WO1992008804A1 - Erythrocytes et thrombo-erythrocytes utilises comme agents a specificite de cible - Google Patents

Erythrocytes et thrombo-erythrocytes utilises comme agents a specificite de cible Download PDF

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Publication number
WO1992008804A1
WO1992008804A1 PCT/US1991/008430 US9108430W WO9208804A1 WO 1992008804 A1 WO1992008804 A1 WO 1992008804A1 US 9108430 W US9108430 W US 9108430W WO 9208804 A1 WO9208804 A1 WO 9208804A1
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Prior art keywords
erythrocyte
thrombo
erythrocytes
molecule
polypeptide
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PCT/US1991/008430
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English (en)
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Barry S. Coller
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The Research Foundation Of The State University Of New York
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Priority to KR1019930701399A priority Critical patent/KR930702341A/ko
Priority to AU90588/91A priority patent/AU651643B2/en
Priority to JP4502274A priority patent/JPH06504535A/ja
Publication of WO1992008804A1 publication Critical patent/WO1992008804A1/fr
Priority to NO93931696A priority patent/NO931696L/no
Priority to FI932114A priority patent/FI932114A/fi

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
    • A61K35/12—Materials from mammals; Compositions comprising non-specified tissues or cells; Compositions comprising non-embryonic stem cells; Genetically modified cells
    • A61K35/14—Blood; Artificial blood
    • A61K35/18—Erythrocytes
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/48—Preparations in capsules, e.g. of gelatin, of chocolate
    • A61K9/50—Microcapsules having a gas, liquid or semi-solid filling; Solid microparticles or pellets surrounded by a distinct coating layer, e.g. coated microspheres, coated drug crystals
    • A61K9/5005—Wall or coating material
    • A61K9/5063—Compounds of unknown constitution, e.g. material from plants or animals
    • A61K9/5068—Cell membranes or bacterial membranes enclosing drugs
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P7/00—Drugs for disorders of the blood or the extracellular fluid
    • A61P7/04—Antihaemorrhagics; Procoagulants; Haemostatic agents; Antifibrinolytic agents
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/745—Blood coagulation or fibrinolysis factors
    • C07K14/75—Fibrinogen
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides

Definitions

  • the present invention is directed to a new 0 composition of matter called thrombo-erythrocytes, which have the ability to bind selectively to activated platelets but not to unactivated platelets.
  • the thrombo-erythrocytes are useful in controlling 5 bleeding in thrombocytopenic (blood platelet deficient) mammals, and for the uptake and delivery of labels, therapeutic agents and genetic materials to selected targets.
  • the invention further relates to targeted erythrocytes and their uses in the uptake and Q delivery of compounds.
  • the activation of platelets is a complicated process, which includes producing or exposing receptors for the plasma protein fibrinogen on the platelet surface. Fibrinogen has multiple binding sites, and binds two or more platelets simultaneously, initiating the aggregation.
  • a platelet receptor that is present on the surface of platelets and becomes exposed during the activation process is GPIIb/IIIa. Patients with low platelet counts often require transfusions of platelets in order to control bleeding.
  • Ryback and Renzulli incorporated a deoxycholate extract of platelet membranes containing 15 proteins, including GPIb, GPIIb/IIIa, and GPIV, into small (50-200 nm) unilamellar liposomes prepared from either sphingomyelin:phosphatidylcholine:monosialyloganglio- side or egg phosphatide (Blood Suppl. 1:473a, abstr). Intra-arterial injections of both preparations decreased bleeding in thrombocytopenic rats to the same extent as human platelets did, but neither produced complete normalization of the bleeding time.
  • liposomes containing GPIIb/IIIa alone were ineffective (Rybak and Renzulli, 1990, Blood Suppl. 1:473a, Abstr.).
  • This approach may provide important mechanistic information but as a therapeutic intervention it potentially suffers from the generic problems of liposomes, including the possibility of short in vivo survival and potential blockade of the reticuloendothelial system (Kahn et al., 1985, Blood 66:1-12, Abstr.).
  • platelets remain the starting material, problems of platelet procurement and the risks of transmitting infectious diseases may not be eliminated.
  • platelets have class I HLA antigens (McFarland and Aster, 1991, In Principles of Transfusion Medicine. Williams & Wilkins, Baltimore, pp. 193-204) , and some platelet glycoproteins are polymorphic (Lopez and Ludwig, 1991, Clin. Res. 39:327 a.s.).
  • Agam and Livne took an approach based on their observations that passive, fixed platelets coated with fibrinogen could function to augment platelet aggregation of native, fresh platelets (Agam and Livne, 1983, Blood 61:186; Agam and Livne, 1984, Thro . Hae ostas. 51:145-149; Agam and Livne, 1988, Thromb. Haemostas. 59:504-506).
  • fibrinogen and platelets interact with fibrinogen-coated polyacrylonitrile beads via a mechanism involving fibrinogen receptors on platelet surfaces (see the paragraph bridging pages 177 and 178 in Coller et al., 1980, Blood. 55:169-178).
  • Agam and Livne (1983, Blood 61:186-191) disclosed that fixed platelets to which fibrinogen had been bound participated in the aggregation of activated platelets, by selective reaction with activated platelets. Ruoslahti et al. , U.S. Patent No.
  • radiolabeled molecules In addition, the precise delivery of radiolabeled molecules, diagnostic, and therapeutic agents to specific target tissues is an important laboratory and clinical problem.
  • one objective of the present invention is to solve the problems of obtaining cells from a small amount of blood, particularly autologous blood, that can be used to deliver precisely agents to specific target issues.
  • compositions of matter that are able to bind selectively to activated platelets but not to unactivated platelets n vivo.
  • Activation refers to the process by which platelets become more susceptible to aggregation. The process by which platelets become activated is poorly understood, especially, j-n vivo. It appears that the activation process is induced by a number of agonists, such as ADP, epinephrine, collagen, thrombin and thromboxane A2. Indiscriminate binding of an agent to both activated and unactivated platelets exposes the patient to the risk of thrombosis (blood clots) that can lead to the death of tissues in vital organs, including the heart and brain.
  • the present invention provides new compounds and methods for promoting platelet aggregation, and preventing hemorrhage.
  • the present invention is based on the surprising discovery that erythrocytes conjugated to certain peptides and polypeptides containing an R-G-D (Arg-Gly-Asp) sequence 5 (collectively termed herein "RGD peptides") according to the invention, selectively bind to activated platelets but not to unactivated platelets.
  • R-G-D Arg-Gly-Asp sequence 5
  • the methods and compounds of the invention overcome the problems with prior art platelet substitutes by providing abundant, safe material to promote platelet aggregation, specific for sites of injury.
  • thrombo-erythrocytes are produced which surprisingly, have no significant change in their rheological properties.
  • the thrombo-erythrocytes have the majority of RGD peptide 0 cross-linked specifically to glycophorin A and glycophorin B on the surface of the erythrocyte, producing a thrombo-erythrocyte that has an altered membrane surface that can interact selectively with activated platelets via the platelet GPIIb/IIIa 5 receptor.
  • the N-terminal Arg of the R-G-D sequence should be spaced within 9-50 Angstroms, more preferably 10-40 Angstroms, and most preferably 11-25 Angstroms, from the erythrocyte protein to which the 0 RGD peptide is conjugated.
  • the activated platelets aggregate with the erythrocytes, forming clumps or clots.
  • clumps or clots form in vivo in mammals, including humans, they are helpful in controlling bleeding, and are especially helpful in 5 controlling bleeding from small wounds.
  • the invention is further directed to erthrocytes modified by replacement of their intracellular contents with a composition comprising a label or agent.
  • carrier erythrocytes Such modified erythrocytes are termed herein "carrier erythrocytes".
  • the carrier erythrocytes have use in delivery of such labels or biologically active agents to specific tissues by conjugation to a targeting agent.
  • the carrier erythrocytes are thrombo-erthrocytes , and are thus targeted to a specific tissue, in particular an activated platelet, by conjugation with an RGD peptide in accordance with the present invention.
  • different targeting molecules such as peptides, proteins, antibodies, antibody fragments, lectins, carbohydrates, or steroids can be conjugated to a carrier erythrocyte or, in particular, a carrier thrombo-erythrocyte.
  • FIG. 1 Ektacyto eter analysis of thrombo- erythrocytes and control erythrocytes.
  • Thrombo- erythrocytes were prepared as described, with samples removed after 15 min, 30 min, 60 min, and 120 min of incubation. The thrombo-erythrocytes were then washed in 0.15 M NaCl, 10 mM Tris/HCl, 5 mM KCl, 10 mM glucose, 1% bovine serum albumin, pH 7.4, and resuspended to a he atocrit of ⁇ 33%.
  • erythrocyte controls were prepared: 1) erythrocytes that were just washed in the above buffer, 2) erythrocytes incubated with peptide, but no mal-sac-HNSA, and 3) erythrocytes incubated with mal-sac-HNSA, but no peptide.
  • the deformability index of each sample was measured as a function of shear rate in an isotonic medium of 22 cp viscosity. All of the thrombo-erythrocyte samples and control samples gave virtually superimposable curves, and so for simplicity only the washed erythrocyte control and the 120 min thrombo-erythrocyte sample are shown.
  • the gel was subsequently dried and placed a cassette with X-ray film at -70°C for 7 days.
  • the P.A.S. stain revealed 3 major bands of Mr 87,000, 42,000, and 22,000, which corresponded to the radioactive bands identified by fluorography.
  • FIG. 4 Platelet-thrombo-erythrocyte co-aggregation assay.
  • Thrombo-erythrocytes and control erythrocytes were prepared as described in Section 8.1 and adjusted to a 10% hematocrit.
  • Citrated platelet-rich plasma was prepared (-500,000 platelets/ ⁇ l) and incubated with antibody 7E3 (anti- GPIIb/IIIa + anti- ⁇ vjS 3 vitronectin receptor; 40 ⁇ g/ml final concentration) , EDTA (10 mM final concentration) , RGDF (300 ⁇ g/ml final concentration) or buffer (0.15 M NaCl, 0.01 M Tris/HCl, 0.05% Na azide pH 7.4) for 30 min at 22°C.
  • the assay was performed by adding 50 ⁇ l of PRP to microtiter wells, followed by 10 ⁇ l of ADP to selected wells, and finally 5 ⁇ l of the thrombo-erythrocytes.
  • the microtiter plate was then rotated at 270 rpm at 22°C for approximately 6 min and then the plate was photographed. Note the absence of platelet aggregation or platelet-erythrocyte co-aggregation in the samples without ADP.
  • ADP treatment With ADP treatment, the thrombo-erythrocytes enter into mixed aggregates with the platelets. Careful inspection of the sample of control erythrocytes with ADP stimulation shows small white aggregates of platelets, indicating that platelet activation and aggregation occurred, but the control erythrocytes did not enter into the aggregates.
  • FIG. 1 Platelet-thrombo-erythrocyte interactions. After performing the platelet-thrombo- erythrocyte co-aggregation assay, samples were spread on a glass slide, air-dried, and stained with a Wright stain. Light microscopy was performed at 1,000X magnification with an oil immersion lens. Note the intimate association between the platelets and the thrombo-erythrocytes, with the platelets interdigitated between the thrombo-erythrocytes.
  • Figure 6 Interactions of control erythrocytes and thrombo-erythrocytes with gel-filtered platelets.
  • Gel-filtered platelets 450 ⁇ l; 340,000/ ⁇ l
  • control erythrocytes or thrombo-erythrocytes (20 ⁇ l; 10% hematocrit) were stirred in an aggregometer cuvette and then ADP (4.3 ⁇ M final concentration) was added.
  • ADP 4.3 ⁇ M final concentration
  • thrombo-erythrocytes do interact with the ADP-activated platelets, resulting in a dramatic decrease in optical density.
  • the thrombo-erythrocytes do not, however, interact with unactivated platelets despite stirring at 37°C.
  • preincubating the platelets with antibody 10E5 which reacts with GPIIb/IIIa, blocks the platelet-platelet and platelet-thrombo-erythrocyte interactions.
  • a mixture of gel-filtration buffer (450 ⁇ l) and control erythrocytes (20 ⁇ l) was used to establish the full scale deflection.
  • Figure 7 Interactions of control erythrocytes and thrombo-erythrocytes with platelets adherent to collagen. Gel-filtered platelets were allowed to form a dense lawn on collagen-coated microtiter wells and then, after washing, control erythrocytes or thrombo- erythrocytes (50 ⁇ l; 10% hematocrit) were added to the wells for 1 hour at 22°C. Finally, non-adherent control erythrocytes and thrombo-erythrocytes were removed by washing. With control erythrocytes, the dense lawn of platelets can be seen with only a single adherent erythrocyte in the field.
  • thrombo-erythrocytes bound extensively to the adherent platelets.
  • the binding of thrombo- erythrocytes to the adherent platelets was inhibited by antibody 10E5 (20 ⁇ g/ml) , which is specific for GPIIb/IIIa, or the peptide RGDF (400 ⁇ g/ml) .
  • the experiment shown is representative of more than 12 separate experiments.
  • FIG. 8 A. Rating of (G) n - RGDF bead agglutination. Platelet-rich plasma (PRP; 70 ⁇ l) was reacted with G 9 -RGDF beads (5 ⁇ l containing 0.22 mg beads) as described in Section 9.1 and rotated at 260 rpm. With increasing time, the agglutination became more extensive. The examples shown were selected at different time intervals to demonstrate the semiquantitative scale used for judging the extent of agglutination. Also shown are the platelet counts in the supernatant fluid after allowing the bead agglutinates to settle for 3 minutes. B. (G) n -RGDF bead agglutination in PRP.
  • the experiment was conducted as above using G ⁇ RGDF, G 3 -RGDF, and G 9 -RGDF beads.
  • the reaction was stopped at the time points indicated on the left and the microtiter plate was photographed.
  • the grading of the extent of agglutination is indicated on the right of each well. Note the minimal agglutination with the Gj-RGDF beads, the modest agglutination with the G 3 -RGDF beads, and the extensive agglutination of the G 9 -RGDF beads over the first 8 min.
  • FIG. 9 A. Agglutination of (G) n -RGDF beads by PRP.
  • the values plotted are the mean ⁇ SEM.
  • FIG. 11 A. Effect of decreasing the bead surface density of (G) 9 -RGDF peptides on agglutination by PRP.
  • the G 9 -RGDF peptide was coupled to beads at the different millimolar concentrations indicated on the graph. The efficiency of coupling was similar for all of the peptides (see Table IV for coupling efficiencies and Table V for maximal mean distances between peptides) .
  • the agglutination of the beads by PRP was then tested as indicated in the text. The values plotted are the results of a single experiment.
  • FIG. 12 Agglutination of G 5 -RGDF and G ⁇ -RGDF beads by gel-filtered platelets (GFP) in the presence and absence of ADP.
  • the values plotted are mean ⁇ SEM.
  • Figure 13 A.
  • the present invention relates to thrombo- erythrocytes, which are erythrocytes conjugated as provided herein to a RGD-containing peptide or polypeptide ("the RGD peptide") , and which are able to bind selectively to activated, but not to unactivated, platelets, causing co-aggregation of the activated platelets and thrombo-erythrocytes.
  • This highly selective binding to activated platelets is in contrast to the behavior of RGD peptides in solution or long RGD peptides on the surface of beads, which bind to both activated and non-activated platelets with much less selectivity.
  • the specificity of the thrombo-erythrocytes for activated platelets can exhibited in vitro in the absence of an exogenous activating agent (see Section 8, infra ) .
  • the thrombo- erythrocytes of the invention overcome the problems associated with prior art platelet substitutes by providing an abundant, safe material to promote platelet aggregation in vivo, specific for sites of injury. Thus, bleeding can be controlled, and hemorrhage can be prevented.
  • the possibility of infectious agent transmittal and adverse allo-immune reactions present in prior art methods are thus avoided.
  • the thrombo-erythrocytes bind to activated platelets via a specifically spaced R-G-D sequence in a peptide conjugated to the erythrocytes.
  • the distance from the erythrocyte to the N-terminal end of Arg within the RGD peptide influences the binding profile, and is preferably about 9 to about 50 Angstroms, more preferably about 10 to about 40 Angstroms, and most preferably about 11 to about 25 Angstroms.
  • the distance is estimated by considering the crosslinker and peptide sequence at the N-terminus of Arg as a linear molecule using standard bond lengths, assuming an extended conformation for the amino acids in the polypeptide.
  • the distance represents the length of the segment from the covalent bond between the erythrocyte and linker molecule, including the bond length, to the N-terminal end of Arg in the RGD peptide.
  • the thrombo-erythrocytes also exhibit rheological properties which do not significantly differ from those of untreated red blood cells. Furthermore, in a preferred aspect of the invention, the thrombo-erythrocytes surprisingly have the majority of the RGD peptides cross-linked to glycophorin A and glycophorin B on the erythrocyte cell surface. In this aspect, the specific binding to glycophorin provides important advantages because it is present in very high copy number on the erythrocyte surface (600,000 to 1 million per red blood cell), which allows highly effective binding of the RGD peptide-linker.
  • targeted thrombo-erythrocytes and targeted erythrocytes are provided, by conjugation of a targeting molecule such as an antibody or physiological ligand to the thrombo- erythrocytes or erythrocytes.
  • the targeted erythrocytes are carrier erythrocytes, which have been treated to release their contents (form erythrocyte "ghosts") and then incorporate an agent before resealing of their membrane, so that they can be used as in vivo delivery vehicles for their internalized agent.
  • form erythrocyte form erythrocyte "ghosts”
  • targeting molecule refers to a molecule that can be conjugated to an erythrocyte (or thrombo-erythrocyte) and that binds specifically to a molecule found in vivo, such as a receptor or other recognition molecule or a molecule specific to a cell or cells, etc.
  • the targeting molecule is a peptide, e.g., a peptide containing the sequence Arg- Gly-Asp (R-G-D) .
  • the cell is a targeted thrombo-erythrocyte.
  • a targeted thrombo-erythrocyte will react with activated platelets in a thrombus, allowing imaging of the thrombus or the delivery of therapeutic agents to the thrombus.
  • a targeted erythrocyte need not be a thrombo-erythrocyte.
  • the targeting molecule is an antibody, or fragment of an antibody, a lectin, a steroid or a carbohydrate. More than one targeting molecule can be used, for example, by using two different molecules to target " an erythrocyte to the same in vivo location.
  • thrombo-erythrocytes In order to produce thrombo-erythrocytes, a polypeptide according to the present invention is prepared and covalently conjugated to an erythrocyte through a polyfunctional molecule according to methods described infra. However, it should be noted that upon completion of the conjugation reaction, the thrombo-erythrocytes should be tested for their unusual ability to retain both normal rheological properties and the platelet's specificity for forming thrombi at sites of vascular injury, i.e., for their ability to interact selectively with activated platelets.
  • the lack of significant difference in the rheological properties displayed by the thrombo- erythrocytes of the invention and those of untreated erythrocytes can be observed by detecting a lack of significant difference between the thrombo- erythrocytes and untreated red blood cells in one or more of the following characteristics: surface/volume ratio, internal cell water, and/or membrane shear rigidity, as tested by laser diffraction ektacytometry as described in Example 8, infra. or by other methods known in the art. Examples of .in vitro assays that can be used to demonstrate the ability of the thrombo- erythrocytes to bind selectively to activated platelets are described in Sections 8.1.5. and 8.1.8. infra) .
  • the thrombo- erythrocytes of the invention are mainly conjugated via glycophorin A and glycophorin B on the cell surface, and have the ability, via their conjugated RGD peptides to interact with the GPIIb/IIIa receptor on activated platelets.
  • Erythrocytes may be purified and concentrated by methods that are known in the art. Typically, by way of example but not limitation, blood is removed from a patient and added to an anti-coagulant such as citrate. The blood is then centrifuged, and the plasma supernatant is removed with a pipet, leaving the erythrocytes.
  • an anti-coagulant such as citrate
  • Buffer of about pH 6 to about pH 8 and about 0.15 N ionic strength preferably phosphate buffered saline (PBS)
  • PBS phosphate buffered saline
  • the polypeptide and the erythrocytes are then each covalently bonded to a polyfunctional molecule. All operations are preferably performed in aqueous solution in order to avoid lysing the erythrocytes, which are sensitive to organic solvents.
  • the pH should be between 6 and 8, preferably between 6.5 and 7.5. It is preferable to use a heterobifunctional cross-linking reagent that reacts directly with each type of group.
  • a heterobifunctional reagent that works well is Mal-Sac-HNSA (N-maleimido-6-aminocaproyl ester of l-hydroxy-2-nitrobenzene-4-sulfonic acid sodium salt) , which may be obtained from Bachem Biosciences, Inc., Philadelphia, PA.
  • cross ⁇ linking agents known in the art can be used, and are described in Section 5.1.2 infra, as long as the resultant cell is tested for the retention of rheological properties and specificity of binding to activated platelets associated with the thrombo- erythrocytes of the invention.
  • the dynamics of the one-step reaction described in Section 6 showed that the thrombo-erythrocytes of the present invention can be prepared and that they bind to activated platelets. However, the dynamics of the one-step reaction indicate that this one-step method is unpredictable.
  • a sulfhydryl (thiol) group of the erythrocyte could react with the Mal-Sac- HNSA linker, rather than the desired reaction between the sulfhydryl (thiol) groups of the peptide and the Mal-Sac-HNSA linker.
  • Cross-linking of erythrocyte cell-surface proteins would result. This potentially competing and undesirable reaction may damage the erythrocytes, and would make less linker available for binding to the peptide.
  • the RGD polypeptide-linkers are prepared first separately, and then subsequently reacted with proteins on the erthrocyte.
  • this can be carried out as follows: the erythrocytes are maintained in a buffer solution at about pH 7.4. This prevents any osmotic damage to the erythrocytes.
  • the polypeptide-linker is prepared separately at a pH of about 6.0. After conjugating the peptide sulfhydryl of the linker, the pH of the reaction solution is raised to a pH of about 7.4.
  • the peptide-linker complex in solution at pH 7.4 can be added to an erythrocyte suspension, thus allowing free amino groups on the erythrocyte proteins to react with the second reactive group on the linker.
  • the peptide linker complex can be lyophilized and stored for later use.
  • a peptide-linker complex is chemically synthesized by attaching the cross-linking group as a subsequent step after peptide synthesis, by standard chemical methods.
  • This complex is suitable in the present invention as long as the linker which is attached to the peptide has an attachment point that is available for linking to reactive functional groups of the erythrocyte.
  • the RGD peptide-linker intermediates can be stored for later use in conjugation to erythrocytes.
  • the peptide must be added in great molar excess to the erythrocytes.
  • the molar excess of RGD peptides added to the erythrocytes should be approximately 0.5 x 10 8 to approximately 20 x 10 8 , preferably approximately 1 x 10 8 to approximately 10 x 10 8 , and more preferably approximately 3 x 10 8 to approximately 7 x 10 8 .
  • the number of polypeptides attached to each erythrocyte should be approximately 0.05 x 10 6 , preferably approximately 1 x 10 6 , to approximately 20 x 10 6 , although it is possible that as few as 0.01 x 10 6 attached polypeptides will yield a functional thrombo-erythrocyte.
  • excess cross-linker is removed by thorough washing.
  • albumin or autologous serum can be added during the washing procedure to react with any remaining reactive sites, and then be removed in the wash step.
  • the erythrocytes have both amino and sulfhydryl groups exposed on their surfaces. Either of these groups may be used to form the covalent bond to one of the functional groups of the polyfunctional molecule. Alternatively a carboxylic acid group can be used to form a covalent bond to one functional group of the polyfunctional molecule, e.g., via carbodiimide activation. Another functional group of the polyfunctional molecule is covalently bonded to the RGD peptide. Preferably, an amino group will usually form the bond to the polyfunctional molecule. In a specific aspect, when the site of attachment on the RGD peptide is cysteine, either the amino group or the sulfhydryl group may be bonded to the polyfunctional molecule. Where bonding is to the sulfhydryl group, the amino group should be protected, e.g., by acetylation.
  • the RGD peptide for conjugation to erythrocytes in accordance with the present invention includes a sequence of amino acids, preferably naturally occurring L-amino acids and glycine, having the following formula (I) : R,-Arg-Gly-Asp-R 2 I in which R y represents an amino acid or a sequence of more than one amino acid; . in a specific embodiment, R- represents
  • R 2 represents OH or NH 2 ; or any amino acid; or a sequence of more than one amino acid.
  • R 2 represents an amino acid other than serine, threonine or cysteine or the amide thereof; in another specfic embodiment, R 2 is more than one amino acid, the first amino acid in the sequence, which is attached to asp, being other than serine, threonine or cysteine, or the amide of any free carboxyl groups.
  • R j and R 2 may be any amino acid or sequence thereof.
  • the amino acids are preferably naturally occurring. The most common naturally- occurring amino acids are shown in Table I:
  • R- and R 2 in Formula I are not limited to the 20 natural amino acids.
  • Rj and R 2 can be non-classical amino acids or cyclic peptides or peptidomimetics (chemical " peptide analogs) .
  • Non-classical amino acids include but are not limited to the D-isomers of the common amino acids, ⁇ -amino isobutyric acid, 4-aminobutyric acid, hydroxyproline, sarcosine, citrulline, cysteic acid, t-butylglycine, t-butylalanine, phenylglycine, cyclohexylalanine, /3-alanine, designer amino acids such as ⁇ -methyl amino acids, C ⁇ -methyl amino acids, N ⁇ -methyl amino acids, and amino acid analogs in general.
  • Arg, and/or Asp in the RGD sequence can be the D (dextrarotary) or L (levorotary) amino acid.
  • X can be any amino acid, and specifically need not be valine.
  • X represents a naturally occurring amino acid, and most preferably cysteine or glycine.
  • Y can represent any amino acid, and specifically need not be threonine.
  • Y represents a naturally occurring amino acid, and most preferably glycine.
  • Z can represent any amino acid, preferably a naturally occurring amino acid.
  • R 2 can represent OH or NH 2 .
  • R 2 may represent an amino acid, preferably a naturally occurring L-amino acid or glycine; in a specific embodiment, R 2 does not represent serine, threonine or cysteine or the amide thereof.
  • R 2 represents phenylalanine or the amide of phenylalanine.
  • R 2 can represent a sequence of more than one amino acid, in particular, the first amino acid in the sequence, which is attached to the carboxyl functional group of Asp, being other than serine, threonine or cysteine, or the amide of any free carboxyl groups in the sequence.
  • R 2 is a sequence of amino acids
  • the polypeptide for conjugation to erythrocytes can be any size, and encompasses what might otherwise be called an oligopeptide or a protein.
  • the polypeptide will have no more than about 1,000 amino acids.
  • R j and R 2 may represent a sequence of the amino acids discussed above.
  • R 2 is not serine, threonine or cysteine.
  • X represents cysteine or glycine
  • Y represents glycine
  • Z represents glycine
  • X represents cysteine or glycine
  • Y represents glycine
  • Z represents glycine
  • R 2 represents phenylalanine or the amide of phenylalanine.
  • X represents cysteine
  • Y represents glycine
  • Z represents glycine
  • R 2 represents the amide of phenylalanine.
  • R t is XY(Z) n
  • X, Y, and Z may represent any tripeptide sequence.
  • the tripeptide need not be Val- Tyr-Gly.
  • the polypeptide may be prepared by methods that are known in the art.
  • solid phase peptide synthesis consists of coupling the carboxyl group of the C-terminal amino acid to a resin and successively adding N-alpha protected amino acids.
  • the protecting groups may be any known in the art or those described in Section 5.1.2 infra. Before each new amino acid is added to the growing chain, the protecting group of the previous amino acid added to the chain is removed.
  • the coupling of amino acids to appropriate resins is described by Rivier et al., U.S. Patent No. 4,244,946.
  • Such solid phase syntheses have been described, for example, by Merrifield, 1964, J. Am. Chem. Soc. 85:2149; Vale et al. , 1981, Science 213:1394-1397; Marki et al., 1981, J. Am. Chem. Soc. 103:3178 and in U.S. Patent Nos. 4,305,872 and 4,316,891.
  • polyfunctional molecule encompasses molecules having one functional group that can react more than one time in succession, such as formaldehyde (although formaldehyde is not indicated for use due to its potential carcinogenicity) , as well as molecules with more than one reactive group.
  • reactive group refers to a functional group on the crosslinker that reacts with a functional group on a peptide, protein, or carbohydrate so as to form a covalent bond between the cross-linker and peptide or protein.
  • the term "functional group” retains its standard meaning in organic chemistry.
  • the polyfunctional molecules which can be used are biocompatible linkers, i.e., they are noncarcinogenic, nontoxic, and substantially non-immunogenic in vivo. Polyfunctional cross-linkers such as those known in the art and described herein can be readily tested in animal models to determine their biocompatibility.
  • the polyfunctional molecule is preferably bifunctional.
  • the term "bifunctional molecule” refers to a molecule with two reactive groups.
  • the bifunctional molecule may be heterobifunctional or homobifunctional.
  • the bifunctional molecule is heterobifunctional, allowing for vectorial conjugation of the RGD peptide and erythrocyte.
  • the polyfunctional molecule prefferably be sufficiently soluble in water for reactions with the polypeptide and with the substrate to occur in aqueous solutions such as in aqueous solutions buffered at pH 6 to 8.
  • the polyfunctional molecule covalently bonds with an amino or a sulfhydryl group on X of the polypeptide and on the surface of the erythrocytes.
  • polyfunctional molecules reactive with other functional groups, such as carboxylic acids or hydroxyl groups are contemplated in the present invention.
  • the homobifunctional molecules have at least two reactive functional groups, which are the same.
  • the reactive functional groups on a homobifunctional molecule include, for example, aldehyde groups and active ester groups.
  • Homobifunctional molecules having aldehyde groups include, for example, glutaraldehyde and subaraldehyde. The use of glutaraldehyde as a cross-linking agent was disclosed by Poznansky et al., Science 223 f 1304-1306 (1984).
  • Homobifunctional molecules having at least two active ester units include esters of dicarboxylic acids and N-hydroxysuccinimide.
  • N-succinimidyl esters include disuccinimidyl suberate and dithio-bis-(succinimidyl propionate) , and their soluble bis-sulfonic acid and bis-sulfonate salts such as their sodium and potassium salts. These homobifunctional reagents are available from Pierce, Rockford, Illinois.
  • the heterobifunctional molecules have at least two different reactive groups.
  • the reactive groups react with different functional groups on the peptide and on a protein on the surface of the erythrocyte.
  • These two different functional groups of the peptide and of the erythrocyte protein that react with the reactive group are usually an amino group, e.g., the epsilon amino group of lysine, and a sulfhydryl group, i.e., the thiol group of cysteine.
  • the carboxylic acid and hydroxyl functional groups on the peptide and the erythrocyte protein can also react with the crosslinker.
  • the covalent bond will usually be an amido or imido bond.
  • the reactive group that forms a covalent bond with amino groups may, for example, be an activated carboxylate group, a halocarbonyl group, or an ester group.
  • the preferred halocarbonyl group is a chlorocarbonyl group.
  • the ester groups are preferably reactive ester groups such as, for example, an N- hydroxy-succinimide ester group or that of Mal-Sac- HNSA.
  • the other functional group typically is either a thiol group, a group capable of being converted into a thiol group, or a group that forms a covalent bond with a thiol group.
  • the covalent bond will usually be a thioether bond or a disulfide.
  • the reactive group that forms a covalent bond with a thiol group may, for example, be a double bond that reacts with thiol groups or an activated disulfide.
  • a reactive group containing a double bond capable of reacting with a thiol group is the maleimido group, although others, such as acrylonitrile, are also possible.
  • a reactive disulfide group may, for example, be a 2-pyridyldithio group or a 5,5'-dithio-bis-(2-nitrobenzoic acid) group.
  • heterobifunctional reagents containing reactive disulfide bonds include N- succinimidyl 3-(2-pyridyl-dithio)propionate (Carlsson, et al., 1978, Biochem J.. 173:723-737), sodium S-4- succinimidyloxycarbonyl-alpha-methylbenzylthiosulfate, and 4-succinimidyloxycarbonyl-alpha-methyl-(2- pyridyldithio)toluene. N-succinimidyl 3-(2- pyridyldithio)propionate is preferred.
  • heterobifunctional reagents comprising reactive groups having a double bond that reacts with a thiol group include succinimidyl 4-(N- maleimidomethyl)cyclohexahe-1-carboxylate and succinimidyl m-maleimidobenzoate.
  • heterobifunctional molecules include succinimidyl 3-(maleimido)propionate, sulfosuccinimidyl 4-(p-maleimido-phenyl)butyrate, sulfosuccinimidyl 4-(N-maleimidomethyl- cyclohexane)-l-carboxylate, maleimidobenzoyl-N- hydroxy-succinimide ester.
  • the sodium sulfonate salt of succinimidyl m-maleimidobenzoate is preferred.
  • Many of the above-mentioned heterobifunctional reagents and their sulfonate salts are available from Pierce.
  • the 2-pyridyldithio group may be cleaved, for example, with dithiothreitol, forming the corresponding thiopropionyl (TP) amide (II) .
  • SPDP is then treated with an erythrocyte having at least one amino group, forming the corresponding PDP amide (III) .
  • III which has a group which forms a covalent bond with a sulfhydryl group, namely a pyridyldithio group, yields a compound wherein the peptide is covalently bonded to the substrate (erythrocyte-protein) through two polyfunctional molecules (IV) .
  • a polypeptide in accordance with the invention is treated with succinimidyl 4-(N-maleimido- methyl)cyclohexane-l-carboxylate (SMCC), forming the corresponding N-maleimidomethylcyclohexane-1- carboxylate amide (V) .
  • SMCC succinimidyl 4-(N-maleimido- methyl)cyclohexane-l-carboxylate
  • V N-maleimidomethylcyclohexane-1- carboxylate amide
  • Mal-Sac-HNSA is used, instead of SMCC.
  • a sulfhydryl group from the polypeptide of the invention to the polyfunctional reagent. This would occur when X represents cysteine.
  • the free amino group of the cysteine residue is protected if the polyfunctional molecule is a heterobifunctional molecule that reacts with amino groups as well as with sulfhydryl groups.
  • the protecting group can be any of the large number of protecting groups known in the art. For example, an acetyl group can be added to the free amino group by treating the polypeptide with acetic anhydride. Alternatively, a carbobenzoxy group can be added by treating the polypeptide with carbobenzoxy chloride.
  • N-protecting groups that are useful include the formyl, L-butoxycarbonyl-, trifluoroacetyl-, tosyl-, p-nitrocarbobenzoxy-, cyclopentyloxycarbonyl-, and phenoxycarbonyl- groups.
  • FITBBO-ERYTHROCYTES TO CONTROL BLEEDING The thrombo-erythrocytes of the invention may be used to control bleeding in vivo.
  • the thrombo-erythrocytes may be used to control bleeding from small wounds in thrombocytopenic mammals, including humans.
  • the thrombo- erythrocytes are administered autologously to control bleeding.
  • the administration is allogeneic.
  • approximately 0.286- 3.57 ml of blood per kg of the mammal is removed.
  • the erythrocytes are then washed and concentrated.
  • the washed erythrocytes are then covalently bonded to a RGD peptide through a polyfunctional molecule as described in Section 5.1, supra.
  • the resulting thrombo-erythrocyte is then introduced into the mammal by means of standard transfusion techniques.
  • the thrombo-erythrocytes of the invention are used in the treatment of thrombocytopenia, ie., to augment a deficiency in platelet levels in a patient.
  • the thrombo-erythrocytes of the invention are introduced into a mammal, including a human, to help control bleeding, e.g., after trauma or during surgery.
  • the thrombo-erythrocytes for administration to a mammal are preferably formulated in a pharmaceutical composition, as described in Section 5.4, infra.
  • the thrombo-erythrocytes can be administered to a mammal by intravenous or intra-arterial bolus injection or by intravenous drip.
  • the number of thrombo-erythrocytes to be administered i.e., the dose, depends upon the degree of thrombocytopenia in the mammal, and can be determined on a case-by-case basis by one skilled in the art.
  • the number of thrombo- erythrocytes augments the number of platelets in proportion to the amount absent from the thrombocytopenic individual relative to a normal individual.
  • erythrocytes or in particular thrombo-erythrocytes, prepared in accordance with the present invention can be modified for delivery, to various target tissues, of labels or biologically active agents that have been incorporated into the erythrocytes (i.e. taken up by erythrocyte ghosts) to form carrier erythrocytes.
  • the carrier erythrocyte is a carrier thrombo-erythrocyte (i.e., a thrombo-erythrocyte whose intracellular contents have been replaced by a composition comprising a label or agent, and then whose membrane is resealed) .
  • the carrier erythrocytes have advantages over liposomes by virtue of their larger size, which avoids the problem of non-specific endocytosis of liposomes by scavenging cells such as macrophages, and because of the presence of an extensive cytoskeleton, that, for example, protects the erythrocytes from complete osmotic lysis under hypotonic conditions.
  • the cell surface integral membrane proteins of erythrocytes provide a convenient scaffold for cross ⁇ linking targeting molecules.
  • the carrier erythrocytes are more likely also to be biocompatible.
  • imaging agents can be incorporated in the carrier erythrocyte.
  • Imaging agents include but are not limited to heavy metal contrast agents for x-ray imaging, magnetic resonance imaging agents, and radioactive nuclides (i.e., isotopes) for radio-imaging.
  • the carrier erythrocyte can be loaded with one or more therapeutic agents.
  • the therapeutic agent can be a chemotherapeutic, an enzyme, a neurotoxin, a growth factor, a neurotrophic factor, a hormone, a thrombolytic agent, or any drug.
  • specific targeting of a drug to the site where it is needed results in more effective therapy because a larger therapeutic dose can be delivered than could be tolerated systemically.
  • larger doses of a chemotherapeutic can be delivered locally to a tumor than can be tolerated systemically by an organism, e.g., a human.
  • a thrombolytic agent can be administered to the site of thrombosis in a concentration that would lead to uncontrollable bleeding if administered systemically.
  • the carrier erythrocyte can be loaded with nucleic acid sequences.
  • the nucleic acids can be anti-sense RNA or DNA for delivery to a target cell.
  • the nucleic acids can be genetic information, such as a gene for gene therapy or an entire genome for fertilization.
  • the carrier erythrocyte can be loaded with sperm, or fused with sperm to obtain the sperm haploid genome.
  • the carrier erythrocyte can contain plasmids, or modified virus or viral nucleic acids targeted for delivery.
  • the instant invention provides for conjugating targeting molecules to the erythrocytes or erythrocyte ghosts.
  • Targeting molecule shall mean a molecule which, when administered in vivo, localizes to desired location(s) .
  • the crosslinkers for the conjugation of peptides to erythrocytes described in Section 5.1.2, supra can be used to conjugate the targeting molecule to the erythrocyte; furthermore, carrier erythrocytes and carrier thrombo- erythrocytes need not retain the rheological properties of control red blood cells, in contrast to non-carrier thrombo-erythrocytes.
  • the targeting molecule can be conjugated to the erythrocyte either prior to or subsequent to the introduction of a material into the carrier erythrocyte.
  • the targeting molecule can be a peptide or protein, antibody, lectin, carbohydrate, or steroid.
  • the targeting molecule is a peptide ligand of a receptor on the target cell.
  • the targeting molecule is a peptide sequence described in Section 5.1.1, supra. or variants thereof that bind RGD receptors on the surface of cells such as endothelial cells, cancer cells, or ova, e.g., human ova that have receptors that recognize the RGD sequence.
  • the targeting molecule is the peptide R ⁇ RGD-R 2 attached as described supra, and the erythrocyte targeting agent is loaded with a thrombolytic agent.
  • a thrombo-erythrocyte is useful for the treatment of thrombosis, particularly since it is targeted to activated platelets.
  • the targeting molecule is an antibody.
  • the targeting molecule is a monoclonal antibody.
  • the antibody can be reduced to two heavy and light chain heterodimers, or the F(ab') 2 fragment can be reduced, and crosslinked to the erythrocyte via the reduced sulfhydryl.
  • the carbohydrate portion of the antibody can be directly, or via a derivative, utilized for attachment to the erythrocyte or thrombo- erythrocyte.
  • Antibodies for use as targeting molecule are specific for cell surface antigen.
  • the antigen is a receptor.
  • an antibody specific for a receptor on cancer cells such as melanoma cells, can be used.
  • antibodies specific for leukocyte surface antigens such as lymphocyte antigens, CD (clusters of differentiation) antigens, and receptors (e.g., T cell antigen receptors) can be conjugated to the erythrocyte ghosts. Any antibody known in the art that is specific for a cell antigen can be used as a targeting molecule.
  • the antibody can be prepared.
  • Various procedures known in the art can be used for the production of antibodies specific for a target antigen that can be used to modify erythrocytes to prepare targeted erythrocytes.
  • Such antibodies include but are not limited to polyclonal, monoclonal, chimeric, single chain. Fab fragments and an Fab expression library, although monoclonal antibodies or a fragment thereof are preferred.
  • various host animals including but not limited to rabbits, mice, rats etc., may be immunized by injection with a target antigen marker.
  • target antigen is conjugated to an immunogenic carrier.
  • a target antigen epitope e.g..
  • a hapten is conjugated to a carrier, such as keyhole limpet hemocyanin.
  • a carrier such as keyhole limpet hemocyanin.
  • an "epitope" is a fragment of an antigen capable of specific immunoactivity, e.g. , antibody binding.
  • Various adjuvants may be used to increase the immunological response, depending on the host species, including but not limited to Freund's (complete and incomplete) , mineral gels such as aluminum hydroxide, surface active substances such as lysolecithin, pluronic polyols, polyanions, peptides, oil emulsions, dinitrophenol, and potentially useful human adjuvants such as BCG (bacille CaImette-Guerin ) and Corynebacterium parvu .
  • Monoclonal antibodies to a target antigen can be prepared by using any technique that provides for the production of antibody molecules by continuous cell lines in culture. These include but are not limited to the hybridoma technique originally described by Kohler and Milstein, (1975, Nature 256: 495-497), the more recent human B-cell hybridoma technique (Kozbor et al., 1983, Immunology Today 4:72) and the EBV- hybridoma technique (Cole et al., 1985, Monoclonal Antibodies and Cancer Therapy. Alan R. Liss, Inc., pp. 77-96) .
  • monoclonal antibodies specific for a target antigen can be produced in germ-free animals utilizing recent technology (PCT/US90/02545) .
  • human antibodies can be used and can be obtained by using human hybridomas (Cote et al., 1983, Proc. Natl. Acad. Sci.. U.S.A. 80:2026-2030) or by transforming human B cells with EBV virus in vitro (Cole et al., 1985, in Monoclonal Antibodies and Cancer Therapy. Alan R. Liss, pp. 77-96) .
  • techniques developed for the production of "chimeric antibodies” (Morrison et al., 1984, Proc. Natl.
  • Antibody fragments that contain sites specific for target antigen can be generated by known techniques.
  • such fragments include but are not limited to: the F(ab') 2 fragments, which can be produced by pepsin digestion of the antibody molecule and the Fab' fragments, which can be generated by reducing the disulfide brides of the F(ab') 2 fragments.
  • reduced fragments are used, since these can be conjugated to erythrocyte protein via their sulfhydryl groups.
  • This invention further provides for the use of other targeting molecules, such as lectins. carbohydrates, proteins and steroids, conjugated to erythrocytes.
  • any procedure known in the art can be used to make the erythrocytes leak their contents (i.e., prepare erythrocyte ghosts) and then take up new molecules before being resealed.
  • Such methods are described in the following references: (editorial) , 1988, Lancet pp.1437-1438; Brearley et al., 1990, J. Pharm. Pharmacol. 42:297-301; Tonetti et al., 1990, Biotech. Appl. Biochem. 12:261-269; Updike and Rokania, 1983, J. Lab. Clin. Med. pp. 679-691; Ramsey et al., 1986, Clin. Res. 34:468A.
  • Resealed erythrocytes are prepared by a gel- filtration method similar to that ' described by Kaplan in "Sodium pump-mediated ATP:ADP exchange: The sided effect of sodium and potassium ions", Journal of General Physiology 80:915-937 (1982); and Sachs in Volume-sensitive K influx in human red cell ghosts". Journal of General Physiology. 92:685-711 (1988).
  • the modified cells are separated from plasma and washed with a 150 mM choline-chloride solution that contains 0. l mM EDTA (ethylenediamine tetraacetic acid) and 10 mM PIPES (piperazine-N,N'-bis (2-ethanesulfonic acid) adjusted to pH 5.5 with Tris (Tris
  • the cells are washed repeatedly until the pH of the cell suspension is 6.0. The cells are then brought to 50% hematocrit in the wash solution and stored on ice until run into a column.
  • the column is 45 x 10 cm and is filled with Bio Gel A50 beads (Bio Rad Inc. , Rockville Center, NY); the bed volume is 3.5 liters.
  • the column is enclosed in a water jacket and maintained at about 1°C.
  • the gel is equilibrated with a solution that contains 10 mM PIPES, 11.2 mM choline chloride, and 0.1 mM EDTA; the solution is adjusted to pH 6.0 with Tris (buffer A) .
  • To prepare thrombo-erythrocyte ghosts 200 ml of solution identical to buffer A except that the choline-chloride concentration is 150 mM (buffer B) is run into the column followed by 75- 100 ml of cell suspension.
  • the cells hemolyse (leak their content of hemoglobin and other materials) on the column and intracellular contents are retained by the beads.
  • ghosts are eluted with buffer B and collected on ice. They are concentrated by centrifugation (40,000 g for 10 min) and aspiration of the supernatant, collected in one or two tubes, and resuspended in buffer A. The ghosts are again centrifuged, the supernatant removed, and the ghosts distributed to resealing solutions.
  • Tris HEPES (4-[2-hydroxymethyl]-l- piperazineethanesulfonic acid) solution (500 mM HEPES adjusted to pH 8.0 at 37° C with Tris), 0.5 mM Tris EGTA (ethyleneglycol bis-[(S- aminoethylether] N,N'-tetraacetic acid), 50 mg/100 ml albumin, and the molecules designed for incorporation into the thrombo-erythrocytes.
  • Tris HEPES 4-[2-hydroxymethyl]-l- piperazineethanesulfonic acid
  • Tris EGTA ethyleneglycol bis-[(S- aminoethylether] N,N'-tetraacetic acid
  • 50 mg/100 ml albumin 50 mg/100 ml albumin
  • Ghosts account for 10-40% of the volume of the suspension.
  • the ghost suspension is kept at 0° C for 5 min and then incubated at 37° C for 60 min.
  • the resealed thrombo-erythrocyte ghosts are separated from the suspension by washing 3 times in 0.15 M Nacl, 0.1 M NaP0 4 , 1 mg/ml human albumin, pH 7.4, and are then ready for use in vivo or in vitro.
  • the lipid composition of the red blood cell can be manipulated by known methods to destabilize the cell membrane or treated by other methods (e.g., heating or removal of surface sialic acid residues) to reduce in vivo half-life of the carrier erythrocyte.
  • the present invention provides for administering the targeted carrier erythrocytes to a subject via any route known in the art.
  • the erythrocyte targeting agents can be administered via any route used to administer liposomes.
  • erythrocyte targeting agents can be administered intraventricularly, intraperitoneally, intramuscularly, subcutaneously, intravenously, and intraarterially, to mention but a few routes.
  • the administration is intravenously or intraarterially.
  • the targeted carrier erythrocytes are administered in a pharmaceutical composition comprising the targeted erythrocytes and a pharmaceutically acceptable carrier or excipient (see Section 5.4.1, infra) .
  • the present invention contemplates administering thrombo-erythrocytes or targeted carrier erythrocytes to a mammal, preferably in admixture with a pharmaceutically acceptable carrier or excipient.
  • a pharmaceutically acceptable carrier or excipient preferably in admixture with a pharmaceutically acceptable carrier or excipient.
  • Such admixtures comprise a pharmaceutical composition of the invention.
  • a pharmaceutically acceptable carrier or excipient for use in the invention should comprise an aqueous solution having the following characteristics: pH of between about pH 6 and about pH 8; ionic strength of about 0.15 N to maintain the appropriate osmotic environment for the modified erythrocytes; and physiological compatibility.
  • the pharmaceutically acceptable carrier or excipient should not disrupt or solubilize the modified erythrocytes, e.g., contain oils, emulsifiers, detergents, or surfactants at concentrations lytic to the cell membrane.
  • the pharmaceutically acceptable carrier or excipient can comprise dextrose. glucose, starch, lactose and the like in aqueous solution or suspension.
  • Blood (10 ml) was drawn from a human by syringe and a 19 gauge needle and placed into a polypropylene tube containing 0.1 ml 40% trisodium citrate. The blood was centrifuged at approximately 2,000 X g for 10 min at 22°C and the supernatant plasma removed. The erythrocyte pellet was washed three times with buffer A (0.15 M NaCl, 0.05 M phosphate, 5 mM glucose, 2 mM KC1, pH 7.4) by repetitive centrifugation at approximately 2,000 X g for 10 min at 22°C.
  • buffer A (0.15 M NaCl, 0.05 M phosphate, 5 mM glucose, 2 mM KC1, pH 7.4
  • thrombo-erythrocytes were resuspended in buffer A to a hematocrit of 10%.
  • a control sample of erythrocytes was treated identically but no peptide or Mal-Sac-HNSA was added.
  • the assay contained 50 ⁇ l of citrated platelet- rich plasma (prepared by centrifuging whole blood anticoagulated with 0.01 volume of trisodium citrate at 700 X g for 3.5 min at 22°C and adjusting the count to 3.0 X 10 8 platelets per ml with plasma free of platelets) and 5 ⁇ l of the thrombo-erythrocytes with, or without, adding 5 ⁇ l of adenosine diphosphate (ADP) (100 ⁇ M stock solution) to activate the platelets.
  • ADP adenosine diphosphate
  • the thrombo- erythrocytes produced 0-1+ agglutination in the absence of ADP and 4+ agglutination in the presence of ADP. These values remained unchanged for the remaining 6 min of observation.
  • the control erythrocytes did not agglutinate.
  • the dynamics of the one-step reaction described in Example 6 illustrated that the thrombo-erythrocytes of the present invention can be prepared and that they bind to activated platelets. However, the dynamics of the one-step reaction provide reason for believing that this one-step method is at best unpredictable. First, the sulfhydryl (thiol) groups of the erythrocyte could react with the Mal-Sac-HNSA linker, rather than the desired reaction between the sulfhydryl (thiol) groups of the peptide and the Mal- Sac-HNSA linker. This potentially competing and undesirable reaction may damage the erythrocytes, and would make less linker available for binding to the peptide.
  • erythrocytes - Blood (10 ml) was drawn from a human by syringe and a 19 gauge needle and placed into a polypropylene tube containing 0.1 ml 40% trisodium citrate. The blood was centrifuged at approximately 2,000 X g for 10 min at 22°C and the supernatant plasma removed. The erythrocyte pellet was washed three times with buffer A (0.15 M NaCl, 0.05 M phosphate, 5 mM glucose, 2 mM KC1, pH 7.4) by repetitive centrifugation at approximately 700 X g for 5 min at 22°C. An aliquot of 0.5 ml of the washed erythrocytes in the same buffer at a density such that 60% of the volume was comprised of erythrocytes (60% hematocrit) was removed.
  • buffer A (0.15 M NaCl, 0.05 M phosphate, 5 mM glucose, 2 mM KC1, pH 7.4
  • the peptide-linker complex was adjusted with a o.l M solution of sodium hydroxide (NaOH) to a pH of 7.4.
  • NaOH sodium hydroxide
  • the 0.5 ml aliquot containing the linked peptide was added and mixed with the 0.5 ml aliquot of erythrocytes prepared as described above and the mixture was rocked for 120 minutes at 22°C and pH of 7.4.
  • thrombo-erythrocytes were washed three times in buffer A. The resulting thrombo-erythrocytes were resuspended " in buffer A to a hematocrit of 10%. A control sample of erythrocytes were treated identically but no peptide or Mal-Sac- HNSA was added.
  • the assay contained 100 ⁇ l of citrated platelet- rich plasma (prepared by centrifuging whole blood anticoagulated with 0.01 volume of trisodium citrate at 700 g for 3.5 min at 22°C and adjusting the count to 3.0 X 10 8 platelets per ml with plasma free of platelets) and 10 ⁇ l of the thrombo-erythrocytes with, or without, adding 10 ⁇ l of adenosine diphosphate (ADP) (100 ⁇ M stock solution) to activate the platelets.
  • ADP adenosine diphosphate
  • Agglutination of erythrocytes was graded from 1- 4+ based on microscopic examination after rotating the samples in a microtiter plate at 260 rpm at 22°C for various periods of time. At 2-3 min, the thrombo- erythrocytes produced 0-1+ agglutination in the absence of ADP and 4+ agglutination in the presence of ADP. These values remained unchanged for the remaining 6 min of observation. The control erythrocytes did not agglutinate.
  • the present example shows that erythrocytes coated with RGD-containing peptides interact with platelets, and critically, that the interactions are selective for activated platelets, a prerequisite for diminishing the risks of indiscriminate thrombus formation.
  • Double couplings were performed with the phenylalanine in three of the syntheses and arginine in all of the syntheses.
  • the amino-terminus was acetylated while the peptide was still on the resin by reaction with acetic anhydride.
  • Cleavage of the peptide from the resin was accomplished with anhydrous HF in the presence of dimethylsulfide, parathiocresol, and anisole, starting at -10°C. After HF cleavage, the peptide-resin mixture was washed with ether alone (first 2 syntheses) or ether and dichloromethane (last 3 syntheses) , and then extracted into acetic acid before lyophilization.
  • HPLC analysis (C-8 column, 220 X 4.6 mm, Applied Biosystems 300 RP) demonstrated a single dominant peak in each synthesis representing 45-57% of the total absorbance at 220 nm.
  • the peptide was purified by HPLC before use.
  • Fast atom bombardment mass spectrometry xenon gun parameters: 7 kV, 1 mA, 0.4 mA ion current; mass spectrometer parameters: acceleration potential 6 kV, mass range 132-1172, resolution 1,500, scan speed 10 sec/decade; lyophilized sample transferred to glycerin or thioglycerin matrix
  • the peptide concentration for the coupling experiments was determined by titrating the free sulfhydryl groups with 5,5'-dithio-bis-(2-nitrobenzoic acid) (Ellman's reagent; Pierce Chemicals, Rockford, IL) using cysteine as a standard.
  • a radiolabeled peptide was prepared by performing the peptide acetylation reaction (0.3 mg of resin) with 0.05 mmol (25 mCi) of 3 H-acetic anhydride (Amersham Corp.
  • a pH of 6.0 was chosen for the first reaction.
  • a pH of 7.4 was chosen for the second reaction.
  • the buffy coat layer was then removed and discarded, and the erythrocytes were brought up to 50 ml with buffer A (140 mM NaCl, 5 mM KC1, 10 mM glucose, 10 mM Na phosphate, pH 7.4).
  • the erythrocytes were then washed 3 times in buffer A and resuspended to a hematocrit of 10% in the same buffer.
  • a 3 ml sample was transferred to a small polypropylene tube and centrifuged at 700 x g for 5 min at 22°C; 2.5 ml of the supernatant buffer was then removed, leaving 0.5 ml of a 60% hematocrit solution (3.3 X 10 9 erythrocytes).
  • a slightly different buffer was employed (150 mM NaCl, 50 mM Na phosphate, 2 mM KC1, 5 mM glucose, pH 7.4) and the results were the same.
  • the Ac-CGGRGDF-NH 2 peptide was then dissolved in buffer B (150 mM NaCl, 10 mM Na phosphate, pH 6.0) at -2.0 mg/ml (2.6 mM) and the solution was readjusted to pH 6.0 with 1 M NaOH.
  • the mal-sac-HNSA was then dissolved at 10 mg/ml in buffer B, and 0.5 ml of the peptide solution (1.3 ⁇ mol) and 0.05 of the mal-sac- HNSA (1.1 ⁇ mol) were incubated at room temperature for 10 min.
  • the pH of the solution was then increased to 7.4 with 0.1 M NaOH, and the solution was immediately added to the 0.5 ml of erythrocytes in buffer A.
  • the tube was then gently rocked at 22°C for various periods of time, usually up to 2 hr, but in some cases 18 hr.
  • the reaction took place in one step, with the peptide, crosslinker, and erythrocytes incubated together at pH 7.4-7.5.
  • the thrombo-erythrocytes were washed X 3 in buffer A. Thrombo-erythrocytes were used immediately or stored at 4°C.
  • QUANTIFICATION OF PEPTIDE BINDING To determine the number of peptide molecules crosslinked to each thrombo-erythrocyte, the radiolabeled peptide was used in combination with unlabeled peptide. At selected time intervals, thrombo-erythrocytes were removed from the incubation mixtures, washed X 3 in buffer A, and then subjected to hypotonic lysis to produce erythrocyte ghosts.
  • the erythrocyte ghosts were solubilized in 0.1 - 0.4 ml 1% sodium dodecyl sulfate (SDS) , and this solution was added to 6 ml of scintillation fluid (Ultima Gold; Packard) and counted in a liquid scintillation counter (Packard 1900CA, Downers Grove, IL) .
  • SDS sodium dodecyl sulfate
  • the number of peptide molecules bound per erythrocyte was then calculated from the radioactivity specifically incorporated into the thrombo-erythrocytes [i.e., radioactivity associated with the ghosts after reaction with the full thrombo- erythrocyte incubation mixture (erythrocytes + peptide + crosslinker) minus the radioactivity associated with the ghosts of the nonspecific control (erythrocytes + peptide) ] .
  • the 10% and 1% lysis buffers contained the protease inhibitors PMSF (1 mM) , leupeptin (0.5 mM) , and EDTA (0.5 mM) .
  • the gel was then stained with the periodic acid-Schiff method by fixing overnight in 25% isopropanol-10% acetic acid, washing with 10% acetic acid, incubating with 1% periodic acid in 3% acetic acid for 60 min, washing with water X 4, reacting with Schiff stain for 60 min in the dark, and washing with 1% Na 2 S 2 0 5 in 0.1 M HC1 X 3.
  • the gel was then stored overnight in 7% acetic acid at 4°C, photographed, stained with Coomassie blue, destained, and rephotographed.
  • the gel was prepared for fluorography by fixing in 30% methanol, 10% acetic acid for 30 min X 3, incubating in a precipitating reagent (solution A) of a fluorography preparation kit (Entensify, New England Nuclear Research Products, Boston, MA) for 30 min, and incubating- in an aqueous fluorescent reagent (solution B) for 30 min.
  • solution A a precipitating reagent
  • solution B aqueous fluorescent reagent
  • Platelet-rich plasma was prepared from blood anticoagulated with 0.01 volume 40% sodium citrate and adjusted to a platelet count of 3.5 X 10 8 /ml with platelet-poor plasma. Aliquots (50 or 100 ⁇ l) of the PRP were added to microtiter wells and then 5 or 10 ⁇ l of ADP (100 ⁇ M stock solution) was added to selected wells, followed by the addition of 5 or 10 ⁇ l of thrombo-erythrocytes (10% hematocrit in buffer A) .
  • PRP Platelet-rich plasma
  • microtiter plate was then rotated at 270 rpm at 22°C for variable periods of time between 0.5 and 20 min and the extent of platelet-thrombo-erythrocyte coaggregation was assessed visually on a scale from 0-4+ with the aid of a magnifying mirror.
  • the PRP was preincubated with 10 mM EDTA, 300 ⁇ g/ml of the peptide RGDF, or 20 ⁇ g/ml of an antibody directed against both the GPIIb/IIIa receptor and the ⁇ .
  • v j8 3 vitronectin receptor that blocks fibrinogen binding to activated platelets (7E3) Coller, 1985, J. Clin. Invest.
  • Blood smears were made from the samples in some experiments and stained with a standard Wright stain (Hemastain, Geometric Data, Wayne, PA) .
  • PRP was prepared from whole blood anticoagulated with ACD-A (8.5:1.5) and gel-filtered over a column of Sepharose 2B (Pharmacia) using a modified Tyrodes buffer (140 mM NaCl, 3 mM KC1, 12 mM NaHC0 3 , 0.4 mM NaH 2 P0 4 , 10 mM HEPES, 2 mM MgCl 2 , 0.2% bovine serum albumin, 5 mM glucose, pH 7.4).
  • Tyrodes buffer 140 mM NaCl, 3 mM KC1, 12 mM NaHC0 3 , 0.4 mM NaH 2 P0 4 , 10 mM HEPES, 2 mM MgCl 2 , 0.2% bovine serum albumin, 5 mM glucose, pH 7.4
  • Samples consisted of 450 ⁇ l of gel- filtered platelets + 20 ⁇ l of thrombo-erythrocytes (10% hematocrit) or control erythrocytes (i.e., erythrocytes incubated with peptide but no crosslinker) . Maximal transmission was set with 450 ⁇ l of buffer + 20 ⁇ l of control erythrocytes. Platelets were activated with ADP (4.3 ⁇ M final concentration) or epinephrine (10 ⁇ M) .
  • OSMOTIC FRAGILITY Thrombo-erythrocytes, control erythrocytes, and untreated erythrocytes were added to NaCl solutions of various concentrations. After 20 min at 22°C the samples were centrifuged and the optical density of the supernatant fluid assessed at 540 nm. Results were expressed as the percent hemolysis, with 100% hemolysis defined as the optical density of a sample of erythrocytes added to water.
  • the first stage of the assay involving the adhesion of platelets to purified type 1 rat skin collagen, was performed as described previously, but without radiolabeling the platelets (Coller et al., 1989, Blood 74:182-192).
  • a sample of gel- filtered platelets 100 ⁇ l; 5.5 X 10 8 /ml
  • 2 mM MgCl 2 was added to microtiter plate wells precoated with collagen and the platelets were allowed to adhere for 1 hour at 22°C.
  • the wells were then emptied and washed X 3 with buffer (0.15 M NaCl, 0.01 M Tris/HCl, 0.5% bovine serum albumin, 5 mM glucose, pH 7.4) .
  • Control erythrocytes or thrombo- erythrocytes (50 ⁇ l; 10% hematocrit) were then added to the wells in the same buffer, which was now supplemented with 2 mM MgCl 2 . After 60 min, the wells were emptied and washed X 3 as above. The wells were then visually inspected at 40OX magnification with the aid of a microscope with Nomarski optics. The effect of 20 ⁇ g/ml of an antibody to GPIIb/IIIa that blocks fibrinogen binding and platelet aggregation (10E5)
  • the samples were then placed in the instrument and the deformability index (a measure of the change in cell shape from circle to ellipse) was measured continuously as the cells were subjected to increasing shear rates (0 - 1,037 s "1 ) .
  • Each reaction mixture contained —3.3 X 10 9 erythrocytes.
  • the assay was also performed with PRP anticoagulated with heparin (4 U/ml) or hirudin (10 U/ml; Sigma) and similar results were obtained, although as expected, thrombin-induced activation did not occur with these anticoagulants. It is important to note that since these assays contain normal plasma, fibrinogen is available for binding to activated GPIIb/IIIa receptors; thus, the thrombo- erythrocytes were able to compete effectively with fibrinogen for the GPIIb/IIIa receptors.
  • FIG. 6 depicts the results of an experiment demonstrating that thrombo- erythrocytes, but not control erythrocytes, interact with ADP-activated platelets. The thrombo-erythrocytes did not interact with unactivated platelets despite the stirring and 37°C temperature.
  • 10E5 a monoclonal antibody to GPIIb/IIIa that blocks the binding of fibrinogen to platelets and partially blocks the 5 interaction of platelets with RGD-coated beads (Coller et al., 1983, J. Clin. Invest.
  • Hemostasis in vivo is thought to be initiated by adhesion of platelets to subendothelial proteins, in particular collagen, when blood vessels are damaged (Coller et al., 1989, Blood. 74:182-192). Platelets 0 then aggregate on top of the adherent platelets, presumably as a result of the GPIIb/IIIa receptors on the lumenal surface of the adherent platelets undergoing the transformation that allows them to bind adhesive glycoproteins such as fibrinogen and von 5 Willebrand factor with high affinity (Plow and
  • the peptide appeared to be selectively crosslinked to glycoproteins that are present in the PAS-1, PAS-2, and PAS-3 regions, making it most likely that it is crosslinked to glycophorin A (whose di eric form is largely responsible for PAS-1 and whose monomeric form is largely responsible for PAS-2) , and the related glycoprotein, glycophorin B (which is largely responsible for PAS-3) (Anstee, 1990, Vox Sang. 58:1- 20). It is interesting that there are an estimated 0.2 - ⁇ o X 10 6 glycophorin A molecules per erythrocyte and -0.25 X 10 6 glycophorin B molecules per erythrocyte (Anstee, 1990, VoxSang. 58:1-20.), raising the possibility that there is 1:1 stoichiometry between the number of crosslinked peptide molecules and the number of glycophorin A + glycophorin B molecules.
  • Thrombo-erythrocytes were analyzed in several ways.
  • the crosslinking reaction itself produced only slightly more hemolysis than simply washing the erythrocytes.
  • Laser diffraction ektacytometry a technique that is sensitive to changes in the erythrocyte membrane and the hydration state of the cytoplasm of the erythrocyte, has been a useful tool in analyzing erythrocytes altered in vitro and erythrocytes from patients with a variety of disorders (Mohandas et al., 1980, J. Clin. Invest.
  • Thrombo-erythrocytes are able to selectively 0 interact with platelets activated with ADP, epinephrine, or thrombin to produce large aggregates containing mixtures of platelets and erythrocytes.
  • Studies wich monoclonal antibodies to GPIIb/IIIa and fluid phase RGD peptides indicate that the RGD peptides
  • the interactions are not limited to platelets in citrated PRP since platelets in PRP prepared from blood anticoagulated with either heparin or hirudin are also able to interact with the thrombo-erythrocytes.
  • GPIIb/IIIa receptors are positive indicators of the utility of the thrombo-erythrocyte as a potential alternative to fresh platelets. Since there are 20 times as many erythrocytes as platelets in the circulation of normal individuals, conversion of the erythrocytes contained in 50 ml of blood into thrombo- erythrocytes would produce as many thrombo-erythrocytes as there are platelets in 1 liter of blood, or approximately 2 conventional units of platelets. Moreover, since erythrocytes are 9 times as large as platelets, the 50 ml of blood would yield the equivalent of 18 conventional units of platelets by mass.
  • erythrocyte washing is already standard practice in blood banks and the cross-linking reaction can be carried out within 1-2 hours, depending upon the density of peptides selected.
  • thrombo- erythrocytes can function as an autologous, semi- artificial platelet alternative.
  • platelets make other contributions to enhancing hemostasis and so it is appropriate to question whether thrombo-erythrocytes might also serve to enhance the hemostatic response.
  • One of the functions platelets serve is to act as a surface on which coagulation reactions take place
  • the erythrocyte membrane can also serve to accelerate coagulation reactions under certain circumstances and so it is possible that thrombo-erythrocytes may also be able to facilitate thrombin formation (Zwaal et al., 1989, Molec. Cell Biochem. 91:23-31).
  • the recent discovery that erythrocytes can enhance platelet activation via cooperative biochemical interactions with platelets involving eicosanoid metabolism provides another potential mechanism by which thrombo-erythrocytes may enhance the function of residual platelets.
  • Platelets release ADP from their dense granules when stimulated, leading to ADP-induced platelet activation; erythrocytes are rich in ADP and so it is possible that ADP may leak from thrombo-erythrocytes that become enmeshed in hemostatic plugs.
  • nitric oxide produced by cells in the blood vessel wall as a potent inhibitor of platelet activation suggests another potential mechanism by which thrombo-erythrocytes may enhance platelet function since free hemoglobin and hemoglobin in erythrocytes have been demonstrated to neutralize the effect of nitric oxide (Houston et al., 1990, Blood 76:953-958).
  • Platelet-rich plasma was prepared by centrifugation at -700 X g for 3.5 min at 22°C and adjusted to 3 X 10 n /l with platelet-poor plasma (PPP, prepared by centrifuging for 10 min at 35 1600 X g at 22°C).
  • PRP platelet-poor plasma
  • GFP Gel-filtered platelets
  • RGDF RGDF
  • Double couplings were used for arginine, phenylalanine, the fifth and all subsequent glycine residues in the first synthesis, and the fourth and all subsequent glycine . residues m the second synthesis.
  • the peptides were cleaved from the resin with HF in the presence of anisol and dimethylsulfide (10:1:1 by volume); the starting temperature was -10°C and the temperature was maintained below -2°C throughout the cleavage by adding ice to the ice-salt mixture.
  • the peptides were washed with ethyl ether and then extracted twice with 30% HAc and twice with 10% HAc.
  • G 19 RGDF exhibited the poorest homogeneity as judged by HPLC (56%) , it was purified to >95% homogeneity with preparative HPLC on a larger column of the same material (10 X 250 mm) .
  • the functional activities of the crude and purified G 19 RGDF peptides in the bead agglutination assay were the same.
  • a fibrinogen 7-chain dodecapeptide (amino acids 400-411) containing an added amino-terminal tyrosine (Y- HHLGGAKQAGDV) was a gift from Dr. Ellinor Peerschke, state University of New York at Stony Brook, NY.
  • the snake venom peptide trigramin which contains an RGD sequence and inhibits fibrinogen binding to GPIIb/IIIa, (Huang et al., 1987, J. Biol. Chem. 262:16157) was a gift of Dr. Stephan Niewiarowski, Temple University, Philadelphia, PA.
  • RGDF RGDF
  • the mass spectrometry probe was precoated with l ⁇ l of 50% glycerine/50% thioglycerine matrix and then 1 ⁇ l of the peptide solution was added; for the G 13 .
  • 19 RGDF peptides the probe was precoated with 1 ⁇ l of thioglycerine matrix and then 1 ⁇ l of the peptide solution was added.
  • the fast atom bombardment mass spectra were generated on a Kratos MS890/DS90 mass spectrometry system (Ramsey, NJ) .
  • a saddle field ion source (Ion Tech, Middlesex, England) was used as a source of fast xenon atoms; it produced 1 mA of ion current when operated at 7 kV.
  • the mass spectrometer was operated at 6.8 kV and the mass range was calibrated with cesium iodide in the positive ion mode using a 10 sec/decade scan speed after adjusting for the 1 amu added to the molecule by protonation.
  • the observed molecular weights of all peptides prepared in the second synthesis (G M9 RGDF) matched precisely the predicted molecular weights.
  • fibrinogen bead agglutination assay was performed as previously described (Coller, 1980, Blood
  • fibrinogen (lot number PR2548, Cutter Laboratories, Berkeley, CA) purified according to the method of Mosesson (Mosesson, 1962, Biochim. Biophvs. Acta 57:204) was coupled to 1.3 ⁇ polyacrylonitrile beads containing N-hydroxysuccinimide groups at a ratio
  • G,, G 3 , G 5 , G 7 and G 9 RGDF peptides were also coupled to beads under these circumstances and these beads were similar in coupling efficiency and platelet agglutinating activity to those coupled in the acetate buffer (see below) .
  • Bovine serum albumin (BSA, essentially globulin free, Sigma) at 30 ⁇ M was coupled to 1 ml of the beads as a control.
  • BSA bovine serum albumin
  • G 3 RGDF and G 9 RGDF peptides were diluted to concentrations between 0.4 ⁇ M and 4.05 mM before coupling.
  • MONOCLONAL ANTIBODIES Table VI lists the antibodies, their specificities and the concentrations used. They have all been characterized previously: antibodies 10E5 (Coller et al., 1983, J. Clin. Invest. 72:325), 7E3 (Coller B.S., 1985, J. Clin. Invest. 76:101), 6D1 (Coller et al., 1983, Blood 61:99), and 6F1 (Coller et al., 1989, Blood 74:182) are from the laboratory; antibody A 2 A 9 (Bennet et al., 1983, Proc. Natl. Acad. Sci. U.S.A. 80:2417) was a gift of Dr.
  • ⁇ 5 uptake was terminated by adding 5 ⁇ M imipramine (Sigma) and stirring was continued for another 5 min. Total uptake under these conditions was 70-80% of the added serotonin.
  • the agglutination assay was performed in quadruplicate in the same way as described above except
  • the RGDS or LGGAKQAGDV(R) 8 RGDV peptide was incubated at 22°C with citrated PRP (-3 x l0 8 /ml) for 1 to 5 min at various concentrations and then non- saturating concentrations of the antibodies (1.5 - 2.5 ⁇ g/ml) were added for 1 to 2 min.
  • the bound antibody was separated from the free by centrifugation of the PRP through 20% sucrose and both the platelet pellet and the supernatant were counted. Results were expressed as either an increase or decrease in antibody binding as compared to a buffer control.
  • the data in Figure 8 and 9 show the extent of agglutination of (G) n -RGDF beads by PRP as a function of time and the number of glycine residues. Both the total platelet agglutinating activity and the speed of agglutination increased dramatically as the number of glycine residues increased from l to 13 and then the activity decreased as the number of glycines increased further to 19.
  • the G 7 -RGDF beads gave values in between those produced by the G 5 -RGDF and G 9 -RGDF beads, and the G ⁇ -RGDF and G ⁇ 5 -RGDF beads gave values that were similar to those of the G 13 -RGDF beads.
  • the immobilized G 9 -RGDF peptide was much more potent in agglutinating platelets than the immobilized smaller peptides.
  • the decrease in platelet agglutinating activity of the longest peptides (G 17 - and G 19 RGDF) was notable and perhaps suggests that the peptides have sufficient freedom to fold back on themselves or interact with each other.
  • the fibrinogen 7-chain dodecapeptide derivative also inhibited agglutination of the (G) n -RGDF 5 beads; on a molar basis, the potency of the inhibition of the ⁇ -chain peptide was slightly less than that of the RGDF peptide (Table VI) .
  • Monoclonal antibodies 10E5, A 2 A 5 , 7E3, and PAC-l Monoclonal antibodies 10E5, A 2 A 5 , 7E3, and PAC-l
  • 35 aggregation is not required for agglutination to occur.
  • Antibody 7E3 also can inhibit a v /IIIa function, and preincubation of platelets with 7E3 decreases the 5 binding of 125 I-LM 609, suggesting that 7E3 and LM 609 may bind to nearby sites on a v /IIIa.
  • PAC-l which effectively inhibited the 5 agglutination of Gj-RGDF and G 3 -RGDF beads, had much less inhibitory activity than the other 3 antibodies when tested with the longer beads. With the G 9 -RGDF beads, for example, it produced only 8% inhibition at 30 min (Table VI) . Thus, even though convincing evidence indicates that PAC-l binds to the RGD-binding site (Taub et al., 1989 J. Biol. Chem. 264:259), it was much less inhibitory than the other antibodies.
  • Trigramin at 3-4.5 ⁇ g/ml (-0.6 ⁇ M) which is approximately twice the concentration reported to nearly saturate platelet GPIIb/IIIa receptors (Huang et al., 1987, J. Biol. Chem. 262:16157), inhibited the agglutination with the shorter beads, but with the longer beads it inhibited only the early phase, such that at 30 min no inhibition was observed (Table VI) .
  • a similar inhibitory pattern was observed with the soluble RGDF peptide at -200 ⁇ g/ml (-400 ⁇ M) , consistent with data for Huang et al.
  • the longer peptides in solution were less potent than the shorter ones in inhibiting the interaction between platelets and fibrinogen-coated beads. This indicates that the immobilized longer peptides do not show enhanced interaction with platelets as a result of an intrinsic affinity advantage conferred by the increased number of glycine residues; in fact, they had to overcome an intrinsic disadvantage with regard to affinity. Differences in peptide density on the beads also cannot account for the observed differences because the peptides were all immobilized at approximately the same density and dilutional studies indicated little effect of minor differences in peptide density.
  • the longer peptides were more effective because they could more easily gain access to the RGD binding sites in the receptors. Both the increased length and increased flexibility of the longer peptides could contribute to this enhanced ability to interact with the receptors.
  • the minimal ability of the shortest peptides to interact with platelets under any conditions indicates that the RGD binding sites may be recessed from the surface of the receptors by at least several angstroms. The gradient of increased interactions as the peptide length increased suggests that the RGD binding sites may be arrayed at various depths, either because the receptors themselves are at variable distances from the platelet surface or because the RGD binding sites are variably recessed in the receptors.
  • the increased response after ADP-activation indicates that activation either causes the RGD binding sites to move closer to the platelet surface or increases the affinity of the receptor for the RGD peptides, perhaps by decreasing steric hindrance.
  • activation either causes the RGD binding sites to move closer to the platelet surface or increases the affinity of the receptor for the RGD peptides, perhaps by decreasing steric hindrance.
  • the reduced agglutination response produced by PGEl suggests either that it causes the RGD binding sites to become more recessed or to have lower affinity, perhaps as a result of greater steric hindrance. It is important to emphasize, however, that even with PGE X pretreatment, the longer beads were able to produce substantial agglutination.
  • PGE X an agent
  • the receptors are in a dynamic equilibrium between conformations that can interact with RGD-containing ligands and others that cannot.
  • the binding of a platelet via a receptor in the proper conformation to an RGD peptide on a bead would then allow the platelet to linger at the bead surface while additional receptors transiently adopt the proper conformation.
  • the density of RGD peptides is so high, it is very likely that the receptor will find an RGD peptide to interact with even if the length of time the receptor stays in the proper conformation is brief. In this way, each interaction makes the platelet linger longer, facilitating additional interactions and encouraging the process to continue to full agglutination.
  • the length and flexibility of the longer peptides may allow them, with sufficient time, to insinuate themselves i.nto the RGD bindi.ng sites of otherwise i.naccessi.ble receptors.
  • the extraordinarily high density of peptides on the beads would also favor even such low affinity interactions.
  • various targeting molecules can be coupled to erythrocytes, to produce targeted erythrocytes, specifically targeted carrier erythrocytes.
  • the present Example demonstrates targeting of erythrocytes to platelets by coupling a monoclonal antibody to the erythrocytes.
  • MONOCLONAL ANTIBODY 10E5 A 1.1 mg/ml solution of platelet glycoprotein GPIIb/IIIa-specific monoclonal antibody 10E5 was prepared (see Sections 8 and 9, supra) . To approximately 1 ml of the 10E5 solution was added 10 ⁇ l of 125 I-labeled 10E5 antibody (21 ⁇ g/ml) . The cold and radiolabelled 10E5 antibody solution was dialyzed using 12,000-14,000 molecular weight cut-off dialysis tubing. Buffer C was degassed with N 2 bubbling and equilibrated with 10 ml of 10-DG chromatography support (BioRad Econopac) .
  • the 10E5 was reduced (into a variety of forms including a pair of heavy chain-light chain univalent molecules) by adding 1 ⁇ l (about 14 mM) of 2- mercaptoethanol and incubating at 22°C for 60 min.
  • the reduced 10E5 was chromatographed on the 10-DG column eluted with Buffer C. Twenty fractions of 0.5 ml each were collected. Fractions #5-8 (2.2 ml) were radioactive and contained 0.53 mg of antibody (1.77 x 10 5 cpm) . From these pooled fractions, 75 ⁇ l was removed for gel electrophoresis and Ellman's assay.
  • erythrocytes Three ml of erythrocytes (10% hematocrit) were centrifuged at 430 x g for 4 min at 22°C. The pellet was resuspended to 30% hematocrit (1 ml) in Buffer C. To the erythrocytes was added 0.5 mg of freshly prepared 10 mg/ml mal-sac-HNSA in 50 ⁇ l of Buffer C. The reaction mixture was rocked for 120 min at 22°C, then washed four times in Buffer C and suspended to 1 ml in Buffer C. A 250 ⁇ l aliquot of the erythrocytes was then reacted with 0.53 mg (2.1 ml) of reduced 10E5 antibody for 30 min at 22°C; the reaction mixture was overlayed with N 2 .
  • Control erythrocytes coupled with mal-sac-HNSA were reacted with 2.1 ml of buffer C (no antibody) for 30 min at 22°C. After reacting with 10E5 or buffer alone, cells were centrifuged 4 min at 430 x g at 22°C. The supernatant was removed and stored frozen. The cells were washed three times with Buffer C, and the radioactivity determined. Calculations based on the specific activity of the 10E5 antibody i.ndicated that approximately 1,360 antibody molecules bound per erythrocyte. 10.1.4. ACTIVITY ASSAYS Platelet co-agglutination assays were performed.
  • erythrocytes (10% 5 hematocrit; 10E5 conjugated, mal-sac-HNSA controls and unmodified controls) were mixed and incubated for 10 min. The mixed cells were rotated for 8 min and microscopic cell association of the fresh samples was immediately determined using a 400X phase contrast 10 microscope. In addition, blood smears were prepared, stained, and viewed microscopically at 1000X using an oil immersion lens.
  • erythrocytes can be targeted to a specific cell by conjugation with on ⁇ a targeting molecule.
  • the targeting molecule was reduced univalent monoclonal antibody 10E5, which is specific for the glycoprotein GPIIb/IIIa on platelets.

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Abstract

Procédés et composés nouveaux favorisant l'agrégation plaquettaire et permettant de contrôler le saignement. Cette invention se fonde sur la découverte surprenante que des érythrocytes conjugués à certains peptides et polypeptides renfermant une séquence R-G-D (Arg-Gly-Asp) (décrite ici sous le nom collectif de 'peptides RGD') se fixent de manière sélective sur des plaquettes activées et ne se fixent pas sur des plaquettes non activées. Etant donné la double nature des érythrocytes dérivés, on leur donne le nom de thrombo-érythrocytes. Les thrombo-érythrocytes ne subissent pas de modification significative au niveau de leurs propriétés rhéologiques. Dans une version préférée, les thrombo-érythrocytes ont la majorité du peptide RGD qui est réticulé de manière spécifique à de la glycophorine A et à de la glycophorine B à la surface de l'érythrocyte. Dans les thrombo-érythrocytes de cette invention, l'Arg à terminaison H de la séquence R-G-D doit être de préférence espacé de 9 à 50 Angströms, préférablement de 10 à 40 Angströms et idéalement de 11 à 25 Angströms, de la protéine d'érythrocyte.
PCT/US1991/008430 1990-11-09 1991-11-12 Erythrocytes et thrombo-erythrocytes utilises comme agents a specificite de cible WO1992008804A1 (fr)

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KR1019930701399A KR930702341A (ko) 1990-11-09 1991-11-12 타겟 특이물질인 적혈구와 혈전-적혈구
AU90588/91A AU651643B2 (en) 1990-11-09 1991-11-12 Erythrocytes and thrombo-erythrocytes as target specific agents
JP4502274A JPH06504535A (ja) 1990-11-09 1991-11-12 ターゲット特異的物質としての赤血球及びトロンボー赤血球
NO93931696A NO931696L (no) 1990-11-09 1993-05-10 Erytrocytter og trombo-erytrocytter som maal-spesifikke midler
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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
US5672585A (en) * 1990-04-06 1997-09-30 La Jolla Cancer Research Foundation Method and composition for treating thrombosis
US6451976B1 (en) 1997-03-20 2002-09-17 Trigen Limited Bi-or multifunctional molecules based on a dendroaspin scaffold
EP1045637A4 (fr) * 1998-01-06 2004-08-18 Univ Boston Globules rouges a structure modifiee
EP1045637A1 (fr) * 1998-01-06 2000-10-25 Trustees Of Boston University Globules rouges a structure modifiee
DE102004054536A1 (de) * 2004-11-06 2006-05-11 Capsulution Nanoscience Ag Multimodal veränderte Zellen als zellulare Darreichungsformen für aktive Substanzen und als diagnostische Zellpartikel
WO2007083949A1 (fr) * 2006-01-19 2007-07-26 Eyegene Inc. Composition pharmaceutique à base de peptide pour le traitement de maladies vasculaires
EP1849482A1 (fr) * 2006-04-25 2007-10-31 Capsulution Nanoscience AG Cellules multi-modalement modifiées comme formes pour l' administration de substances actives et comme particules diagnostiques
WO2008044846A1 (fr) * 2006-10-10 2008-04-17 Caregen Co., Ltd Peptides à activités de facteur de croissance épidermique et utilisations de ceux-ci
EP2078035A1 (fr) * 2006-10-10 2009-07-15 Caregen Co., Ltd. Peptides à activités de facteur de croissance épidermique et utilisations de ceux-ci
EP2078035A4 (fr) * 2006-10-10 2010-09-08 Caregen Co Ltd Peptides à activités de facteur de croissance épidermique et utilisations de ceux-ci
US8188049B2 (en) 2006-10-10 2012-05-29 Caregen Co., Ltd. Peptides having activities of epidermal growth factor and its uses

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FI932114A0 (fi) 1993-05-10
AU9058891A (en) 1992-06-11
EP0558645A1 (fr) 1993-09-08
KR930702341A (ko) 1993-09-08
EP0558645A4 (en) 1994-07-06
AU651643B2 (en) 1994-07-28
FI932114A (fi) 1993-07-01
CA2095925A1 (fr) 1992-05-10
JPH06504535A (ja) 1994-05-26

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