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WO1992010520A1 - Anticorps monoclonal op-g2 et procede d'utilisation - Google Patents

Anticorps monoclonal op-g2 et procede d'utilisation Download PDF

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Publication number
WO1992010520A1
WO1992010520A1 PCT/US1991/009278 US9109278W WO9210520A1 WO 1992010520 A1 WO1992010520 A1 WO 1992010520A1 US 9109278 W US9109278 W US 9109278W WO 9210520 A1 WO9210520 A1 WO 9210520A1
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WIPO (PCT)
Prior art keywords
monoclonal antibody
gpiib
iiia
platelets
binding
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PCT/US1991/009278
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English (en)
Inventor
Toshiaki Tomiyama
Thomas J. Kunicki
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The Blood Center Research Foundation
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Publication of WO1992010520A1 publication Critical patent/WO1992010520A1/fr

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C07K16/2839—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the integrin superfamily
    • C07K16/2848—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the integrin superfamily against integrin beta3-subunit-containing molecules, e.g. CD41, CD51, CD61
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/30—Immunoglobulins specific features characterized by aspects of specificity or valency
    • C07K2317/34—Identification of a linear epitope shorter than 20 amino acid residues or of a conformational epitope defined by amino acid residues

Definitions

  • the present invention is directed to a murine monoclonal antibody (Mab) OP-G2 which, when added to blood, helps to prevent clotting.
  • the present invention is specifically directed to the use of the monoclonal antibody OP-G2 and derivatives of OP-G2 that incorporate information inherent in its primary amino acid sequence and/or structure.
  • the antibody competes with fibrinogen and other adhesive proteins for RGD recognition site(s) on the platelet membrane glycoprotein (GP) Ilb-IIIa protein.
  • Mammalian blood contains small cells known as platelets.
  • platelets In the "resting" phase, platelets circulate freely though blood vessels. They are generally non- adhesive and exhibit little or no interaction with tissue or with other blood cells.
  • platelets In the process of hemostasis, i.e., blood stoppage, platelets can be transformed from free floating, resting cells to an activated adhesive and aggregated mass. The transformation is generally in response to an insult to the blood vessel, which requires the aggregation of platelets at the point of damage in order to prevent blood loss.
  • the activation of blood platelets is generally initiated by a change in the protein structure of the platelet cell surface. This change can be stimulated by exposure of the cell to various blood components, such as adenosine-5'-diphosphate (ADP) , epinephrine, thro bin, collagen or serotonin acting on certain proteins on the platelet cell surface.
  • ADP adenosine-5'-diphosphate
  • epinephrine epinephrine
  • thro bin thro bin
  • collagen or serotonin acting on certain proteins on the platelet cell surface.
  • glycoprotein (GP) Ilb- Ilia complex is a noncovalently associated, divalent cation-dependent heterodimer, and a member of the supergene family of adhesive protein receptors called integrins (Hynes, R.O., 1987, "Integrins: A Family of Cell Surface Receptors," Cell, 48:549-554; Ruoslahti, E. , and M.D. Pierschbacher, 1987, “New Perspectives in Cell Adhesion: RGD and Integrins," Science 238:491- 497) .
  • GPIIb-IIIa serves as a receptor for fibrinogen, von Willebrand factor, fibronectin and other adhesive proteins (Phillips, D.R. , et al. , 1988, "The Platelet Membrane Glycoprotein Ilb-IIIa Complex," Blood, 71:831- 843) .
  • Each of these adhesive proteins contains an Arg- Gly-Asp (RGD) sequence, and the interaction of these proteins with GPIIb-IIIa appears to be mediated, at least in part, by this RGD recognition sequence (Gartner, T.K. , and J.S. Bennett, 1985, "The Tetrapeptide Analogue of the Cell Attachment Site of Fibronectin Inhibits Platelet Aggregation and Fibrinogen Binding to Activated
  • GPIIb-IIIa In comparison to other integrins, GPIIb-IIIa has two unique features. First, GPIIb-IIIa is an activation-dependent receptor. Although it is present on the surface of non-stimulated platelets, its receptor function becomes apparent only after stimulation of platelets with agonists, such as ADP, epinephrine, collagen and thrombin (Bennett, J.S., 1985, "The Platelet-Fibrinogen Interaction," In Platelet Membrane Glycoproteins, J.N. George, A.T. Nurden, and D.R. Phillips, editors, Plenum Press, New York, 193-214) .
  • agonists such as ADP, epinephrine, collagen and thrombin
  • GPIIb-IIIa is the only integrin that also recognizes an amino acid sequence at the carboxy- ter inus of the fibrinogen ⁇ A chain (Kloczewiak, M. , et al., 1984, "Platelet Receptor Recognition Site on Human Fibrinogen. Synthesis and Structure-Function
  • H12 HHLGGAKQAGDV
  • GPIIb-IIIa has recognition sites that bind to adhesive proteins, such as fibrinogen. GPIIb-IIIa mediates the binding of fibrinogen, which is necessary for the aggregation (cohesion) of platelets. Fibrinogen has an arginine- glycine-aspartic acid (RGD) amino acid sequence which is a binding site recognized by Ilb-IIIa.
  • RGD arginine- glycine-aspartic acid
  • GPIIb-IIIa In order to inhibit platelet aggregation, one can block the RGD recognition site of GPIIb-IIIa. This can be done by the introduction of a peptide which competes for the RGD recognition site on GPIIb-IIIa. If it is successful, it will inhibit GPIIb-IIIa from interacting with fibrinogen thus preventing the platelet aggregation phenomenon.
  • anti-thrombotic proteins which will react with GPIIb-IIIa to inhibit fibrinogen-mediated platelet aggregation.
  • a promising class of anti- thrombotic proteins are in the form of monoclonal antibodies having the proper amino acid sequence for effectively binding to the RGD recognition site of GPIIb- IIIa in order to competitively inhibit fibrinogen binding and, thus, platelet aggregation.
  • monoclonal antibodies include AP2, PAC-1 and 7E3.
  • the present invention is directed to a monoclonal antibody or derivative thereof which is specific for the GPIIb-IIIa complex.
  • the monoclonal antibody known as OP-G2
  • OP-G2 binds to GPIIb-IIIa. Its binding ability is inhibited by RGD peptides.
  • OP-G2 has a binding affinity or dissociation constant (Kd) of about 25 nanomolar (nM) .
  • Kd dissociation constant
  • the antibody has been characterized as having an isotype of IgG. ] , kappa.
  • the monoclonal antibody OP-G2 has several advantages over other known monoclonal antibodies.
  • the differences between OP-G2 and the monoclonal antibody PAC-1 relate to size of the monoclonal antibody, binding efficiency and binding location.
  • the PAC-1 monoclonal antibody is an IgM molecule having a molecular weight of approximately 900,000.
  • OP- G2 is an IgG molecule, which is much smaller having a molecular weight of approximately 150,000. There are certain advantages in using a smaller molecule for therapeutic purposes.
  • PAC-1 an IgM mAb
  • 7E3 an IgG mAb, binds equally to nonactivated and ADP-activated platelets after an one hour incubation. However, 7E3 exhibits an enhanced rate of binding following ADP-activation (Coller, B.S., 1985, "A New Murine Monoclonal Antibody Reports an Activation- Dependent Change in the Conformation and/or Microenvironment of the Platelet Glycoprotein GPIIb-IIIa Complex," J. Clin . Invest. 76:101-108).
  • PAC-l also requires prior activation of GPIIb-IIIa before the PAC-l monoclonal antibody will bind to the glycoprotein. This may have something to do with the larger size of the PAC-l monoclonal antibody.
  • OP-G2 has no requirement for prior activation possibly because it is a smaller molecule.
  • the location of binding also differs between the two antibodies. It is known that there exists a second site on GPIIb-IIIA which is termed a fibrinogen gamma chain dodecapeptide recognition site. It has been determined that PAC-l is blocked by both the dodecapeptide and the RGD peptide. On the other hand, OP-G2 is only blocked by the RGD peptides.
  • OP-G2 Another advantage of OP-G2 is its lower affinity compared to other known monoclonal antibodies. For example, OP-G2 has an affinity (Kd) of about 25 nM as opposed to the monoclonal antibody AP2, which has an affinity (Kd) of about 0.5 nM - 1.0 nM.
  • OP-G2 contains, within the amino acid sequence of the variable region of its heavy chain, the tripeptide sequence arginine-tyrosine-aspartic acid (RYD) , which likely mimics the RGD binding sequence. Therefore, if 0P-G2 is present in the blood system, adhesive proteins, like fibrinogen, that contain the RGD peptide cannot bind and platelet aggregation will not occur. While the same region of the PAC-l heavy chain also contains RYD, the upstream and downstream flanking sequences within the OP- G2 D gene region are different from those of PAC-l. These amino acids differences may contribute to the differences in binding behavior of OP-G2 compared to PAC- 1.
  • RYD arginine-tyrosine-aspartic acid
  • a further advantage is that the OP-G2 monoclonal antibody is easier to remove from GPIIb-IIIa because of its lower affinity relative to other antibodies. Once the GPIIb-IIIa is freed of OP-G2, the platelets can resume clotting. On the other hand, the monoclonal AP2, once bound to GPIIb-IIIa, remains on the platelet surface until the platelet is removed from the circulation.
  • the monoclonal antibody can be placed into an antithrombotic kit containing a sterile package with the monoclonal antibody and a known pharmaceutically acceptable carrier.
  • OP-G2 has another advantage which relates to the current lack of insight into the three-dimensional structure of GPIIb-IIIa.
  • the three-dimensional structure of a protein can be determined after X- ray crystallography.
  • X-ray crystallo ⁇ graphy has been used to successfully determine the three- dimensional structure of small or simple proteins, like myoglobin or lysozyme or the thrombin-hirudin complex
  • very large protein complexes like GPIIb-IIIa
  • One other class of proteins that has been and can be successfully analyzed by X-ray crystallography is antibody proteins (immunoglobulins) .
  • GPIIb-IIIa Since it is now difficult to resolve the complete 3-D structure of GPIIb-IIIa, it may be possible to ascertain the structure of a portion of GPIIb-IIIa, namely, the portion that functions as the RGD recognition site.
  • OP-G2 a protein that can be readily resolved by X-ray crystallography, contains a sequence that acts like RGD, the structure of OP-G2 can help to deduce the structure of that part of GPIIb-IIIa that recognizes and binds to RGD and RGD-containing peptides or proteins, e.g., fibrinogen.
  • the RGD-recognition region or site of GPIIb-IIIa should be a mirror image of the RGD-like sequence of OP-G2.
  • OP-G2 is an effective RDG-like ligand whose complete structure (in 3-D) can be determined readily by X-ray crystallography.
  • the complete structure of 0P-G2 represents an effective framework that can then be used to engineer RGD-like ligands which, differ in specificity for different integrins, affinity for a given integrin, and relative efficacy in vivo, taking into consideration such factors as half-life in vivo, target of action, etc.
  • Figure 1 is an indirect immunoprecipitation of platelet surface proteins.
  • the total protein in a Triton X-100 extract of 125 I-labeled platelets is illustrated in lane 1.
  • Surface-labeled platelets were incubated with 0P-G2 (lane 2) or nonimmune mouse IgG (lane 3) , washed, then incubated with goat anti-mouse IgG.
  • the position of MW markers (in Kd) is indicated at the left. Bands corresponding to glycoprotein lb, lib and Ilia are indicated.
  • Figure 2 is crossed immunoelectrophoresis of Triton X-100-solubilized platelet protein. Depicted are a Coomassie blue-stained gel (A) , and autoradiographs of identical gels wherein the second dimension contained: 125 I-OP-G2 (B) ; I25 l-Hil-1 (C) ; or 125 I-AP3 (D) in the intermediate gel.
  • PMI-1 and AP-3 are murine monoclonal antibodies that bind to GPIIb-IIIa, respectively.
  • Figure 3 illustrates four graphs showing the effect of OP-G2 Fab fragments on platelet aggregation.
  • Citrated platelet-rich plasma (3 x 10 8 platelets/ml) was preincubated with the indicated concentration ( ⁇ g/ l) of OP-G2 Fab for 3 min at 37°C with stirring prior to addition (arrow) of ADP (5 ⁇ M) , epinephrine (10 ⁇ M) , collagen (1 ⁇ M) or ristocetin (1.3 mg/ml) .
  • Percent light transmission (ordinate) as a function of time (abscissa) is plotted. Bars indicate one minute.
  • Figure 4 illustrates two graphs showing the inhibition of fibrinogen binding to ADP-activated platelets by 0P-G2 Fab fragments.
  • Figure 4A illustrates suspensions of washed platelets that were preincubated with buffer (o) or OP-G2 Fab fragments at a concentration of 40 ⁇ g/ml (•) for 5 min.
  • buffer (o) or OP-G2 Fab fragments at a concentration of 40 ⁇ g/ml (•) for 5 min.
  • FIG. 4B illustrates double-reciprocal plots of the data from (A) .
  • Figure 5 illustrates two graphs showing the effect of synthetic peptides on OP-G2 and AP2 binding to purified GPIIb-IIIa. Microtiter wells coated with purified GPIIb-IIIa were incubated with 50 ⁇ l of a solution containing peptide for 60 min. The concentration of peptide is indicated on the abscissa.
  • Figure 6 illustrates four graphs showing the binding of 0P-G2 to nonactivated and activated platelets, as determined by flow cytometry.
  • the binding of 0P-G2 was detected with FITC-goat anti-mouse IgG.
  • Figure 6A illustrates the binding of nonimmune mouse IgG (40 ⁇ g/ml) (open peak) and OP-G2 IgG (40 ⁇ g/ml) (solid peak) to nonactivated platelets.
  • Figures 6 B, C, and D illustrate the binding of 0P-G2 IgG (40 ⁇ g/ml) ( Figure 6B) , 0P-G2 Fab (40 ⁇ g/ml) ( Figure 6C) , or AP2 IgG (2 ⁇ g/ml) ( Figure 6D) to nonactivated platelets (open peaks) or thrombin- activated platelets (solid peaks) .
  • Log fluorescence is plotted on the abscissa.
  • Figure 7 illustrates two graphs showing the binding of 125 I-OP-G2 to nonactivated and activated platelets.
  • Figure 7A illustrates various concentrations of 125 I-OP-G2 ( ⁇ g/ml; abscissa) were added to nonactivated (o) and thrombin-activated ( ⁇ ) platelets while the final platelet concentration was maintained at 2.5 x 10 8 /ml. After a 60 min incubation at ambient temperature, bound 125 I-OP-G2 was measured (ordinate) .
  • Figure 7B illustrates the analysis of the binding data shown in Figure 7A by the method of Scatchard.
  • Figure 8 illustrates the sequence of the heavy chain variable gene region of OP-G2 identified as: SEQ ID NO:l.
  • Complimentary-DNA (cDNA) corresponding to the heavy chain variable gene region was obtained using 0P-G2 messenger RNA and the polymerase chain reaction (PCR) . Individual codons are indicated and the deduced amino acid is noted above each codon using the single letter amino acid code.
  • the regions corresponding to the Variable-Heavy (V H ) gene sequence, the D-gene sequence. and the J-heavy (J H ) gene sequence are shown. All three contribute to the total variable domain of the heavy chain.
  • the three regions of hypervariability or complementarity-determining regions (CDR) are also indicated. Within the center of the D gene region is the sequence RYD.
  • IgG monoclonal antibody 0P-G2
  • OP-G2 Fab fragments inhibit fibrinogen-mediated platelet aggregation induced by ADP, epinephrine, collagen and thrombin in a dose-dependent manner, but do not inhibit ristocetin-induced platelet agglutination which involves the interaction of von Willebrand factor with GPIb.
  • OP- G2 Fab fragments competitively inhibit ADP-induced binding of 125 I-fibrinogen to washed platelets, and 0P-G2 binding to purified GPIIb-IIIa is inhibited by RGD- containing peptides.
  • RGD- containing peptides Within the sequence of its heavy chain variable domain, OP-G2 contains the tripeptide RYD which likely mimics RGD.
  • the murine mAbs OP-G2, AP2 (anti-GPIIb-IIIa complex) and AP3 (anti-GPIIIa) were developed according to the procedures described in Newman, P.J., et al. , 1985, "Quantitation of Membrane Glycoprotein Ilia on Intact Human Platelets Using the Monoclonal Antibody, AP3," Blood 65:227-232; Pidard, D. , 1983, "Interaction of AP2, a Monoclonal Antibody Specific for the Human Platelet Glycoprotein Ilb-IIIa Complex, With Intact Platelets," J. Biol . Chem . 258:12582-12586; and
  • Purified IgG was labeled with 125 I using the chloramine T method. Free 125 I was separated from the sample by filtration through a Biogel P2 column. A specific activity of 400 - 800 cpm/ng IgG was routinely obtained.
  • OP-G2 Fab fragments For the preparation of OP-G2 Fab fragments, monoclonal IgG was dialyzed against 0.01 M PBS (pH 7.4) and adjusted to a concentration of 4 mg/ml. After adding 10 ml cysteine and 2 mM EDTA, 0P-G2 was digested with mercuripapain (Sigma Chemical Company; St Louis, MO) at a 1:99 ratio of papain to protein for 4 hr at 37°c. The reaction was terminated by adding iodoacetamide to a final concentration of 10 mg/ml. The Fab fragments were separated from Fc fragments and undigested IgG by chromatography on Protein A-Sepharose CL-4B.
  • ELISA was performed as described in Kunicki, T.J., et al., 1990, "Human Monoclonal Autoantibody 2E7 is Specific for a Peptide Sequence of Platelet Glycoprotein lib. Localization of the Epitope to Hb 23 i_ 238 with an Immunodominant Trp 235 ,” J. Autoimmunity (In press), which is incorporated herein by reference.
  • Microtiter wells coated with purified GPIIb-IIIa were incubated with 50 ⁇ l of a solution containing peptide for 60 min at ambient temperature. Fifty ⁇ l of OP-G2 or AP2 was then added to the wells, and the plates were incubated an additional 60 min.
  • the wells were washed 6 times with PBS-0.05% Tween, 50 ⁇ l of alkaline phosphatase-conjugated goat anti-mouse IgG (1:1000 dilution in PBS-0.05% Tween) was added to each well, and the plates were incubated for 60 min at ambient temperature.
  • the wells were washed 6 times, the substrate (p-nitrophenylphosphate in 100 mM Tris, 100 mM NaCl, 5 mM MgCl 2 , pH 9.5) was added, and absorbance at 405 nm was recorded.
  • Experiment 1 was designed to determine to molecular weights of proteins to which 0P-G2 binds by indirect immunoprecipitation. Indirect immunoprecipitation was performed as described in Tomiyama, Y., et al., 1990, "Identification of the Platelet-Specific Alloantigen, Nak a , on Platelet Membrane Glycoprotein IV," Blood 75:684-687, which is incorporated herein by reference, with minor modifications. In brief, eight ⁇ g of OP-G2 IgG were incubated with 100 ⁇ l 125 I-labeled platelet suspension (10 8 platelets per ml) for 60 min at ambient temperature.
  • the platelets were washed three times, and then 10 ⁇ l of goat anti-mouse IgG, (Cooper Biomedical Inc. , Malvern, PA) diluted 1:100, was added. After a 60 min incubation, the antibody-sensitized platelets were washed three times and solubilized in 1.2 ml of 0.01 M TBS containing 1% Triton X-100, 2.5 mM KI, 10 mM EDTA and 1 mM PMSF (Sigma) at 4°C. One ml of each radiolabeled platelet lysate was incubated with 50 ⁇ l of washed protein A-bearing
  • Staphylococcus aureus (EcSorb; E «Y Laboratories, San Mateo, CA) for 20 min at 4°C.
  • the S . aureus were then washed five times, and the immune complexes absorbed by S. aureus were subjected to electrophoresis in a 7.5% polyacrylamide slab gel according to the method of
  • Fig. 1 illustrates that 0P-G2 binds to two radiolabeled proteins having apparent molecular weights of 140 and 92 kD under nonreduced electrophoretic conditions. These proteins correspond to GPIIb and GPIIIa.
  • Experiment 2 was designed to further characterize the OP-G2 epitope on GPIIb-IIIa.
  • Triton X- 100-soluble platelet protein was prepared in the presence or absence of 5 mM EDTA and was analyzed by Crossed Immunoelectrophoresis (CIE) employing radiolabeled 0P-G2, PMI-1 or AP3 in the intermediate gel.
  • CIE Crossed Immunoelectrophoresis
  • CIE was performed as described in Kunicki, T.J. , et al., 1981, "The Formation of Ca 2+ -Dependent Com ⁇ plexes of Platelet Membrane Glycoproteins lib and Ilia in Solution as Determined by Crossed Immunoelectrophoresis," Blood 58:268-278, which is incorporated herein by reference. Briefly, 100 ⁇ g of Triton X-100 solubilized platelet protein were electrophoresed at 10 V/cm at 16°C for 75 min in a first dimension gel consisting of 1% agarose dissolved in 38 mM Tris, 0.1 M glycine, 0.5% Triton X-100, pH 8.7.
  • Second dimension electrophoresi ⁇ was performed at 2 V/cm for 18 h against an intermediate gel containing 1 x 10 6 cpm of 125 I-monoclonal IgG followed by an upper gel containing rabbit anti-whole platelet antibody. Precipitin arcs containing 125 I- monoclonal IgG were revealed by autoradiography of the CIE plate.
  • Experiment 3 was performed to determine whether the portion of 0P-G2 that binds to its antigen, i.e., the Fab fragment, inhibits platelet aggregation.
  • PRP was obtained from blood (9 vol) anticoagulated with 1 vol ACD-A and washed twice with ringer's citrate dextrose (hereinafter also referred to as "RCD") containing 20 ng/ml of PGE lf pH 6.5.
  • RCD ringer's citrate dextrose
  • the platelets were resuspended in 5 mM Hepes, 0.3 mM NaH 2 P0 , 12 mM NaHC0 3 , 5.5 mM glucose, 1 mM MgCl 2 , 2 mM CaCl , 2 mM KC1, 137 mM NaCl, pH 7.4.
  • 0P-G2 Fab fragments or synthetic peptides were measured by preincubating the PRP with antibody or peptides for three min at 37°C with stirring before the addition of aggregation-inducing agents.
  • ADP, epinephrine, collagen and ristocetin were used as aggregating agents.
  • Thrombin, 0.1 u/ml was added to washed platelet suspension without added fibrinogen.
  • 0P-G2 Fab fragments were found to inhibit ADP (5 ⁇ M)-, epinephrine (10 ⁇ M)- or collagen (1 ⁇ g/ml)-induced platelet aggregation in a dose-dependent manner.
  • 0P-G2 Fab fragments also were found to inhibit thrombin-induced platelet aggregation (data not shown) .
  • a slight inhibitory effect of OP-G2 Fab fragments upon ristocetin-induced aggregation was also observed probably reflecting inhibition of secondary, secretion-dependent aggregation.
  • 0P-G2 Fab When PRP was preincubated with 5 mM EDTA for 2 min, 0P-G2 Fab had no effect on ristocetin-induced agglutination (data not shown) . 0P-G2 Fab fragments did not affect shape change of platelets and did not themselves induce platelet aggregation or agglutination.
  • Experiment 4 was designed to examine the effect of 0P-G2 Fab fragments on fibrinogen binding to ADP-stimulated platelets.
  • Fibrinogen binding to washed platelets was measured as described in Kunicki, T.J., et al.,1985, "Human Platelet Fibrinogen: Purification and Hemostatic Properties," Blood 66:808-815, which is incorporated herein by reference.
  • 10 ⁇ M ADP was added to the suspension. After 5 minutes without stirring at ambient temperature, the platelets were sedimented through 30% sucrose dissolved in resuspension buffer as described above for binding of monoclonal antibodies. Nonspecific binding was determined in parallel tubes that contained 10 mM EDTA.
  • 0P-G2 Fab fragments The effect of 0P-G2 Fab fragments on ADP-stimulated fibrinogen binding was determined by preincubating platelets with OP-G2 Fab at a concentration of 40 ⁇ g/ml for 5 min before initiating the fibrinogen binding assay. Suspensions of washed platelets were incubated with 0P-G2 Fab at a concentration of 40 ⁇ g/ml at ambient temperature for 5 min. Various concentrations of 125 I-fibrinogen and 10 ⁇ M ADP were then added to the suspensions. After an additional 5-min incubation at ambient temperature, the bound fibrinogen was measured.
  • Fig. 4A prior incubation of platelets with 0P-G2 Fab resulted in specific inhibition of fibrinogen binding.
  • Fig. 4B the examination of the binding data by using double- reciprocal plots revealed that 0P-G2 Fab fragments are a competitive inhibitor of fibrinogen binding.
  • the mean Ki from two experiments using platelets from different normal donors is 68 nM.
  • experiment 5 was designed to determine the effect of synthetic peptides on OP-G2 binding to purified GPIIb-IIIa.
  • the RGD-containing peptides (dextrorotatory-Arg)-Gly-Asp-Trp (hereinafter referred to as "(+)RGDW”, CG(+)RGDWGY (hereinafter also referred to as "(+)RGD-8") and YAVTGRGDSPASSK
  • H12 dodecapeptide
  • HHLGGAKQAGDV dodecapeptide
  • H12 dodecapeptide
  • the peptides were synthesized using a Milligen/BioResearch Labs 9050 Automated Pepsynthesizer (San Rafael, CA) employing PepSyn KA resins and F oc-amino acids. Peptides were cleaved from the resin with trifluoroacetic acid and purified by reverse-phase high performance liquid chromatography (Beckman System Gold, Beckman Instruments, Inc. , Alex Division, San Ramon, CA) using Vyadec C18 preparative columns (The Sep/a/ra/tions Group, Hesperia, CA) .
  • (+JRGD-8 CG(+)RGDWGY;
  • Citrated platelet-rich plasma was preincubated with various amounts of peptides for 3 min at 37°C with stirring before the addition of 5 ⁇ M ADP.
  • Experiment 6 was designed to evaluate the effect of the peptides described in experiment 5 on OP-G2 and AP2 binding to purified GPIIb-IIIa.
  • OP-G2 and AP2 was used at concentrations of 2 ⁇ g/ml and 20 ng/ l, respectively. These were the antibody concentrations which resulted in roughly 50% of maximum binding to GPIIb-IIIa in the same ELISA.
  • Fig. 5B none of these synthetic peptides had an effect on AP2 binding to GPIIb- IIIa.
  • OP-G2 binding to GPIIb-IIIa was inhibited by the RGD-containing peptides, but not by RGEW or ALPLGS, as illustrated in Fig. 5A.
  • the order of potency with respect to inhibition of OP-G2 binding was exactly the same as the order of potency with respect to inhibition of ADP-induced aggregation.
  • H12 did not inhibit OP-G2 binding to GPIIb-IIIa, although the inhibitory effects of H12 and Fnl4 on ADP- induced aggregation were of the same order of magnitude.
  • OP-G2 may be able to overcome such an allosteric, conformational change induced by H12.
  • Experiment 7 was designed to compare the binding of 0P-G2 to nonactivated and activated platelets.
  • Thrombin activation of platelets was performed by incubating 2.8 x 10 8 platelets with 1 u/ml of human ⁇ - thrombin (Sigma) at 37°C for 15 min, and the reaction was stopped by adding hirudin (Sigma) at a final concentration of 2 u/ml.
  • hirudin Sigma
  • Various concentrations of 125 ⁇ - 0P-G2 were added to the platelet suspension while the final platelet concentration was maintained at 2.5 x 10 8 /ml.
  • triplicate 100 ⁇ l samples were layered onto 200 ⁇ l of 30% sucrose in buffer, in 400 ⁇ l microcentrifuge, polypropylene tubes. Tubes were centrifuged at 7000 g for 10 min. The supernatants were aspirated and the radioactivity of both supernatants and pellets was measured in a gamma counter.
  • Thrombin activation of platelets was performed as described above, except that platelets were resuspended in RCD-PGE ⁇ After thrombin activation, the platelets were washed once more with RCD-PGE lf and the platelet count was adjusted to 5 x 10 8 /ml. The platelet suspension was incubated with OP-G2 (40 ⁇ g/ml) , AP2 (2 ⁇ g/ml) or nonimmune mouse IgG (40 ⁇ g/ml) for one hour at ambient temperature.
  • the platelets were then pelleted, resuspended in RCD-PGE- L , and incubated with a 1:20 dilution of FITC- conjugated goat anti-mouse IgG (Zymed Laboratories Inc., South San Francisco, CA) for 1.5 hours.
  • the platelet suspension was then fixed with 1% paraformaldehyde and analyzed in a flow cytometer (Becton-Di ⁇ kinson FACS Star pluB , Mountain View, CA) .
  • 0P-G2 bound to nonactivated platelets as illustrated in Fig. 6A.
  • thrombin-activation of platelets markedly increased the binding of OP-G2 IgG to platelets.
  • Fig. 6D the binding of AP2 to nonactivated platelets was essentially the same as that to activated platelets.
  • Experiment 8 was designed to evaluate the increase in OP-G2 binding more precisely.
  • Direct binding assays using 125 I-OP-G2 IgG were performed.
  • Binding data were analyzed by the method of Scatchard, G., 1949, "The Attractions of Proteins for Small Molecules and Ions," Ann. N. Y. Acad . Sci . 51:660-672, which is incorporated herein by reference. Based on an analysis of six normal donors, the number of.
  • OP-G2 molecules bound per platelet was determined to be 49800 ⁇ 8180 (mean ⁇ SD) with a dissociation constant (Kd) of 25.4 ⁇ 5.6 nM (mean ⁇ SD) .
  • Kd dissociation constant
  • Experiment 9 was designed to explore the basis for the apparent increased affinity of 0P-G2 by kinetic binding studies.
  • the dissociation rate constant (K 2 ) was determined by measuring the displacement of 125 I-0P-G2 from the platelets at 1 min after adding a 100-fold excess of unlabeled 0P-G2.
  • the value of the association rate constant (K- ⁇ was determined by measuring 125 I-0P-G2 binding after 1 min.
  • K 2 for thrombin-activated platelets was calculated to be 2.66 x 10 "2 min -1 .
  • K K 2 /K_.
  • the Kd for nonactivated platelets could not be determined
  • the Kd for thrombin-activated platelets determined from kinetic studies was 4.6 nM, in excellent agreement with the value determined from the equilibrium binding studies.
  • Experiment 10 was designed to ascertain the nucleotide sequence of the gene that encodes the variable region of the heavy chain of OP-G2.
  • Antigen recognition and hence antibody specificity resides in the variable region of either or both the heavy and light chains of the antibody molecule.
  • the extent to which either or both the heavy and light chains contribute to antigen binding is empirical and variable from one antibody to the next.
  • antigen binding is known to be inherent in one or more of three sequence regions in each of the heavy chain and light chain variable regions. These are areas of hypervariability that are the sequences most unique to any given heavy or light chain. These sequence regions are commonly called complementarity-determining regions and are each abbreviated CDR.
  • cDNA of immunoglobulin-specific (IgG) RNA was produced using the reverse transcriptase enzyme and an oligonucleotide primer corresponding to the hinge region of the urine gamma chain gene (ATTGTGCCCAGGGATTGTACTAGTAAGCCT) .
  • OP-G2 heavy chain immunoglobulin cDNA was then amplified using the Taq 1 polymerase chain reaction (PCR) and an oligonucleotide primer corresponding to the consensus sequence of the 5• fra ework region of the murine variable heavy gene III family (CTGCTCGAGTCTGGAGGAGGCTTG) .
  • Amplified cDNA was prepared such that it contained 5• Xhol and 3• Spel restriction sites (these sites were included in the 5' and 3' oligonucleotide primers employed in the PCR) .
  • OP- G2 heavy chain cDNA was purified, digested with Xhol and Spel and subcloned into the plasmid pGEM-11 (Promega Biotech, Inc., Madison, WI) . The sequence of the subcloned OP-G2 heavy chain was then determined using the Sequenase reaction (Version 2.0, United States Biochemical Corp., Cleveland, OH).
  • variable region of the heavy chain of OP-G2 is identified as SEQ ID NO:l and is depicted in Figure 8.
  • D gene employed by OP-G2 which comprises a large portion of CDR3 of the heavy chain, has in its center the nucleotide sequence that encodes the tripeptide RYD. This sequence, akin to RGD, likely accounts for the specificity of OP-G2.
  • OP-G2 is the first murine monoclonal IgG antibody to be described that binds so close to the RGD recognition site of GPIIb-IIIa that antibody binding can be completely inhibited by RGD peptides.
  • 0P-G2 itself contains the sequence RYD within CDR3 (D gene) of its heavy chain. This likely accounts for the specificity of 0P-G2.
  • This antibody has permitted us to study more closely the availability of the RGD recognition site as a function of platelet stimulation.
  • Our results with OP-G2 provide strong support for the hypothesis that significant conformational changes occur within the GPIIb-IIIa complex during activation that include the RGD-recognition site.
  • OPG2 Heavy Chain Gene Sequence (Includes V H gene plus D gene plus J H gene).
  • cDNA sequence obtained by PCR from OPG2 hybridoma RNA. Protein sequence deduced from cDNA.
  • GGC ATG TCT TGG GTT CGC CAG ACT CCG GAG AAG AGG CTG GAG TGG GTC Gly Met Ser Trp Val Arg Gin Thr Pro Glu Lys Arg Leu Glu Trp Val

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Abstract

Un anticorps monoclonal IgG murin, OP-G2, a été caractérisé, et est spécifique pour le complexe glycoprotéique (GP) IIb-IIIa. OP-G2 reconnaît un épitope au niveau du site de reconnaissance (RGD) Arg-Gly-Asp. La chaîne lourde de OP-G2 contient dans CDR3 de sa région variable la séquence RYD qui peut justifier sa spécificité pour les sites de reconnaissance RGD de GPIIb-IIIa. La liaison de OP-G2 au complexe purifié GPIIb-IIIa est inhibée par les peptides contenant RGD mais pas le peptide à terminaison carboxy de la chaîne fibrinogène ηA.
PCT/US1991/009278 1990-12-12 1991-12-10 Anticorps monoclonal op-g2 et procede d'utilisation WO1992010520A1 (fr)

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Cited By (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5672585A (en) * 1990-04-06 1997-09-30 La Jolla Cancer Research Foundation Method and composition for treating thrombosis
US5780303A (en) * 1990-04-06 1998-07-14 La Jolla Cancer Research Foundation Method and composition for treating thrombosis
US6017877A (en) * 1990-04-06 2000-01-25 La Jolla Cancer Research Foundation Method and composition for treating thrombosis
US6521594B1 (en) 1990-04-06 2003-02-18 La Jolla Cancer Research Foundation Method and composition for treating thrombosis

Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP0206533A2 (fr) * 1985-06-14 1986-12-30 The Research Foundation Of State University Of New York Fragment d'anticorps monoclonal inhibant les plaquettes
EP0368486A2 (fr) * 1988-11-10 1990-05-16 Merck & Co. Inc. Inhibiteurs de l'attachement des plaquettes
WO1990006134A1 (fr) * 1988-12-01 1990-06-14 Centocor, Inc. Anticorps humains specifiques de plaquettes
WO1991001380A1 (fr) * 1989-07-25 1991-02-07 Institut National De La Sante Et De La Recherche Medicale Anticorps monoclonaux diriges contre des proteines impliquees dans les fonctions plaquettaires, leur application en tant qu'agent diagnostique et therapeutique

Patent Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP0206533A2 (fr) * 1985-06-14 1986-12-30 The Research Foundation Of State University Of New York Fragment d'anticorps monoclonal inhibant les plaquettes
EP0368486A2 (fr) * 1988-11-10 1990-05-16 Merck & Co. Inc. Inhibiteurs de l'attachement des plaquettes
WO1990006134A1 (fr) * 1988-12-01 1990-06-14 Centocor, Inc. Anticorps humains specifiques de plaquettes
WO1991001380A1 (fr) * 1989-07-25 1991-02-07 Institut National De La Sante Et De La Recherche Medicale Anticorps monoclonaux diriges contre des proteines impliquees dans les fonctions plaquettaires, leur application en tant qu'agent diagnostique et therapeutique

Non-Patent Citations (2)

* Cited by examiner, † Cited by third party
Title
Chemical Abstracts, vol. 104, no. 3, 20 January 1986, (Columbus, Ohio, US), V.T. TRAPANI: "Independent modulation of von Willebrand factor and fibrinogen binding to the platelet membrane glycoprotein IIb/IIIa complex as demonstrated by monoclonal antibody", see page 345, abstract no. 18049y, & J. CLIN. INVEST. 1985, 76(5), 1950-8, see abstract *
The Journal of Biological Chemistry, vol. 264, no. 1, 5 January 1989, R. TAUB: "A monoclonal antibody against the platelet fibrinogen receptor contains a sequence that mimics a receptor recognition domain", pages 259-265, see page 259: "Introduction" *

Cited By (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5672585A (en) * 1990-04-06 1997-09-30 La Jolla Cancer Research Foundation Method and composition for treating thrombosis
US5780303A (en) * 1990-04-06 1998-07-14 La Jolla Cancer Research Foundation Method and composition for treating thrombosis
US6017877A (en) * 1990-04-06 2000-01-25 La Jolla Cancer Research Foundation Method and composition for treating thrombosis
US6521594B1 (en) 1990-04-06 2003-02-18 La Jolla Cancer Research Foundation Method and composition for treating thrombosis

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