WO1999000145A1 - Modulation antigenique de particules virales - Google Patents

Modulation antigenique de particules virales Download PDF

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Publication number
WO1999000145A1
WO1999000145A1 PCT/US1998/013198 US9813198W WO9900145A1 WO 1999000145 A1 WO1999000145 A1 WO 1999000145A1 US 9813198 W US9813198 W US 9813198W WO 9900145 A1 WO9900145 A1 WO 9900145A1
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cell
immunogenic
cell surface
viral
cells
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PCT/US1998/013198
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English (en)
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WO1999000145A9 (fr
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Mark D. Scott
John W. Eaton
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Albany Medical College
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Priority to AU79878/98A priority Critical patent/AU7987898A/en
Publication of WO1999000145A1 publication Critical patent/WO1999000145A1/fr
Publication of WO1999000145A9 publication Critical patent/WO1999000145A9/fr

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/87—Introduction of foreign genetic material using processes not otherwise provided for, e.g. co-transformation
    • C12N15/88—Introduction of foreign genetic material using processes not otherwise provided for, e.g. co-transformation using microencapsulation, e.g. using amphiphile liposome vesicle
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/69—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit
    • A61K47/6901—Conjugates being cells, cell fragments, viruses, ghosts, red blood cells or viral vectors
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/85—Vectors or expression systems specially adapted for eukaryotic hosts for animal cells
    • C12N15/86—Viral vectors
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2710/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA dsDNA viruses
    • C12N2710/00011—Details
    • C12N2710/10011—Adenoviridae
    • C12N2710/10311—Mastadenovirus, e.g. human or simian adenoviruses
    • C12N2710/10341—Use of virus, viral particle or viral elements as a vector
    • C12N2710/10343—Use of virus, viral particle or viral elements as a vector viral genome or elements thereof as genetic vector

Definitions

  • the present invention relates generally to antigenic modulation of cells, and more particularly to non-immunogenic cellular compositions comprising cells modified with a non-immunogenic compound, and uses of such non- immunogenic cells.
  • Acute tissue rejection can be observed in two major clinical situations: 1) blood transfusions; and 2) organ transplantation. In both situations, to be described in greater detail below, antibody binding and complement fixation are the two major mechanisms underlying the destruction of the donor tissue (the donor tissue referring to blood or organs).
  • Previous means of attempting to control acute rejection have centered on tissue matching and pharmacologic interventions. Despite these measures a significant number of often life- threatening acute tissue rejection reactions continue to occur.
  • Blood transfusions are a crucial component in the treatment of a number of acute and chronic medical problems. These range from massive blood loss following traumatic injury to chronic transfusions to treat diseases such as thalassemia and sickle cell anemia. In most acute injuries simple blood typing (ABO/rh) is sufficient to identify appropriate donors.
  • transplantation of organs (such as kidneys and livers) from one human to another is often made difficult by a lack of exact immunologic identify between donor and recipient.
  • organs such as kidneys and livers
  • the transplanted organ is subject to direct attack by the immune system of the recipient even before a secondary immunologic response has had time to occur.
  • This so-called 'hyperacute rejection' is often life threatening and, obviously, prevents the effective integration of the transplant into the recipient. Therefore, a need exists for methods and agents which may prevent immediate recognition of the endothelial surfaces of organ transplants, thereby moderating or stopping the process of acute graft rejection.
  • transplantation of organs from one species to another (“xenotransplantation") faces even more daunting immunologic barriers and would be greatly facilitated by methods for blocking immunologic recognition of the foreign endothelial surface.
  • Proteins have been modified by the covalent attachment of soluble polymers such as polyvinyl alcohol, carboxymethyl cellulose (Mitz and Summaria 1961), and polyvinylpyrrolidone (von Spect et al. 1973).
  • soluble polymers such as polyvinyl alcohol, carboxymethyl cellulose (Mitz and Summaria 1961), and polyvinylpyrrolidone (von Spect et al. 1973).
  • PEGs polyethylene glycols
  • Abuchowski et al. (1977a) disclose the modification of purified bovine serum albumin (BSA) by covalent attachment of methoxypolyethylene glycol, rendering the BSA non-immunogenic.
  • BSA bovine serum albumin
  • Islets of Langerhans have been microencapsulated in semipermeable membranes in order to decrease immunogenicity of implanted islets (Lacy et al. 1991; Lim 1980).
  • PEG was not directly incorporated into the islet cell membranes but rather the cells were surrounded by the PEG- containing hydrogel.
  • Zalipsky and Lee (1992) discuss the use of functionalized polyethylene glycols for modification of polypeptides, while Merrill (1992) and Park and Wan Kim (1992) both disclose protein modification with polyethylene oxide.
  • U.S. Patent No. 4,179,337 of Davis et al. discloses purified polypeptides, such as enzymes and insulin, which are coupled to polyethylene glycol or polypropylene glycol having a molecular weight of 500 to 20,000 daltons to provide a physiologically active non-immunogenic water soluble polypeptide composition.
  • the polyethylene glycol or polypropylene glycol protect the polypeptide from loss of activity and the composition can be injected into the mammalian circulatory system with substantially no immunogenic response.
  • U.S. Patent No. 5,006,333 of Saifer et al. discloses a biologically persistent, water-soluble, substantially non-immunogenic, substantially non- antigenic conjugate of superoxide dismutase, prepared by coupling purified superoxide dismutase to one to five strands of a polyalkylene glycol which is polyethylene glycol or polyethylene-polypropylene glycol copolymer, wherein the polyalkylene glycol has an average molecular weight of about 35,000- 1,000,000.
  • U.S. Patent No. 5,013,556 of Woodle et al. discloses a liposome composition which contains between 1 -20 mole percent of an amphipathic lipid derivatized with a polyalkylether, as exemplified by phosphatidylethanolamine derivatized with polyethylene glycol.
  • U.S. Patent No. 5,214,131 of Sano et al. discloses a polyethylene glycol derivative, a purified peptide modified by the polyethylene glycol derivative, and a method for production thereof.
  • the polyethylene glycol derivative is capable of modifying the guanidine groups in peptides.
  • the peptides modified by the polyethylene glycol derivative are extremely stable, are considerably delayed in biological clearance, and retain their physiological activities over a long period.
  • PCT Application PCT WO 95/06058 published on March 3, 1995 describes the reaction of a sulfonate (sulphonate) ester-activated polymer with a target material.
  • Representative polymers are recited in claims 14-15 and include methoxypolyethylene glycols (MPEG), representative targets are described in claim 16, and target molecules that are parts of cells are disclosed at page 38, lines 28-34.
  • Example 7 shows the reaction of human erythrocytes with activated PMEGF, with covalent bonding of the polyethylene glycol to the erythrocytes, which remain intact.
  • U.S. Patent No. 5,529,914 describes a method and materials produced by that method which comprises encapsulation ( and adherence of) of a biologic material, including a cell, by photopolymerizing an unsaturated macromer, such as a polyethyleneglycol multiacrylate to form a biocompatible membrane (e.g., around the cell).
  • a biologic material including a cell
  • an unsaturated macromer such as a polyethyleneglycol multiacrylate
  • the membrane is described as being immunoprotective (column 6, lines 4-11).
  • the invention provides a method for modulating the antigenicity and aggregation of mammalian, preferably human, cells.
  • the subject invention provides for the covalent binding of a non-immunogenic compound to intact cells.
  • Cells that can be effectively modified in accord with the invention include anucleate cells (platelets and red blood cells) and nucleated cells (epithelial cells, endothelial cells, and lymphocytes).
  • the non-immunogenic compound is polyethylene glycol (PEG) or a derivative thereof.
  • PEG- derivatized red blood cells to diminish transfusion reactions arising from mismatched blood or sensitization to minor blood group antigens due to chronic transfusions
  • PEG-derivatization of the vascular endothelium of donor tissues prior to transplantation to prevent/ diminish acute tissue rejection to prevent/ diminish acute tissue rejection
  • implantation of PEG-derivatized cells to correct enzyme deficiencies, other inborn errors of metabolism, or other types of defective cellular functions to transfusion of derivatized RBC into malaria-infected individuals to correct the accompanying acute anemia and prevent the infection of the transfused cells.
  • red blood cells modified by PEG have normal in vitro and in vivo survival when compared to control cells.
  • Covalent linkage of non-immunogenic compounds e.g., PEG or PEG- derivatives, such a methoxypolyethylene glycol or PEG-like compounds such as polyethylene oxide
  • PEG or PEG- derivatives such as methoxypolyethylene glycol or PEG-like compounds such as polyethylene oxide
  • transfusion reactions to both major and minor red blood cell antigens
  • these transfusion reactions actually result from minor surface antigens not routinely measured by blood banks.
  • PEG-modified red blood cells can be employed to diminish/prevent the recognition of red blood cell antigenic determinants
  • the application of this invention can also lead to procedures for modification of animal red blood cells which can then be used for transfusion into humans, or into animals of the same or other species.
  • the application of this invention can further lead to procedures for modification of red blood cells to prevent malarial invasion or opsonization by factors such as complement.
  • the scope of this invention extends well beyond blood banking to other areas where foreign tissues are manipulated or introduced in vitro or in vivo.
  • One area of primary interest is the use of PEG-modified tissues (especially covalent modification of the vascular endothelium) for tissue transplantation.
  • PEG-modified tissues especially covalent modification of the vascular endothelium
  • many organ transplants fail as a result of immediate tissue rejection. This rejection reaction occurs primarily at the level of the vascular endothelium and results in vessel occlusion, tissue hypoxia ischemia and ultimate loss of the organ transplant.
  • the invention thus provides a non-immunogenic cellular composition
  • a non-immunogenic cellular composition comprising: a cell having a cell surface and antigenic determinants on the cell surface; and a non-immunogenic compound covalently attached to the cell surface directly or by means of the linking moiety, which linking moiety can be derived from a linker molecule, as discussed below.
  • the non-immunogenic compound acts to block recognition of the antigenic determinants on the cell surface.
  • the linking moiety is covalently attached directly to the antigenic determinant on the cell surface.
  • the linking moiety may be covalently attached to a non-antigenic site of the cell surface, the antigenic site on the cell surface is camouflaged or masked by virtue of the long chain length of the non-immunogenic compound.
  • the invention further provides a method of producing a non- immunogenic cell.
  • the method comprises: covalently attaching a non- immunogenic compound to the surface of the cell directly, or by means of a linking moiety, so that non-immunogenic compound blocks recognition of antigenic determinants on the cell surface to produce a non-immunogenic cell.
  • a non-immunogenic cell produced by this method is also provided by the subject invention.
  • the concept of the subject invention can also provide a method of decreasing phagocytosis of a cell.
  • This method comprises: selecting a cell for introduction into a subject, the cell having a cell surface and antigenic determinants on the cell surface; covalently attaching an amount of a non- immunogenic compound to the cell surface directly or by means of a linking moiety, so that the attached non-immunogenic compound blocks recognition of antigenic determinants on the cell surface to produce a non-immunogenic cell; and introducing the non-immunogenic cells into a subject, wherein phagocytosis of the non-immunogenic cell is decreased as compared to phagocytosis of the cell prior to modification.
  • a method of decreasing an adverse reaction to a transfusion comprising: selecting a red blood cell for transfusion into a subject, the red blood cell having cell surface and blood group antigenic determinants on the cell surface; covalently attaching a non-immunogenic compound capable of blocking the blood group antigenic determinants on the cell surface, to the cell surface directly or by means of a linking moiety, so as to produce a non-immunogenic red blood cell; and transfusing a subject with the non-immunogenic red blood cell, wherein adverse reaction to the transfusion of the non-immunogenic red blood cell is decreased as compared to transfusion of the red blood cell prior to modification.
  • Also provided is a method of decreasing rejection of a transplanted cell comprising: selecting a cell for transplantation into a subject, the cell having a cell surface and antigenic determinants on the cell surface; covalently attaching a non-immunogenic compound capable of blocking the recognition of the antigenic determinants on the cell surface, to the cell surface directly or by means of a linking moiety, so as to produce a non-immunogenic cell; and transplanting the non-immunogenic cell into a subject, wherein rejection of the transplanted cell is decreased as compared to rejection of the cell prior to modification.
  • the invention provides a method of decreasing aggregation of nucleated and anucleate cells such as that induced by antibodies or by other cell: cell interactions.
  • the method comprises: covalently attaching non-immunogenic compounds capable of blocking recognition of antigenic determinants on a cell surface to the cell surface of each of a plurality of cells directly or by means of a linking moiety, so as to produce non-aggregating cells, wherein antibody- induced aggregation of the non-aggregating cells is decreased as compared to antibody-induced aggregation of the cells prior to modification.
  • linking moiety refers to an at least divalent organic group that covalently, or by complexation or chelation binds to both the non-immunogenic molecule and the cell surface, to attach at least one non-immunogenic compound to at least one functional group or structure on the cell surface.
  • the linking moieties can be derived from reactive linker molecules, as described hereinbelow.
  • Fig. 1 is a schematic depiction of the preparation of certain embodiments of the non-immunogenic cellular compositions according to the subject invention
  • Fig. 2 is a schematic depiction of a further embodiment of a non- immunogenic cellular composition according to the subject invention.
  • the non-immunogenic compound is polyethylene glycol or a derivative thereof and the activated PEG (PEG-linker) is covalently attached to antigenic determinants on the cell surface (directly blocking antigenic sites) and also covalently attached to non-antigenic sites on the cell surface (indirectly blocking antigenic sites due to their long chain length);
  • Fig. 3 is a graph showing that monomethoxypoly(ethylene glycol) (mPEG) modification of red blood cells causes a dose-dependent inhibition of anti-A antibody induced RBC aggregation defined turbidometrically;
  • Fig. 4 is a bar graph showing that mPEG modification of red blood cells only slightly increases red blood cell lysis
  • Fig. 5 is a graph showing the mPEG modification of red blood cells has no effect on red blood cell osmotic fragility
  • Fig. 6 is a bar graph showing that mPEG-modified type A red blood cells bind significantly less anti-A antibody
  • Fig. 7 is a bar graph showing that mPEG-modified sheep red blood cells are significantly less prone to phagocytosis by human peripheral blood monocytes;
  • Fig. 8 is a graph showing no significant differences in the in vivo survival of control mouse red blood cells and mouse red blood cells modified with activated PEG;
  • Fig. 9 is a graph demonstrating that sheep red blood cells (solid symbols) enter and survive within the circulatory system of a mouse whereas unmodified sheep red blood cells (open symbols) do not.
  • Fig. 10 is a graph depicting the Donor A peripheral blood mononuclear cell (PBMC) response to antigenically foreign Donor B PBMC (Panel A) and the Donor B PBMC response to Donor A (Panel B) in MLC analysis of control and mPEG derivatized PBMC.
  • PBMC peripheral blood mononuclear cell
  • Fig. 11 is a graph demonstrating that platelet aggregation is prevented by the covalent modification of platelet surfaces with mPEG.
  • Fig. 12 is a bar graph demonstrating that mPEG-modification of epithelial cells blocks antibody recognition of surface antigens.
  • Figure 13 is a blot demonstrating that Simian Vacuolating virus 40 (SV40) shows a shift in coat protein electrophoresis.
  • SV40 Simian Vacuolating virus 40
  • the present invention provides a non-immunogenic cellular composition
  • a non-immunogenic cellular composition comprising: a cell having a cell surface and antigenic determinants on the cell surface; a linking moiety covalently attached to the cell surface; and a non- immunogenic compound covalently attached to the linking moiety and capable of blocking recognition of the antigenic determinants on the cell surface.
  • the non-immunogenic compound can be bound directly to the cell surface, if it comprises groups such as carboxylic acids, aldehydes, ketals or acetals that are reactive with NH 2 or SH groups on the cell surface.
  • the present invention is differentiated from the invention of copending U.S. Patent Application 08/671,452, identified above, in that the present invention is specifically directed towards another class of cell which can be readily modified by the same materials used on red blood cells, lymphocytes, etc., that class of cell being viruses (more appropriately "virus particle”).
  • Simian Vacuolating virus 40 SV40
  • adenovirus as a vector in gene therapy.
  • Primary inoculation (via inhalation) of adenovirus in cystic fibrosis patients have demonstrated effectiveness in ameliorating disease severity.
  • subsequent administrations of the adenovirus vector are progressively less effective due to initiation of an effective immune response against the viral coat proteins by the patient.
  • a balance can be achieved between viral inactivation (i.e., impaired cell invasion) and decreased immune recognition of the viral coat proteins by the patients immune system.
  • Sufficiently coated viruses may avoid detection inactivation by the immune system while still giving rise to productive therapeutic invasion of the lung epithelial cells.
  • mPEG modification of the external aspect of the cell membranes effectively "hides" major and minor antigenic determinants on a large variety of cell types.
  • viral particles can also be readily derivatized. This camouflage occurs as a consequence of both decreased antibody binding and alteration in celkcell interaction.
  • nonimmunogenic materials may have significant clinical implications. These include, but are not limited to: 1) derivatized RBC to diminish transfusion reactions arising from mismatched blood or sensitization to minor blood group antigens due to chronic transfusions; 2) derivatization of the vascular endothelium of donor tissues prior to transplantation to prevent/diminish acute tissue rejection; 3) implantation of derivatized cells to correct enzyme deficiencies; 4) transfusion of derivatized RBC into malaria-infected individuals to correct the accompanying acute anemia and prevent infection of the transfused cells; (5) viral infectivity (e.g., SV40, Adenovirus, HIV) may be dramatically decreased (due to altered celkcell interaction) by modification of the viral coat proteins; (6) use of mPEG (or other nonimmunogenic tails) may host prevent
  • the cell can be any suitable cell with accessible antigenic determinants on the cell's surface.
  • suitable cells include anuclear cells, for example, hematopoietic cells, i.e., red blood cells or platelets, or nucleated cells, for example, vascular endothelial cells, PBMCs, hepatic cells, neuronal cells, pancreatic cells, or epithelial cells.
  • the antigenic determinants on the cell surface can be due to the presence of antigenic proteins, antigenic carbohydrates, antigenic sugars, antigenic lipids, antigenic glycolipids, antigenic glycoproteins, etc.
  • Antigenic can also be involved in malarial invasion of a cell, or opsonization of a cell.
  • red blood cells have antigens on their surface which determine ABO/rh blood types. These antigens are often referred to as blood group antigenic determinants. These antigens are recognized by an incompatible host and the donor cell will be rapidly destroyed. This can involve the enhancement of natural immunity (through phagocytes, such as macrophages, neutrophils, and natural killer cells) or the stimulation of specific or acquired immunity
  • the cell is recognized as foreign and elicits an immune response.
  • the subject invention involves modification of the antigenicity of the cell. This modification is accomplished by attaching a non-immunogenic compound to the cell.
  • Suitable non-immunogenic compounds for use in the subject invention are non- immunogenic compounds capable of blocking recognition of antigenic determinants on the cell surface.
  • the compounds are generally long chain compounds, wherein the long chain can sterically block the antigenic determinants.
  • non-immunogenic compounds include polyalkylene glycols such as polyethylene glycol, polypropylene glycol, mixed polypropylene- polyethylene glycols, or derivatives thereof (including monomethoxypoly ethylene glycol), certain polysaccharides such as dextrans, cellulosics, Ficoll, and arabinogalactan, as well as synthetic polymers such as polyurethanes.
  • polyalkylene glycols such as polyethylene glycol, polypropylene glycol, mixed polypropylene- polyethylene glycols, or derivatives thereof (including monomethoxypoly ethylene glycol), certain polysaccharides such as dextrans, cellulosics, Ficoll, and arabinogalactan, as well as synthetic polymers such as polyurethanes.
  • Useful molecular weights ofthese compounds can range from about 100-500 to 100,000-200,000 Daltons or above.
  • the presently preferred non-immunogenic compound according to the subject invention is polyethylene glycol or a derivative thereof.
  • the polyethylene glycol or derivative thereof is a molecule with a very long chain length.
  • the non-immunogenic compound e.g., polyethylene glycol or derivative thereof
  • the long chain of the non- immunogenic compound e.g., polyethylene glycol or derivative thereof
  • the non-immunogenic compound e.g., polyethylene glycol or derivative thereof
  • the linker molecule is generally referred to as an "activated" polyethylene glycol or derivative thereof.
  • Polyethylene glycols (PEG) and derivatives thereof are well known in the art.
  • Polyethylene glycol has the formula H(OCH 2 CH 2 ) n OH wherein n is greater than or equal to 4, with a molecular weight of up to about 20,000 Daltons.
  • PEGs and derivatives thereof are available having molecular weights of 200,000 Daltons and above, and can be used in the practice of the present invention, alone, or in combination with lower m.w. materials.
  • polyethylene glycol comprise substitutes for the H or OH end groups, forming, for example, polyethylene glycol ethers (such as PEG-O-R; PEG-O-CH 3 ; CH 3 -PEG-OH or "mPEG”; 2,4-dinitrophenyl ethers of PEG), polyethylene glycol esters (such as PEG-O 2 C(CH 2 ) 14 CH 3 ; PEG- O 2 CCH 2 CH 2 CO 2 -atropine), polyethylene glycol amides (such as PEG- O 2 C(CH 2 ) 7 CONHR; mPEG-O 2 CCH 2 CH 2 CONH(CH 3 )CHCH 2 C 6 H 5 ; PEG- O 2 CCH 2 CH 2 CONHCH 2 CH 2 -NAD + ), polyethylene glycol amines (such as PEG- NH 2 ; PEG-NH(CH 2 ) 6 NH 2 ; PEG-OCH 2 CH 2 NH 2 ; mPEG-NH
  • these non-immunogenic compounds are covalently attached to the cell surface by means of a linking moiety.
  • linking moieties can be prepared by reaction of the polyethylene glycol or derivative thereof with suitable linker molecules that are also well known in the art, and include, for example, cyanuric chloride, imidazolyl formate, succinimidyl succinate, succinimidyl glutarate, N-hydroxysuccinimide, 4-nitrophenol, and 2,4,5- trichlorophenol.
  • linker molecules 'activate" the PEG, a term also well known in the art. For a description of activation of PEG, with examples of known linking moieties and molecules, see Harris 1985.
  • linker molecules listed above are exemplary only, and the invention is not intended to be limited to those particular examples.
  • the linking molecules disclosed hereinabove and on Figure 1 react with a reactive group such as a hydroxy of the non-immunogenic compound, e.g., the PEG or MPEG, and also react with an NH 2 or, in some cases, SH, group of a peptidyl or other amino acid residue on the cell surface to covalently join them, whereby the linking molecule is converted in one or more steps into a divalent linking moiety such as shown on Table 1, below.
  • a number of "activated" methoxypolyethylene glycols are commercially available, in which mPEG (m.w. 5000) has been bound to a linking molecule at the hydroxyl terminus. These include, methoxypolyethylene glycol (mPEG) para-nitrophenyl carbonate, mPEG cyanuric chloride, mPEG- succinimidyl succinate, mPEG tresylate, and mPEG imidazolyl carbonyl.
  • mPEG methoxypolyethylene glycol
  • mPEG methoxypolyethylene glycol
  • mPEG methoxypolyethylene glycol
  • mPEG methoxypolyethylene glycol
  • mPEG methoxypolyethylene glycol
  • the invention thus further provides a method of producing a non- immunogenic cell.
  • the method comprises: covalently attaching a non- immunogenic compound capable of blocking recognition of antigenic determinants on a cell surface, to a cell surface, directly, or by means of a linking moiety, so as to produce a non-immunogenic cell.
  • the cell is a red blood cell
  • the method can further comprise transfusing a subject with the non-immunogenic cell. Since the antigenic determinants, such as the blood group antigenic determinants, on the red blood cell are blocked by the non-immunogenic compound, the transfused non-immunogenic red blood cell will not elicit an immune response.
  • this method can be very useful when red blood cells need to be transfused quickly without the availability of complete blood typing or cross-matching, or when unmatched from a subject is available.
  • the method can further comprise transplanting the non-immunogenic tissue or organ into a subject. Since the antigenic determinants on the tissue or organ, such as the vascular endothelial cells which form an exposed antigenic surface of the tissue or organ, are blocked by the non-immunogenic compound, the transplanted non-immunogenic tissue or organ will not elicit an immune response. As discussed above, this method is very useful to avoid severe rejection reactions, or graft vs. host disease, when organs or tissues are transplanted.
  • the invention further provides a non-immunogenic cell produced by the above method.
  • the concept of the subject invention can also provide a method of decreasing phagocytosis of a cell. This method comprises: introducing the non- immunogenic cell into a subject, wherein phagocytosis of the non-immunogenic cell is decreased as compared to phagocytosis of the cell prior to modification.
  • the non-immunogenic cell can be prepared by a process comprising: selecting a cell for introduction into a subject, the cell having a cell surface and antigenic determinants on the cell surface; covalently attaching to the cell surface, directly or by means of a linking moiety, a non-immunogenic compound that blocks recognition of the antigenic determinants on the cell surface, so as to produce a non-immunogenic cell.
  • this method can prevent phagocytosis of the "foreign" red blood cell, by rendering the red blood cell non-immunogenic.
  • the "foreign" red blood cell may be from another human, or may be from another non-human subject. In either case, the body's response would be to attempt to eliminate the "foreign” red blood cell including by phagocytosis.
  • a method of decreasing an adverse reaction to a transfusion comprising: transfusing a subject with the non- immunogenic red blood cell, wherein adverse reaction to the transfusion of the non-immunogenic red blood cell is decreased as compared to transfusion of the red blood cell prior to modification.
  • the non-immunogenic red blood cells are prepared by selecting a red blood cell for transfusion into a subject, the red blood cell having a cell surface and blood group antigenic determinants on the cell surface; covalently attaching to the cell surface a non-immunogenic compound in an amount capable of blocking the blood group antigenic determinants on the cell surface; wherein the compound is covalently attached to the cell surface directly or by means of a linking moiety, so as to produce a non-immunogenic red blood cell.
  • the red blood cell could be from another human or from a non-human mammal.
  • Also provided is a method of deceasing rejection of a transplanted cell comprising: transplanting a non-immunogenic modified cell into a subject, wherein rejection of the transplanted modified cell is decreased as compared to rejection of the cell prior to modification.
  • the cell is prepared by a process comprising: selecting a cell for transplantation into a subject, the cell having a cell surface and antigenic determinants on the cell surface; covalently attaching a non-immunogenic compound to the cell surface directly or by means of a linking moiety, so that the non-immunogenic compound blocks the recognition of the antigenic determinants on the cell surface, to produce a non- immunogenic cell.
  • a preferred method of carrying out the covalent attachment is to perfuse the tissue or organ with a solution of an activated polyethylene glycol or derivative thereof (i.e., the polyethylene glycol or derivative thereof is first attached to the linker molecule, forming an activated PEG, which is then perfused over the tissue or organ).
  • an activated polyethylene glycol or derivative thereof i.e., the polyethylene glycol or derivative thereof is first attached to the linker molecule, forming an activated PEG, which is then perfused over the tissue or organ.
  • the activated PEG covalently attaches to the cell surface via a linking moiety.
  • the invention provides a method of decreasing antibody-induced aggregation of cells, the method comprising: covalently attaching to the cell surface non-immunogenic compounds capable of blocking recognition of antigenic determinants on the cell surface; wherein the compounds are covalently attached to the cell surface of each of a plurality of cells, directly or by linking moieties, so as to produce non-aggregating cells, wherein antibody- induced aggregation of the non-aggregating cells is decreased as compared to antibody-induced aggregation of the cells prior to attachment of the compounds.
  • This method is particularly applicable where the cells are red blood cells, and where the antigenic determinants on the cell surface comprise blood group antigenic determinants.
  • a linker molecule can be first reacted with the non-immunogenic compound (forming an "activated" compound) and then the linker molecule can be reacted with the cell surface.
  • the order ofthese steps can be reversed, and any reference to the two steps is intended to cover the two steps in either order.
  • the linker molecule can also be attached to the cell surface, then the non-immunogenic compound can be reacted with the linker molecule to bind it to the cell surface via a thus- formed linking moiety, in accordance with the claims and disclosure herein.
  • PEG modification of the external aspect of the red blood cell membrane effectively 'hides' major antigenic determinants such as ABO blood group substances. This is evident in the (1) lack of gross antibody-induced agglutination, (2) significantly decreased antibody-induced aggregation, and (3) diminished phagocytosis by heterologous macrophages. Treated red blood cells remain intact, exhibiting only minor spontaneous hemolysis, and demonstrate normal osmotic fragility over at least 48 hours in vitro incubation. The "normal" nature of the modified mouse red blood cell is further demonstrated by normal in vivo survival.
  • the PEG modification procedure is surprisingly well tolerated by the cells, yielding a product which survives normally in the circulation.
  • the derivatized cells are antigenically disguised and not recognized by blood group antibodies or by phagocytes. Perhaps most surprisingly, treated red blood cells from one species survive much longer than do untreated red blood cells in the circulation of another species.
  • the invention thus provides for (1) derivatization of human red blood cells to permit transfusions into people difficult to match (because they have pre- existing antibodies to minor blood groups); (2) derivatization of human red blood cells to permit transfusions into people of unknown blood groups who may even differ in major (e.g., ABO) blood groups from the donor; (3) derivatization - by perfusion of activated mPEG solutions - of human organ grafts to prevent unexpected hyperacute rejection episodes; (4) derivatization - by perfusion of activated mPEG solutions - of organs from non-human animals to prevent hyperacute rejection and to improve the chances of ultimate successful engraftment in humans.
  • major blood groups e.g., ABO
  • red blood cells erythrocytes
  • isotonic alkaline phosphate buffer PBS; 50 mM K 2 HPO4 and 105 mM NaCl, pH about 9.2.
  • Cyanuric chloride-activated methoxypolyethylene glycol Sigma Chemical Co.
  • Cell derivatization can also be done under other pH and temperature conditions with comparable results to those presented. For example, red blood cells derivatized at pH 8.0 for 60 minutes at 22°C demonstrated virtually identical characteristics to those derivatized at pH 9.2 for 30 minutes at 4°C.
  • Typical activated mPEG concentrations used range from 0 to 8 mg per ml of red blood cell suspension.
  • the typical activated mPEG concentration to be used on other anuclear (i.e., platelets) and various nucleated cells e.g., vascular endothelial, hepatic, hematopoietic, neuronal, pancreatic cells, epithelial cells, etc.
  • nucleated cells e.g., vascular endothelial, hepatic, hematopoietic, neuronal, pancreatic cells, epithelial cells, etc.
  • mPEG covalent binding of mPEG to the membrane proteins of intact red blood cells prevents red blood cell agglutination. This is apparent at the gross level using agglutination induced by ABO antibodies, and at a finer level using a platelet aggregometer modified to measure red blood cell aggregation (Fig. 3).
  • Type A red blood cells were treated with 0, 3, or 6 mg cyanuric chloride-activated mPEG (m.w. 5000) per ml of blood and incubated at 4°C for 30 minutes. The cells were washed 3 times with isotonic saline and resuspended to a 40% hematocrit in saline.
  • agglutination For gross agglutination, equal volume of a RBC suspension of hematocrit 40% and a commercially available anti-A blood typing antibody (Carolina Biological Supply) were mixed and photographed. Increasing amounts of bound mPEG effectively inhibited the agglutination reaction. In the absence of derivatization, a typical blood typing response was observed. In contrast, with increasing amounts of covalently bound mPEG, a dose-dependent decrease in sera-induced agglutination of RBC was observed. Indeed, at 6 mg mPEG/ml RBC, no detectable agglutination was observed at the gross level.
  • Fig. 3 shows red blood cell microaggregation as measured at 37°C in a platelet aggregometer. As shown, mPEG modification caused a dose-dependent inhibition of anti-A antibody induced red blood cell aggregation.
  • Agglutination response is measured macroscopically with a 4+ s rating being the strongest and 1 + w being the weakest agglutination response.
  • mPEG- modification virtually abolished its detection (e.g., 4 + to l +w ).
  • the degree of activated mPEG derivatization used in this study was relatively low (6 mg/ml) in comparison to the levels which can be used (up to approximately 30 mg mPEG/ml RBC) while exhibiting no adverse effects on the RBC. Indeed, based on the mPEG-dose dependency noted in Fig. 3, it is very likely that higher degrees of derivatization will likely further suppress antigen detection.
  • red blood cell lysis was slightly increased by the attachment of mPEG.
  • red blood cell lysis of the RBC during mPEG modification followed by 24 hours storage at 4°C or after incubation at 37°C was less than 5%.
  • osmotic fragility of the mPEG-treated red blood cells was also unaffected.
  • mPEG-modified red blood cells bind significantly less anti-A antibody (Fig. 6).
  • an ELISA assay of mPEG-treated human blood type A" red blood cells demonstrates significantly less antibody binding by mPEG-modified red blood cells.
  • the control and mPEG red blood cells were mixed with an IgG anti-A antibody incubated for 30 minutes. The samples were extensively washed and a secondary antibody (anti-human IgG conjugated with alkaline phosphatase) was added to quantitate bound anti-Blood group A antibody.
  • EXAMPLE IV Inhibition of Phagocytosis of Foreign Cells: mPEG-modified sheep red blood cells are significantly less prone to phagocytosis by human peripheral blood monocytes (Fig. 7). As would be indicated by decreased antibody binding (Fig. 6), mPEG-modified sheep red blood cells are significantly less susceptible to IgG-mediated phagocytosis by human peripheral blood monocytes. mPEG-modified sheep red blood cells were incubated with human peripheral blood monocytic cells for 30 minutes. The uningested red blood cells were removed by hypotonic lysis and the number of monocytes containing sheep red blood cells, as well as the number of sheep red blood cells ingested, were determined microscopically. EXAMPLE V mPEG-Derivatized Mouse Red Blood Cells Have Normal In Vivo Survival:
  • EXAMPLE VI mPEG-Derivatization of Sheep Red Blood Cells Results in Enhanced In Vivo Survival in Mice: Comparable numbers of mPEG-modified sheep red blood cells (mPEG- sRBC) were injected i.p. into BALB/C mice. As shown in Fig. 9, mPEG-sRBC showed a greater rate of entry into the peripheral circulation and demonstrated longer in vivo survival in mice. In vivo survival of mPEG-sRBC in mice was determined using a fluorescent fatty acid label (PKH-26; Sigma Chemical Company). Blood was obtained from a donor sheep and treated with 0 or 6 mg/ml activated mPEG and washed thrice.
  • PSH-26 fluorescent fatty acid label
  • the mixed lymphocyte culture is a very sensitive measure of histocompatibility between donor and recipient. Indeed, though time consuming, this assay is perhaps the best indicator of the probability of tissue transplant survival in the organ recipient. Primarily the MLC measures the antigenic variance between the HLA complex (the primary antigens responsible for tissue compatibility in transplants) between two individuals. As shown in Figure 10, covalent modification with mPEG of lymphocytes from either donor results in a virtually complete inhibition of recognition of the antigenically foreign lymphocytes. Shown is the proliferation, measured by 3 H-thymidine incorporation into DNA, of responder cells in response to a fixed concentration (2.5 x 10 5 PBMC) of stimulator (i.e., cells irradiated to prevent cell replication).
  • stimulator i.e., cells irradiated to prevent cell replication.
  • Panel A demonstrates PBMC Donor A's response to antigenically foreign Donor B PBMC.
  • Panel B demonstrates Donor B's response to Donor A.
  • the population of responder (i.e., nonirradiated) cell expands tremendously in response to control irradiated PBMC (peripheral blood mononuclear cells).
  • mPEG platelet rich plasma
  • ADP control unactivated platelets
  • mPEG derivatized platelets do not aggregate in response to activation by ADP (5 ⁇ M). While control platelets are fully aggregated within approximately 2 minutes, mPEG-modified platelets remain unaggregated even after 7 minutes of exposure to ADP.
  • the loss of aggregation is mediated by disruption of celhcell interaction (i.e., preventing platelet interaction and microaggregate formation). Indeed, alteration in celhcell interaction is a primary event due to the covalent modification of cell surfaces with non-immunogenic materials.
  • a breast carcinoma epithelial cell line (MCF7) was examined.
  • ESA epithelial specific antigen
  • Mouse anti-human ESA binding was quantitated using a BD-FACScan.
  • FITC-conjugated goat anti- mouse antibody was used to detect bound ESA.
  • Epithelial cell concentration was 5 x 10 5 cells/ml with a 1 :6000 titre of anit-ESA antibody.
  • Epithelial cells were derivatized using a modification of the RBC-derivatization protocol.
  • confluent monolayers of MCF7 cells were scraped from tissue culture flasks and suspended in RPMI media.
  • the cell suspensions were incubated with increasing concentrations of activated mPEG at pH 8.0 and incubated at room temperature for 60 minutes.
  • the cells were then washed 3 x with culture media prior to the antibody binding assay.
  • non- immunogenic materials e.g., mPEG
  • covalent modification of the external cell membrane with non- immunogenic materials effectively "hides” both major and minor antigenic determinants on a large variety of nucleated and anucleated cells.
  • non-immunogenic materials e.g., RBC, endothelial cells, epithelial cells, pancreatic ⁇ cells, etc.

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Abstract

L'invention se rapporte à une composition cellulaire non immunogène qui comprend: une cellule, notamment une particule virale ou un virus, possédant une surface cellulaire et portant sur cette surface des déterminants antigéniques; éventuellement, une molécule de liaison liée par covalence à la surface cellulaire; et un composé non immunogène (tel que du polyéthylèneglycol ou un dérivé de celui-ci) lié par covalence à la molécule de liaison ou directement fixé à la cellule. Dans un mode de réalisation, la molécule de liaison est directement liée par covalence au déterminant antigénique sur la surface cellulaire. Dans un autre mode de réalisation, la molécule de liaison est liée par covalence à un site non antigénique sur la surface cellulaire et, en même temps, camoufle le déterminant antigénique sur ladite surface cellulaire. L'invention concerne également divers usages de la cellule virale non immunogène obtenue de cette manière, notamment une méthode permettant de lutter contre la phagocytose d'une cellule virale, une méthode permettant d'atténuer les effets indésirables d'une transfusion, une méthode permettant de lutter contre le rejet d'une cellule virale, d'un tissu ou d'un organe transplanté et une méthode permettant de lutter contre l'agrégation de cellules induite par des anticorps.
PCT/US1998/013198 1997-06-26 1998-06-25 Modulation antigenique de particules virales WO1999000145A1 (fr)

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Cited By (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP2114138A2 (fr) * 2007-02-09 2009-11-11 Canadian Blood Services Stockage à froid de plaquettes modifiées
EP2163261A1 (fr) * 2008-07-14 2010-03-17 Canadian Blood Services Stockage à basse température de plaquettes modifiées au PEG
US8048620B2 (en) * 2006-12-22 2011-11-01 Canadian Blood Services Method for enhancing the detection of contamination in a cellular blood product by covalently binding a hydrophilic polymer to the cell membrane
US8067151B2 (en) 2007-02-09 2011-11-29 Canadian Blood Services Cold storage of pegylated platelets at about or below 0° C.
WO2016109306A1 (fr) * 2015-01-02 2016-07-07 Cellics Therapeutics, Inc. Utilisation de nanoparticules revêtues avec des membranes d'érythrocytes pour permettre une transfusion sanguine

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WO1996041606A2 (fr) * 1995-06-08 1996-12-27 Therexsys Limited Compositions pharmaceutiques ameliorees utilisees pour la therapie genique
WO1997028254A1 (fr) * 1996-02-01 1997-08-07 Biomedical Frontiers, Inc. Modulation antigenique de cellules
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JEONG S T ET AL: "DECREASED AGGLUTINABILITY OF METHOXY-POLYETHYLENE GLYCOL ATTACHED RED BLOOD CELLS: SIGNIFICANCE AS A BLOOD SUBSTITUTE", ARTIFICIAL CELLS, BLOOD SUBSTITUTES, AND IMMOBILIZATION BIOTECHNOLOGY, vol. 24, no. 5, 1996, pages 503 - 511, XP000673685 *
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Cited By (8)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US8048620B2 (en) * 2006-12-22 2011-11-01 Canadian Blood Services Method for enhancing the detection of contamination in a cellular blood product by covalently binding a hydrophilic polymer to the cell membrane
EP2114138A2 (fr) * 2007-02-09 2009-11-11 Canadian Blood Services Stockage à froid de plaquettes modifiées
US7964339B2 (en) * 2007-02-09 2011-06-21 Canadian Blood Services Cold storage of modified platelets
US8067151B2 (en) 2007-02-09 2011-11-29 Canadian Blood Services Cold storage of pegylated platelets at about or below 0° C.
EP2114138A4 (fr) * 2007-02-09 2012-01-18 Canadian Blood Services Stockage à froid de plaquettes modifiées
EP2163261A1 (fr) * 2008-07-14 2010-03-17 Canadian Blood Services Stockage à basse température de plaquettes modifiées au PEG
WO2016109306A1 (fr) * 2015-01-02 2016-07-07 Cellics Therapeutics, Inc. Utilisation de nanoparticules revêtues avec des membranes d'érythrocytes pour permettre une transfusion sanguine
US10434070B2 (en) 2015-01-02 2019-10-08 Cellics Therapeutics, Inc. Use of nanoparticles coated with red blood cell membranes to enable blood transfusion

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