polyclonal or monoclonal, with the monoclonal antibody being a preferred embodiment.

This invention provides ~onoclonal antibodies directed to atherosclerotic plaque antigens which include the monoclonal antibody produced by hybridoma 15H5 (ATCC Accession No.
~B9839); the monoclonal antibody produced by hybrido~a Z2D3 (ATCC Acc~ssion No. HB9840) and Z2D3/3E5 (ATCC Accession No.
HB 10485), an IgG, which is a class switc~ variant of Z2D3, which is an IgM, as well as other daughter cell lines of Z2D3 such as Z2D3/5C5 (an IgG); and the ~onoclonal antibody produc~d by hybridoma 17H3 ~ATCC Accession No. EB 10189).
The monoclonal antibody produced by hybridoma QlOE7 (ATCC
Ac~ession No. HB 10188) is directed toward normal artery.
Hybrido~as 15H5, Z2D3, Z2D3/3E5, 17H3 and QlOE7 were deposit~d pursuant to, and in satisfaction of, the requirements of ~h2 Budape~t Treaty on the International Recognition of the Deposit ~f ~icroorqan~ 8ma for the Purposes of Patent Procedure with the American ~ype Culture Collection (ATCC), 12301 Parklawn Drive, Rockville, Maryland ` 20852 under A~CC Accession Nos. HB 9839, HB 9480, HB 10485, HB 10189, and HB 10188, respectively.
. ".
~he invention provides a recombinant polypeptide which comprises an amino acid sequence which is substantially the same as ~he amino acid seguence of the hypervariable region of monoclonal anti~ody Z2D3, Z2D3/3E5 and other daughter ; cell3 lines of Z2D3 or QlOE7. One may also obtain such a polypeptide by nonrecombinant methods, such as ~or example, proteolytic digestion.

A chimeric antibody or a fragment thereof comprising such a recombinant polypeptide is also provided, particularly a chim~ric antibody comprising the amino acid ~equences of a hum~n ~ramework region and o~ a constant region from a human wo~l/02~s2 PCT/~S90/04272 . .
-21- ~ ~iJ~ 6~J~

antibody 80 a~ to "humanize" or render nonimmunogenic the hypervariable region of the mouse Z2D3, Z2D3/3E5 or QlOE7.
~180 provided i8 the polypeptide or chimer~c antibody or fragment derived by site-directed mutagenesi6, ~specially R~te-directed mutagenesis which con~ers eguivalent or better binding properties. The fragments of the chimeric antibody include Fab, F(ab)2, Fv and V~ fragments.
'~.',:
The ~ubject învention also provides for an atherosclerotic plaque antigen bound to a solid support and an antibody which specifically binds to an atherosclerotic plaque antigen bound to a solid support. In ~ pre~erred embodiment, the monoclonal antibody produced by hybridoma 15~5 is bound to a solid support.
j 15 ;~ Anti-plaque antibody or the atherosclerotic plague antigen may be bound to an insolu~le support by conventional process~s. Procedures ~or binding o~ antibodies to insoluble supports are described in U.S. Patents 3,551,555, 3,553,310, 4,048,298 and RE-29,474, ~or example. Binding o~
antibodies to polystyrene by adsorption has been described , in U.S. Patents 3,646,346 and 4,092,408, for example.
Binding o~ protein containing antigens to a variety of insoluble supports has been described in U.S. Pate~t 3,720,760.
,. . ~
A variety o~ materials may be used as the insoluble support, the primary consideration being the binding characteristics o~ the anti-plaque antibody or the plaque antigen to the ~ 30 sur~ace, the absenc~ o~ inter~erenc2 with ~he anti-plaque : .
~ antibody and plague antigen con~ugating reaction or with ~ . . .
other reactions which may be employed to determine the presence snd extent of the conjugating reaction. Organic and inorganic polymers, both natural and synthetic, can be used as the insoluble support. Examples o~ ~uitable polymers include polyethylene, polypropylene, polybutylene, .
,. .
.. . ~ .
., , Wo9l/0-2s2 PCT/US90~042~2 poly(4-methylbutlyene~, ~utyl rubber, silastic polymer6, ^~ polyesters, polyamides, cellulose and cellulose derivatives ; (such a6 cellulose acetate, nitrocellulose and the like), acrylate~, methacrylates, vinyl polymers (~uch a polyvinyl ; 5 acetate, polyvinyl chloride, polyvinyl~dene chlorid~, polyvi~yl fluoride, and the like), acrylate6, methacrylates, s:: vinyl polymers (such a~ polyvinyl acetate, polyvinyl . chloride, polyvinylidene chloride, polyvinyl fluoride, and the li~e), polystyrene and ~tyrene graft copolymer6, rayon, ~i 10 nylon, polyvinylbutyrate, polyformaldehyde, etc. Other ~:~ materials which can be used as the insoluble suport can ~he ~ latexes of the above polymers, sil$ca gel, silicon wafers, `~ glass, paper, insoluble protein, metals, metalloids, m~tal t` oxides, magne~ic materials, ~emi-conductive materials, ~- 15 cermets and the like. In addition are included ~ubstance~
which for~ gels, such as proteins such as gelatins, lipopolysaccharides, silicates, agarose, polyacrylamides or i polymers which form several aqueous phases such a~ dextra~s, i polyalkylene glycols (alkylene w~th 2 to 3 carbon atoms) or surfactant6, e.g. amphophilic compounds ~uch as ;~ pohospholipids, long chain (~2-24 carbon atoms) alkyl ~:. ammonium salts and the like.
. ~
. One diagnostic support comprises polystyerene, styrene copolymers, or polyolefins such as polyethylene .or `.~ polypropylene, and acrylate and methacrylate polymers and copoly~ers. The anti-plaque reagent antibody or the plaque antigen can be bound to the i~soluble support by adsorption, . ~onic bonding, van der Waals adsorption, electrostatic bonding, or other non-covalent bonding, or it can be bound : to the insoluble support by covalent bonding. A
~ particularly advantageous support for ~his procedure .~ comprises a microtiter plate having a plura}ity o~ wells.
The well surface or plastic cup inserts therein can constitute the antigen or antibody support. I~ the determination will require the use of fluorometric ::~

,.', ~
"

'~ W091/02252 PCT/US90/04272 ~23- 2~Q.~t...

:~ measurements, the microtiter plat~ or the well in erts are advantageously opaque to light 80 that ~xcltation light , applied to a well does not reach or in~luence conte~ts of 3- the 6urrounding wells.
, 5 . Procedure6 for non-covalent bonding are describ~d ln U.S.
Patent 4,528,267. Procedur~s ~or covalently banding antibodies and antigens to insoluble supportQ are described by Ichiro Chibata ~Immobilized Enzymes, Halsted Pres.: New Yor~ (1978)] and A. Cuatrecasa, [J. Bio. Che~., ~ 3059 (19~0)], the entire contents of which are hereby incorporat~d by reference. The surface can be coated with : a protein and coupled with the antibody or antigen using : procedures described in U.S. Patent 4,210,418 using glutaraldehyde as a coupling agent, ~or exa~ple. In a stlll `~ further procedure, the well can be coated with a layer having free isocyanate groups such a~ a pelyether isocyanate, and application of the antibody or antigen in aqueous solution thereto e~ects the requisite bonding. In ~ `20 a ~till further procedure, the antibody or antigen can be ; coupled to a hydroxylated material by means of cyanogen i bromide as described in U.S. Patent ~,720,760.
: .
The subject invention also provides a method for detecting in a biological sample an antigen present in, and indicative o~ the presence of, atherosclerotic plaque which comprises . contacting the biological fluid with the antibody which : specifically binds to the atherosclerotic plaque antigen under conditions such that the antibody bind~ to the antigen to form a detectable complex, detecting the co~plex 80 ~ormed and thereby detecting any antigen in the biological sample.
.., :. In a pre~erred method the biological sample is a tissue sample. Tissue samples may be used in a variety of histological techniques lncluding but not lim~ted to ~hose ,`. ' , .
,, , '' ~
:

. w~9l/022~ ~ PCT/US~7/0~72 ;,~
. illustrated ~hroughout ths application.
'''~' In another embodiment the biological ~ample i5 a biologlcal ;,,s fluid. It is preferred that the biological fluid comprises ; 5 blood, plasma, or ~erum. However, in the more preferred ~: embodiment the biological fluid i5 serum. To fur~her aid in detecting the complex it is preferrQd that the antibody :::. which binds specifically to the athero6clerotic plaque :i.
antigen is labeled with a detectable marXer. The choice of ~` 10 mark@r is readily determinable to one skilled in the art.
x . In one embodiment of the subject method, the antibody is a monoclonal antibody and `more preferably the monoclonal .antibody is produced by hybridoma lSHS (ATCC Accession No.
' H~9839). To further aid in detecting the complex, it i8;. 15 pre~erred that the antibody be bound to a 601id 6upport.
$. one preferred solid æupport is a bead ~ormed og an inert ~ polymer a~d another is a microwell.
,,, :
The sub; ect invention also provides a method ~or ~: 20 quantitatively determining in a sample of a biological ~luid ,. the concentration of an antigen which is present in, and .
'3 ~ indicative of the presence o~, atherosclerotic plaque which comprises: a) contacting a solid support wi~h an exce~s o~
an antibody which binds specifically to an atherosclerotic ::. 25 plague antigen under ~onditions permitting ~he antibody to ~j.,.
i,. attach to the surface of the solid support; b) removing unbound antibody; c) contacting ~he resulting solid ~upport ~. to which the antibody i~ bound with the sample of the .. biological ~luid under condition~ such that any antig~n 5: 30 present ~n the sample binds to the bound antibody ~nd for~s :.
a complex therewith; d) removing any antigen which i8 not bound to the complex; e) contacting any complex 80 formed . with an excess of a detectable reagent which specifically binds to any antigen present in the comp}ex so as to form a s~cond complex which include~ the antibody, the antigen, and .-the detectable reagent; f) removing any detectable reagent Y' ,.,~ ' .

''; ' .

. .. , ... ... - . . . : 7 . .. . ..
, ~ . .

wo9l/n225~ PCT/US90/0~272 -25- 2~ r~

which iY not bound in the eco~d complex; g) quantitativaly ~-determining the concentration of detectable reagent present in the second complex; and h) thereby quantitatively datermining the concentration of antigen in the biological fluid.
:' One embodiment of this method is wherein the biological fluid comprises blood, plasma, or ~erum. ~ore preferably, the biological fluid is 6erum.
, In one embodiment the solid support is a bead formed of an inert 2olymer and in another the solid support is a microw~ll.
~' ' In one embodiment the reagent is labeled with a detectable marker, the choice of ~arkar being determinable by one skilled in the art.
~ ' In a preferred e~bodiment, the marker i5 an enzyme, a paramagnetic ion, biotin, a ~luorophore, a chromophore, a heavy metal, or 2 radioisotope. .~.lthough any reagent capable o~ detecting tho atherosclerotic plaque antigen may ;~
be employed, it is preferred that the reagent is ~n antibody lab~led with a detectable marker. Again, it i8 preferred 25 that the marker is an enzyme, a paramagnetic ion, biotin, a fluorophore, a chromophore, a heavy metal, or a radio~sotope. More prf2rably, the marker i8 an en2yme, particularly e~ective anzymes being horseradish perosidase alkaline phospha~dse.
The sub~ect invention al80 provides for the above method wherein the detecta~le reagent is labeled with an enzyme and step (g) comprises contacting the second complex with a specl~ic ~ubstrate ~or the enzyme under conditions such that the enzyme reacts with the substrate to form a detectable product.

, . .. .. ,. .. ~ . - -. . ~ ~

w09l/0~252 PCT/US90/04272 ` ' ~'3 `~ 26-~noth~r provision of the 6ubject invention i8 a m~thod for d~ ecting in a biological sample an antibody whi~h specifically ~orms a complex with an antigen present in, and indicative of the presence of, atherosclerotic plaque which comprises contacting the biological sample with an atherosclerotlc plaque antigen under condition~ ~uch that the antigen binds to the antibody in ~he biolo~ical ~ample and det~cting the antigen bound to the antlbody and therQhy detecting the antibody in th~ biological ~ample.

In a preferred embodiment the biological sample is a ti~sue sample. Ti~sue samples mày be used in any histological tachnique known to one skilled in the art to detect ~nd guantify the amount of antibody in the sample. The methods include, but are not limited to, the illustrations provided throughout the application.

A preferred embodiment of the above-described method is wherein the biological sample is a biological fluid. In one preferred embodiment the biological fluid comprises blood, plasma, or serum. More preferably the biological fluid ~8 serum. ~o aid in detecting the complex formed, it is preferred that the antigen is labeled w~th a detectable marker. Another embodiment of the invention i8 wherein the antigen is bound to a solid support. Thi~ allows the complex to be readily separated from the biological fluid and be detected. One preferr~d embodiment is wher~in solid support i5 a bead formed o~ an inert polymer, and another is wherein the solid support is a microwell.
The subjeat invention also provides a method for quantitatively determining in a sample o~ a biological ~luid the concentration of an antibody which specifically forms a complex w~th an antigen present in, and indicative of the presence of, atherosclerotic plaque which comprises: a) contaGting a solid support with an Qxcess of an " . ~

W~9l/~22~2 PCTtUS9~/04272 -27- 2 ~ ~ ~ "~

athero~clerotic plaque antigen under conditions permitting the antigen to attach to the surface o~ the solid support;
~) removing unbound antigen; c) contacting the re~ulting solid support to which the antigen is ~ound with the sample of the biological fluid under conditions ~uch that any antibody pr~æent in the sample binds to ~he bound antigen and forms a complex therewith~ d) removl~g a~y antibody which is not bound to ~he complex; e) contacting any complex 50 formed with an exces o~ a detectable reagent which specifically binds to a~y antibody present in the complex ~o as to ~or~ a seoond complex which includes the antigen, the antibody, and the detectable reagent; f) re~oving any detectable reagent which is not bound in the second complex;
g) quantitatively determining the concentration of detectable raagent present in the second complex; and h) thereby quantitatively determining the con~entration o~
antibody in ~he biological ~lu~d.

In one preferred embodiment the biological fluid co~prises blood, plas~a, or serum. Mor~ pre~erably, the biological fluid is ~erum.

To better detect the complex ~ormed it is preferr~d that the reagent i5 labeled with a detectable marker. Preferably, the marker is an enzym~, a paramagnetic ion, biotin, a ~luorophorQ, a chromophore, a heavy metal, or a radioisotope. More preferably, the marker is ~n enzyme.
The most pre~erred embodiment is when the enzyme is horseradish peroxidase or alkaline phosphatase.
A ~urther embodiment o~ the above-described method i8 wh~rein the detectable reagent is labeled with an enzy~e and step (g~ comprises contacting the second co~plex with speciric substrate to the enzyme under conditions such that the enzyme reacts with the substrate to form a detectable product.

., j :: . - - ~ - , .. ..

W091/0225~ PcT/usso/o4272 j~Ss- -28 The ~ubject invention discloses a method for quantltatively determining in a sample of a biological ~luid the concentratlon of an antigen whlch is pres~nt in, and indicative of the presence of, atherosclerotic pl3qua which comprise3: a) contacting a solid 6upport with a pr~determin~d amount of an antibody which bind~ specifically to atherosclerotic plaque under conditions permitting the antibody to attach to the surface o~ the ~upport; b) removing unbound antibody; c) contacting the resulting ~olid ~upport to which the antibody is bound with a predetermined amoun of antigen labeled with a detectable ~arker and wit~
a sample of the biologieal fluid under condition~ such ~hat the antigen binds to the antibody bound to the solid 6upport and for~s a complex therewith; d) removing labeled antigen ~hich is not bound to the complex; e) quantitat~vely determining the concentration of labeled antigen bound to the solid support; and f) thereby quantitatively determining tha concentration of antigen in the biological ~luid.

In one e~bodiment the biological fluid comprises blood, plasma, or serum. However, the presently preferred ~;
biological fluid is serum.

One preferred solid support is a bead formed of an inert polymer, and another is wherein the solid support i8 a microwell.

In one e~bodiment of the subject method the detectable marker ~ 8 an enzyme, a paramagnetic ion, biotin, a ~luorophore, a chromophore, a heavy metal, or a radioi~otope. Pre~erably, the marker is an enzyme, and more preferably the enzyme is horseradish peroxidase or alkaline phosphatase.

In a ~urther embodiment of the above-described ~ethod, the antigen is labeled ~ith an enzyme and step (e) comprises ?

wosl~o22s2 PCT/US9OtO4272 -29- 2~ J'-~

contacting the labeled antigen bound to the ~olid ~upport with specific substrate to the enzyme under condition6 such that the enzyme reacts with the ~ub5tr3te to form a : detectable product.
The subject invention further provides a method for quantitat$vely determining ln a sample of a biological fluid the concentration of an antigen which is presen~ in, and indicativ~ of the presence of~ a~herosclerotic plaque which comprises: a~ contacting a so}id support with a predetermined amount of an antibody which specifically binds to an atherosclerotic pla~ue antigen under conditions permitting the antibody to attach to the surface of the support; b) removing any antibody not bound to the ~upport;
c) contacting the solid support to which the antibody i~
bound wi~h the sample of ~he biolo~ical fluid under conditions such that any antigen present in the sample bind~
to the bound antibody and forms a complex therewith; d) ramoving any antigen which i5 not bound to the complex; e) contacting the complex so formed with a predeterminQd amount of plague antigen labeled with a detectable marker under conditions such that the labeled antigen competes with the antigen from the biological fluid for binding to the antibody; f) quantitatively determining the concentration of labeled antigen not bound to the solid support, and g) thereby quantitatively determining the conc~ntration of antigen in the biological fluid.

One embodiment of this method is wherein the biological ~luid comprises blood, plasma, or serum. Preferrably, the biological ~luid is serum.

In one embodiment, the solid support is a bead formed of an inert polymer, and in another, the solid ~upport i5 a microwell.

. ., .. ,.. , . . . .. ,.. ... ..... . -. .

WO gl/o~5~ ,~ PCl/US9~/04272 ~ ;J ~J '_ ' ~ -30-A~ discussed hereinabove, the choice o~ markar i~ readily determined by one ~killed in the art. Ho~aver, in a prsf~rred embodiment the marker i8 an enzyme, a para~agnetio ion, biotin, a ~luorophore, a chromophore, a heavy metal, or a radioisotope. More preferably, the marker i~ an enzyme.
Although many enzymes produce a detectable product, pre~errad enzymes are horseradish peroxidase and alkaline pho6phatase.

In a ~urther embodiment of this method, the antigen i labeled with an enzyme and step (f) comprise~ remoYing the labeled antigen which was not bound to ~he ~olid ~upport and contacting ~t with a speoific substrate to the enzy~e under condi~ions such that the enzyme reacts with the substrate to form a d~tectable product.

Another me~hod provided ~or the subj~ct inv~ntion i8 a ~ethod ~or quantitatively determining in a sample o~ a biological ~luid the concentration of an antibody which specifically forms a complex with an antigen which i8 present in, and indicatiYe of the presence o~, atherosclerotic plague which comprises: a) contacting a solid support with a predetermined amount of an atherosclerotic plague antigen under conditions permitting the antigen to attach to the surface of the ~upport; b) removing unbound antigen; c) contacting the resulting solid support to which the antigen is ~ound with a predeterminod amount of an antibody labeled with a detectable marker and with the sample of biological fluid under ~ondition~ such that the antibody binds to the antigen bound to the solid support and for~s a complex therowith; d) removing any antibody which is not bound to the complex; e) guantitatively determining the concentration of labeled an~ibody bound to the solid ~upport; and f) thereby quantitatively determining the concentration of antibody in the biological Pluid.

wosl/o22s2 PCT/US90/04272 -31 ~ 3 ~ 3 One embodiment of this invention is wherein the biological fluid comprises blood, plasma, or serum. More preferably, the biological fluid is ~erum.

In one pr~ferr~d embodiment, the ~olid 6upport i8 a bead for~ed o~ an inert polymer, and in another, ~he ~olid ~upport is a microwell.

T~e choice of detectable marker may be readily determined by one skilled in the art. It is preferred, however, that the detectable marker is an enzyme, a paramagnetic ion, biotin, a fluorophore, a chromophore, a heavy metal, or a radioisotope. More preferably, the marker is an enzyme, and mo8t preferably the enæy~e is horseradish peroxida3e or alkaline phosphatase.

In a ~urther embodiment of this method, the ant~body i8 labeled with an enzyme and step(e) comprises contacting the labeled antibody which was displacèd ~rom the solid ~upport with a specific substrate to the enzyme under conditions ~uch that the enzyme reacts with the substrate to form a detectable product. :~

The subject inYention ~urther dlscloses a method for guantitatively determining in a sample of a biological flu~d the Goncentration of antibody which speci~ically forms a ~omplex wi~h an atherosclerotic antigen which is present in, and indicative of the presence of, atheroscleroti~ plaque which comprises: a) contacting a solid support with a predetermined a~ount of an athQrosclerotic plaque antigen under conditions permitting the antigQn to attach to the surface of the support; b) removing any antigen which is not bound to the support; c) contacting the solid support to which the antigen is bound w~ith th~ sample of the blological ~luid under conditions such that any antibody present i~ the sample binds to the bound antigen and forms a complex ~09l/~2252 PCT/US90/04272 ~ 32-therewith; d) removing any antibody ~hich is not bound to the complex; e) contacting the complex so formed with a predetermined amount of a pla~ue antibody labeled with a detectable marker under conditions such that the labeled antibody competes with the antibody in the biological fluid for binding to the antigen; f) quantitatively deter~ining the concentration of labeled antibody not bound to the ~ol~d support; and g) thereby quantitatlvely determining the concentration of antibody in the biological fluid.

In one embodiment thP biological fluid comprises blood, plasma, or serum. However, the preferred biological fluid is serum.

The choice of solid ~upport may be readily determined by one ~killed in the art. In one preferred method, the solid support is a bead formed of an inert polymer, ~n another the solid support is a microwell. The markers used in the above-described method are a matter of choice to one skilled in the art. It is preferred that the detectable marker is an enzyme, a paramagnetic ion, biotin, a fluorophore, a chromophore, a heavy metal, or a radioisotope. More preferably, the marker is an enzyme, and most preferably, the enz~me is horseradish peroxidase or alkaline phosphatase.

A ~urther embodiment of this method is wherein the antibody is labeled with an enzyme and step ~) comprises removing the labeled antigen which was not bound to the 601id support and contacting it with specific substrate to the enzyme under conditions such that the enzyme reacts with the substrate to form a detectable product.

The subj2ct invention also provides a method for monitoring the progression o~ atherosclerosis which comprises determining the amount of atherosclerotic plaque antigen W091/02252 PCT/US90/0~272 ~33~ 2~
present in a ~ample o~ biological ~luld of pati~nt, and comparing the amount determined with the amount detarmined at sarliar points of time, any cha~ge ln the amount of antigen indicating a change in the ext~nt of atheroscl~rotic plaque. , Anoth~r provision o~ this invention i8 for a method for monitoring the ~fficacy of treatment of athero~clerosis which comprises determining the æmount o~ the atherosclerotic plaque antigen pr~sent in a ~ample of a biological ~luid of a patient and comparing khe amount determined at earlier points in time with a change in the amount of antigen indicating a change in the extent of a~herosclQrotic plaque. .
Further di~closed ~y the invention is a reagent for use in imaging atherosclero~ic plaque which comprises an antibody which binds specifically to atherosclerotic plaque antigen labsled with a detectable marker. This invention al80 provides a composition comprising an amount of thi~ reagent and a physiologically acceptable carrier.

The detectable marker used is a matter o~ choice to one skilled in the art. It is pre~erred that the marker be a radioactive isotope, an element which is opague to X-rays, a paramagnetic ion, or a chelate of a paramagnetic ion.

~adioactive isotopes are commonly used in medicine and are well known to tho~e skilled in the art. It is presently prs~err~d that the markar be I-123, I-125, I-128, I-131, or a chelated metal ion o~ chromium-51, cobalt-57, gallium-67, indium-111, indium-1~3m, mercury-197, selenium-75, thaliu~-201, technetium-99m, lead-203, strontium-85, strontiu~-87, gallium-68, samarium-153, europium-171, ytterbium-169, zinc-62, rhenium-188, or mixtures thereof.
Preferably, the marker is technetium, iodine, indiu~ or a -~

wosl/02~s2 PCT/US90/04272 ?.

metal ion chelate ther~to.
'' In another embodiment o~ the above-id~ntl~ied UQthod, the marker i5 a paramagnetic ion. Paramagnetic ion~ ~re also commonly used in medicine. Examples of ~uch markers included ch~lated m~tal ion of chromium (III), manganese ~II), iron (III), iron (II~, cobalt ~II), nickel (II), copper ~II), praseody~ium ~III), neodyminum (III), ~a~ariu~ (III), gadolinium (III), terbium (III), dysprosium (III), hol~iu~
(III), erbium (III), ytterbium (III), or mixtures thereo.

The subject invention also provides a method ~or imaging atherosclerotic plaque which comprises contacting the atherosclerotic plaque to be imaged with a reagent which bind6 specifically to the atherosclerotic plaque antigen described above, under conditions such that ~he reagent binds to the atherosclerotic plaque and detecting the reagent bound thereto, thereby imaging the atherosclerotic plaqu~.
Also provided is a ~ethod for imaging atherosclerotic plaque and ad;acent normal tissue which comprisea ~ontacting the normal lumen to be imaged with an antibody which speci~ically binds to normal intima and/or media and which ~8 labeled with a detectable marker; contacting the atherosclerotic plaque with a reagent described above under condition~ such that the reagent binds to the atherosclerotic plaque; and detect~ng the reag~nts bound to the atheroscleroti~ plaque and ad~acent normal ti6SUQ, th~reby imaging the atherosclerotic plaque and ad~acent normal tissue. The antibody which specifically binds to normal intima and/or media is a purified antibody whi~h spscifically binds to an antigen characterized by being synthesized by, or present in, normal smooth muscle cells and normal connective tissue surrounding arterie3. In a preferred embodiment, the antibody is a monoclonal antibody W(~ 91/Q~252 PCT/US~0/~4272 --3 5-- 2 J ~

produced by hybridoma QlOE7 (ATCC Accession No. HB 10188).

The subject invention provides reagents for use ~n the method described above for imaging normal intima and/or media comprising an antibody labeled with a detectable marker a~ well as a composition comprising an e~fective imaging amount of ~uch reag~nts and a phy~iologically acceptable carrier.

As described for the reagent for use in imaging atherosclerotic plaque, the detectable marker u~ed is a matter of choice ~o one skilled in the art. It i8 preferred that the marker be a radioactive isotope, an element which i8 opaque to X-rays, a paramagnetic ion, or a chelate of a para~agnetic ion. Markers that may be used in imaging normal ti~sue correspond to those described above ~or imaging atherosclerotic plaque.

Another provision of the subject invention i~ a method for monitoring the progression of atherosclero~is which compris~6 determining the amount of an atherosclerotic plaque speci~ic antigen presant in a patient'~ blood vessels and comparing the amount determined with the amount determined at earlier points in time, a chang~ ~n the amount of antigen indicating a change in the extent of atherosclerotic plaque.

Further provided is a method for monitoring the e~icacy o~
treatment o~ atheros~lerosis which comprises determlning the amount o~ an atherosclerotic plaque speci~ic antigen present in a patient'~ blood vessels and comparing the amount determined with the amount determined at earlier points in time, a change $n the amount of antigen indicating a change in the extent of atherosclerotic plaque.
. . :
Also provided for is a method ~or imaging atherosclerot$c ..
., , ~

Wo91/02252 PCT/US90/04272 . & . ~, .~J .J -36-plaque in a subj~ct which comprises: a) contacting the blood vessel wal}s containing atherosclerotic plaqu~ with the above-described reagent for imaging plaque; b) detecting the reagsnt bound to th~ atherosclerotic plaque; and c) imaging the a~herosc}erotic plaque~ ;

A meth~d for imaging atherosclerotic plaque and adjacent normal tissue in a su~ject which comprises contacting the nor~al lumen to b~ imaged with an antibody which speci~ically blnds to normal intima and/or ~edia and which is labeled with a detectable marker; contactinq the blood vessel walls containing atherosclerotic plaque and surrounding area to be imaged with the reagent o~ claim 118 under conditions such that the reagent bind~ to the atherosclerotic plaque; and detecting the reagents bound to the atheroscl~rotic plaque and adjacent normal tissue, thereby i~aging the atherosclerotic plaque and adjacsnt nor~al tissue. In a preferred embodiment, the antibody which speci~ically binds to normal intima and/or media i9 a monoclonal antibody produced by hybridoma QlOE7 (ATCC
Accession No. HB 10188).

Imaging may be done through any of the methods known to one skilled in the art. These methods include but are not limited to X-ray, CAT scan, PET scan, NMRI, and ~luoroscopy.

An alternative approach to removing plaque is by en~yme digestion. The sub~ect invention provides a reagent ~or use in digesting atherosclerotic plaque which co~prises an antibody which binds specifically to atherosclerotic plaqu~
bound to an enzyme capable of digesting a component o~ `~
atherosclerotic plaque. One such reagent comprises the monoclonal antibody produced by hybridoma Z2D3 or Z2D3/3E5 .
or other daughter cell lines and another comprises the 15~5 or 17~3 monoclonal antibody. ~ .
' "

' :' .

wosl/o22s2 PCT/US90/0427~

31 f ~
Another ~uch reagent comprises the chimeric antibody described above or a fragment thereof comprising the recombinant polypeptide which comprises an ~mino acid sequence which is substan~ially the ~ame as the amino acid sequence o~ the hypervariable region of the monuclonal antibody produced by hybridoma Z2D3 or by Z2D3/3E5.
Further, the antibody may comprise the amlno acid ~equences of a human framewor~ region and o~ a constant region fro~ a human antibody. Such chimeric antibody ~ay be a genetically engineered hybrid neomolecule conjugated to the enzy~e or to the proenzy~e, such that the neomolecu}e is partially an antibody and partially an enzyme. The chimeric antibody ~ay also be a bifunctional antibody. The bifunctional ~ntibody i8 usually produced by a quadroma. In a preferred emb~diment, the quadroma is derived from the ~usion o~ a hybridoma c~ll line Z2D3 or Z2D3/3E5 and a hybr~doma sscreting a ~onoclonal antibody binding an enzymQ.

The enzyme may be any enzyme capable of digesting a ; 20 component of the plaque. In a preferred embodiment, the enzyme is a proteinase, an elastas~, a collagenase, or a saccharidase. In a particularly preferred embodiment, the enzyme i8 f ibroblastic collagenase, gelatinase, polymorphonuclear collagenese, granolocytic collagenase, stro~ely~in I, stromelysin II, or elastase.
/

The subject invention also provides a composition comprising an amount o~ the above-described reagent e~fective to digQst a component of atherosclerotic plaqu~ and a physiologically acceptable carrier. `

The sub~ect invention provides a method gor reducing the amount of atherosclerotic plaque in a blood ves~el which comp~is~s contacting the atherosclerotic pla~ue with the rQagQnt ~or digesting ~therosclerotic plaque described above, under conditions and in an amount ~uch that the - . , ~ , . . ..

W~91/02~52 PCT/US90/042~2 reaqent binds to and digests, a component of plaque~

The subject invention also provides a method for rQducing the amount of atherosclerotic plague in a blood vessel which comprises: a) contacting normal lum~n with an antibody which spec~fically binds to normal intima and/or media and ha~
bound thereto an inh~bitor of an enzyme under conditions such that the antibody bind~ to normal intima and/or media;
and b) contacting ~he atherosclerotic plague wi~h the reagen~ for digesting atherosclerotic plaque under conditions ~uch that the reagent bind~ to the a~herosclerotlc plaque. The antibody which ~pecifically binds to normal intima and/or media is provided in a reagent for use in protecting normal axterial tissue from an enzy~e capable of digestion of atherosclerotic plaqueO Such reagent, which is bound to an inhibitor of an enzy~e capable of digesting atherosclerotic plaque, comprises an antibody which binds an antigen synthesized or present in such normal tissue, such as th~ monoclonal antibody produced by hybridoma QlOS7, as well as a recombinant polypeptide which comprises an amino acid seguence which is substantially the same as the amino acid ~equence of the hypervar$able region of the monoclonal antibody produced by hybridoma QlOE7, or a chimeric or humanized antibody or ~ragment thereof comprising the recombinant polypeptide.

This invention further provides a method for reducing the amount of atherosclerotic plaque in a blood vessel which comprises: a) contacting the atherosclerotic plague with a reagent under conditions such that the reagent binds to the plaque so as to form a reagent-plaque complex, which reag~nt i~ capable of sp2cifically binding to both the plaque and to a proenzyme which, when cleaved, is converted into an enzyme whose substrate is a connective tiss~e present in atherosclerotic plaque, and which enzyme is capable of dis~olving a component of the plaque; b) contacting the W~9l/02252 PCT/US90/04272 -39~ {~ J

reagQnt-plaque complex with the proenzyme to which the reagent specifically binds under conditions ~uch that the prOQnZyme i8 bound to the reagent 60 as to form a proenzyme-raagent-plaque complex; and c) contacting the proenzyme-reagent-plaque complex with an ag~nt which i8 capable of 6peci~ically cleaving the proenzyme ~o that the proenzyme is converted into the enzyme under condition~ cuch that the enzyme digests the plague.

The subject invention further provides a ~ethod ~or reducing the amount o~ atherosclerotic plague in a blood vessel ~hich compris~s a) contac~ing the atheroscl~rotic plaque ~ith a reagent ~uch as the reagent described above ~or digesting atherosclerotic plaque under conditions ~uch that the reagent binds to the plaque 80 as to form a reagent-plaque complex, which reagent is bound to both the plaque and to a proenzyme which, when cleaved, is converted into an enzy~e who~e ~ubstrate i8 a connective tissue present in atherosclerotic plaque, and which enzyme iB capable o~
dissolving a component o~ the plaque; and b) contacting the proenzyme-reagent-plague complex with an agent which is capable o~ speci~ically cleaving the proenzy~e so that the proenzyme is converted into the enzyme under conditions such that the enzyme digests the plague.
In a preferred embodiment the reagent i~ a bifunctional antibody. The bifunctional antibody may be produced ~y any me~hod known in the art including chemical linkage o~
~ragments, and recombinant genetic enginQering. In a presently pre~erred embodiment, the bifunctional antibody is produced by a quadroma, wherein the quadroma i8 derived ~rom the ~usion o~ a hybridoma cell line comprising the monoclonal antibody produced by hybridoma Z2D3 or Z2D3/3E5 or related cell line and a hybridoma secreting a ~onoclonal antibody binding an enzyme. To digest the plague e~iciently, it i8 preferred that the proenzyme be a . .

Yosl/022s2 P~T/US90/0427 ~;!3~if'~ -40-pro~nzyme o~ granulocytic collagenase, ~ibroblastic collagenas~, or stromelysin. It is preferred that the agent of step ~c) i6 plasmin. The plasmin may be obtained by treating the subject with tissue plasmi~ogen activator under such conditions ~o as to cleave plasmino~en into plasm~n.

Turnlng now to radiant energy treatment of atherosclerotic plaque, the subject invention provides a reagent for use in ablating atherosclerotic plaque which comprises an antibody which specifically binds to atherosclerotic plaque bound to a chromophore capable o~ absorbing radiation having a plaque ablating wa~elength.

In one embodiment of this method the antibody i8 a msnoclonal antibody such as that produced by hybridoma 15H5 or 17H3and more preferably, the monoclonal antibcdy i~
produc~d by hybridoma Z2D3 or hybridoma Z2D3/3E5 or related daughter cell line. In another embodiment the chromophore ab~orbs light having a wavelength o~ from about 190 nm to about 1100 n~. Such chromophores are well known in the art.
Accordingly, th~ choice o~ chromophore i8 readily d~terminable to one skilled i~ the art although a preferred ~mbodiment i6 wAerein the chromophore i6 fluorescein;
rhodamine, tetracycline, or hematoporphyrin.
2~
The subject invention further provides a composition comprising an amount of tha above-described reagent effective for use $n ablating atherosclerotic plaque and a physiologically acceptable carrier.
This invention provida~ a method for ablating atherosclerotic plaque which comprises: a) contacting atheroscl~rotic plaque with an effective amount of the reagent for use in ablating atherosclerotic plaque described hereinabove 80 that the reagent binds to the atherosclerotic plaque for~ing an atherosclerotic plaque-reagent complex; b) - ;

..: ..., . -. ~ - ,- . -W~9l/0225~ PCT/US90/04272 -41- 2 ~ ;J~

exposing the rasulting complex to radiation haYin~ a plaque ablating wav~length under condltion~ such that the light is absorbed by the chromophore at a sufficiQnt energy to ablate the athero~clerotic plaque.
The subject i~vention further provides a method for ablating a~herosclerotic plaque pr~sent in a blood ~essel whieh comprises: a~ contactinq th~ normal lum~n with ~n antibody which ~pecifically binds to normal intima and/or media and has bound thereto a moiety capable of reflecting radiation :~
of the plaque ablating wavelength; b) contacting the atherosclQrotie plague with the reagent ~or u~e in ablating atherosclerotic plaque described hereinabove under conditions such that the reagent bind~ to the :~
atherosclerotlc plaque; and c) exposing the atherosclerotic plaque to radiation having plaque ablating wavelength~
there~y ablating the plaqu~O

In a preferred embodiment o~ this ~ethod, the antibody which speclfically ~inds to normal intima and/or~media i8 ~
monoclonal antibody produced by hybridoma QlOE7 (ATCC
Accassion No. ~8 10188).

The choice of moiety for reflecting light is readily deterfflinable to one skilled in the art.

The ~ubject invention also provides a reagent for use in treating atherosclerosis which comprises an antibody which binds speci~ically to atherosclerotic plaqu~ ~ound to drug use~ul in treating atherosclerosi3. In a pra~erred e~bodiment the antibody is the monoclonal antibody produced by hybridoma Z2D3 (ATCC Accession No. HB9840). These rea~ents can be used in a method o~ treating atherosclerosis in ~ sub~ect which comprises ad~inistering to the sub~ect an amount o~ such reagent e~fective to trQat atherosclerosis.

'. ~
.`~ ':' - ~ -. . . .. ; ~ .. i. . .. ... ..

wosl/02~s~ PCT/VS9~/04~72 ~ 42-2'3 ~
Further, the subject invention provides a method o~ treating ~thero~clero6i6 in a subject which comprise~ a) administering to the subject an antibody whic~ speci~ically binds to normal intima and/or media and which has bound thereto an inhibitor of a drug useful in treatlng atherosclerosis; and b) administering to the subject an amount of the reagent described above ef~ective to treat athero clerosis. In a preferred embodiment o~ thi8 method, the antibody for use in protscting normal arterial ti~ue lo ~rom a drug useful in treating atherosclerosis i~ a monoc}onal antibody produced by hybridoma QlOE7 tATCC
Accession No. H3 10188) which has bound thereto an inhibitor of a drug useful in treating atharosclerosis.

The subjQct invention provides a ~ethod o~ treatlng a~herosclerosis which comprises blocking the synthesis o~ an athero6clerotic plaque speci~ic antigen. ~he blocking of ~he atherosclerotio plaque antigen may be accomplished in saveral ways. One embodiment of this method is ~herein the synthesis o~ the antigen is blocked by using an antisense nucleic acid which specifically binds to a nucleic acid encoding the antigen, the expression of which i8 associated with synthesis of the antigen. In another embcdiment of thi8 method the synthesis o~ the antigen is blocked by inhibiting an enzyme involved in the synthesis of the ant~gen.

~he subject invention also provides a method o~ tr~ating atharosclerosis which comprises blocking the binding of an antibody, such as an auto-antibody to the atherosclerotic antibody plaque antigen.

This method may encompass any o~ the ~ethods known to one skilled in the art. One erbodiment of this method comprises blocking the binding of the auto-antibody to the antig~n by contacting the antigen with an excess of antibody. Another ' ..... . . . ..

.. .. . -.,, . -: .. : . : . . . . -. - . ~ . . :,:: ,. .. . .. ~ : . ...... . .. : ,. ... . . .. .. .. . . .
... ~ -: : . , . . ~. - - . - .. ... ,.. ,, .. ; .. , wo91/02252 PCTtUS90/04~72 ~43~ 2 ~ ~r3 ~

embodiment af this method comprises blocking th~ binding of the auto-antibody to the antigen by contacting ~he auto-antibody with an excess o~ an anti-idiotype antibody made therQto.

The ~ubject invention ~urther provides a method for diagnostic analysis comprising ~he step~ o~: a) obtaining a ~alue ~or the body masC index (BMI) of ~ pati~nt; b) obtaining a value f or the concentration of an antigen or other ~erum or plasma analyte~ associated with a pathological condition or an antibody which binds with the antigen; c) plotting the body ~ass index of the patient against the antigen or antibody concentration of ~he same patien~; and d) comparing ~he resulting value again~t a ~et o~ re~erence Yalues to determine whether the r~sulting value exc~eds the reference value and thereby indicat~s the presenc~ of a pathological condition. Thi~ ~ethod i5 a generic method ~ay be used instead of conventional methods which ~ust reveal positive or negative results in testlng whether a patient ha~ a predisposition toward such pathological conditions as cancer and atherosclerosi~. In d~termining whether a patient has a predisposition toward atherosclerosis, the method is preferable used wherein the antigen is an antigen ~ynthesized by, or pre~ent in, atheroscl~rotic plaque (Figure 24), or wherein the antibody is an antibody which specifically binds to such an antigen (Figure 23). The body mass index (BMI) i8 obtained by dividing a subject's weight by their height2.

. , . ., ~ . :

WO91/022s~ PCT/US9~/0427 ' ~44~

The Experi~ental Detail section i~ arranged a~ ~ollows:
I. PREPARATION OF ATHEROSCLEROTIC PLAQUE ANTIGENS
II. CHARACTERIZATION OF ATHEROSCLEROTIC PLAQUE ~ IGENS
III. PROCEDURES FOR AN~}BODY ISOLATION AND PREPARATION :~
IV. PROCEDURES FOR IMMUNOASSAYS .
V. PROCEDURES FOR ANT B ODY LABELING
VI. PROCEDURES FOR IMAGING ATHEROSCLEROTIC PLA~UE ;:~
VII. PROCEDURES FOR HISTOLGGY
VIII. ~ET%ODS OF TREATING ATHEROSCLEROTIC PL~QUE

I. PREPARATION OF T~E ATHEROSCLEROTIC PLAQUE ANTIGEN5 . ' ~.`

(Solubilization) Tissue handling and antig~n solubilization were done a~
described below:
1~ Obtain athero~clerotic arteries ~rom human autop~y within 24 hours of death or ~rom surgical procedures.
2. Remove sample and wash in multiple changes o~ 20 mM
phosphate buffer 0.15 M NaCl/pH 7.3/0.02~ NaN3 (PBS) to remove blood components.
3. Fr~eze lesions at -80C until use.

4. Whén ready to process lesions, remove them ~ro~ -80C
~reezer and let thaw at room temperature~ ;`

5. Rinse thawed lesions in cold PBS; care~ully peel and retain atherosclexotic lesion from normal artery remnant. Discard artery remnant.

6. Weigh athersclerotic lesions and record.

7. Using shaxp, surgical scissors, cut lesions into 5x5 pieces. Keep them moist ~n cold PBS (enough solution to ~ust cov~r ~ragments).

8. Homogenize lesion ~ragments in lce-Fold PBS adding 5 ml "

W~9l/Q22S2 PCT/US90/042~2 ~45~ 2~t~
of the cold PBS to 1 gram o~ lesien. ~Polytron; 2 to ~ minutes.)

9. Centr~fuge homogenate at 10-15,000 RPM, 4C for 30 minutes. Retain supernate. Discard lipid layer.

10. Resuspend pellet in cold PBS (5 ml/gm) and re-homogenize.

11. Repeat ~tep ~9 and pool both supernates.

12. Aliquot and freeze plaque supernate at -89C unle~s remainder of purification i5 then carried out.
(Affinity Purification) Crude or partially purified plague or ~erum antigen was m$xed with 15H5 antibody coupled resin for 2 hrs. at room te~perature (R.T.) ~batch method] or applied to a colu~n of resin at a Plow rate of about 1 ml/min., the run-through was reapplied to the same column ~column method]. All samples and resins were equilibrated with lOmM NaPO~/150~M NaCl/pH
7.2 (P~S).

After 8ample loading, the resin was then washed extensivQly with PBS ~nd the bound antigen was then releas~d by the addition o~ 0.1 ~ glycine at pH 2.5 or 3.5 M NaSCN in PBS.
Fractions were collected, dialyzed in PBS at 4C, tested for antigen reactiv~ty by ELISA, and appropriate tubes poolsd and 6tored for subsequent characterization.
. ~, ~p~ on Q~ Mono~lonal Antibody A~initY ~hro~a~oqra~hy Matrix Mouse a8cit~8 containing monoclonal antibody 15H5 or Z2D3 were passed through a 0.45 ~ ~ilter and then fractionated by HPLC gel filtration. Ten mls of ascites fluid was applied to a Bio-Gel TSK-400 Column (600 X 21.5 mm, Bio-Rad) and eluted in 0.1 M potassium phosphate pH 7Ø Fractions, of appropriat~ size to IgM, were pooled.

:,:

.. . . : . . . . : .. ~ ..

WO9l/02252 PCT/US90/04272 ~ f; :~ -46-Coupling of antibody to Affi-Gel resin (Bio-Rad) wa~ done in the presence of 0.1 M KPO~ buffer at pH 7 with 1 mg antibody/l ml packed gel. Gel preparation, antibody ~
coupling (4 hr~. at R.T.) washing, and bloc~ing with ' S ethanolamine were done as per manufacturer'6 specificat$ons.

Affinity Puri~ic~tion of ~ntiae~ From Positive S~rum/~13sma The fol}owing method i5 a one-step procedure for antigen a~finity purification by means of 15H5 Ab gel: ~ .
:;.
1. 15H5 Ab Gel, 7.5 ml, is uspended with 10 mM PBS pH -~
7.0, 32.5 ml. Final volume is 40 ml. Dispense 80 ~1 of :~
strong positive serum or plasma as determined by Antigen Capture Assay into 40 ~1 15H5 anti~ody gel.

2. Gently mix on ~haker at R.T. for 4 hr~. and then continuously mix it at 4~C overnight.

3. Centrifuge at 3000 R.P.M for 10 minutes. Discard supernatant. The 15H5 Ab gel is washed with 10 ~M PBS
pH 7.0 (40 ml/time) for three times.

4. Add 20 ml 0.1 M Glycine HC1, p~ 2.5, into 15H5 antibody gel and mix well on shaker at R.T. for two ~inutes in order to disasso~iate antigen from 15H5 Ab gel~ ~:

5. Centrifuge at 3000 RPN ~or 10 minutes. Collect ~upernatant and immediately neutralize it with 2 ml of 1.0 M Tri3 HCl pH 8.25 bu~fer.

6. Purified Ag solution is dialyzed against 10 mM PBS, pH
7.0, at 4C overnight ~change buffer once). :

Purif iç~iQn ~f ZZD3 and 010~7 Anti~e~
Ho~oaenizati~n o~ Atherosclerotiç ~laque ~ .

W09l/02252 PCTtUS9~/04272 2 ~ J
~othod ~:

we report the isolation and characterization of a specific extracellular antigen ~ound in the atherosclerotic plaques of humans, monkeys, pigs, and rabbits.

The plaque-matrix-specific antigen (Z2D3 Ag) wac discovered when it~ identifying monoclonal antibod~ , Z2D3, was screened positiYe by immunohistology on athero~clerotic human coronary arteri~s. The Z2D3 antibody stained the plaque matrix without staining normal artery. The lmmunogen usQd in the hybridoma program that produced this antibody was affinity-purified material obtained from homogenized human plaque using the monoclonal antibody 15H5. The 15H5 antibody recognizes the carbohydrate autoantigen produced by no~mal and plaque-derived smooth musc}e cells tLamaziere, J.~., et al. AtherosclerosiS (Ireland) Z4 ~1-2!: 11S :
tl988) ) . ,.
.
The Z2D3 antibody was further screened on a ~ariety of human tissues using 5~ un~ixed ~rozen tissue sections (Calenof~, E., et al., unpublished results). The plaques of all diseased human coronary arteries and aortae 6tained positive. All nor~al tissue~; with the exception of spleen fi~romyocy~es and focal cell clusters of ovary and sebaceou~
~lands failed to ~tain with this antibody (Table 7). The normal tissue ~taining was c~nfined to the cytosol without extracellular manifestations. In contrast, the vast portion of staining within atherosclerotic plaque was extracellular, di~fusely manifest throughout the connective tissue ~atrix in addition to staining the cytosol of the plaque smooth muscle cells. In fibro~atty lesions, area~ of ~acrophage involvement stained less strongly than areas with only connective tissue and/or smooth muscle cell involvement.
The macrophages themselve~ ~ailed to ~tain with the Z2D3 antlbody.

02252 PCT/US90/0~2~2 ~ 48-The Z2D3 antibody also stained the atherosclexotic plaques of macaque monkeys, Watanabe rabbits, New Zealand white r~bbits, and hypercholesterolemic pigs (Frashly dissected tissues were washed in cold normal 5aline 601ution and snap S frozen. Five ~ sections were cryostat cut and applied onto gelatin-coated slides. No fixation was done. ~he ABC
immunoperoxidase method was employed a~ per the ;~
manufacturer's instructions ~Vector, Burlingame, CA)). In the case of the monkey tissues, ~everal phase~ of lesion growth were studied. The plaques of the monXeys that had been maintained on a 2% cholesterol diet ~or a period exceeding one year stained with the Z2D3 antibody. ~ore interesting, however, was the observation that benea~h the early fatty streaks of monXeys that had been maintained on the cholesterol diet for only months, the Z2D3 antibody stained the cytoplas~ and immediate pericellular regions of the medial ~mooth muscle cells located immediately beneath the elastic lA~ina of those areas of the artery wall that were thus involved. This appeared within thQ tlme sequence corr~sponding to the migration of both macrophages and lymphocytes to this early lesion (Rapacz, J., et al. Science ~ 1573 (1986)). Slightly later i~ time, the 8~00th muscle cell6 were seen to penetrate the elastic lamina and migrate into the fatty strezk area.
All attempts at isolating and purifying the Z2D3 p}aque antigen under aqueous conditions were generally unsuccess~ul. The observation that organic solvent fixation o~ the ~rozen tissue sections employed in immunohistochemistry resulted in a total loss of ~taining led the applicant toward organic solvent extractions o~ the antigen from plaque tissue (Masuda, J. and Ross, R., Arte~iosclerosis 10(2): 164 and 178 (1990)).
.

Tha extracted antigen was further purified by a sequence of chromatographic steps that included gel sieve sizing, ion-w091/02252 PCT/US~/04272 _~9_ 27~ r,~t~

exchange chromatography, and thin-layer chro~atography.
Fresh plaque was peeled away ~rom normal artery remnant and washed ln cold normal 6alins. ~he plaque was then cut into 2 x 4mm fragments and freeze-dried into small Plakes (to remove as much free water as possible). The flaXe~ were embedded in o.C.T. medium (~ s Labs, Elkhart, IN), blocked, and finap frozenr The ~rozen tissue block was mounted in a tissue ~ectiQn cryostat and 5~ ti~sue section~
were cut. The tissue sections were mixed with 10 part~
acetonitrile in a glass beaker. The glass beaker was tightly covered and the contents stirred ~igorously for 18-24 hours at room kemperatur~. The ~tirred contents were then centrifuged at 20,000 g, 40C for 1 hour and the ~ ;
precipitat~ discarded. The acetonitrile ~olution was ~ilterad through a 0.22~ nylon filter (~illipore, Bedford, MA) yielding pale, yellow colored supernatant.

An ELISA immunoassay was utilized to determine the various chromatographic fractions for antigen content.
Gel Siev~_Çh~oma~QqraphY. ~he acetonitrile in the antigen solution was evaporated and the remaining residue redissolved in 100% ethanol. The pale, yellow solution was t~en ~ixed with 1 gram activated charcoal per 100 ~1 6upernatant and the resulting 81urry stirred ~or 1 hour at room teDperatur~. The slurry was then filter~d through the 0.22~ $ilter yielding a clear solution. This solution was then sized through a C26/100 column (Pharmacia, Piscataway, NJ) packed with ethanol-equilibrated lipophilic Sephadex LH-60 (Sigma, St. Louis, M0). The antigen positive ~ractionswere pooled.

Ion ~xchanae Chromato~raphy. Ion-exchange chromatography was per~ormed on a Mono Q HPLC column (Pharmacia) as per the ~ethod o~ Nansson (J.E. Mansson, B. Ros~ngren, L.
Svennerholm, J. Chromatography ~, 465 (1985)), W091/022~ PCT/US90/04272 2~ 50-substituting ethanol for methanol a~ the solvent of choice.
The Z2D3 antigen passed through the HPLC column in the void volume, unbound.

~ TLC was performed utilizing Whatman LX2 Linear-K cellulose plates (Ca~. ~4825-620). The plates were cut in half yielding two 5 x 10 cm plates. The fraction containiny the Z2D3 antigen w~s applied to the plate, 1-10 uL per lane, using a gla~s capillary tube. (Sample was applied in multiple 0.5 uL
aliquots per lane, and dried each time with a hair dryer.) The loaded plate was then placed in a container containing the mobile phase (chloroform, methanol, glacial acetic acid and water in the volume ratios 25:15:4:2) When the mobile phase was approximately 3mm from the top o~ the plate, the plate was removed and allowed to air dry at ambient temperature. The plates from various TLC rUllB were stained individually with iodine staining or immunoperoxidase staining employing the Z2D3 antibody. The iodine staining wa~ done by placing iodine crystals in a covered glass container and then incubated ~or 10-30 ~in. at 37C to produce the iodin~ vapor. The dry TLC plate was placed in the container. When the iodine reactive spots reached the desir¢d degree of darkness, the plate was remov~d and inspected. A single spot at the upper edge of the mo~ile phase wa~ observed. It was confirmed to be ~he Z2D3 antigen by running duplicate plates through an immunoperoxidase staining procedure employing the Z2D3 antibody and a negative control antibody.
I~munoperoxidase Dete~tion o~ Z2D3 Antiaen on TLC. Two dr$ed TLC plates from abov~ were blocked in a 0.2% casein/50 mM Tri3-HCll150 mM NaCl buffer (TBS), pH 7.6 ~or 45 min.
on~ plate was then transferred to a 5 ug/mL solution of Z2D3 IgM MAb in thQ ca~ein buffer and incubated ~or 18 hours at ~`
room temperature with gentle agitation. The other plate was .

..... , ... . , ,.,., .,, .,, ~ ,, :.

W~91/02~2 PCTlUS~OtO42~2 -51~ J)~, incubat~d in the negatl~e control antibody 801ution.
Unbound antibody was removed by washing the plates ~or 15 minutes per wash X3. The washed plates were than incubated in a l:Soo dilution o~ goat anti-mouse IgM/horse radi~h peroxidase conjugate (Tago, Burlingame, CA.), for 3 hour~
with gentle agitat~on. Th~ plat2s wer~ then washed in casein bu~fer followad by 3 washes in TBS. The washed plates ~ere then immersed ~n the substrate ~lution which consi~ted of 8ml o~ 3 mg/mL 4-chloro-naphthol in ~ethanol, 32 mL of TBS and 20 uL 30% ~202. The plates were de~eloped for 10-20 minutes and the reaction stopped by rinsing the plates with deionized water. A developed spot wa~ ~een at the mobile phase line on the Z2D3 plate but non~ on the non-speci~ic antibody plate.

The Z2D3 antigen appears to be a small, lipid containing molecule. It is probably not a sphingolipid becausQ o~ its resistance to the usual acid hydrolysis conditions but i8 perhaps a neutral lipid or a proteolipid. Variou~ antigen Practions in either ethanol or other organic ~olvents were applied in lOO uL samples into the well~ Q~ I~mulon 4 microtiter plates (Dynatech, Chantilly, Va.) and the organic solvents evaporated by blowing gently with bottled n$trogen gas or air. The dried plates were then blocked by ~pplying 200 uL of the caseinlTBS solution (wash buffer) for 1 hour.
The plate~ were then washed 4x and lOO uL aliquot~ of 5 ug/~L Z2D3 antibody diluted in wash buffer applied to each test well. The plates were covered and incubated at 37 ~or l hour. Th~y were washed 4x and lOOu~ o~ goat anti-mouse IgM/peroxidase conjugate, diluted l:lOO applied. The plates were incubated for 1 hour at 37 and then washed. lOO ul of TMB substrate ~Rirkegaard and Perry, Gaithersburg, ND.) wa~
applied to each well and incubated ~or 1 hour at room temperature. 50 uL of lM HCl was applied to 2ach well to ~top the substrate catalysis and the plates read at 450 nm W091/0~252 PCT/~S90/04272 ?.~ 52-on a Molecular DeYic~s ELISA reader (Molecular Davices, Menlo Park, CA).

One benefit o~ having uncovered this antigen is its scientific utility in marking the altered smooth muscle cell~ which are directly responsible for the ~ynthesi~ of these arterial lesions. From applicant's ani~al ~tudie~, it i5 obvious tha~ the transition of the smooth ~uscle cell~
from normal to pathologic can be seen by immunohistologic means from the earliest phases of atherosclerosis to its end-stage.

The other benefit of having identified this molecular marker of atherosclerotic plaque is that it makes possible t~e development of novel diagnostic and therapeutic reagents for improYing the clinical care of patients afflicted with coronary and/or cerebrovascular atherosclero~is. ~he~e take the ~orm of imaging agents comprising Fab fragments labeled with radionuclides such as technetium to be used in nuclear imaging or those labeled with paramagnetic molecules to be used in MRI imag~ng. Targetable therapeutic reagents such as neomolecules could be constructed which posseæs antibody~
like targetin~ and enzymatic activity which would yield a controlled catalysis and reduction o~ the connective tissue content in atherosclerotic plaque, thereby relieving focal arterial obstruction and preventing myocardial infarction a~d/or stroke (Xates, N., Techniques of Lipidology tElsevier, New York, ed. 2, 1986)).
, Mathod 2:
T~ssue handling and antigen solubilization were done as described below:
1. Obtain atherosclerotic arteries ~rom human autopsy with 24 hours of death.
2. Remove sample and wash in multiple changes of 20mM

. ~

.. . . .: . ;, .................. .. . . :
- ~. ~ . . -....... . . .

WO 91 ~0i!252 2 rl J ~3 ~J ;~ J ~)J04272 PBS/0.15M NaCl/pH 7.3/ 0.2% NaN3 (PBS) to remove blood components.

3. Fre~ze lesions at -80c until use.

4. When ready to process lesions, remove them from -80C
freezer, and let thaw at room temperature.

5. Rinse ~hawed lesions in cold P8S; carefully peel and retain atherosclerotic lesion from normal artery remnant. Discard artery remnant.

6. Weigh atherosclerotic lesions and record.

7. Using sharp surgical sci~sors, cut lesions into 5 x 5 m~ pieces. ~eep them moist in roo~ temperature (RT) 8M
urea, adding 2 ml of the 8~ urea to 1 gxam of lesion.
Polytron 30 seconds.

8. Homogenize lesion fragments in RT 8M urea, adding 2 ~1 of the 8~ urea to 1 gram of lesion. Polytron 30 seconds.

9. Centrifuge homogenate at 15,000 RPM, 10-15C for 30 25~ ~inu~es. Retain supernate. Discard lipid layer.

10. Resuspend pellet in 8M urea (2 ml/gm) and re-homogenize for 30 seconds.

11. Repeat steps 9 and 10 as needed, and pool supernates.

12. Aliquot and ~reeze plaque supernate at -80C unless re~ainder of purification is then carried out.

pr~aratio~ of Cs~l Gradiçnt Frac~ionated PL~g~ç ~x~ac~
,~

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W091/02252 PCTJUS90/04~2 ~ t ~; ` ' ,' --54--,~ ~
1. Sampl~ is made 3M CsCl by adding O.5 gm CsC1 per ml of 8M urea extract. Stir until CsCl is dissolv4d. This requires approximately 30 minutQs. (~ixing i8 an endothermic reactlon, and the 3M CsCl/sample may at first appear as a slush. Continue 6tirring until CsCl goes into solution).

2. Dispense 3M CsCl/sample fiolution into ultracentrifuge tubes, and ~ap. Be siure tubes are ~illed completely and e~aled tight. Place tubes in TY70 rotor ~eckman).

3. Spin 50,000 rpm at B~C for 65 hour~ in Beckman ultracentrifuge.

4. At end of run, remove tubes from rotor and carefully pump out gradient ~olution from bottom of tubes using peristaltic pump. Collect equal ~ractions from each tube. ~ool corresponding fractions. Fraction one l~i the bottom most ~raction and fraction is the topmost fraction.

5. Store ~ractions at -80C until needed.

D~AE I~n E~h n~ h~ eg~raphy 1. Either CsCl fraction n~mber one (Z2D3) or fraction number two (QlOE7) is/are dialyzed in 3500 MM tubing against 20 ~M Tris-Hcl/7M Urea, pH 7.5, to achieYQ a 50,000x dialysis e~ect.
2. Datermine O.D.2~ ~ sample.

3. Load sample on appropriate Bio-Gel DEAE-5-PW ~PLC
columns (Bio-Rad): -Analytica} column - 1 mg protein load range S~mi-prep column a 10-lgO mg protein load range wogl/0~2s2 PCT/~S~o/~4272 -55- ~ 3 4~ The eluting buffers are 20mN Tris-HC1/7~ Urea, pH 7.5 (A), and 20mM Tris-HCl/ 7M Urea/LM NaCl, pH 7.5 (B).

5. The elution pro~iles programmed into HPLC are:
ANALYTICAL COLUMN- .
l~lL=inl ~a $~ 5L~2.~c~
O 1.0 100 0 Linear 1.0 100 0 Linear l.o 50 50 ~inear SE~I-PREP DEAE COLUMN:
~ Elow lml/min) %A%B Gradien~ ~rofilç
0 4.0 lOo O Linear 10 4.0 100 0 Linear 55 4.0 50 50 Linear 6. One ~1 fractions are collected the in analytical run and 4 ml fractions are collected in the semi-prep run.

7. 50 ~L of each sa~ple ~rom each tube are diluted in 20 ~M Tris-HCl/3.SM Urea, pH 7.5, and used to coat wells on microtiter plate corresponding to ~ach tube coll~cted. Plates are incubated overnight at R~.

8. Plates are tested by employing the ELISA procedure described herein for determining Z2D3 activity (CsCl ~raction one) or QlOE7 activity ~C~Cl ~raction ~our).
Sae illustrations b and c.

9~ Those tubes whose ~ontents gave a positive ELISA signal ~or the antigen in question are pooled together.

3S 10. This Z2D3 or QlOE7 antigen-containing ~ixture is :, ' , ; ~ ~: ; . : . i .

WO 91/OZ~52 PCI`/US90/04272 ~, ~ r r~ 5 6-dialyzed against 20 mM Tris-HCl/0.15M NaCl, pH 7.4, to remove the urea, using dialysis tubing with 3500 MW
pores.

Af~initv Chromatoara~hy 1. The antigen-containing mixture is added to agarose gel, coupled to Z2D3 or QlOE7 monoclanal antibody (depending `
on which antigen requires purification).
O
2. This agarose gel/antigen mixture is gently mixed in a shaker at 4C overnight.

3. The gel is then loaded onto an appropriately-sized S glass column.

4. The gel is washed with 20 gel volumes of 20mM
Tris-HC1~0.15M NaCl, pH 7.4(Tris).

5. The antiqen is eluted with O.lM glycine HCl buffer, pH
2.5.
: ';-6. The eluted antigen is dialyzed against Tris buffer.

Gel_Sizing ~;

- 1. The affinity purified antigen is concentrated using an Amicon concentrator with 10,000 MM filter.

2. The concentrated antigen is filtered using 0.~5~ filter and loaded onto a Bio-Sil TSK-400 column (Z2D3 antigen) or Bio-Sil TSX-250 column (QlOE7 antigen) equilibrated with 0.1~ potassium phosphate buffer, pH 7.0 (Bio-Sil O
columns are sold by Bio-Rad). d `
d 3. The major macromolecules of Z2D3 antigen are eluted at ~;
~ ~' wosl~o22s2 P~T/US9V/04272 ~57~ 2;~ s~
greater than 200,000 MW from the ~io-Sil TSK-400 column. The QlOE7 antigen is eluted fro~ the Bio-Sil Tsx-2so column at greater than 150,000 M~.

II . C~a~AC~ER~Z;~TIO~I OF AT}IE:~05C~E~OT$C PI-AQUE A~ G~18 ~lndinq Studies_~inq ~tins and ~ommercial Antibodies Affinity pu*i~ied atherosclarotic plaque antiqen was coated onto polystyrene microtiter plates (Immunlon II). Sample was diluted in 100 ~M NaPO4/400 mM NaC1/pH 6.9 and 100 ~1 was zpplied to each well and then incubated overnight at 4C.

Plates ~ere blocked and then washed with PBS containing 0.1%
Tr$ton X-100 and 0.05% Tween-20 (for lectin study) or casein buff~r (~or commercial antibody study). Biotinylatad lectins (from Vector or Sigma) were dilut~d to a ~inal concentration of 1-10 ~g/ml and 100 ~1 appli~d to wella coatad w~th athero-antigen ~or 2 hrs at 37C. Bound lectins were detected using an Avidin-Peroxidase con~ugate [ABC ~rom Vector Labs]. Commercial polyclonal and monoclonal antibodies were diluted with casein buffer (1/100 to 1/2000) and incubated with coated antigen (prepared as above) for 2 ~rs. at 37C. Appropriate peroxidase con~ugated second antibodies (Tago) were then applied to detect binding of commercial antibodies to the coated athero-antigen.

~olve~t Ex~r~çtio~/PreciPi~tions tTCA] .
Partially puri~ied atherosclerotic plaque antigen in PBS
buf~er was brought to a ~inal concentration o~ 5% ~wt./vol.) of trichoroac2tic acid (TCA), incubated on ice ~or 30 minutes and then centri~uged to separate acid soluble and insoluble ~ractions. The insoluble material was dispersed `:
: . , W09l/0225~ PCT/VS~o/04272 ~ t~ -58-into PBS and tested for remaining antigen by ELISA. The TCA
supernatant fraction was neutralized by addition of 1~ Tri~
pH 9, dialyzed against PBS and then assayed by ELISA.

~Acetone]
One ~olume of partially purified atherosclerotic plaque antigen was mixed with nine volu~es of ice cold acetone, mixed, left on ice for 30 minutes, and then centrifuged.
The pellet was air dried, resuspended in ltC original volume of PBS and assayed by ELISA. The acetone supernatant was discarded.

~Chloroform~
One volu~e of partially purified athero clerotic plaqueantigen was ~ixed vigorously with one volume of chloroform, and centrifuqed 5 minutes at 2000 RPM. The upper aqueous layer was removed and extractad again with chlorofor~. A~ter a second centrifugation, the aqueous layer was assayed by ELISA.
;
En~y~e Di1çsts o~ Athe~Q~lerotiç Ptaq~ Antigen . .
Affinity purified atherosclerotic plaque antigen from plague or serum was mixed with a wide array of hydrolytic enzymes, the specific reaction buffers were thDse suggested by the manufacturer. All reactions were done in a total volume of 1.0 - 1.5 ~1, incubated overnight at 37, then boiled ~or 5 minutes to stop the reaction. Samples were ~iltered ~0.45 ~) and injected onto an HPLC column (TSK-400 600 m~ x 7.5 mm Bio-Rad) ~or molecular sieve fractionation in 0.1 M KPO4, pH
7Ø Individual ~ractions (25 drops each~ were tested by ELISA in both antibody capture and coated antigen for~ats for changes in the elution profile of atherosclerotic plaqu~
antigen relative to control (undigested) samples. Molecular weight standards ~Bio-Rad: thyroglobulin, immunoglobulin, ovalbum~n, myoglobin, and vitamin B-12] were u6ed to .':

~: J ' ` . : :. . ' : ' :: '' ~ , :: ': ` ': - . ': -: :,'' '. ,.. , .... : , : ' . . . : , Wog1/0~252 PCT/US~0104272 ~ 7;~
calibrate the TSXo400 colu~n elution.

Colorim~tric ~s~

a) Total hexosamine was mQasured by th~ ~thod o~
Blumenkrantz and Asboe-HansQn ~Clin. Bioch~m., ~:264 (1976)]. Briefly, antig~n ~ample (0~4~l) wa6 ~ix~d with 0.3 ml o~ 3.5% acetylaceton~ in phosphate/tetraboronata bu~er ~nd heated to 109C for 30 minut~s. The ~ixtur~ was cool~d, 1 ml of Ehrlich'~
reagent was added, and the re~ulting absorbance (at 535 ~ wave length) was measured. D (+) glucosa~ine was used as a ~tandard.

b) Uronic acid was ~easured using the method of Blu~enkrantz and Asboe-Hansen ~nal. Bioche~., ~:484 (1973)1. Briefly, to O.l ~l o~ antigen ~ample was added 0.6 ~l of sul~uric acid/~QtraboronatQ reagent followed by ~ixing and incubation 3t 100C ~or five minute~. After cooling, O.Ol ml of m-hydroxydlph2nyl reagent wa~ added, ~he tubes mixed, and aPt~r five minutes, absorbanc2 at 520 nm was r~corded.
Chondroitin ~ul~ate waR used as a re~erence ~tandard.

Molecul~r_C~3~æ~ taLL~atiQn Initial ef~orts to determine ~he isoelectric pH ~pI) of th~
ath2rosclerotio plaque antigen ulilized mixed bed ion--exchange resin reagents chromatofoc~sing system ~rom Pharmacia. Conditions used were a3 per manu~acturer'~
instruction~; antigen sample was dialyzQd into high pH
buffer ~11.0) and 1 ml was applied to Polybu~fer Exc~ange 118 resin (10 ml bed). The elution gradient was then developed with a pH 8.0 buf~er. The pH of individual ~ractions wa~ measured, then they were dialyzed into PBS pH
7.2 and assayed ~y ELIS~.

WO9l/022S2 PCT/~S90/04272 2 ~1 S ~ 60-Add~tional ion exchange bind~ng studies wer~ done using QAE-Sepharose (anio~ exchanger) and S-Sepharose ~catlon exchanger) over a pH range of 7-12 according to the method of Lang and Langer [Anal. Bioche~ 148 (1985)~.
Brle~ly, partially purified antigen wa~ dialyz~d into 5 mM
NaP0~ buffer at pH 7, 8, 9, 10, 11, and 12. Aliguots of :
ion-exchange raain 0.5 ml packed gel equ$1ibrated at the same pHs were ~ixed with 1 ml of antigen for 30 ~inutQs at 25C. The samples were centrifuged at 2000 RP~ ~or S
minutes, ~he supernatants re~oved and ~iltered to remove gel frag~ents, and then assayed by ELISA to quantitate unbound antigen. Cytochrome C and myoglobin were used aB a high pI
(10.2) ctandard and mid ran~e pI (7.4) standard, ~ :
respectively, to validate the procedure.
MisceLl~neQ~ Tr~at~ent~ ;

tchaotrope~3 ,, Ath~rosclerotic plaque antigen was ~xposed to the following .:
list o~ denaturants and then returned to its original ~uffer ; `
tPB8~ by dialysis.
Agents: 8M urea in PBS for 24 hrs. at R.T `~ :
6M Guanidine HC1 $n PBS for 24 hr6 . at R.T.
2~ ~riflouroacetic acid for 30 ~inute ~t R.T.
3.5 M NaSCN in PBS for 8 hr8. at R.T. :~
0.1 M Glycine pH 2.S for 2 hrs. at R.T.

0.19 SDS in PBS for 1 hr. at R.T. (recovQr antigen by precipitation with acetone) .
~Alkylation-Reduction~
Partially purified atherosclerotic plaque antigen (0.5 ml in 0.1 M KP0~, pH 7.0) was mixed wi~h 0.44 guanidine-HCl (7M
~inal concentration) and 250 ~g of dithiothrQitol. The pH
was adju~ted to 8.6 and the sample left ~or 1 hr. at R.T.
~hen 16 mg of iodoacetamide was slowly added and pH :

W091/02~52 PCT/US9~/04~72 ~ 2 ~ ~r J~

maintained at 8.5 wlth NaOH as needed. A~ter 1 hx. at R.T.
thi5 6ampl~ was run on a TSK-400 HPLC gel filtra~ion column (as dascribed above) and individual fractions were tested for atherosclerotic plaque antigen by ELISA and compared to fractions collected from unreacted antigen.

The following outline describes the procedure~ used for carbohydrate analysis of ~arious polysaocharide hydrolates a~ Vasocor.

Antigen and control samples were prepared by a~inity chr,~matcgraphy. A~finity resins were prepared by coupling Vasocor ~onoclonal antibody 15H5 to Bio-Rad A~igal 10 using published methods.

Plasma samples were incubated with the resin in ~ batchwise procedure. After washing, the bound antigen was ~luted with o.1 ~ glycine bu~er pH 2.5. The antigen solution was then neutralized and dialyzed against PBS.

Sample Prep~ o~:

Salts were re~oved by extensive dialysis against puri~ied water at 4C. Each sample was concantratQd by lyophilization and the residue redi~solv~d in a minimal volume of puri~ied water.

Standar~ p~para~lon:

Ultra high purity monosaccharide standards wer~ obtained ~rom P~ansteiehl Laboratories Inc., Waukegan, IL. Standard solutions and dilutions thereof were all prepared in wosl/0~2s2 PCT/US90/04272 ~ 3 -62-purified water. Aliquots o~ each ~tandard wer~ ~tored at -80C until us~.

HydrolYsis:
Concsntrated tri~luoroac0tic acid (Pierce, ~ock~ord, IL) wa~
added to the aqueous sample solution to a ~inal concentration of 2M. The vial was flu6hed thoroughly wi~h : filtered nitrogen and capped with a thermo tabl~, tsflon-lin~d cap. The vial was placed in a sand bath at 104 4~C
~or four hour~. After hydrolysis, the vial ~as cooled (lO
~inutes) and the solvents evaporated under a ~tream of filtered nitrogen.

~arbohydr~_~n~lYl:

The Dionax instrument was expressly con~igur~d ~or monosaccharide analysis, consisting o~ ~ reagQnt delivery module, micro-in~ection valve, pulsed amperometrlc detector with gold electroda, and CarboPac PA-l analytical column.
Data were collected on a Dionex 4270 Integrator.

For each run, ~he column was thoroughly equilibrated in 15 mM NaOH in puri~ied water. Hydrolysate residue were redissolved in purified water just prior to in~ection. Th~
bound ~onosaccharides were eluted from the Car~oPac column with a linear gradient o~ NaOH in puri~ied water.

Results:
Figure 20 illustrates a chromatographic blank run with ~ust distilled water.

Figure 21 illustrates a chromatographic run with sevan 3tandard ~ono~accharides.

Wo~l/02252 PCT/U~0/04272 -63- 2~?~

Figure 22 illustrate~ a chromatographic blank run with the auto-antigen affinity purification with the 15H5 ~onoclonal antibody.
.

III . ~PROCED~ 58 ~OR AN~I!IBODY I~30I-ATIO~ 7D PRE:PA~a~IO~

~ntibody Con~uqation to_Sepharos~
: -Freeze-dried C~Br-Sepharose 4~ powder tPharmacia) i~ swelled for 15 min in 1 mM HCl. The gel is washed on a sintered glass filter (porosity G-3) with a total of 200 ~l of 1 ~M
HCl per gram of gel (dry wt.) This is done in several aliquots, the supern~tant being suctioned off ~etween ~5 successive additions.
:
5 mg of protein to be coupled per 1 ml of gel is dissolved in Coupling Buffer (0.1 M NaHC03, pH 8.3, containing 0.5 Nacl). The gel is washed with Coupling Buffer, the excess is removed by suction, and the protein solution i~ mixed with the gel. The mixture is allowed to stand overnight at 4C with stirrlng. The gel is then placed in a Blocking Buffer containing 1 M ethanolamine, pH 8.0, for 2 hr at rm ~;
temp. The gel is then washed with the Coupling Buffer containing 1 M ethanolamine, pH 8.0, for 2 hr at rm temp.
The gel is ten washed with the Coupling Buffer, 0.1 M
Acetate Bu~fer, pN 4.0, containing 0.5 M NaC1, and washed twice with Coupling Buffer. The protein-Sepharose con~ugate is now ready for use and can be stored at 4 to 8C.
Cyanogen bromide can be added to the bu~er ~olution as a bacteriostat.

IaG Antibodv A~_orption from Plaoue_Sunernat nt A column i9 packed with 25 ml of Sepharose gel conjuga~ed to anti-IgG antibody prepared in accordance with the above ', ;:' , :

.

VO9l/02251 PCT/US90/~4272 2 ~S~ 4-procedure containing a total of about 129 mg of anti IgG
antibody. The column is equilibrated with ~rom 2 to 3 volumes of buffer (0.15 ~ PBS, pH 7.2), and the sample is then applied to the column.
The flow rate of eluting buffer ~0.15 M PBS, pH 7.2) ic 125 ml/hr. The eluted fractions containing antibody are collected until peak activity disappears.

~he column is then washed with sodium acetate buffer solution, pH 4.0 (Eluting Buffer) ~o desorb immunoaffinity bound IgG antibody. The colu~n is eluted a rate of 15-20 ml/hr, collecting the eluted samples and retaining peak fractions. The peak fractions are dialyzed against 0.15 M
PBS, pH 7.2, for 24-36 hr at 4C with multiple buffer changes.
.
IgE Anti~dy ~dso~ption from Plaque Supernatan~

The above procedure is repeated with a column packed with 7.5 ml of Sepharose gel conjugated to anti-IgE antibody ;~
prepared as stated above. The flow rate of Eluting Buffer ;
is 15-20 ~l/hr.

I3A Antibody Adsorption f~om Plaaue Supernatan~

The above procedure of is repeated with a column packed with 7.5 ~l of Sepharose gel conjugated to anti-IgE antibody prepared in accordance with ~he procedure as stated above.
The flow rate of Eluting Buffer is 15-20 ml/hr.

IaM Antibody_Adsorption from Plaque Su~e~natant The above procedure is repeated with a column packed with 7.5 ml o~ Sepharose gal conjugated to anti-IgE antibcdy prepared in accordance with the procedure shown above. The ;

woslto22s2 PCT/US90/04272 -65- 2~ J~ 3 flow rate of Elutinq Buf~er i~ lS-20 ml/hr. ~

.
Polyclonal An~i-Plaoue Antibodies ;

Polyclonal antiserum against atheroslerotic plaque antigen is elicited in rabbits using the immunization techniques and schedules described in the literature, e.g. tStollar, Methods of Enzymology, 70:70 ~1980)]. The anti~erum ig then screened in a solid phase assay similar to that used for monoclonal antibodies, e.g. [Lange et al., Clin. Exp.
Immunol., 25:191 (1976) and Pisetsky et al., J. Immun.
Methods. 41:187 (1g81).] The initial screening criterion would be binding to atherosclerotic plaque antigen.

Polyclonal anti-plaque antibody must be prepared ~ follows:
Rabbits may be injected intramuscularly with a mixture of 0.5 ~g of plaque antigen prepared by the procedure described ~`
hereinabove in 0.2 ml of 0.15 M sodium chloride ~olution and 0.8 ml o~ complete Freund's adjuvant. The immunization is repeated for 1~ days and then each week ~or 3 weeks. After a further lO days have passed, blood is removed ~rom ~he rabbits, and antiserum is recovered from the blood by allowing it to coagulate and removing the clot.

Repeating the above procedure but replacing the antibody reagent with the plaque antigen yields horseradish peroxidase or alkaline phosphatase labeled plaque antigen.

The IgG fraction of the antisera is purified further by a~inity chromatography on a column containing a resin on which the anti-plaque antigen i9 immobilized.

Monsclonal Anti-Plaque Antibody Using the purified atherosclerotic plaque antigen, mouse monoclonal antibodies to the plaque antigen are obtained .;~ .
~, .

WO 91/02252 PCr/US90/04272 using standard procedures of ~alfre and Hilstein, ~Methods in Enzym., 73:1 (1981)]. The monoclonal antibodies are screened using a modification of the techniques described in the literature, e.g., [Lange et al., Clin. Exp. Immuno., ~:191 (1976)] and Pisetsky et al. ~J. Immun. Methods., 41:187 (1981)~.

To be use~ul for the assay of serum plaque antigen (or immunocomplexes thereof), a monoclonal antibody should bind to the plaque antigen with high affinity (pre~erably, XA 10~
~5-1 ) .

Mouse monoclonal antibody is purified in a two step procedure. The neat ascites fluid is applied to a column of Affi-Gel Blue resin (Bio-Rad Laboratories, Rich~ond, CA~ `~
equilibrated with 10 ~M Tris-HCl, 0.15 M NaC1, p~ 8.0, and eluted with the bu~fer. This step remov~s albumin, which is retained on the column. The final step in the purification is application to a DEAE-Sepharose (Pharmacia Fine Chemicals, Piscataway, NJ) and elution with a linear gradient of 10 mM Tris-HCl, pH 8.0, to 10 mM Tris-HCl, 100 mM NaCl. ~his gives purified mouse monoclonal antibcdy ~ree from contamina~ing serum proteins such as albumin and transferrin. ~`
~ ;
.
Successful Isolation of a Class $witch Variant Within ~he Hvbridoma Cell Line Z2D3 The class switch was from an IgM isotype cell line ZaD3 (ATCC Accession No. 9840) with speci~icity for atherosclerosis plaque antigen, to an IgG isotype cell line (Z2D3/5C5) with the same specificity. Z2D3/5C5 is an example o~ several daughter cell lines of Z2D3. Such daughter cell lines also include Z2D3/3E5 (~TCC Ac~ession No. HB 10485).

W09l/02252 PCT/US90/0427~ ~
-67- 2 ~

The IgM isotype Z2D3 hybridoma cell line wa~ prepared by fusing Balb/c splenocytes with the SP2 Myeloma cell line.
(See Journal of Immunology, Vol. 131 No. 2 August 1983.
Isolation of Immunoglobulin Class with Variants from Hybridoma lines secreting Anti-Idiotope ~ntibodies by sequential sublining. Christa E. Muller and Xlaus ~ajewsky;
Journal of I~munology Methods, Vol. 74, 1984, pg. 307-315.
The Identification of Monoclonal Class Switch V2riants by Sib Selection and an ELISA Assay, Gad Spira, Autonio Bargellesi, Jean-Luc Teilland, Matthew D. Scharff.) Z2D3 was scr~ened initially for IgG producing cells. 100 cells were plated/well ~n 96 well Falcon plates for a total of 10 plates. At day 8 supernatants were collected and tested for IgG. 96 wells were coated overni~ht at 4C with 50ng/well of goat anti-mouse IgG. (~ chain specific) reagent Zymed 62-6600). Wells were washed X 4 with PBS with 0.05%
Tween (wash buffer), and 50 ~l of supernatant from the plated cells was added. After incubating two-to three hours at room temperature, plates were washed X4 with the wash buffer and 50 ~1 1/1000 dilution of the Alkaline Phosphatase conjugated - Goat anti-mou~e IgG (~ chain specific) reagent added. (Zymed 62 - 6622). After two hours incubation at room temperature, plates were washed X4 with the wash bu~fer and 100 ~l of four-methylu~belliferyl phosphate ~ubstrate solution (Sigma No. M8883) was added to each well. After 60 minutes at room temperature, the plates were read using a FlouroPast 96 well Flourometer. (3M Diagnostics, Santa Clara, CA).

The sensitivity of the assay enabled one positive cell in 100 to be detected easily. Initially 3 positive wells were r detected. The well (8G2) producing the highest ~ignal was ~urther enriched by subcloning as follows:

wosl/o 2sz PCT/USsO/04272 ~. ~3 ,S' ;~ -68-Thi6 positive well was then resuspended in 100 ml of medium containin~ 9% Fetal Cal~ Serum, and plated ln 5, 96-well plates at 200 ~ltwell. Supernatants fr~m these wells were tested as above 8 days later, and 70~ of the well~ were positive for IgG. The well (lA12) with the highest si~nal for IgG was chosen for additional subclonlng. Cells in the well were suspended by pipetting and 20 ~1 of the ~uspension was diluted into lOo ml of medium with 9~ Fetal Calf Serum.
The suspension was plated 200 ~llwell in 5 plates, with approximately 3 cells/well.

After 8 days the supernatants were test~d for I~M and IgG
using the protocol described above but using a Goat anti IgM
(~ chain specific) reagent (Tago. 4142) to coat the wells 50 ng/well overnight and Goat anti-IgM (~ chain specific) reagent (Tago 4652) as the Alkaline phosphatase conjugate, for the assay to detect IgM. The three with the highest IgG
Signal were retested by doing dilution curves to more accurately determine amounts of ~ and ~ chains. 7D10 had ~he highest ~ and the lowest ~. This well (7D10) was then subcloned at 0.5 cells/well in 6 plates for the final derivation of a cloned line.

Single clones were identified visually and tested with IgM
and IgG reagents. Several ~ producin~ clones were chosen, of which 5C5 was further grown and studied. This clone is designated Z2D3/5C5.

Supernatants from the ~-producing Z2D3 cloned llne and ~he ~-prsducing Z2D3/5C5 clonal line show identical specificity as tested by the following:

1. Z2D3 IgM and Z2D3/5C5 IgG, when used in Immunohistological staining of frozen sections of human and rabbit atherosclerotic plaque show identical histological locali2ation, and on normal tissu~ give identical negative Wo'~ )22~2 PCT/US90/0427 results.

2. When tested (ELISA) ~or binding to antisen (alcohol extracted from human atherosclerotic plaque) both antibodieQ
bind specl~ically, whereas other antibodies of the ~ame : .
classes give negative results.

The IgG class was confirmed and the subGlas~ deter~ined using a SubIsotyping Ki~ (Hyclone E05051~ 2D3/5~5 is IgG1.

IY o P~OC~DIJR13B ~OR I~iJNOA88AY8 Antibody Assav Procedu~e 1. Add 10 ~l sa~ple or control (positive and negative) into 2 ml sample diluent in glass tubes.
`:
2. Incubate at 4 C overnight.

3. The foIlowing morning take antigen-coated plate and aspirate antigen coating solution out of each well.
Then add 200 ~l 0. 2% casein buf~er into each well to block wells at R.T. ~or 30 minutes. After that, aspirate and wash with 0.2% casein buffer once.
:.:
4. Apply 100 ~l sample or control into each well according to prepared plate map (sample or control run `.
duplicate).

5. Cover pïate with parafilm and incubate at ~ or two hrs.

35 6 . Aspirate and wash plate with 0.2~ casein buffer three ti~n2s.

.. i. - i ~, , . . ~ ~ .. .. .; , ...... . .;, . . .

Wo ~1~02252 PCr/US50/04272 t~; 3 .,.70_ :

7. Add 100 ,ul working dilutior~ o~ anti-human IgG coniugate or anti-human IgA conjugate anti-human IgA conjugate into each well. Incubate at R.T. for 2 hrs.

8. Wash plate with 0.2~ casein buffer four time~.

9. Prepare TMB substrate (using equal volume6 ~ix TMB
6ubstrate with peroidase solutio~ B).

10. Add 100 ~1 substrate into each well and react at R.T.
for 60 mimutes.

11. Read pla~e at 6S0 nm on ELISA reader first, and then ~:;
add 50 ~1 lM HC1 into each well to st~p reaction.
After that, read plate at 450 nm again. :
';
12. The optical density ~O.D.) number i~ directly ~;
proportional to the concentration of antibod~ in tested sample.
Antiq~ Ca~ture Assay ~rocedure 1. Add 250 ~1 sample or control (positive or negative) :~:
into 250 ~1 sample diluent in glass tube. :~
: ;:
2. Incubate above mixture at 37C for four hrs. ~
., .
3. Aspirate buffer from 15H5 Ab-coated plate (200 ~l/well) and wash plate with 100 mM PBS/Tween/Triton buf~er once.

4. Apply 200 ~1 sample or control into each well according to prepared plate map (sample or control run :~
duplicate).
5. Cover plate with parafilm and incubate at R.T~
'~ ~
,~:

w~sl/0~2~2 PCTtUS90/~42~2 -71~

~vernight.

6. The following morning, aspirate samples out o~ ~ells.
Wash plate with 0.2~ casein buffer three times.
7. Add 200 ~1 working dilution of 17H3 Ab-peroxidase conjugate into each well.

8. Cover plate with parafilm and incubate at 37C for four lo hrs.

.
9. Wash p}ate with 0.2% casein buffer four times.

10. Prepare TMB substrate ~using equal volumes mix TMB
substrate with ~eroxidase solution B).
~ ~.
11. Add 200 ~1 substrate into each well and react at R.T.
for 60 minutes~

12. Read plate at 650 nm on ELISA Reader first, and then add 50 ~1 lM HCl into each well to stop reaction.
After that, read plate at 450 nm again.

13. The optical density (O.D.) number is directly proportional ~o the concentration of antigen in the tested sample.
. ~.
In~bition_~ssaY Protocol .
1. Apply 100 ~1 different concentration of HCAD or in-house monoclonal antibody ~17H3 Ab., Z2D3i~b., 15H5 Ab., and normal mouse IgM as a mono Ab. control .into antigen-coated wells in order to pre-block wells. At the same time, add 100 ~1 10 mM PBS buffer into antigen-coated wells as a noninihibition control). The plate is covered with parafilm.

.:

W~91/02~52 PCT/US90/04272 ~ 2.- -72-2. Incu~ate plate at R.T. ~or two hrs., and then at 4C
, overnight.

3. The following morning, aspirate each well and wash with 0 . 2 % casein buf f er three times . ;

4. Add lOO ~1 of optimal concentration of ~ach monoclonal ~;
antibody into each HCAD pre-blocked wells, or 100 ~l o~
optimal dilution o~ }ICAD into each ~onocloanl ~
pre-bIocked wells. The plate is covered w1th parafilm. ~;

5~ Incubate at R.T. for two hrs, 6 . Aspirate and was with 0.2% casein buffer four times.
7. Add 100 ,ul conjugate (1:2 K goat anti-mouse Igm peroxidase conjugate for HC~D pre-blocked wells and 1: 400 mouse anti-HulgG-peroxidase conjugate for monoclaonal preblocked wells) into each well including PBS control wells. Cover the plate with para~ilm.
.,' 8 . Incubate at R . T . l f or two hrs . ~ -9 . Aspirate and wash with 0 . 2% casein buffer four times.
;

10. Add lO0 ~l l~B substrate into each well. React at R.T.
for one hr.
~ .
11. Read plate at 650 nm and then add 50 ,ul 1.0 M HCL0 stop solution into each well, read plate at 450 nm again.

12. Calculation:

Inhibition % = 100~6 - Assay well mean O . p, x 100% PBS
control well Mean O. D .

WO~)1/02~52 PCT/US90/04272 Im~unoas$av Procedure for Atherosclerotic Plaqu Q~ti~ens To each microtiter plate coated with anti-plaque antibody, 90 microliters/well of the serum sample of the patient being tested, mixed with 10 microliters/well of noraml mouse serum, is applied. The plates are covered to prevent drying and incubated overni~ht. The sera mixture is removed, and the plate is washed 3 times with casein wash buffer.

100 microliterstwell of horseradish peroxidase conjugated antibody or alkaline phosphatase conjugated anti~plaque antibody prepared in accordance with the procedure described hereinabove is applied to each well, and the plates are covered to prevent drying and incubated for 2 hours. The lS enzyme labeled antibody solution is removed, and the plates are washed 4 times with casein wash buffer.

100 Microliters/well of either tetramethylbenzidine, in the case of horseradish peroxidase, or 4-methylumbelliferyl phosphate solution (3M Diagnostics Systems~, in the case of alkialine phosphatase, is then applied to the well. The microtiter plates are then read in either a colorimetric reader (Molecular Devices) or a fluorometer (3M Diagnostics) every 10 minutes until the the maximum reading or 1 hour is reached.

Immunoassay Procedure for Antibodies Which Bind S~eciically to Atherosclerotic Plaque ~o each microtiker plate coated with atherosclerotic plaque antigan, 100 microliters/well of human serum is appliad ~Sample may be diluted). The plates are covered to prevent drying and incubated for 2 hours, and the residual so~ution is removed, and the plates washed three times with casein wash buf~er.

''' ' : ' . ' - ' ~ :~: : . '' ' ' w091/0225 7 PC~ S90/04272 100 Microliters/well of a ~olution of af~inity purified goat anti-IgG, IgM, IgA, or IgE conjugated to either horseradish peroxidase or alkaline phosphatase (appropriately diluted) is applied to each well. The plates are covered to prevent drying and incubated for 2 hrs. The anti-IgG, IgM, IgA, or IgE 601ution is removed and the plates washed three times with casein wash buffer.

100 Nicroliters/well of either a tetramethyl benzidine solution, in the case o~ a horseradish peroxidase conjugated antibody, or a 4-methylumbellif~ryl phosphate solution (3M
Diagnostic Systems), in the case o~ an alkaline phosphatase conjugated antibody, is then applied to the well. The microtiter plates are then read in ~ colorimetric reader (Nolecular Devises) or a fluorometer (3M Diagnostics) every 10 minutes until the first maximum readinq or 1 hr is reached.

~l~gue Anti~en Coated Microtiter Plate PreP~ration 100 Microliters of prepared dilutions of plaque antigen are applied to the sur~ace of IMMULON II microtiter plates (Dynatech). The coating solution dilutions are 1:10, 1:100, 1:1000 and 1:10,000. The plates are tapped gently and to insure the coating solution covers the bottom of each well completely. The well are incubated at 4C overnight in a covered, humidified box.

The coating solution is discarded and 200 microliters PBS is added per well. The wells are then incubated at room temperature for 1 hr in a humidity box, then washed with 200 microliters o~ Wash Buf~er (PBS, 0.5% Tween and 0.02% ~odium azide), and stored in a humidity box at 4C until use.

Antibodv Coated ~ic~o~iter Plate wo 9 1 /()22s2 PCT/USgO/~4272 _75_ ?~ 3 oo Microliters o~ prepared dilutions of anti-plaque antibo~y ars applied to the ~ur~ace of IMMnLoN II ~icrotiter plates ~Dynatech). The coatlng solution concentrations are selected to be from 1-5 micrograms/well but can be varie~ up or down depending upon the selection of other reagents and immunoassay procedures to be followed. The plates are tapped gently and to insure the coating solutin covers-the bottom of each well completely. ~he wells are incubated at 4C overnight in a covered, humidified box. ~
,-The coating solution is discarded, and 200 microliters of lS
BSA in PBS is added per well. The wells are then incubated at rm temp ~or 1 hr in a humidity box, and the BSA solution ~;
is removed. The wells are the washed with 4 times with 200 microliter~ of Wash Bu~fer ~PBS, 9.5% TWEEN, and 0.~2%
~odiu~ azide), and stored in a humidity box at 4C until use.

V. PROCEDURES FOR ANTIBODY LABELING
15H5 antibodies and 17H3 antikodies Peroxidase CQni~g~te The following method is an one-step procedure for coupling peroxidase to a monoclonal antibody (e.g. the monoclonal antibody produced by hybridoma 15~5 or 17H3):

1. x mg purified 15H5 Ab or 17~3 Ab is dialyzed against lOmM PBS pH 6.8 at 4C overnight.

2. 2x mg peroxidase enzyme is dissolved into the above Ab solution.

3. Add dropwise glutaraldehyde (1% solution) into above mixture. The ratio of glutara}dehyde solution to (antibody peroxidase mixture) is 1:20, then gently mix it on shaker at room temperature for two hours.

'''~

w09l~0~252 PCT/U~90/04272 ~ ~ C~; `'3 ~ 76-Dialyze conjugate against 10 mM PBS pH 7.2 (change buffer three times) at 4OC overnight.

5. conjugate is filtered by 0.2 ~ filter.

1-L3~ Labçled Antibo~v ~qe~a~

The antibody can be labeled with I-131 by the Pierce Iodobead ~ethod described by Rosebrough, s. (supra, p 575)~
100 ~l (microliters) of 0.2 M PBS, pH 7.0, i5 added to 150 ~g of antibody, fo~lowed by the addition is incubated for 10 min. The solution is removed with a pipette and reserved, and the beads are washed with 100 ul of 0.2 M PBS, pH 7Ø
The solution and wash buffer ~rom the beads are combined.
To ~eparate the ~ree iodine, the solukion is ~ashed exhaustively with a CENTRICON C-30 ~ilter. Approximately 60% of the original I-131 is bound to the antibody.

DTP~ Labeled An~ibody D~PA is coupled to antibody by the method of Hnatowich, D.
et al. ~Journal of I~munolical Methods, ÇS:147 tl983)~. The bicyclic anhydride of DTPA is prepared as described by Hnatowich D. et al., tInt. J. Appl. Radiat. Isot. 33:327 (1982~] and is stored as the solid in a desiccator at R.T.
suspension o~ the anhydride in dry chloroform or ether (O.01 ~g/ml) is prepared and an aliquot evaporated under nitroqen in a clean, dry teat tube. from 10-20 ~l of the antibody solution in 0.05 M bicarbonate buffer in saline, pH
7.0 - 7.5, is i~mediately added and the contents agitated for 30 to 60 sec. If the coupled antibody i5 to be puri~ied be~ore labeling, the preparation is diluted to about 0.2 ml with the above buffer and purified on a 5 cm gel filtration colu~n tG-50; Roche Diagnostics, Nutley, NJ) using saline eluant. The purification takes about 5 min and provides a product which is approximately 95% pure.

.. .

:: `

.. .. .. ; .

Wo 91/02252 PCl/US90/04272 In-lll h~beled AntibodY

Chelation grade I--111 (Medi Physics, Emeryville CA) in 0.5 M acetate bu~fer, pH 6.o, is added to the DTPA-antibody coniugate solution described above, in ~toichometric quantity. Thi~ yields the In-111 chelate-antibody conjugate.
The product can be purified by conventional chromatography.

Flu-Q~ sc~ dy-~aent Antibody is dialyzed overnight against pH 9.5 carbonate/bicarbonate buffer solution. The concentration is determined (for example by otical density at 280nm. A
801ution of luorescein isocyanate (1.0/mg/ml) in DMS0 is prepared, and the desired volume (1-10% of total protein solution volume) is added to the antibody solution dropwise, with stirring. The reaction proceeds for two hours, shielded from light. The product is purified by gel filtration on SEPHADEX G-25 gel in PBS containing 0.1% NaN3 to separate the unreacted or hydrolyzed fluorochrome. The absorbance of the conjugate is measured at 280 nm and at 495 nm to yield a solution of ~luorescein labeled antibody.

Rhodamine Labeled Antibody ?
Antibody is dialyzed overnight against pH 9.5 carbonate/bicarbonate buffer solution as described in Example 7. A solution of rhodamine isocyanate (10.0 mg/ml) in DMS0 is prepared, and the desired volume tl-10% of total protein solution volume) is added to the protein solution dropwise, with stirring. The reaction proceeds for two `~
hours, shielded from light. The product is purified by gel ~ltration on SEPHADEX G-25 gel in PBS containing 0.1% NaN3 to separate the unreacted or hydrolyzed fluoroschrome. The absorbance of the conjugate is measured at 280 nm and 550 nm to yield rhodamine labeled antibody.

w o 91/02252 Pc~r/US90~04272 ~ou~arin Labeled Antibody Antibody which binds specifically to atherosclerotic plaque (1 m mole) is dialyzed st 4C against a buffer ~olution of 0.01 M PBS, pH 6.~ overnight. To this solution i~ added 50 nmole of 3-carboxy-7-hydroxycoumarin. The solution i8 added 50 mnole of 3-carboxy-7-hydroxycoumarin. The solution i~
cooled in an ice bath and added with 50 nmole of l^ethyl-3-(3~dimethylaminopropyl)carbodi~mide hydrochloride.
After addition the mixture was stirred at 4~C ~or one hour and chromatographed on a 2.5 c 5U cm column of SEPHAEX G-50.
The absorbance of the conjugate is monitored at 345 nm to yield a solution of coumarin labeled antibody.

~ile Blue A Labeled Ant bodY

Nile Blue A (350) mg) is diazotized according to the procedure described above. The solution containing tha diazonium salt o~ Nile blue A is add~d dropwise to anti-plaque antibody (0.05 m mole) in on 0.1 M PBS, pH 8Ø
After addition the mixture is puri~ied on a 2.5 x 50 c~
SEPHADEX column. The absorbance of the eluate is monitored at 628 nm to yield the Nile blue A labeled antibody.

Hematopor~hvrin Con~uaated Antibody 1-Ethyl-3-(3-dimethylaminopropyl) carbodiimide (50 m mole) is added to a mixture of hematoporphyrin (50 m mole) and anti-plaque antibody (1 m mole) in 0.01 M PBS, pH 6.8.
After addition, the mixture is stirred at 4C for 2 hours.
The mixture is then purified with a SEPHADEX G-50 column to yield the hematoporphyrin conjugated antibody.

Tetracycline Coniugated Antibo~y The NHS ester of carboxymethyltetracycline is prepared as WO~ 22~2 P~T/US90/04272 -79~

described ab~ve. To the anti-plaque antibody solution in O.1 M PBS, pH 8.0, is added portion wise the NHS ester vf t2tracycline. ~ft~r addition, the mixture is allow~d to ~it at 4OC for 2 hours. Purification of the mixture on a SEPHADEX G-50 column yields the tetracyclin~ con~ugated antibody.

EnzYme Labeled Anti-Plaque Ant~bo~y ~nti-plaque antibody may be conjugated wi~h alkaline phosphatase ~ollowing the modified prooedure o~ O'Sullivan, M. et al. tAnalytical Biochem., loQ:loo (1979)].

Horseradish peroxidase is conjugated to anti-plaque antibody in accordance ~ithe procedure of Nygtren, H. et al. ~edical Biology, 57:187-191 (1979)] as follows: Hor~radi~h peroxidase (HRP, Type II or TYPE VI, Sig~a) i5 dissolved in 0.05 M carbonate: bicarbonate bu~fer, pH 9.5, containing 0.25% glutaraldehyde (GA, Polaron). A~ter 2 hr at room ~0 t~mperature, the excess GA is separated ~rom t~ GA-HRP on a Sephadex G-25 column (0.7 12 cm, Pharmacia) equilibrated with 0.15 H NaCl. The GA-HRP complex is, in a second step, mixed with the antibody in 0.05 M carbonate: bic~rbonate buf~er, pH 9.5, containingg 0.15 M NaCl, at different IgG:HR~ ratios for 16-64 hr at 4C. The reaction is stopped by the addition of lysine to a final ~oncentration of 0.02 M.

Trvpsin-labeled ~ntibody mlMaleimidobenzoyl N-hydroxysuccinimide in dry dimethyl~ormamide (100 ul, m mole/1) is added to the purified antiplaque antibody. The resulting ~ixture is stirred at room temperature for 30 min. The antibody solution i5 fractionated on a SEPH~DEX G-50 colu~n with PBS
a3 oluant. To the pooled antibody solution is added trypsin ;' "

~'0'~l/0225~ Pcr/uss~/

tl3 mg) at room temperature. After addition, the mixture is stirred for anoth~r 2 hr. 2-Mercaptoethanol i8 a added to a fi~al concentration of 2 mM/1, and the solution stirred for a further 30 min. The conjugate i5 dialyzed overnight against PBS (3 x 2 liters) to yi~ld the tryp~in-labeled antibody.

~pal~ ~ab~led An~ib~lY

Equimolar amount of purified the anti-plaque antibody in papain are mixed in a solution of 0.1 M sodium-potassium.
To this solution is added 1 vol~% glutaraldehyde solution in phosphate buf~er. A~ter addition, the mixture is stirred at r~om ~emperature for 3 hours and finally dia}ysed overnight against 0.1 M. P~S, pH 8rO~ at 4C. The mixture is further purified on a SEPHADEX G-50 colu~n to yield the papain-labeled antibody.

Hvaluronidase Labeled Antibodv To a solution of hyaluronidase (5 mg) in 1.0 ml of 0.3 M
bicarbonate buffer, pH 8.0, is added phenyl isothicocyanate to protect the free amino groups on the hyaluronida-~e molecule. After addition, the solution is stirred gent}y at room temperature for 1 hour. A solution of sodium periodate (0.06 ~ in distilled water) is added and the ~ixture ~tirred gently for 30 minutes. One ml of 0.16 M of ethylene glycol in distilled water is added, and the solution stirred for another 1 hour at room temperature. After dialyzing against 0.01 M of sodium carbonate buffer, pH 9.5, at 4C (3 x 1 liter), the mixture is mixed with purified anti-plaque antibody. The reaction mixture is stirred for 2-3 1 hours and treated with 5 mg of sodium borohydride. The mixture is allowed to stand at 4C overnight. Following dialysis against PBS bu~fer, the dialyaia against PBS buffer, the mixture is chromatographically purified with a 1.5 x 85 cm .. . .

Wo 91/02252 PCJ/US9~/~4272 -81- ?~

(BN) SEPHP.DEX G-100 column to yield th~3 hyaluronidase antib~dy .

Kallikrein Labeled Antibodv Equimolar amount of purified anti-~normal vascul~r epithelium) antibody and urinary kallikrein are mixed in 0.1 M ~odium-potassium phosphate buffer, p~ 6.S. The antibody and kallikrein are coupled following the procedure of Example 12. The final reaction mixt~rP is purified with a 2.5 x 50 cm SEPHADEX G-200 colu~n to yield kallikrein conjugat~d anti-(normal vascular epitheli~m) antibody.

~olla~enase ~abele~ Antibod~
Collagenase I (65 lysines) collagenase II (50 lysines) is dissolved in 0.05 M phosphate bu~fer, pH 8Ø To this solution is added m maleimidobenzoly N-hydroxysuccinimide in anhydrous di~ethylformamide ~100 ul, 8 m mole/liter).
After addition, the mixture is stirred for 30 minutes. The enzyme solution is first fractionated SEPHADEX G-50 column with PBS as eluant and teated with purlfied anti-plaque antibody. The mixture is stirred fcr 2 hours at room temp~rature and added with 2-mercaptoethanol to a final concentration o~ 2 m mole/l. The mixture is 6tirred for a further 30 minutes and chromatographically purified on a SEPHADEX G-50 column to yield collagenase conjugated anti-pla~ue antigen antibody.

Beta-1 ~nticollaqenase Labeled An~ibody Beta~ anti-collagenase and anti-(normal vascular epithelium) antibody are coupled with glutaraldehyde by the procedure described hereinabove. The final reaction mixture is puri~ied with a 2.5 x 50 cm SEPHADEX G-200 column to yield the beta1 anticollagenase labeled anti-~normal vascular W09~ 2~, PCT/US90/04272 ~pithelium) antib~dy.

VI. P~OCEDURES FOR IMAGING AT~EROSCLEROTIC PL~QUE

DPTA ~ouplina of Z2D3 1. Preparation of mixed Anhydride o~ DTPA tKrejcarek and Tucker, BBRC, 77:5~1 (1977)]
a) lO0 mg of triethylammonium-DTPA ~ 2 ml acetonitrile b) Cool to 4C
c) Add isobutylchloroformate d) ~ixed anhydride of DTPA formed at 4C.

2) Modification of Z2D3 a) 1.69 Z2D3 in 0.45 ml 0.l M NaHC03 (2 x l0g mole~) b) Add 15 ~l of carboxycarbonic anhydride of DTPA (2 x 10-7 moles c) React at RT for l hr.
d~ Dialyz2 in 6 ~ 0.15 M NaCl at 4C, overnight 3) In-lll labeling of D~PA-Z2D3 a) 0.2S mg DTPA-Z2D3 in l5l ~l and l mCi 111In-Cl3 in equi-volume o~ 1 M citrate pH 5.5 b) Incubate 30 min at RT
c) Separate ~re~ from antibody bound 111 In by sephadex G-25 column chromatography. The 111In-DTPA-Z2D3 was eluted with 0.15 M NaCl. The activity in the void volume was pooled and used ~or in vivo studies.

4) In Vivo Studies Rabbits with approximately 6 week old denuded descending aortic endotheliu~ were injected with 0.5 - l.0 mCi 111In-DTPA-Z2D3 intravenously via ear vein administration.

WO~l/0~52 PCT/US90/04272 -83- 2~,S~

The rabbits were anesthetized with ~etamine and Rompum, and imagad with a gamma camera (Ohio Nucl~ar, Sigma 410 or 1003 equipped with a medium energy collimator. Anterior images were obtained soon aft~r IV administration and at 24 H. The animal~ were then infusQd intravenously with 5 ml of 5~
Evans Blue, followed by euthanization with IV pentobarbitol.

The descending aorta segment from the thorax region was used as normal control relative to the de-endothalialized abdominal segment of descending aorta. The aortic segments were cleaned of blood and dissected enfaced.

These segments were weighed and then counted in a gamma counter. The segments were then used to obtained macro-autoradiographs. The ~egments were }aid enfaced on mammography film and allowed to develop ~or 1 to 2 weeks.
Subsequently the ~ s ~ere developed and color photographs of the segments made for comparison to the autoradiographs.

ocalization of ExE~erimental-atherosclerotic LesiQ~ hL~
Monoclonal Antibody Monoclonal antibody Z2D3-5C5 F(ab')2 fragments specific ~or an atheroma connective tissue antigen were used for non-invasive imaging of atheromatous lesions in an experimental rabbit mode} produced by balloon catheter de-endothelialization of the descending aorta followed by a high cholesterol and fat diet. Seven weeks later, 3 animals were injected intravenously with In-lll Z2D3. I-125 Z2D3 and I-125 nonspacific monoclonal F(ab')2 were also in~Qcted ln each one o~ these animals. Images were recordQd at 15 min, 24 H, and 48 H. ~he normal (N) and lesioned (L) segments were weighed, counted by gam~a scintigraphy and expresssd as mean percent injected dose per gram. Lesions could be visualized in 1 rabbit with In-lll Z2D3 and in another with I-125 Z2D3. Macroautoradiography of the ex-wosl/02~5~ PCT/US90/~42~2 , 84-jJ 3 vivo aorta de~onstrates that the uptake of Z2D3 was ~uperior to that of nonspecific antibody. The In-lll Z2D3 uptake was ;:
0.035 ~ 0.0001 in L as compared to 0.0008 + 0.0003 in ~.
The I-125 Z2D3 uptake was 0.026 and 0.005 and the I-125 nonspeci~ic F(ab')z uptake was 0.008 and 0.003, ~espectively. This study indicates the potential feasibility of non-invasive visualization of atheromatou6 lesions of aorta with monoclonal antibodies.

~II. PROCED~R~S FOR ~I8~0~0GY

Histolo~ica.l Counter-Stainina With Hemotoxvlin ~erner-l ~eagents and Su~Plies Absolute ethanol (Gold Shield Chemical-Proof 200).
Hemotoxy}in Lerner-~ (SP ~S7737-l or equivalent~. Xylene SSP ~8668-4, Mallinckrodt or equivalent). Coverbond" :;
Mounting Media (SP ~763904 or equivalent).
Deionized water.
Coplin jars (SP #S7655-1 or equivalent).
Staining dish~s (SP ~S7675-1 or equivalent).
Coverslip (SP ~M6020 or equivalent). ;

Stainina Procedurç
Bg999n~ ~rocedure.

1. Hemotoxylin Lerner-1 2 dips (8 counts each) 2. D.I. water (3 times) l dip each 3. 70% ethanol 15 ~ip8 4. 95% ethanol 15 dips 5. Absolute ethanol (2 times) 20 dips each 6. Xylene (2 times) 1 minute each :
7. Place 3 drops of Coverbond mounting media on coverslip.
~:
~istoloaical Stainlna with Vectorn (Avidin-Bio~in_Complex~, :

'' ' .. ' '~

wo9l/02 s2 ~CT/US90/0~27 Reaaent and Su~Plies ~ -Reaaent and SUP~1 1 es Unfixed fro~in tissue section ~5-6 ~m thick) preferably freshly cut and stored overnight at -80C.
Bovine S~rum Albumin ~SA ~Sigma ~A-7030).
Normal Horse Serum (Vector - Peroxidose Mouse IgG PR 4002). ::
Primary antibody.
Biotinylat~d horse antimouse IgG (Vector - P2roxidose ~ouse lo IgG PK 4002).
Hydrogen peroxide 30~ (Sigma ~H-loos or equivalent).
~ethyl alcohol absolute low acetone (Mallinc~rodt ~3016-4 or equivalent).
3,3'-Diaminobenzidine - DAB (Sigma ~D-9015).
Trizma base (Sigma ~T-1503 or equivalent).
Sodium chloride (Nallinckrodt t7581 or equival~nt).
lN HCl (Prepared from ~icca chemical company ~3740 or equivalent).
Sodium phosphate, dibasic anhydrous (Mallinckrodt t7917 or .
equivalent).
Potassium phosphate, monobasic, anhydrous ~Hallinckrodt ~7100 or equivalent).

Eoui~men~
Lab-line orbit shaker . ~. :
Fume hood :
Covered, dark humidified container . .~ . .
Reagent Prçparation 30 Phosphate-buf~ered saline (PBS) pH 7.2, 1 liter Sodium chloride NaCl 7.2 g Sodium phosphate Na2HP04 1.48g dibasic, anhydrous Potassium phosphate XH2P04 0.43g monobasic, anhydrous .:
D.I. water 0.5 to 1000 ml.

WO91/02252 PCT/US9~/04272 PBS ~ 0.1~ BSA, pH 7.2, 1 liter BSA lg PBS, pH 7.2, dissolve in 1000 ml.
Make fresh every time Tris-HCl/saline buffer (0.05 M Tris-HCl ~ 0.15 M NaCl), pH
7.6 0.5 Tris ~Cl pH 7.6 (stock solution) Trizima base.
D.I. water, dissolve in HCl; adjust to pH 7.6 D.I. water 0.5 to lO00 ml.
O.9% NaCl (normal saline). ~; :
NaClO
D.I. water, dissolve in ~000 ml.
Tri~ ~Cl/saline buffer (working solution).
~ix 1 part of 4.3.1 with nine parts 4.3.

Prepare fresh every time.
3,3'-Diaminobenzidine tetrahydrochloride (DAB).
O.5% DAB (stock solution).
(1 vial contains 0.1 g lyophilized DAB).
Tris/saline buffer, pH 7.6. Disso}ve and aliquot 1 into vials and store at -20C.
, 0.05% DAB and 0.01% H202 working solution, Stock DAB 1 ml ~ris/NCl saline buffer 9 ml 30% H202 Vectastain ABC Reagent (1 drop - 50~1 Reagent A 2 drops Reagent B 2 drops QS with PBS/BSA to 10 ml ~ .
Mix immediately and allow to stand for 30 minutes before ... .

~ ! . . ~ . .

wogl/02~5~ PCT/USg~/04272 use.

P~o~çdure Immunoperoxidose staining 1. Remove slides from freezer and dry for 10 ~inutes.
2. Wash in PBS/BSA on shaker (very gently) for 20minutes. s Note: A~ter this step, sections should not be allowed to dry out during any of the remaining procedur~s. Drying out can lead t~ misleading results.
3. Incubate with 3% normal horse serum ~or 20 minutesO
4. Blot excess serum from section. ;~
5. Incubate section with appropriate dilution of pri~ary antibQdy for 30 minutes, or longer if required, in ~`~
hu~i~i~ied_con~aiQ~
6. Wash ~lides for lO~inutes in PBS/BSA on shaker.
7. Incubate section with antimouse Ig& biotinylated antibody (1:50 dilution in PBS/BSA) for 30 minutes.
8. Wash slides for lQ minutes in PBS/BSA on shaker.
9. ~lock endogenous peroxidases with 0.3% H2o2 in methanol ~`~
for 10 minutes on shaker. ~::
10. Wash slides for lOminutes in PBS/BSA on shaker.
11. Incubate with Vectastain ABC for 20 minutes.
12. Wash slides for lOminutes in PBS/BSA on shaker.` :
13. Incubate with DAB for 7 minutes. ;.

14. Wash ~ection for 5 minutes in D.I. water.

15. Perform counter staining with hemotoxylin. .~

~III. nBT~OD8 OF TR9A~ING AT~9R08C~ERO~C PLAQU~ :
.Enzym~tic_Reduction o~ Atherosclero~c Plaque by P~oenz~
Tar~etinq_with Plaque-S~eci~ic Antibody F~aoments :~

1. FAB2 fragments having the ~ollowing properties are intravenously administered (see Fiqure 30A):

WO gltO22~;~ PCr/US90/0427;~

a. Bl~unctional antibody with one hypervariable region binding Z2D3 antigen, and the other binding the propeptide of the fibroblast collagenase proenzyme.
S
b. Bifunctional antibody with one hypervariable region binding Z2D3 antigen, and the other binding the propeptide of the neutrophil collagPnase proenzyme.

c. Bifunctional antibody with one hypervariable region binding Z2D3 antigen, and the other binding the propeptide of the type IV/V
collagenase proenzyma.
d. BLfunctional antibody with one hypervariable region binding Z2D3 antigen, and the other binding the propeptide of the stromelysin proenzyme.
e. A mixture o~ the four FAB2 fragments above (a-d), labelled with radionuclide x, and representing a ~inor component of the overall pool of FAB2 fragments. -2. The patient is scanned with a gamma camera, attuned to radionuclide x, 24 to 48 hours a~ter administration of the F~b2 fragments, and an estimate is made of the quantity o~ FAb2 fragments localized ln the target lesions, based on the amount of radiolabelled FAb2 ~ragments detected. (see Figure 30B) ;
3. An appropriate mixture of fibroblast collagenase, neutrophil collagenase, type IV/V collagenase, and stromely~in proenzymes is intravenously administered, in proportion to the number of receptive FAb2 fragments :

W09lt02~52 PCTlUS90/04272 calculated to be localized in the targe~ lasio~s. A
6mall portion o~ each proenzyme is labelled with radionuclide. (see Figure 30C) 4. u6ing a gamma camera attuned to radionuclide y, the proenzyme mixture i5 administered in incremental doses, until the desired amount is localizQd in the lesions.
The desired amount is that which will dissolve enough plaque ot relieve the arterial obstruction, without causing aneurysm formation or per~oration in severely diseased vessals. (see Figure 30D) 5. Tissue plasminogen acitivator (TPA) in intravenously administered in an amount sufficient to generate enough circulating plamsin to cleave ~he functional enzymes ;~
~rom their bound propeptides, yet insuf~icient to -~
create a hemorrhagic diathesis. (see Figure 30E) 6. Once released from the FAb2 fragments localized in the plaque, the collagenase and stromelysin enzymes immediately bind and begin degrading their adjacent substrates:
a. collagen type I (neutrophil collagenase) b. collagen type III (fibroblast collagenase~
c. collagen type IV/V (type IV/V collagenase) ~ ;
d. proteoglycans/fibronectin tstromelysin).

This invention is ~urther illustrated by the following 6pecific, but non-limiting exampl~s. Temperatures are given in degrees Centigrade and percents as weight percent~ unless otherwise specified. Examples which are constructively reduced to practice herein are presented in the prese~t tense, and examples representing laboratory experi~ents previously reduced to practice are presented in the past tense. ;
Plaque Treatment ;~

-~

W091/(3225~ PCr/US90/04272 9 o-A representative treatment protocol can be as ~ollows:

1. Catheterized artery (coronary ostia, carotid, aorta or peripheral vessels).
2. Visually examine Yessel lumen with contrast agent.

3. Inject antibody-enzyme inhibitor conjugate in a physiologically acceptable solution.
4. Allow excess olearance time. ~-5~ Inject a plaque antibody~enzyme conjugate in a physiologically acceptable solution in which the enzyme r~mai.ns active.
~, 6. Let circulate for sufficient time to permit perfusion (e.g. 60 min.) 0 7. Visually examine vessel intima and/or media with contrast agent under fluoroscopy.

8. Repeat Steps 5-7 until lumen restoration is satisfactory.
9. Inject enzyme inhibitor in physiologically acceptable solution to stop all enzyme reaction of any given therapeutic episode.
0 10. Repeat Step 7 after a few minutes or after clearance of reaqents. I~ no ~urther enzymatic reduction o~ vessel is seen, terminate procedure. Otherwise repeat Step 9 and 10, or introduce inhibitor by IV.
5 ~iscussion WO 9ItO22:~ Pcr/US9O/0427~
~91 2~"~
Atherosclerosis i5 characterized by the presence of one or more of the atherosclerotic plaque specific antigens disclosed in the subject invention. Because of the cyclical nature o~ the immune response, either the plaque antigen or antibody which specifically binds to the antigen ~ay be detected at any one point in time. Accordingly, the presence of either the antigen or antibody thereto is indicative of atherosclerotic plaque. Figure 5 show~ a comparison of lev~ls of IgA specific to atherosclerotic plaque antigen present in the sera of normal persons less than 35 years of age, and persons diagnosed as having coronary artery disease (CAD). Ae indicated in the fiqur~, 70 of 207 persons under 35 years of age, and 21 of 121 normal persons over 35 years o~ age, had elevated levels of IgA in their ser~m. Normal persons are defined as apparently healthy individuals not ~nown to have CAD.
Figure 6 shows a comparison of levels of atherosclerotic plaque specific antigen present in the sera normal persons greater than 35 years of age, and persons diagnosed as having CAD. As with levels of IgA, levels of IgG which specifically binds to the atherosclerotic plaque specific antigen~ are higher in persons with CAD. Of the persons afflicted with CAD, 45 o~ 125 tested showed elevated levels of antigen, whereas only 4 of 25 normal persons under 35 years of age, and 8 of 49 normal persons over 35 years of age showed elevated levels of antigen.

The amount of antigen expressed was studied as a function of age and severity of disease. Figure 7 shows a plot of antigen level vs. patient age for apparently healthy individuals. In contrast, Figure 8 shows the same plot for individuals having 50% or greater occlusion of their coronary artery, and Figure g shows the same plot for individuals having mild CAD. As is graphically illustrated, the amount of antigen present in sera is less dependent upon age than upon severity of CAD. Atherosclerosis is ~herefore .. ,.. ,.. ., .... . .. :- , , , ~091/02252 PC~/US90/04~72 indica~ed by the presence of the atherosclerotic plaque specific antigen.

The prevalence of antibodies which bind speci~ically to the atherosclerotic plaque antigen appear to increase with age in normal persons. However, this increase is small relative to the levels of antibodies detected in patient~ with CAD.
(Figures 10-14).

Figure 15 depicts positive prevalence for the antigen in a -~
population based on age. The persons tested were from 31 to 75 years o~ age.

Initial studies of the atherosclerotic plaque antigen involved phosphate buffered saline (PBS) extraction of human atherosclerotic plaqu~. The extracts were then run through HPLC-DEAE fractionation procedures and fraction~ were tested ~ ;
to determine i~ they would react with serum ~rom patients having coronary artery disease (CAD) (See Figure 2). There existed a large amount of binding in the fractions wh~ch elut~d just after the void voluma. In the presence of normal serum (i.e. that obtained form patients under 35 years of age without CAD~, there was no antigen-antibody binding.
The fractions which immediately followed the void volume of the column showed the highest levels of binding to CAD
serum, and were used to immunize Balb C mice. Splenocytes ~rom mice that produced antibodies were then fused with the immortal cell line SP2. One such fusion produced hybridoma 15H5.

The 15H5 monoclonal antibody was then covalently coupled to separose/agarose. This solid support antibody complex was then used in a number of assays to determine levels of antigen in various samples. Further, by contacting a PBS

, :, . ..
` `:

W09l/02252 PCT/US90/04272 -93~

extraction of human atherosclerotic plaque with the 15H5 monoclonal antibody-solid support complex, it was possible .
to remove the auto-antigen from other extracted materials.
The resulting complex was then washed and purified auto-antigen was then eluted Srom the complex. :-:

'~ ' .

w~/022s~ PCT/US90~427 2 ~
Tabl~ 1 Plaque Serum Ch~r~ct~ristiç ~ a~ a~
1. Reacts with coronary + +
artery patient serum 2. Reacts with 15H5 Ab + +
Reacts with 17H3 Ab ~ +
Reacts with Z2D3 Ab - _ Reacts with QlOE7 Ab 3. I~munoreactivity + +
following boiling for 1 hr.
- 4. Solubility in trichloroacetic + +
acid 5. I~munoreactivity a~ter + +
trichloroacetic acid ~
6. Im~unoreactivity after TFA . +
7. Immunoreactivity after TFA - -and heat 8~ Molec~lar weight estimate >500,000 mw ~500,000 ~w by gel sieve chromatography 9. Molecular charge by ion exchange chromatography, ion exchange gels:
- DEAE sepharose - QAE sepharose - 5ulfopropyl sepharoseneutralneutra 10. Glycosidase sensitive+(limited) +(l~ted) ;
11. Protease resistant + +
:
12. Acetone precipitable yes yes 13. Extractable with chloro~orm no no 14. Detectable primary amino no no groups ..
lS. U.V. absorbance none none

16. Sensitive to chaotropes,no no ;.~ :
S~S, or alkylation-reduction ` :

17. Sensitive to periodate treatment yesyes ., .

W09l/022s2 PCT/US~0/0427~
~95~ 2 ~ 3

18. Sensitive to urea no no To further characterize the antigen, antibodies and antisera which specfically bind to the antigen were reacted with various components found in atherosclexotic plaque (see Table 2). ~ :
-w~l/022~2 PCT/US90/0427Z

Ta~le ANTI-SERA, MONOCLUNAL ANTIBODY REACTIVITY

Anti~odv/Anti-Sera Immuno-~ctlvit Apolipoprotein A-I
Apolipoprotein A-II
Apolipoprotein B
Apolipoprotein C~
Apolipoprotein E
- Human Collagen Type I
Human Collagen Type II
Human Collagen Type III
Human Collagen Type IV
Human Collagen Type V
Human Collagen TypP VI
Fibronectin Keratin - :
Laminin Tenascine Vitronectin '','.

:
, ''. ~ ' W09l/0225~ P~/US9~/04272 -97~ v As shown in Table 2, antibodies which bind specifically to the various components of atherosclerotic plaque do not bind to the 15H5 antigen showing that the antigen is not one of the components listed in Table 2.

To further characterize the antigPn the antigen was reacted with various enzymes to determine if the antigen is suscepti~le to degradation. It was found that proteinases, deoxyribonucleases, lipases, and ribonucleases did not degrade the 15H5 antigen. However, the 15H5 antigen was partially degraded by certain glycosidases, especially ~-amylase, B-amylase, and glycoamylase. This suggests that the 15H5 antigen comprises a structure which has a carbohydrate nature. The results obtained with indi~idual enzymes are shown in Table 3.

WO9l/02252 PCT/US90/04272 3 ÇJ' ~ 9~3 _ ~able 3 ENZYME REAC~ TY WI~H 15H5 pl~OUE ANTIGEN
Enzyme Reactivi~y Proteinases Bromelain Collagenase (Achromoba-ter) Collagenase/Dispase Collagenase Type I
Collagenase Type II
Collagenase Type III
Chymotrypsin Dispase - 15 Elastase Endoproteinase E
Endoproteinase Lys-C
Papain Pepsin Plasmin Pronase Proteinase X
Staphylococcal Protease V-8 Thro~bin Trypsin Glycosidases Chondroitinase ABC
Chondroitinase AC I
Chondroitinase AC II
Chondroitinase B
Heparinase Heparinitase Hyaluronidase Keratanse ~-amylase (+
~-amylase (+
~-mannosidase B-mannosidase a-galactosidase Endo-~-N-Acetylgalactosaminidase Endo-B-galactosidase ~-fucosidase ~-glucoronidase B-N-Acetylhexoseaminidase N-Acetyl-D-glucosaminidase Endoglycosidase D
Endoglycosidase F
Endoglycosidase H
Glucoamylase . .. . .... .~ -. . . , . ~ :

: . ,-. .... ... ., ~ :

~Osl/022s2 PCT/US90/04Z72 _99_ ~ :
6~ . :
Invertase _ Neuraminidase Other Deoxyribonuclease Lipase (~acteria}) -Lipase (Yeast) , ribonuclea~e - .

Further characterization of the antigen was accomplished by measuring lectin binding to the auto-antigen. For example, Conayalia ensiformis and Triticum ,Vulaaris showed strong .' 15 binding to the 15H5 antigen, whereas.Lens cuLinaris, Ricinus commonis, and Tetra~onolobus pur~ureai showed moderate binding, and other lectins showed no binding. These results :~ are summarized in Table 4.

'~
'"
:~

, , ~
'.

.
.

W~l/1)2~52 PCT/US90/04272 ~r~~
9 ~ ,b -: -100-~able 4 LECTIN BINDING_PROFILE OF 15H5 ATHEROSCLEROSIS ANTIGEN

Plaque Extracted :~
e5~a Antiaen_ Arachis hypgaea (Peanut) - ;
Bandeiraea simplicitolia Conavalia ensifo~miis (Con A) ~++
Dolichos biflorus ~Horse gram) Glycine max (Soybean) - `
Lens culinaris (Lentil) ++ ~ :
Limulus polyphenus (Limulin) Phaseolus vulgaris-E
(Phytohemaglutinin) ~
.
Phaseolus vulgaris-L
(Phytohemaglutinin) ~isum sativum (Pea) -Ricinus commonis (RCA I) ++
Sophova japonica - .
(Pagoda Tree) Tetragonolobus purpureas +~
(Lotus) Triticum vulgaris (Wheat germ) Ulex europaeus (UEA-I) Ulex europaeus (UEA-II) Vicia villosa (Isolectin B4) The antigen was al30 characterized by hydrolysis using 2M
: .

.. . . . . . ... . ..

w09l/0225~ PCT/US90~04272 trifluoroacetic acid heated for 104C for 4 hrs follow~d by carbohydrate analysis. (Results are shown under Carbohydrate Analysis of Human Plasma Antigen in the Experimental Details Section).
Accordingly, the atherosclerotic plaque antigen may be characterized by the carbohydrate profile shown in Figures 20-22.

The antibodies produced by hybridomas 15H5, Z2D3, QlOE7, and 17H3 were tested for binding to various tissues to determine if the antigens are plaque specific. The results of this testing, which was performed using the histological methods described in Procedures for Histology in the Experimental ~5 Detail section, are set forth in Table 5. Table 6 further characterizes the antibodies by cross reactivity and inhibition of antigens present in CAD serum. Actual inhibition assay results are depicted in Figure 25. Figure 26 shows the effect of binding inhibition using monoclonal antibodies instead of CAD serum.
.

w09l/02~52 PCT/US90/0~Z72 S,J~ C'JJl ~abl~ 5 IMMUNOHISTOCH~MICAL_SCREENING oE-~oNocLoNAL ANTIBODIES

Tissue 15H5 Z2D3 QloE7 17H3 Cerebellum - - 2-3~*
Cerebral cortex - - 2-3+* -Medulla - - 1-2+*
Spinal cord - - _ _ Dura Peripheral nerve Heart Lung _ _ _ _ .
Trachea - - - + .~:
Bronchus - - - +
Breast - - - l+
Pectoral muscle - - - -Esophagus Diaphraym Stomach - - - +
Liver Spleen - 3~4+*~ ~ +
Pancreas - - - +
S~all Bowel - - - - :~
Colon - ~ ~ ~ .

Ovary - 1-2+*b Uterus ~ ~ ~ ~ ?
Kidney - - - _ Bladder - ~ ~ + :~
Rectum - - - +
Psoas muscle - - - +
Lymphnode - - - + :

: ' .

':

W09~ 2s~ PCT/US90/04272 . V 3 ~ . i Skin - 1-3~*e _ _ Coronary artery lesions.
- early lesions - +-1+ +
- advanced lesions + 3-4+ - ~1+

Nor~al Arteries - - 4~ -* Intracellular staining only a Fibromyocytes only .;
b Focal luteal cells only c Sebaceous glands only WO9l/02~' PCT/US90/04272 Tsbl~ ~

MONOCLONAL ANTIBODY CHARACTERIZATION
15H5 Z2D3 QlOE7 17H3 IsotypeIgM IgM IgGl IyM
pI(5.2-5.9) (5.0-5.7) (5.1-5.8) (4.5-5.1) 1 0 ~ `
Auto-antigen- :~
reactivity + + - +
Z2D3 antigen - + -reactivity QlOE7 antigen reactivity Inhibition of + - - +
auto-antigen specific human antibodies Apparent binding 1O9 109 ~ 101 constant Wo 9 1 tO225~ PCl tUS90/04272 -105- 2~

TAb~,e 7 IkMUNOHISTOCHEMICAL SCREENING OF Z2D3 MONOCLONAL A~TIBODY
Tissue Stainirla Cerebe l lum Cerebral cortex Medulla lo Spinal cord Dura Peripheral nerve Heart Lung _ Trachea -Bronchus Rreast - :
Pectoral muscle Esophagus Diaphragm Stomach - ::
Liver Spleen +*a Pancreas Small bowel Colon Ovary ~*a Uterus Kidney Bladder Rectum Psoas muscle Lymph node Skin ~*c Coronary arteries: --lesions +
-normal artery Aorta:
-lesions +
-normal artery *Intracellular staining only a Fibromyocytes onlyb Focal luteal cells only c Sebaceous glands only Lastly, figures 31-35 demonstrate the histologically specificity of the the Z2D3 and QlOE7 antigens histologically. Figure 31 shows specific binding of the ~
Z2D3 antigen to atherosclerotic plaque in rabbit aorta. ~:

, , , , - , . ; -; . . ,, . ~ . . , . . . ; . ,: . . .

091/022S~ PCT/~S~o/04272 ~ 106-Note that there is no staining in non-plaque areas. Figures 32 ~nd 33 show specific binding o~ the Z2D3 antibody in unfixed sections of human coronary artery obtained postmortem. Note that there is little ~on-specific staining and that in control sections, i.e. those treated with a non-specific monoclonal antibody, there is no staining.

The three photographs in Figure 34 are ~rom consecutive sections of the artery. Figure 34 clearly demonstrates that Z2D3 recognizes a late stage plaque antigen and QlOE7 recognizes an early stagP plaque antigen or a ~ormal section of artery. Figure 34A shows binding of Z2D3 to be in the area of the occlusion.

Figure 34B shows binding of QlOE7 to be in the area opposite the area stained by Z2D3, i.e. in an earlier stage of atherosclerosis or normal remaining artery. Figure 34C
shows that no binding occurs using a non-specific monoclonal antibody.
Figure 35 demonstrates in vivo binding of 1l1In-labeled Z2D3 in rabbit aortas which were treated to produce atherosclerotic plaque. Non-treated sections of the aorta showed only trace amounts of binding.
The in vivo binding of Z2D3 antibody in rabbits indicates that in vivo uses of the antibodies of the subject invention will be useful in therapy and imaging in humans. This is particularly supported by the staining of human coronary artery shown in Figures 32-34.

Claims

NOT TAKEN INTO CONSIDERATION
FOR THE PURPOSES
OF INTERNATIONAL PROCESSING

8. An antigen of claim 3 further characterized as being resistant to degradation by proteinases, deoxyribonucleases, lipases, and ribonucleases.

9. An antigen of claim 3 further characterized by being susceptable to partial degradation by .alpha.-amylase, .beta.-amylase, and glucoamylase.

10. An antigen of claim 3 further characterized as being non-reactive with antibodies which bind to apolipoproteins, human collagen, fibronectin, keratin, laminin, tenascin, and vitronectin.

11. An antigen of claim 3 further characterized by being reactive with the monoclonal antibody produced by hybridoma 15H5 (ATCC Accession No. HB9840) after the antigen has been boiled for one hour.

12. An antigen of claim 3 further characterized by being reactive with the monoclonal antibody produced by hybridoma 15H5 (ATCC Accession No. HB9839) after the antigen has been treated with 8M urea in phosphate buffered saline for 24 hours at room temperature, 6 M guanidine HCl in phosphate buffered saline for 24 hours at room temperature, 2 M
trifluroacetic acid for 30 minutes at room temperature, 3.5 M sodium thiocyanate in phosphate buffered saline for 8 hours at room temperature, or 0.19 M sodium dodecyl sulfate in phosphate buffered saline for one hour at room temperature.

13. A purified antigen indicative of the presence of atherosclerotic plaque characterized as being a lipid-containing molecule which selectively binds to the monoclonal antibody produced by hybridoma Z2D3 (ATCC
Accession No. HB 9840) or by hybridoma Z2D3/3E5 (ATCC
Accession No. HB 10485).

14. An antigen of claim 13, wherein the antigen is further characterized as being synthesized by or present in atherosclerotic plaque connective tissue and plaque smooth muscle cells.

15. An antigen of claim 13, wherein the antigen is characterized by having its ability to be used for histological staining destroyed upon treatment with acetone.

16. An antigen of claim 13 characterized by selectively binding to the monoclonal antibody produced by hybridoma Z2D3/3E5 (ATCC Accession No. HB 10485).

17. A purified antigen, wherein the antigen is characterized by its selective binding to the monoclonal antibody produced by hybridoma Q1OE7 (ATCC Accession No. HB
10188).

18. A purified antigen of claim 17, further characterized by being synthesized by, or present in, normal smooth muscle cells and normal connective tissue surrounding arteries.

19. An antigen of claims 1, 3, 13, 6 or 17 labeled with a detectable marker.

20. An antigen of claim 19, wherein the marker is an enzyme, a paramagnetic ion, biotin, a fluorophore, a chromophore, a heavy metal, or a radioisotope.

21. An antigen of claim 20, wherein the marker is an enzyme.

22. An antigen of claim 21, wherein the enzyme is horseradish peroxidase or alkaline phosphatase.

23. A purified antibody which specifically binds to the antigen of claims 1, 3 or 6.

24. A purified antibody which specifically binds to the antigen of claim 13.

25. A purified antibody which specifically binds to the antigen of claim 17.

26. An antibody of claim 23, 24 or 25 labeled with a detectable marker.

27. An antibody of claim 26, wherein the marker is an enzyme, a paramagnetic ion, biotin, a fluorophore, a chromophore, a heavy metal, or a radioisotope.

28. An antibody of claim 27, wherein the marker is an enzyme.

29. An antibody of claim 28, wherein the enzyme is horseradish peroxidase or alkaline phosphatase.

30. A monoclonal antibody of claim 23.

31. The monoclonal antibody of claim 30 or a fragment thereof produced by hybridoma 15H5 (ATCC Accession No. HB
9839).

32. The monoclonal antibody of claim 30 or a fragment thereof produced by hybridoma 17H3 (ATCC Accession No. HB
10189).

33. A monoclonal antibody of claim 24.

34. The monoclonal antibody of claim 33 or a fragment thereof produced by hybridoma Z2D3 (ATCC Accession No. HB

9840) or by hybridoma Z2D3/3E5 (ATCC Accession No. HB.
10485).

35. A recombinant polypeptide which comprises an amino acid sequence which is substantially the same as the amino acid sequence of the hypervariable region of the monoclonal antibody of claim 34.

36. A chimeric antibody including or a fragment thereof comprising the recombinant polypeptide of claim 35 or such polypeptide modified by site-directed mutagenesis.

37. The chimeric antibody of claim 36 or a fragment thereof comprising the amino acid sequences of a human framework region and of a constant region from a human antibody.

38. A monoclonal antibody of claim 25.

39. The monoclonal antibody of claim 38 or a fragment thereof produced by hybridoma Q1OE7 (ATCC Accession No. HB
10188).

40. A recombinant polypeptide which comprises an amino acid sequence which is subscantially the same as the amino acid sequence of the hypervariable region of the monoclonal antibody of claim 39.

41. A chimeric antibody or a fragment thereof comprising the recombinant polypeptide of claim 40 or such polypeptide modified by site-directed mutagenesis.

42. The chimeric antibody of claim 41 or a fragment thereof comprising the amino acid sequences of a human framework region and of a constant region from a human antibody.

43. The antigen of claim 1, 3, 13, 6 or 17 bound to a solid support.

44. The antibody of claim 23, 24 or 25, bound to a solid support.

45. The monoclonal antibody of claim 31, 32, 34, 35, 39 or 40 bound to a solid support.

46. A method for detecting in a biological sample an antigen present in, and indicative of the presence of, atherosclerotic plaque which comprises contacting the biological sample with the antibody of claim 23 or 24 under conditions such that the antibody binds to the antigen to form a detectable complex, detecting the complex so formed, and thereby detecting any antigen present in the biological sample.

47. A method of claim 46, wherein the biological sample is a tissue sample.

48. A method of claim 46, wherein the biological sample is biological fluid.

49. A method of claim 48, wherein the biological fluid comprises blood, plasma, or serum.

50. A method of claim 49, wherein the biological fluid is serum.

51. A method of claim 46, wherein the antibody is labeled with a detectable marker.

52. A method of claim 46, wherein the antibody is a monoclonal antibody.

53. A method of claim 52, wherein the monoclonal antibody is produced by hybridoma 15H5 (ATCC Accession No. HB 9839).

54. A method of claim 52, wherein the monoclonal antibody is produced by hybridoma 17H3 (ATCC Accession No. HB 10189).

55. A method of claim 52, wherein the monoclonal antibody is produced by hybridoma Z2D3 (ATCC Accession No. HB 9840) or by hybridoma Z2D3/3E5 (ATCC Accession No. HB 10485).

56. A method of claim 48, wherein the antibody is bound to a solid support.

57. A method of claim 56, wherein solid support is a bead formed of an inert polymer.

58. A method of claim 56, wherein the solid support is a microwell.

59. A method for quantitatively determining in a sample of a biological fluid the concentration of an antigen which is present in, and indicative of the presence of, atherosclerotic plaque which comprises:
a) contacting a solid support with an excess of the antibody of claims 23 or 24 under conditions permitting the antibody to attach to the surface of the solid support;
b) removing unbound antibody;
c) contacting the resulting solid support to which the antibody is bound with the sample of the biological fluid under conditions such that any antigen present in the sample binds to the bound antibody and forms a complex therewith;
d) removing any antigen which is not bound to the complex;
e) contacting any complex so formed with an excess of a detectable reagent which specifically binds to any antigen present in the complex so as to form a second complex which includes the antibody, the antigen, and the detectable reagent;
f) removing any detectable reagent which is not bound in the second complex;
g) quantitatively determining the concentration of detectable reagent present in the second complex;
and h) thereby quantitatively determining the concentration of antigen in the biological fluid.

60. A method of claim 59, wherein the biological fluid comprises blood, plasma, or serum.

61. A method of claim 60, wherein the biological fluid is serum.

62. A method of claim 59, wherein the reagent is labeled with a detectable marker.

63. A method of claim 62, wherein the marker is an enzyme, a paramagnetic ion, biotin, a fluorophore, a chromophore, a heavy metal, or a radioisotope.

64. A method of claim 59, wherein the reagent is an antibody labeled with a detectable marker.

65. A method of claim 64, wherein the marker is an enzyme, a paramagnetic ion, biotin, a fluorophore, a chromophore, a heavy metal, or a radioisotope.

66. A method of claim 65, wherein the marker is an enzyme.

67. A method of claim 66, wherein the enzyme is horseradish peroxidase or alkaline phosphatase.

68. A method of claim 59, wherein the detectable reagent is labeled with an enzyme and step (g) comprises contacting the second complex with a specific substrate to the enzyme under conditions such that the enzyme reacts with the substrate to form a detectable product.

69. A method for detecting in a biological sample an antibody which specifically forms a complex with an antigen present in, and indicative of the presence of, atherosclerotic plaque which comprises contacting the biological sample with the antigen of claim 1, 3, 13 or 6 under conditions such that the antigen binds to the antibody in the biological sample and detecting the antigen bound to the antibody and thereby detecting the antibody in the biological sample.

70. A method of claim 69, wherein the biological sample is a tissue sample.

71. A method of claim 69, wherein the biological sample is biological fluid.

72. A method of claim 71, wherein the biological fluid comprises blood, plasma, or serum.

73. A method of claim 72, wherein the biological fluid is serum.

74. A method of claim 69, wherein the antigen is labeled with a detectable marker.

75. A method of claim 71, wherein the antigen is bound to a solid support.

76. A method of claim 75, wherein solid support is a bead formed of an inert polymer.

77. A method of claim 75, wherein the solid support is a microwell.

78. A method for quantitatively determining in a sample of a biological fluid the concentration of an antibody which specifically forms a complex with an antigen present in, and indicative of the presence of, atherosclerotic plaque which comprises:
a) contacting a solid support with an excess of the antigen of claim 1, 3, 13 or 6 under conditions permitting the antigen to attach to the surface of the solid support;
b) removing unbound antigen;
c) contacting the resulting solid support to which the antigen is bound with the sample of the biological fluid under conditions such that any antibody present in the sample binds to the bound antigen and forms a complex therewith;
d) removing any antibody which is not bound to the complex;
e) contacting any complex so formed with an excess of a detectable reagent which specifically binds to any antibody present in the complex so as to form a second complex which includes the antigen, the antibody, and the detectable reagent;
f) removing any detectable reagent which is not bound in the second complex;
g) quantitatively determining the concentration of detectable reagent present in the second complex;
and h) thereby quantitatively determining the concentration of antibody in the biological fluid.

79. A method of claim 78, wherein the biological fluid comprises blood, plasma, or serum.

80. A method of claim 79, wherein the biological fluid is serum.

81. A method of claim 78, wherein the reagent is labeled with a detectable marker.

82. A method of claim 81, wherein the marker is an enzyme, a paramagnetic ion, biotin, a fluorophore, a chromophore, a heavy metal, or a radioisotope.

83. A method of claim 78, wherein the reagent is an antibody labeled with a detectable marker.

84. A method of claim 83, wherein the marker is an enzyme, a paramagnetic ion, biotin, a fluorophore, a chromophore, a heavy metal, or a radioisotope.

85. A method of claim 84, wherein the marker is an enzyme.

86. A method of claim 85, wherein the enzyme is horseradish peroxidase or alkaline phosphatase.

87. A method of claim 78, wherein the detectable reagent is labeled with an enzyme and step (g) comprises contacting the second complex with a specific substrate to the enzyme under conditions such that the enzyme reacts with the substrate to form a detectable product.

88. A method for quantitatively determining in a sample of a biological fluid the concentration of an antigen which is present in, and indicative of the presence of, atherosclerotic plaque which comprises:
a) contacting a solid support with a predetermined amount of the antibody of claim 23 or 24 under conditions permitting the antibody to attach to the surface of the support;

b) removing unbound antibody;
c) contacting the resulting solid support to which the antibody is bound with a predetermined amount of antigen labeled with a detectable marker and with the sample of the biological fluid under such conditions that antigen binds to the antibody bound to the solid support and forms a complex therewith;
d) removing any antigen which is not bound to the complex;
e) quantitatively determining the concentration of labeled antigen bound to the solid support; and f) thereby quantitatively determining the concentration of antigen in the biological fluid.

89. A method of claim 88, wherein the biological fluid comprises blood, plasma, or serum.

90. A method of claim 89, wherein the biological fluid is serum.

91. A method of claim 88, wherein the detectable marker is an enzyme, a paramagnetic ion, biotin, a fluorophore, a chromophore, a heavy metal, or a radioisotope.
92. A method of claim 91, wherein the marker is an enzyme.

93. A method of claim 92, wherein the enzyme is horseradish peroxidase or alkaline phosphatase.

94. A method of claim 88, wherein the antigen is labeled with an enzyme and step (e) comprises contacting the labeled antigen bound to the solid support with a specific substrate to the enzyme under conditions such that the enzyme reacts with the substrate to form a detectable product.

95. A method for quantitatively determining in a sample of a biological fluid the concentration of an antigen which is present in, and indicative of the presence of, atherosclerotic plaque which comprises:
a) contacting a solid support with a predetermined amount of the antibody of claim 23 or 24 under conditions permitting the antibody to attach to the surface of the support;
b) removing any antibody not bound to the support;
c) contacting the solid support to which the antibody is bound with the sample of the biological fluid under conditions such that any antigen present in the sample binds to the bound antibody and forms a complex therewith;
d) removing any antigen which is not bound to the complex;
e) contacting the complex so formed with a predetermined amount of plaque antigen labeled with a detectable marker under conditions such that the labeled antigen competes with the antigen from the biological fluid for binding to the antibody;
f) quantitatively determining the concentration of labeled antigen not bound to the solid support;
and g) thereby quantitatively determining the concentration of antigen in the biological fluid.

96. A method of claim 95, wherein the biological fluid comprises blood, plasma, or serum.

97. A method of claim 96, wherein the biological fluid is serum.

98. A method of claim 95, wherein the marker is an enzyme, a paramagnetic ion, biotin, a fluorophore, a chromophore, a heavy metal, or a radioisotope.

99. A method of claim 98, wherein the marker is an enzyme.

100. A method of claim 99, wherein the enzyme is horseradish peroxidase or alkaline phosphatase.

101. A method of claim 95, wherein the antigen is labeled with an enzyme and step (f) comprises removing the labeled antigen which was not bound to the solid support and contacting it with a specific substrate to the enzyme under conditions such that the enzyme reacts with the substrate to form a detectable product.

102. A method for quantitatively determining in a sample of a biological fluid the concentration of an antibody which specifically forms a complex with an antigen which is present in, and indicative of the presence of, atherosclerotic plaque which comprises:
a) contacting a solid support with a predetermined amount of the antigen of claim 1, 3, 13 or 6 under conditions permitting the antigen to attach to the surface of the support;
b) removing unbound antigen;
c) contacting the resulting solid support to which the antigen is bound with a predetermined amount of antibody labeled with a detectable marker and with the sample of the biological fluid under conditions such that the antibody binds to the antigen bound to the solid support and forms a complex therewith;
d) removing any antibody which is not bound to the complex;
e) quantitatively determining the concentration of labeled antibody bound to the solid support; and f) thereby quantitatively determining the concentration of antibody in the biological fluid.

103. A method of claim 102, wherein the biological fluid comprises blood, plasma, or serum.

104 . A method of claim 103, wherein the biological fluid is serum.

105. A method of claim 102, wherein the detectable marker is an enzyme, a paramagnetic ion, biotin, a fluorophore, a chromophore, a heavy metal, or a radioisotope.

106. A method of claim 105, wherein the marker is an enzyme.

107. A method of claim 106, wherein the enzyme is horseradish peroxidase or alkaline phosphatase.

108. A method of claim 102, wherein the antibody is labeled with an enzyme and step (e) comprises contacting the labeled antibody bound to the solid support with a specific substrate to the enzyme under conditions such that the enzyme reacts with the substrate to form a detectable product.

109. A method for quantitatively determining in a sample of a biological fluid the concentration of antibody which specifically forms a complex with an antigen which is present in, and indicative of the presence of, atherosclerotic plaque which comprises:
a) contacting a solid support with a predetermined amount of the antigen of claim 1, 3, 13 or 6 under conditions permitting the antigen to attach to the surface of the support;
b) removing any antigen which is not bound to the support;

c) contacting the solid support to which the antigen is bound with the sample of the biological fluid under conditions such that any antibody present in the sample binds to the bound antigen and forms a complex therewith;
d) removing any antibody which is not bound to the complex;
e) contacting the complex so formed with a predetermined amount of plaque antibody labeled with a detectable marker under conditions such that the labeled antibody competes with the antibody in the biological fluid for binding to the antigen;
f) quantitatively determining the concentration of labeled antibody not bound to the solid support;
and g) thereby quantitatively determining the concentration of antibody in the biological fluid.

110. A method of claim 109, wherein the biological fluid comprises blood, plasma, or serum.

111. A method of claim 110, wherein the biological fluid is serum.

112. A method of claim 109, wherein the detectable marker is an enzyme, a paramagnetic ion, biotin, a fluorophore, a chromophore, a heavy metal, or a radioisotope.

113. A method of claim 112, wherein the marker is an enzyme.

114. A method of claim 113, wherein the enzyme horseradish peroxidase or is alkaline phosphatase.

115. A method of claim 109, wherein the antibody is labeled with an enzyme and step (f) comprises removing the labeled antibody which was not bound to the solid support and contacting it With a specific substrate to the enzyme under conditions such that the enzyme reacts with the substrate to form a detectable product.

116. A method for monitoring the progression of atherosclerosis which comprises determining the amount of the antigen of claim 1, 3, 13 or 6 present in a sample of a biological fluid of a patient, and comparing the amount determined with the amount determined at earlier points in time, a change in the amount of antigen indicating a change in the extent of atherosclerotic plaque.

117. A method for monitoring the efficacy of treatment of atherosclerosis which comprises determining the amount of the antigen of claim 1, 3, 13 or 6 present in a sample of a biological fluid of a patient and comparing the amount determined at earlier points in time, a change in the amount of antigen indicating a change in the extent of atherosclerotic plaque.

118. A reagent for use in imaging atherosclerotic plaque which comprises an antibody of claim 23 or 24 labeled with a detectable marker.

119. A composition comprising an effective imaging amount of a reagent of claim 118 and a physiologically acceptable carrier.

120. A reagent of claim 118, wherein the marker is a radioactive isotope, an element which is opaque to X-rays, a paramagnetic ion, or a chelate of a paramagnetic ion.

121. A reagent of claim 120, wherein the marker is a radioactive isotope.

122. A reagent of claim 121, wherein the marker is I-123, I-125, I-128, I-131, or a chelated metal ion of chromium-51, cobalt-57, gallium-67, indium-111, indium-113m, mercury-197, selenium-75, thalium-201, technetium-99m, lead-203, strontium-85, strontium-87, gallium-58, samarium-153, europium-171, ytterbium-169, zinc-62, rhenium-188, or mixtures thereof.

123. A reagent of claim 120, wherein the marker is an iodine or a metal ion chelate of iodine.

124. A reagent of claim 120, wherein the marker is a paramagnetic ion.

125. A reagent of claim 124, wherein the paramagnetic ion comprises chromium (II}), manganese (II), iron (III), iron (II), cobalt (II), nickel (II), copper (II), praseodymium (III), neodyminum (III), samarium (III), gadolinium (III), terbium (III), dysprosium (III), holmium (III), erbium (III), ytterbium (III), or mixtures thereof.

126. A method for imaging atherosclerotic plaque which comprises contacting the atherosclerotic plaque to be imaged with the reagent of claim 118 under conditions such that the reagent binds to the atherosclerotic plaque and detecting the reagent bound thereto, thereby imaging the atherosclerotic plaque.

127. A method for imaging atherosclerotic plaque and adjacent normal tissue which comprises:
a) contacting the normal lumen to be imaged with an antibody which specifically binds to normal intima and/or media and which is labeled with a detectable marker;
b) contacting the atherosclerotic plaque with the reagent of claim 118 under conditions such that the reagent binds to the atherosclerotic plaque;
and c) detecting the reagents bound to the atherosclerotic plaque and adjacent normal tissue, thereby imaging the atherosclerotic plaque and adjacent normal tissue.

128. The method of claim 127, wherein the antibody which specifically binds to normal intima and/or media is a purified antibody which specifically binds to an antigen characterized by being synthesized by, or present in, normal smooth muscle cells and normal connective tissue surrounding arteries.

129. The method of claim 127, wherein the antibody is a monoclonal antibody produced by hybridoma Q1OE7 (ATCC
Accession No. HB 10188).

130. A reagent for use in imaging normal intima and/or media comprising an antibody of claim 39 or 40 labeled with a detectable marker.

131. A composition comprising an effective imaging amount of the reagent of claim 130 and a physiologically acceptable carrier.

132. A reagent of claim 130 wherein the marker is a radioactive isotope, an element which is opaque to X-rays, a paramagnetic ion, or a chelate of a paramagnetic ion.

133. A reagent of claim 132, wherein the marker is a radioactive isotope.

134. A reagent of claim 133, wherein the marker is I-123, I-125, I-128, I-131, or a chelated metal ion of chromium-51, cobalt-57, gallium-67, indium-111, indium-113m, mercury-197, selenium-75, thalium-201, technetium-99m, lead-203, strontium-85, strontium-87, gallium-68, samarium-153, europium-171, ytterbium-169, zinc-62, rhenium-188, or mixtures thereof.

135. A reagent of claim 134, wherein the marker is an iodine or a metal ion chelate of iodine.

136. A reagent of claim 132, wherein the marker is a paramagnetic ion.

137. A reagent of claim 136, wherein the paramagnetic ion comprises chromium (III), manganese (II), iron (III), iron (II), cobalt (II), nickel (II), copper (II), praseodymium (III), neodyminum (III), samarium (III), gadolinium (III), terbium (III), dysprosium (III), holmium (III), erbium (III), ytterbium (III), or mixtures thereof.

138. A method for monitoring the progression of atherosclerosis which comprises determining the amount of the antigen of claim 1, 3, 13 or 6 present in a patient's blood vessels and comparing the amount determined with the amount determined at earlier points in time, a change in the amount of antigen indicating a change in the extent of atherosclerotic plaque.

139. A method for monitoring the efficacy of treatment of atherosclerosis which comprises determining the amount of the antigen of claim 1, 3, 13 or 6 present in a patient's blood vessel and comparing the amount determined with the amount determined at earlier points in time, a change in the amount of antigen indicating a change in the extent of atherosclerotic plaque.

140. A method for imaging atherosclerotic plaque in a subject which comprises:

a) contacting the blood vessel walls containing atherosclerotic plaque with the reagent of claim 118;
b) detecting the reagent bound to the atherosclerotic plaque; and c) imaging the atherosclerotic plaque.

141. A method for imaging atherosclerotic plaque and adjacent normal tissue in a subject which comprises:
a) contacting the normal lumen to be imaged with an antibody which specifically binds to normal intima and/or media and which is labeled with a detectable marker;
b) contacting the blood vessel walls containing atherosclerotic plaque and surrounding area to be imaged with the reagent of claim 118 under conditions such that the reagent binds to the atherosclerotic plaque; and c) detecting the reagents bound to the atherosclerotic plaque and adjacent normal tissue, thereby imaging the atherosclerotic plaque and adjacent normal tissue.

142. The method of claim 141, wherein the antibody which specifically binds to normal intima and/or media is a monoclonal antibody produced by hybridoma Q10E7 (ATCC
Accession No. HB 10188).

143. An antibody of claim 23, 24, 34, 35, 36 or 37 conjugated to an enzyme capable of digesting a component of atherosclerotic plaque or to a proenzyme which, when cleaved, is converted to an enzyme capable of digesting a component of atherosclerotic plaque.

144. An antibody of claim 143, wherein the antibody is a monoclonal antibody produced by hybridoma Z2D3 (ATCC

Accession No. HB 9840) or by hybridoma Z2D3/3E5 (ATCC
Accession No. HB 10485).

145. An antibody of claim 36 or 37 conjugated to an enzyme capable of digesting a component of atherosclerotic plaque or to a proenzyme which, when cleaved, is converted to an enzyme capable of digesting a component of atherosclerotic plaque.

146. An antibody of claim 145, wherein the chimeric antibody is a hybrid neomolecule conjugated to the enzyme or to the proenzyme.

147. An antibody of claim 145, wherein the chimeric antibody is a bifunctional antibody.

148. An antibody of claim 147, wherein the bifunctional antibody is produced by a quadroma.

149. An antibody of claim 148, wherein the quadroma is derived from the fusion of a hybridoma cell line Z2D3 or Z2D3/3E5 and a hybridoma secreting a monoclonal antibody binding an enzyme.

150. An antibody of claim 143, wherein the enzyme is a proteinase, an elastase, a collagenase, or a saccharidase.

151. An antibody of claim 143, wherein the enzyme is fibroblastic collagenase, gelatinase, polymorphonuclear collagenese, granolocytic collagenese, stromelysin I, stromelysin II, or elastase.

152. A pharmaceutical composition comprising an amount of the antibody of claim 143 effective to digest a component of atherosclerotic plaque and a physiologically acceptable carrier.

153. A method for reducing the amount of atherosclerotic plaque in a blood vessel which comprises contacting the atherosclerotic plaque with a reagent comprising the antibody of claim 143 under conditions and in an amount such that the reagent binds to, and digests, a component of the plaque.

154. A reagent for use in protecting normal arterial tissue from an enzyme capable of digestion of atherosclerotic plaque which comprises an antibody of claim 25, 39, 40, 41 or 42 bound to an inhibitor of an enzyme capable of digesting atherosclerotic plaque.

155. A reagent of claim 154, wherein the antibody is a monoclonal antibody produced by hybridoma Q10E7 (ATCC
Accession No. HB 10188).

156. A method for reducing the amount of atherosclerotic plaque in a blood vessel which comprises:
a). contacting the normal lumen with an antibody which specifically binds to intima and/or media and has bound thereto an inhibitor of an enzyme capable of digesting a component of atherosclerotic plaque under conditions such that the antibody binds to the normal intima and/or media; and b) contacting the atherosclerotic plaque with the antibody of claim 143 under conditions such that the antibody binds to the atherosclerotic plaque.

157. The method of claim 156, wherein the antibody which specifically binds to normal intima and/or media is a purified antibody which specifically binds to an antigen characterized by being synthesized by, or present in, normal smooth muscle cells and normal connective tissue surrounding arteries.

158. The method of claim 157, wherein the antibody is a monoclonal antibody produced by hybridoma Q10E7 (ATCC
Accession No. HB 10188).

159. A method for reducing the amount of atherosclerotic plaque in a blood vessel which comprises:
a) contacting the atherosclerotic plaque with a reagent under conditions such that the reagent binds to the plaque so as to form a reagent-plaque complex, which reagent is capable of specifically binding to both the plaque and to a proenzyme which, when cleaved, is converted into an enzyme whose substrate is a connective tissue present in atherosclerotic plaque, and which enzyme is capable of dissolving a component of the plaque;
b) contacting the reagent-plaque complex with the proenzyme to which the reagent specifically binds under conditions such that the proenzyme is bound to the reagent so as to form a proenzyme-reagent-plaque complex; and c) contacting the proenzyme-reagent-plaque complex with an agent which is capable of specifically cleaving the proenzyme so that the proenzyme is converted into the enzyme under conditions such that the enzyme digests the plaque.

160. A method for reducing the amount of atherosclerotic plaque in a blood vessel which comprises:
a) contacting the atherosclerotic plaque with a reagent comprising the antibody of claim 143, 145 or 146 under conditions such that the reagent binds to the plaque so as to form a reagent-plaque complex, which reagent is bound to both the plaque and to a proenzyme which, when cleaved, is converted into an enzyme whose substrate is a connective tissue present in atherosclerotic plaque, and which enzyme is capable of dissolving a component of the plaque;
b) contacting the proenzyme-reagent-plaque complex with an agent which is capable of specifically cleaving the proenzyme so that the proenzyme is converted into the enzyme under conditions such that the enzyme digests the plaque.

161. A method for reducing the amount of atherosclerotic plaque in a blood vessel which comprises:
a) contacting the atherosclerotic plaque with a reagent under conditions such that the reagent binds to the plaque so as to form a reagent-plaque complex, which reagent is bound to both the plaque and to a proenzyme which, when cleaved, is converted into an enzyme whose substrate is a connective tissue present in atherosclerotic plaque, and which enzyme is capable of dissolving a component of the plaque;
b) contacting the proenzyme-reagent-plaque complex with an agent which is capable of specifically cleaving the proenzyme so that the proenzyme is converted into the enzyme under conditions such that the enzyme digests the plaque.

162. A method of claim 159 or 161, wherein the reagent is a bifunctional antibody.

163. A method of claim 162, wherein the bifunctional antibody is produced by a quadroma.

164. A method of claim 162, wherein the bifunctional antibody is a quadroma derived from the fusion of a hybridoma cell line comprising the monoclonal antibody produced by hybridoma Z2D3 (ATCC Accession No. HB 9840) or by hybridoma Z2D3/3E5 (ATCC Accession No. HB 10485) and a hybridoma secreting a monoclonal antibody binding an enzyme.

165. A method of claim 159 or 160, wherein the proenzyme is a proenzyme of granulocytic collagenase.

166. A method of claim 159 or 160, wherein the proenzyme is a proenzyme of fibroblastic collagenase.

167. A method of claim 159 or 160, wherein the proenzyme is a proenzyme of stromelysin.

168. A method of claim 159 or 160, wherein the agent of step (c) is plasmin.

169. A reagent for use in ablating atherosclerotic plague which comprises an antibody of claim 23 or 24 having bound thereto a chromophore capable of absorbing radiation having a plaque ablating wavelength.

170. A reagent of claim 169, wherein the antibody is a monoclonal antibody.

171. A reagent of claim 170, wherein the monoclonal antibody is produced by hybridoma Z2D3 (ATCC Accession No. HB 9840).

172. A reagent of claim 169, wherein the chromophore absorbs light having a wavelength from about 190 nm to about 1100 nm.

173. A reagent of claim 172, wherein the chromophore is fluorescein, rhodamine, tetracycline, or hematoporphyrin.

174. A composition comprising an amount of the reagent of claim 169 effective for use in ablating atherosclerotic plaque and a physiologically acceptable carrier.

175. A method for ablating atherosclerotic plaque which comprises:
a) contacting atherosclerotic plaque with an effective amount of the reagent of claim 169 so that the reagent binds to the atherosclerotic plaque forming an atherosclerotic plaque-reagent complex; and b) exposing the resulting complex to radiation having a plaque ablating wavelength under conditions such that the radiation is absorbed by the chromophore at a sufficient energy to ablate the atherosclerotic plaque.

176. A method for ablating atherosclerotic plaque present in a blood vessel which comprises:
a) contacting the normal lumen with an antibody which specifically binds to normal intima and/or media and has bound thereto a moiety capable of reflecting radiation of the plaque ablating wavelength;
b) contacting the atherosclerotic plaque with the reagent of claim 169 under conditions such that the reagent binds to the atherosclerotic plaque;
and c) exposing the atherosclerotic plaque to the radiation having plaque ablating wavelength, thereby ablating the plaque.

177. The method of claim 176, wherein the antibody which specifically binds to normal intima and/or media is a monoclonal antibody produced by hybridoma Q10E7 (ATCC
Accession No. HB 10188).

178. A monoclonal antibody of claim 25 bound to a moiety capable of reflecting radiation of the plaque ablating wavelength;

179. A reagent for use in treating atherosclerosis which comprises an antibody of claim 24 bound to a drug useful in treating atherosclerosis.

180. A reagent of claim 179, wherein the antibody is the monoclonal antibody produced by hybridoma Z2D3 (ATCC
Accession No. HB9840).

181. A method of treating atherosclerosis in a subject which comprises administering to the subject an amount of a reagent of claim 179 effective to treat atherosclerosis.

182. A method of treating atherosclerosis in a subject which comprises:
a) administering to the subject an antibody which specifically binds to normal intima and/or media and which has bound thereto an inhibitor of a drug useful in treating atherosclerosis; and b) administering to the subject an amount of the reagent of claim 179 effective to treat atherosclerosis.

183. A method of claim 181, wherein the antibody for use in protecting normal arterial tissue from a drug useful in treating atherosclerosis is a monoclonal antibody produced by hybridoma Q10E7 (ATCC Accession No. HB 10188).

184. The monoclonal ant body of claim 25 bound to an inhibitor of a drug useful in treating atherosclerosis.

185. A method of treating atherosclerosis which comprises blocking the synthesis of the antigen of claim 1, 3, 13 or 6.

186. A method of claim 185, wherein the synthesis of the antigen is blocked by use of an antisense nucleic acid molecule which specifically binds to a nucleic acid, the expression of which is associated with the synthesis of the antigen.

187. A method of claim 185, wherein the synthesis of the antigen is blocked by inhibiting an enzyme involved in the synthesis of the antigen.

188. A method of treating atherosclerosis which comprises blocking the binding of antibody to the antigen of claim 1, 3, 13 or 6.

189. A method for diagnostic analysis comprising the steps of:
a) obtaining a value for the body mass index (BMI) of a patient;
b) obtaining a value for the concentration of an antigen or other serum or plasma analytes associated with a pathological condition or an antibody which binds with the antigen;
c) plotting the body mass index of the patient against the antigen or antibody concentration of the same patient;
and d) comparing the resulting value against a set of reference values to determine whether the resulting value exceeds the reference value and thereby indicates the presence of a pathological condition.

190. A method for determining a predisposition toward atherosclerosis using the method of claim 189 wherein the antigen is the antigen of claim 1, 3, 13 or 6.

191. A method for determining a predisposition toward atherosclerosis using the method of claim 189 wherein the antibody is the antibody of claim 23 or 24.