A method for producing novel monoclonal antibodies against purified platelets are provided. A monoclonal antibodies which inhibits platelet reactions with collagen or collagenous surfaces is described. A new method of detecting and treating tumor metastatis is described.

The present invention is related generally to the preparation of monoclonal 
antibodies. More particularly, the present invention is related to 
providing anti-platelet monoclonal antibodies which recognize a specific 
heterodimer antigen, composed of two components with molecular weights of 
about 157 and 130 kDa. Such an antibody, designated herein as 176D7, has 
not heretofore been known or described. 
Unless defined otherwise, all technical and scientific terms used herein 
have the same meaning as commonly understood by one of ordinary skill in 
the art to which this invention belongs. Although any methods and 
materials similar or equivalent to those described herein can be used in 
the practice or testing of the present invention, the preferred methods 
and materials are now described. All publications mentioned hereunder are 
incorporated herein by reference. Unless mentioned otherwise, the 
techniques employed herein are standard methodologies well known to one of 
ordinary skill in the art.

MATERIALS AND METHODS 
The following are only illustrative and not limiting. 
Platelet Aggregation and Release Reaction 
Aggregation of platelets in PRP (platelet rich plasma) is measured on an 
aggregometer, an instrument that measures increases in light transmission 
through a cuvette of stirred PRP. Various agents (epinephrine, ADP, 
ristocetin, collagen and the like) are added to the PRP to cause 
aggregation and change in the optical density as an index of platelet 
aggregation is recorded on a graph. 
Following aggregation, platelets release specific chemical substances (ADP, 
ATP, Serotonin, calcium ions and the like). The secretion of ATP is 
measured by detecting the luminescence in the firefly luciferase system. 
Reagents and Material: 
1. Lumi-Aggregometer and recorder. 
2. Glass cuvettes (Chrono-Log Corp.) siliconized in 1% silicon solution 
(Prosil-28 PCR Research Chemicals, Inc.). 
3. Teflon coated stir bars (Chrono-Log Corp.). 
4. Plastic pipettes and tubes. 
5. Device for maintaining pH and controlling humidity (Coller et al: The pH 
Dependence of Quantitative Ristocetin-induced Platelet Aggregation: 
Theoretical and Practical Implication-A New Device for Maintenance of 
Platelet Rich Plasma pH. BLOOD 47:841, 1976). 
6. Epinephrine (Park-Davis Co.) MW183.2. Working solution 0.68 mM. Dilute 
1.0 ml from a liquid ampule (1 mg/ml) 1:8 with 0.15M NaCl. Aliquot to 1.0 
ml and store at -70.degree. C. Thaw fresh sample daily. 
7. ADP (Adenosine 5' Diphosphate Disodium) (Sigma Co.) MW427.2 Stock 
solution 0.225 mM. Dilute 9.6 mg to 100 ml in 0.15M NaCl, aliquot to 2 ml 
and store at -70.degree.. Thaw fresh sample daily before use. Dilute stock 
solution 1:2.5 (.090 mM) and 1:5 (0.045 mM) with 0.15M NaCl for working 
solutions. 
8. Soluble Collagen (Worthington Biochemical Corp.) 7.0 mg/ml protein. 
Store in the cold and when using, keep on ice. Tendrils form at room 
temperature. 
9. Ristocetin Sulphate (Lenau, Copenhagen, Denmark) Working solution of 80 
mg/ml, 60 mg/ml, 40 mg/ml and 20 mg/ml are diluted in 0.01M Tris-0.15M 
NaCl pH 7.4. Store at room temperature (about 22.degree.-24.degree. C.). 
10. Thrombin (purified Human thrombin) Stock solution 100 U/ml diluted in 
0.01M Tris-0.15M NaCl-0.66% PEG 6000 pH 7.35. Store at -70.degree. C. Thaw 
fresh sample daily and dilute to 1.5 & 2.0 U/ml with 0.01M Tris-0.15M NaCl 
pH 7.35. 
11. Luciferace (Sigma) Store powder at -70.degree. C. Working solution 40 
mg/ml diluted in distilled water. Make fresh daily, protect from light and 
keep on ice. 
12. ATP (Adenosine 5'-Triphosphate, Disodium salt from Equine Muscle) 
(Sigma Co.) MW551.2 Working solution 0.34 mM. Dissolve 18.6 mg ATP in 100 
ml NaCl. Aliquot to 2 ml and store at -70.degree. C. Thaw fresh sample 
daily. Final concentration 1.5 .mu.M. 
Procedure: 
Draw 20 cc blood in 38% Na Citrate (0.1 ml/10 ml blood) with a 29 guage 
needle using a two-syringe technique. Perform a platelet count on whole 
blood and keep at room temperature. 
Prepare Platelet Rich Plasma (PRP) by spinning the Whole Blood at 750 g, 3 
min. Spin the remainder of the whole blood at 2,000 g for 10-15 minutes at 
24.degree. C. The plasma is now platelet poor. Save 0.4 ml for a blank 
before adjusting the PRP to 2,000,000/mm. 
Adjust pH of PRP at 200,000/mm to pH 7.7 (.+-..05). If the pH is too high, 
add a small amount of CO.sub.2 to the PRP. If the pH is too low, mix the 
PRP gently between two plastic tubes. 
Aliquot 0.4 ml PRP into siliconized cuvettes and place in a device to 
control pH and humidity. The pH of the platelets will be stable for about 
6 hours. 
Set up the aggregometer according to the instruction manual; allow PRP to 
run for 1 minute without more than a 0.2 unit change. Also record each 
aggregation until the OD changes less than 0.2 units/minute. 
When the PRP has stabilized, add 50 Luciferase. After 1 minute add 
epinephrine, ADP, ristocetin, thrombin or collagen. Avoid bubbles which 
will cause the pen to become very erratic. The first wave of aggregation 
indicates direct contact of reagent on platelets. The second wave 
indicates release of endogenous ADP which will cause the remaining free 
platelets to clump. Platelets lose their sensitivity to epinephrine and 
ADP about 3 hours after drawing. If the patient is abnormal, increase or 
decrease the amount of reagent in an attempt to produce a normal curve. At 
the end of the aggregation and release response add 2 ATP(.34 mM) in order 
to measure the amount of ATP released from the platelet. Calculate the % 
yield of the control and patient: 
##EQU1## 
To test for platelet sensitivity to heparin add 20 of the patient's heparin 
(40 units/ml) to 0.4 ml PRP (control and patient). Record platelet 
activity for 10 minutes. Next, add 50 of the patient's PPP to 0.4 ml of 
the control PRP, allow to stabilize for 1 minute and add 20 heparin (40 
units/ml). Record platelet activity for 10 minutes. Final Concentration of 
reagents 
______________________________________ 
Final 
Working Solution 
Concentration 
______________________________________ 
5 Epinephrine 0.68 mM 7.5 .mu.M 
20 ADP 0.045 mM 1.9 .mu.M 
0.090 mM 3.8 .mu.gM/ml 
15 Collagen 7 mg/ml 226 .mu.gM/ml 
10 Ristocetin 80 mg/ml 1.74 mg/ml 
60 mg/ml 1.30 mg/ml 
40 mg/ml 0.87 mg/ml 
20 mg/ml 0.44 mg/ml 
50 Thrombin 1.5 U/ml 0.15 U/ml 
2.0 U/ml 0.20 U/ml 
______________________________________ 
Normal Results 
Reagents Aggregation 
______________________________________ 
Epinephrine Double Wave 
ADP Large single wave 
or double wave 
Collagen Single wave 
lag time 1-3 min. 
Thrombin Single wave 
or double wave 
Ristocetin Double wave 
or single wave 
______________________________________ 
Preparation of Monoclonal Antibodies: 
The antibody, 176D7, was prepared against purified platelets isolated from 
whole blood by standard procedures using Stractan (arabinogalactan) 
gradients and injected into mice. The third injection into mice was an 
intrasplenic injection. Hybridomas were prepared by standard techniques 
and the multiple clones formed were tested for their ability to inhibit 
collagen-induced platelet aggregation by the methodology described herein 
supra, and a hybridoma secreting antibody 176D7, which showed desirable 
properties, was selected. This hybridoma was deposited with the American 
Type Culture Collection, 12301 Parklawn Drive, Rockville, MD 20852-1776, 
on Feb. 7, 1992 under the accession number ATCC HB 10973. It is noted that 
antibody 176D7 did not interfere with the aggregation of platelets by any 
other agonist. This antibody specifically inhibits the adhesion of 
platelets to type I and III collagen in a dose-dependent fashion in the 
presence of Mg.sup.++ and blocks platelet interaction with collagen 
surface and inhibits it at concentrations as low as 1 microgram per ml 
final concentration (FIG. 1). It has no effect on adhesion of platelets to 
fibronectin or von Willebrand factor. In addition to inhibiting the 
adhesion to matrix, it also inhibits collagen-induced platelet aggregation 
of both purified platelets and platelet-rich plasma. It binds to about 
1,900.+-.300 sites per platelet with a Kd of about 5 nM. Binding of the 
antibody to the platelets of 11 normal subjects gave a Kd of about 
5.1.+-.1.2 nM and the number of sites per platelet was found to be about 
1.9.+-.0.16.times.10.sup.3. Antibody 176D7 also inhibits the adhesion of 
platelets to the subendothelium of umbilical arteries, in a flowing system 
(Baumgartner technique). This technique involves perfusing whole blood at 
flow rates (sheer forces) found in the microvasculature; that is a sheer 
force of approximately 2600 inverse seconds (FIG. 2). 
The antibody 176D7 immunoprecipitates a 157/130 kDA heterodimeric antigen 
from the platelet surface. In the non-reduced state, this heterodimer has 
molecular weights of approximately 136 for the alpha subunit and 105 for 
the beta subunit. After reduction of the disulfide bonds, the alpha 
subunit increases in molecular size to about 156 kDa and the beta subunit 
increases in molecular size to about 130 kDa. The role of this antibody is 
quite important in thrombosis, vessel wall growth, cancer growth and 
metastasis of cancer. 
Antibody 176D7 binds to purified platelets without exogenous proteins and 
to platelets in the presence of plasma proteins. The association constant 
is identical in the presence or absence of plasma proteins, however, the 
number of binding sites per platelet is decreased approximately 15% in the 
presence of plasma proteins. This monoclonal antibody also recognizes the 
a collagen receptor present on endothelial cells, on tissue culture lines 
which include breast cancer cells, lung cancer cells, melanoma cells, 
colon cancer cells (FIGS. 3 and 4). Tests indicate that this antibody also 
inhibits the spreading and migration of fibrosarcoma cells on a collagen 
surface (FIG. 5). 
Antibody 176D7, which recognizes a collagen receptor, is also able to 
totally inhibit all aspects of collagen-platelet interactions. In 
particular, it inhibits the adhesion of platelets to collagen surfaces, 
the aggregation of platelets in the presence or absence of plasma proteins 
to collagen, and inhibits the binding of platelets in a flowing system to 
the vessel subendothelium (FIG. 2). The antibody recognizes a specific 
antigen found on human platelets, human endothelial cells, and a wide 
variety of human tumor cells (FIGS. 3 and 4). 
The antibody of the present invention is quite useful in detecting tumor 
cells which contain the heterodimeric antigen mentioned above. The data 
indicate that, in the presence of this antibody, tumor cells are inhibited 
from migrating onto a collagen surface (FIG. 5). Since collagen plays a 
pivotal role in the ability of cancer cells to metastasize, the collagen 
inhibiting property of 176D7 makes it useful as an inhibitor of tumor cell 
metastasis by inhibiting the interaction of tumor cells with a collagen 
substrate. For the same reason, 176D7 is also useful in the treatment of 
patients who have cardiovascular disease with angina pectoris or acute 
myocardial infarction due to rupture of an atherosclerotic plaque. In 
these conditions, collagen is exposed in the subendothelium and as 
demonstrated herein this antibody would inhibit platelet interaction with 
collagen which, in turn, prevents the formation of new thrombus which 
might partially or totally occlude the vessel. 
A diagnostic kit comprising a container containing the antibodies 176D7 of 
the present invention now makes it possible to identify tumor cells from 
biopsies or other tissues and to ascertain whether the tumor cells contain 
the surface glycoprotein specifically recognized by 176D7. The kit also 
allows the diagnosis of specific platelet defects, both congenital and 
acquired, involving the collagen receptor on platelets. These tests are 
performed by routine immunological, cytological or hematological 
techniques and the like, such as antibody binding, immunofluorescence, 
flow cytometry and the like, well known to one of ordinary skill in the 
art. 
The antibodies of the present invention also allow the determination in 
experimental animals whether, in the presence of the antibody, injected 
tumor cells or tumor cells inoculated into an organ, metastasize or if 
metastasis is inhibited. This would also allow the determination of 
whether the metastasis occurred through the interaction of glycoprotein 
Ia/IIa on the tumor cell surface with collagen. Inhibition of this 
interaction by 176D7 would result in decreased number of individual cells 
metastasizing, growing and forming tumor nodules. This would be 
significant in the formulation of therapeutic modalities in the treatment 
of metastasis in cancer. 
A composition comprises the monoclonal antibodies 176D7 in an effective 
amount to inhibit platelet reactions with collagenous surfaces and 
pharmaceutically acceptable vehicle. 
It is understood that the examples and embodiments described herein are for 
illustrative purposes only and that various modifications or changes in 
light thereof will be suggested to persons skilled in the art and are to 
be included within the spirit and purview of this application and scope of 
the appended claims.