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WO1993006848A1 - IMMUNOADHESINES DE CD36 ET LEUR UTILISATION POUR TUER DE MANIERE SELECTIVE LES ERYTHROCYTES INFECTES PAR $i(PLASMODIUM FALCIPARUM) - Google Patents

IMMUNOADHESINES DE CD36 ET LEUR UTILISATION POUR TUER DE MANIERE SELECTIVE LES ERYTHROCYTES INFECTES PAR $i(PLASMODIUM FALCIPARUM) Download PDF

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Publication number
WO1993006848A1
WO1993006848A1 PCT/US1992/008482 US9208482W WO9306848A1 WO 1993006848 A1 WO1993006848 A1 WO 1993006848A1 US 9208482 W US9208482 W US 9208482W WO 9306848 A1 WO9306848 A1 WO 9306848A1
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WO
WIPO (PCT)
Prior art keywords
irbc
icam
binding
agent
antibody
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Application number
PCT/US1992/008482
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English (en)
Inventor
Donald E. Staunton
Timothy A. Springer
Original Assignee
The Center For Blood Research
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Publication of WO1993006848A1 publication Critical patent/WO1993006848A1/fr

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    • 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
    • C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
    • 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
    • C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
    • C07K14/70596—Molecules with a "CD"-designation not provided for elsewhere
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
    • Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
    • Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change

Definitions

  • the present invention relates to agents which bind to the ICAM-1 or the CD36 binding site on malarially infected eiythrocytes (IRBC).
  • the agents of the present invention include antibodies, peptides, and carbohydrates. These agents are useful in ameliorating the symptoms of malaria since they are capable of inhibiting the binding of an IRBC to either ICAM-1 or CD36 and stimulating the phagocytosis of IRBCs.
  • the present invention further provides methods for the treatment of malaria, methods of preferentially killing an IRBC, methods of stimulating phagocytosis of an IRBC, and a method of diagnosing the presence of an IRBC.
  • IRBC malaria-infected eiythrocytes
  • ICAM-1 intercellular adhesion molecule- 1
  • CD54 Bosset et al, Nature (Lond.) 341:57-59 (1989)
  • CD36 GPIV
  • ICAM-1-coated and CD36-coated surfaces have recently been identified as endothelial receptors for IRBC.
  • Laboratory-adapted IRBC bind to purified ICAM-1-coated and CD36-coated surfaces and the cytoadherent phenotype of these malaria-infected red cells can be modulated by successive panning on ICAM-1 or CD36-coated surfaces (Ockenhouse et al, J. Infect. Dis. 164:163-169 (1991)).
  • ICAM-1 intercellular adhesion molecule- 1
  • CD36 GPIV
  • ICAM-1-specific and CD36-specific monoclonal antibody (MAb) staining of small capillary endothelium from postmortem brain tissue colocalizes with IRBC cytoadherence in patients who have died from complications of cerebral malaria (Barawell et al, J. Clin. Invest. 84:165-111 (1989); Aikawa et al, Am. J. Trop. MedL Hyg. 43:30 (1990)).
  • ICAM-1-specific and CD36-specific monoclonal antibody (MAb) staining of small capillary endothelium from postmortem brain tissue colocalizes with IRBC cytoadherence in patients who have died from complications of cerebral malaria (Barawell et al, J. Clin. Invest. 84:165-111 (1989); Aikawa et al, Am. J. Trop. MedL Hyg. 43:30 (1990)).
  • MAb monoclonal antibody
  • ICAM-1 a member of the imm ⁇ noglobulin-like superfamily, is a monomeric unpaired 90-115 ML. glycoprotein composed of a bent extracellular domain containing five tandemly arranged immunoglobulin- like domains, a transmembrane region, and a cytoplasmic domain (Staunton et al, Cell 52:925-933 (1988); Simmons et al, Nature (Lond.) 331:624-621 (1988)). ICAM-1 is a ligand for the leukocyte integrins, lymphocyte function antigen-1 (LFA-1; CDlla/CD18) (Rothlein et al, J. Immunol 137:1210-1214- (1986); Marlin et al, Cell 52:813-819 (1987)) and
  • Mac-1 (CDllb/CD18) (Diamond et al, J. Cell Biol. 111:3219-3139 (1990); Smith et al, J. Clin. Invest 83:2008-2011 (1989)).
  • the recognition, adhesion, and extravasation of lymphoid and myeloid blood cells through the vascular endothelium is an initial step of host immune response to tissue injury.
  • the CD11/CD18 family of proteins are crucial for leukocyte and myeloid cell adhesion to endothelium, T cell activation, cytotoxic T cell killing, and neutrophil chemotaxis and homotypic aggregation (Larsen et al, Immunol Rev. 114:181 (1990)).
  • ICAM-1 is also subverted as a cellular receptor by the major group of human rhinoviruses (HRV),..lhe etiologic agent of the common cold (Staunton et al, Cell 5 ⁇ 5:849-853
  • red blood cells infected with mature intracellular forms of the malaria parasite bind to a region located within the ammo-terminal immunoglobulin-like domain of ICAM-1 that is distinct from the regions recognized by LFA-1 and rhinovirus (Ockenhouse et al, Cell 68:63-69 (1992); and Berendt et al, Cell 68:11-81 (1992)).
  • ICAM-1 has a restricted distribution in vivo, and its expression is regulated by LPS and the cytokines TNF, TL- l ⁇ , and interferon-gamma
  • TNF up regulate the surface expression of ICAM-1 and support adhesion of malaria-infected eiythrocytes (Berendt et al, Nature (Lond.) 341:51-59 (1989)).
  • individuals with cerebral malaria have higher levels of plasma TNF than individuals with uncomplicated malaria or uninfected controls.
  • an inflammatory response initiated in response to malarial infection is used to the parasites' advantage by selectively modulating the expression of receptors to which parasitized eiythrocytes attach.
  • the receptor binding site on IRBC surfaces should be conserved and selective pressure exerted to maintain minimal structural variation unless compensatory binding to alternate receptors occur.
  • Sequestration of malaria-infected eiythrocytes to host endothelium occurs in all persons infected with the parasite regardless of clinical severity. A small percentage of infected individuals, independent of parasitemia, progress to complicated and severe forms of the disease. The precise factors and mechanisms responsible for severe malaria are unknown. While the majority of parasitized eiythrocytes from naturally-acquired infections bind only to CD36 in vitro, a smaller subpopulation of parasitized eiythrocytes from some isolates bind to ICAM-1 and CD36.
  • IRBC bind to different receptors in different tissues depending upon the genetic regulation of host cellular receptors and the parasite cytoadherent phenotype as expressed by single or multiple counter-receptors. Deleterious effects to the host result from the sequestration o a numerically smaller proportion of IRBC expressing the pertinent counter- receptor within a population of parasitized red cells directing the binding of IRBC to capillary endothelium within the brain leading to cerebral malaria.
  • Antigenically diverse naturally-acquired malaria isolates demonstrate serologically defined infected erythrocyte surface epitopes.
  • the present invention discloses the binding site on ICAM-1 for Plasmodium falciparum-miected erythrocytes.
  • An IRBC binds to the first NH 2 -terminaI domain of human but not mouse ICAM-1.
  • the present invention discloses that small peptides, corresponding to a contiguous sequence of ICAM-1, are capable of inhibiting the binding of an IRBC to ICAM-1.
  • the binding sites within domain 1 reside spatially distant from the recognition sites for LFA-1 and HRV.
  • a therapeutic strategy directed toward reversing parasite sequestration ultimately can protect infected individuals from the deleterious complications of vascular occlusion.
  • anti-receptor soluble ICAM-1 analogues based upon the critical contact residues for IRBC can now be engineered to bind, lyse, and kill sequestered intraerythrocytic parasites in cases of severe and complicated falciparum malaria, as well as diagnosis of the presence of malaria.
  • the two primary sites an IRBC can bind to on a non-infected .cell are ICAM-1 and CD36. Therefore, the binding of an IRBC to an umnfected cell can be inhibited by providing to the cells an agent capable of binding to the ICAM-1 binding site on the IRBC, the IRBC binding site on ICAM-1, the CD36 binding site on the IRBC, or to the IRBC binding site on CD36.
  • the complications arising from malaria can be ameliorated.
  • the agents of the present invention include: (a) agents which are capable of binding to the ICAM-1 binding site on an IRBC, said agents selected from the group consisting of ICAM-1, a fragment of ICAM-1, a functional derivative thereof, a peptide, an antibody, or a carbohydrate;
  • agents which are capable of binding to the IRBC binding site on ICAM-1 said agents selected from the group consisting of a peptide, an antibody, or a carbohydrate
  • agents which are capable of binding to the CD36 binding site on an IRBC said agents selected from the group consisting of CD36, a fragment of CD36, a functional derivative of CD36, a peptide, an antibody, or a carbohydrate;
  • agents which are capable of binding to the IRBC binding site on CD36 said agents selected from the group consisting of a peptide, an antibody, or a carbohydrate.
  • the present invention includes the peptide agent whose amino acid sequence is: GSVLVT (SEQ ID NO 1). This agent is capable of binding to the ICAM-1 binding site of an IRBC.
  • the invention further includes a method for producing a desired hybridoma cell that produces an antibody which is capable of binding to the IRBC binding site on ICAM-1, the ICAM-1 binding site of an IRBC, the IRBC binding site on CD36, or the CD36 binding site of an IRBC.
  • the invention further includes chimeric proteins comprising ICAM, or fragments thereof, or CD36, or fragments thereof, fused to an immunoglobulin or a fragment thereof.
  • ICAM-1 fusion protein herein designated F185G1
  • CD36 fusion protein herein designated D30F492
  • D30F492 consists of soluble-CD36 fused to the hinge region and constant domains CH2 and
  • the invention further includes a method of stimulating phagocytosis of an IRBC in a patient with malaria comprising administering to said patient a therapeutically effective amount of a fusion protein comprising ICAM-1, or a fragment thereof, and/or CD36, or a fragment thereof each of which is fused to an immunoglobulin or a fragment thereof.
  • ICAM-1 by overlapping synthetic hexapeptides. ItG-ICAM IRBC and
  • ICAM-1 hexapeptides (500 ug ml) were added to ICAM-1 coated plates for 60 minutes. The peptides were acetylated at the N-terminus, amidated at the C-terminus. Aba is alpha amino butyric acid and is substituted in sequence for Cys. Results represent the mean ⁇ s.d. of three determinations and are compared to control IRBC binding to ICAM-1 in absence of peptides.
  • FIG. 1 A. Schematic diagram of the F185G1 expression construct (pCDF185Gl) and the F185G1 immunoadhesion.
  • CD36-binding IRBC p eincubated with F185G1 chime a bind to the monocyte surface but are not phagocytosed.
  • ICAM-1-binding IRBC preincubated with F185G1 chimera are phagocytosed and internally degraded by monocytes.
  • D. ICAM-1-binding IRBC in the absence of ICAM-1 immunoadhesin are not phagocytosed by monocytes.
  • Amino acid sequence of CD36 The residue used in generating the chimeras are identified in the sequence.
  • the present invention is based on the identification of the two primary binding sites an IRBC can bind to on a non-infected cell. These sites are contained on ICAM-1 and CD36.
  • the present invention discloses that the binding site on ICAM-1 for Plasmodium falciparum-wizcted eiythrocytes is the first NH 2 -te ⁇ ninal domain between residues Gly ⁇ Ser 22 of human, but not mouse, ICAM-1. Further, it is disclosed herein that a peptides with an amino acid sequence selected from this region, can block the binding of an IRBC to ICAM-1. Utilizing the amino acid sequence of the binding site, the present invention provides agents and methods for the treatment and diagnosis of malaria.
  • the present invention includes:
  • agents which are capable of binding to the ICAM-1 binding site on an IRBC said agents selected from the group consisting of ICAM- 1, a fragment of ICAM-1, a peptide, an antibody, or a carbohydrate;
  • agents which are capable of binding to the IRBC binding site on ICAM-1 said agents selected from the group consisting of a peptide, an antibody, or a carbohydrate;
  • agents which are capable of binding to the CD36 binding site on an IRBC, said agents selected from the group consisting of CD36, a fragment of CD36, a peptide, an antibody, or a carbohydrate; and
  • agents which are capable of binding to the IRBC binding site on CD36 said agents selected from the group consisting of a peptide, an antibody, or a carbohydrate.
  • agents which are capable of stimulating phagocytosis of an IRBC said agents selected from the group consisting of an immunoglobulin, or fragment thereof, fused to ICAM-1, a fragment thereof; CD36, or a fragment thereof.
  • agents are capable of blocking the binding of an IRBC to either ICAM-1 or CD36.
  • the present invention includes functional derivatives of the above described agents.
  • a “functional derivative” of an agent of the present invention is an agent which possesses a biological activity that is substan ⁇ tially similar to the biological activity of the agent it is a derivative of. For example, if the agent is capable of binding to the ICAM-1 binding site of an IRBC, then the functional derivative will possess this binding ability.
  • the term “functional derivative” includes "fragments,” “variants,” and “chimeras” of the parent molecule.
  • fragment of an agent is meant to refer to any subset of the agent it is derived from. Fragments of ICAM-1 or CD36 which contain
  • Soluble fragments of CD36 or ICAM-1 can be rationally designed by one skilled in the ar Generally, soluble fragments are generated by deleting the trans membrane regions of the molecule. Additionally, some of the more hydrophobic regions of the protein can be deleted.
  • a "variant" of a molecule is meant to refer to a molecule substantially similar in structure and function to either the entire molecule, or to a fragment thereof.
  • a molecule is said to be "substantially similar” to another molecule if both molecules have substantially similar structures or if both molecules possess a similar biological activity. Thus, provided that two molecules possess a similar activity, they are considered variants, as that term is used herein, even if the sequence of amino acid residues is not identical.
  • an agent is said to be a "chimeric-agent" if the agent possesses a structure not found in the agent it is derived from.
  • additional structures are added to a parent agent in order to improve one of the agent's physical properties such as solubility, absorption, biological half life, etc., to eliminate or decrease one of the agent's undesirable properties or side effects such as immunogenicity or toxicity, or to add a property to the agent which is not present in the parent agent such as the ability to stimulate a biological effector function such as phagocytosis, complement-dependent cytolysis (CDC), antibody-dependent, cell- mediated cytotoxicity (ADCC), etc.
  • Moieties capable of mediating such effects are disclosed in Remington's Pharmaceutical Sciences (1980).
  • One type of chimeric-agent are "chemical-derivatives.” Chemical- derivatives contain one or more additional chemical moieties which are not part of the naturally occurring agent
  • Toxin-derivatized agents constitute a special class of chemical- derivatives. Toxin-derivatives contain an agent of the present invention covalently attached to a toxin moiety. Procedures for coupling such moieties to a molecule are well known in the art and are generally performed in situ.
  • toxin-derivatized agent The binding of a toxin-derivatized agent to a cell brings the toxin moiety into close proximity to the cell and thereby promotes cell death.
  • Any suitable toxin moiety may be employed; however, it is preferable to employ toxins such as, for example, the ricin toxin, the cholera toxin, the diphtheria toxin, radioisotopic toxins, or membrane-channel-fo ⁇ ning toxins.
  • Protein-derivatized agents constitute another type of chimeric- agent.
  • Protein-derivatives contain one or more additional peptide moieties which are not part of the naturally occurring agent Protein derivatives may be generated in situ using chemical means or in vivo using recombinant DNA techniques.
  • Antibody-derivatized agents constitute a special class of protein- derivative.
  • Antibody-derivatives contain an agent of the present invention covalently attached to an antibody or antibody fragment Procedures for coupling such moieties to a molecule are well known in the art.
  • the binding of an antibody-derivatized agen£ to a cell brings the antibody or antibody fragment into close proximity to the cell.
  • the antibody fragment will promote cell death by stimulating a biological effector function such as phagocytosis.
  • Any suitable antibody or antibody fragment may be employed depending on the effector function which is to be stimulated (see Bruggeman et al, J. Exp. Med 26 " tf:1351-1361 (1987) for a review of effector functions); however, it is preferable to emplo a fragment which contains the constant domain of one of the antibody chains such as the hinge and constant regions CH2 and CH3 of the human IgGl heavy chain.
  • Antibody derivatives of CD36 are, in general, generated byligating a DNA sequence encoding a fragment of the entire CD36 molecule into a vector which contains a signal peptide and sequences encoding the desired antibody fragment
  • the fragments of CD36 which are used in such constructs are preferably deleted for the hydrophobic regions of CD36, residues 6-28 and 439-465. In one aspect of this embodiment residues 1-6 and 466-471 are also deleted.
  • the preferred fragments of CD36 start with an amino acid residue selected from the group consisting of D30, Q34, Q40, G46, or F50 of CD36 and continue to an amino acid residue selected from the group consisting of N416, F429, V433, G435, or L439.
  • Functional derivatives of the peptide agents of the present invention having an altered amino acid sequence include insertions, deletion, and substitutions in the amino acid sequence of the agent These can be prepared by synthesizing a peptide with the desired sequence. While the site for introducing an alteration in the amino acid sequence is predetermined, the alteration per se need not be predetermined. For example, to optimize the performance of altering a given sequence, random changes can be conducted at a target amino acid residue or target region to create a large number of derivative which can then be screened for the optimal combination of desired activity.
  • IRBC binding site on ICAM-1 is made by synthesizing a polypeptide containing an alteration in the amino acid sequence of ICAM-1. The peptide is then screened for the ability to block IRBC binding to immobilized ICAM-1. Additionally, other screening assays known in *he art can be employed to identify a change in a specific characteristic of the agent such as a change in the immunological character, affinity, redox or thermal stability, biological half-life, hydrophobicity, or susceptibility to proteolytic degradation of the functional derivative.
  • soluble derivatives of the agents of the present invention which are especially preferred are soluble derivatives.
  • soluble derivatives of a molecule are generated by deleting transmembrane spanning regions or by substituting hydrophilic for hydrophobic amino acid residues.
  • Another class of derivatives of the agents of the present invention which are based on CD36 which are especially preferred are those agents which lack the normal CD36 collagen binding site.
  • Such derivatives can be created by generating random mutations via site directed or random mutagenesis and then screening the derivatives for their inability to bind collagen.
  • site directed mutagenesis directed to regions suspected of containing the collagen binding site can be performed.
  • the collagen binding site can be identified by, comparing the amino acid sequence of CD36 with other collagen binding proteins to identify regions of homology, analyzing the amino acid sequence of CD36 for regions which from disulfide bridges, or by cross linking collagen to
  • the agents of the present invention may be obtained by: natural processes (for example, by inducing an animal, plant fungi, bacteria, etc., to produce a peptide corresponding to a particular sequence, or by inducing an animal to produce polyclonal antibodies capable of binding to a specific amino acid sequence); synthetic methods (for example- by synthesizing a peptide corresponding to the IRBC binding site on ICAM-
  • the antibodies of the present invention can be generated by a variety of techniques known in the art
  • the antibodies of the present invention include monoclonal and polyclonal antibodies, as well fragments and humanized forms of these antibodies.
  • Humanized forms of the antibodies of the present invention may be generated using one of the procedures known in the art such as chimerization or CDR grafting.
  • the invention provides an antibody, and especially a monoclonal antibody, capable of binding to a molecule selected from the group consisting of the IRBC binding site on ICAM-1, the ICAM-1 binding site on an IRBC, the IRBC binding site on CD36, and the CD36 binding site on an IRBC.
  • An antibody which binds to the IRBC binding site on ICAM-1 can be generated using a synthetic polypeptide whose amino acid sequence is identical to the amino acid sequence of the IRBC binding site on ICAM-1 as an antigen for immunizing an animal.
  • One such peptide for generating an antibody which binds to the IRBC binding site on ICAM-1 has the following amino acid sequence: GSVLVT (SEQ ID NO 1).
  • An antibody which binds to the ICAM-1 binding site on an IRBC can be generated by immunizing an animal with an IRBC. The antisera is then screened for its ability to block an IRBC from binding to immobilized ICAM-1.
  • An antibody which binds to the CD36 binding site on an IRBC can be generated by immunizing an animal with an IRBC. The antisera is then screened for its ability to block an IRBC from binding to immobilized CD36.
  • An antibody which binds to the IRBC binding site on CD36 can be generated by immunizing an animal with CD36. The antisera is then screened for its ability to block an IRBC from binding to immobilized CD36.
  • One skilled in the art will be able to readily obtain both polyclonal and monoclonal antibodies with the above described specificities using procedures known in the art (Lutz et al, Exp. Cell Res. 275:109-124 (1988), Campbell, A.M., Monoclonal Antibody Technology: Laboratory Techniques in Biochemistry and Molecular Biology, Elsevier Science Publishers, Amsterdam, The Netherlands (1984).
  • the polypeptide may be modified or administered in an adjuvant in order to increase the peptide antigenicity.
  • Methods of increasing the antigenicity of a polypeptide are well known in the art Such procedures include coupling the antigen with a heteroiogous protein (such as globulin or ⁇ -galactosidase) or through the inclusion of an adjuvant during immunization.
  • a heteroiogous protein such as globulin or ⁇ -galactosidase
  • the peptides of the present invention can be generated by a variety of techniques known in the art
  • the peptides of the present invention include peptides whose amino acid sequence is substantially homologous to the naturally occurring binding sites disclosed herein as well as peptides generated through rational design which possess a desired binding specificity but differ significantly in amino acid sequence from the naturally occurring binding site.
  • a peptide is said to have an amino acid sequence substantially homologous to another if, due to the presence of common amino acid residence in homologous positions, the two peptides share common biological of physical property.
  • techniques for preparing synthetic peptides with a defined sequence or structure are well known in the art
  • the peptides of the present invention whose amino acid sequences are substantially homologous to the naturally occurring binding site include; the ICAM-1 binding site of an IRBC, the CD36 binding site of an IRBC, the IRBC binding site on ICAM-1, and the IRBC binding site on ICAM-1.
  • SEQ ID NO 1 One such peptide, SEQ ID NO 1, has an amino acid sequence which is homologous to the IRBC binding site on ICAM-1.
  • peptides whose sequence, are substantially homologous to the naturally occurring binding site can readily generate, through rational design, peptides that possesses the ability to bind to a specific amino acid sequence or antigenic epitope (Hodgson, J, Biotechnology 5:1245-1247 (1990)).
  • Computer modeling systems are available that allow one skilled in the art to design a peptide which is able to bind to the specific regions and sequences disclosed herein.
  • the peptide which are made according to this method can be readily screened for a desired specificity and physical properties.
  • carbohydrates can be rationally designed to block protein/protein binding (Hodgson,J. Biotechnology 9:609-613 (1991)).
  • carbohydrate can now be designed to block an IRBC from binding to ICAM-1 or to block an IRBC from binding to CD36.
  • the invention includes the use of the agents disclosed herein a) to inhibit the binding of an IRBC to a non-infected cell, and b) to preferentially kill an IRBC
  • the binding of an IRBC to ICAM-1 can be inhibited by providing an effective amount of an agent capable of binding to either the IRBC binding site on ICAM-1 or the ICAM-1 binding site on a IRBC.
  • the binding of an IRBC to CD36 can be inhibited by providing an effective amount ⁇ _£ an ageat capable of binding to either the IRBC binding site on CD36 or the CD36 binding site on a IRBC.
  • An example of an agent capable of inhibiting the binding of an IRBC to ICAM-1 is a peptide whose sequence is shown in SEQ ID NO 1.
  • An IRBC can be preferentially killed by providing an IRBC with a toxin derivatized agent which is capable of selectively binding the IRBC.
  • a toxin derivatized agent which is capable of selectively binding the IRBC.
  • agents include a peptide of SEQ ID NO 1 or an antibody which is capable of binding to either the ICAM-1 or the CD36 binding site on an IRBC oovalently liked to a toxin such as ricin.
  • the IRBC can be preferentially killed.
  • an IRBC can be preferentially killed by utilizing a mammal's natural defense systems.
  • the constant regions of the antibody moiety of the antibody- derivative agent wiB stimulate biological activities such as phagocytosis, CDC, and ADCC
  • such agents include F185G1 and D30F429 which consist of the hinge region and constant domains CH2 and CH3 of the human IgGl heavy chain covalently linked to a soluble derivative of ICAM-1 or CD36 respectively.
  • agents of the present invention may be administered to a mammal singly or in combination with each other. Most preferably, an agent based on ICAM-1 is administered in combination with an agent based on CD36.
  • the agents of the present invention may be administered intravenously, intramuscularly, subcutaneously, enterally, topically or other non-enteral means.
  • the administration may be by continuous injections, or by single or multiple injections.
  • the agents of the present invention are intended to be provided to recipient mammal in a "pharmaceutically acceptable form" in an amount sufficient to "therapeutically effective.”
  • An amount is said to be therapeutically effective if the dosage, route of administration, etc. of the agent are sufficient to block the binding of an IRBC with a defined molecule or is sufficient to kill a portion of the IRBCs present in the mammal.
  • an agent of the present invention when provided to a mammal to block the binding of an IRBC to ICAM-1 is said to be therapeutically effective if it is provided in sufficient dosage to block IRBC/ICAM-1 binding.
  • the administration of the agents of the present invention may be for either a "prophylactic" or "therapeutic" purpose.
  • the agent When provided prophylactically, the agent is provided in advance of any malaria symptomology.
  • the prophylactic administration of the agent serves to prevent or attenuate any subsequent spread of the malaria parasite.
  • the agent When provided therapeutically, the agent is provided at (or shortly after) the onset of a symptoms of the actual infection.
  • the therapeutic administration of the compound(s) serves to attenuate or ameliorate any actual symptoms.
  • agents of the present invention can be formulated according to known methods of preparing pharmaceutically useful compositions, whereby these materials, or their functional derivatives, are combined with a pharmaceutically acceptable carrier vehicle.
  • Suitable vehicles and their formulation, inclusive of other human proteins, e.g., human serum albumin, are described, for example, in Remington's Pharmaceidical Sciences (16th ed., Osol, A., Ed., Mack, Easton PA (1980)).
  • a pharmaceutically acceptable composition which is suitable for effective ad ⁇ ministration, such compositions will contain an effective amount of an agent of the present invention together with a suitable amount of carrier.
  • the antibodies of the present invention may be supplied in humanized form, through chimerization or CDR grafting. when administered to a human in order that the antibody is in a more
  • Control release preparations may be achieved through the use of polymers to complex or absorb the agents of the present invention.
  • the rate and duration of the controlled delivery may be regulated to a certain extent by selecting an appropriate macromolecule matrix, by varying the concentration of macromolecules incorporated, as well as the methods of incorporation.
  • Another possible method to control the duration of action by controlled release preparations is to incorporate the agents of the present invention into particles of a polymeric material, such as polyesters, polyamino acids, hydrogels, poly(lactic acid) or ethylene vinyl acetate copolymers.
  • microcapsules prepared, for example, by coacervation techniques or by interfacial polymerization, by gelatine or poly(methylmethacylate) microcapsulation, or in colloidal drug delivery systems, for example, liposomes, albumin microspheres, microemulsions, nanoparticles, and nanocapsules or in macroemulsions.
  • the agents of the present invention can be used to; a) diagnose the presence of an IRBC in a mammal, and b) determine the location of the IRBC in a mammal.
  • One skilled in the art can: a) detectably label the agents of the present invention using radioisotopes, affinity labels (such as biotin, avidin, etc.), enzymatic labels (such as horse radish peroxidase, alkaline phosphatase, etc.) fluorescent labels (such as FITC or rhodamine, etc.), or paramagnetic atoms, using procedures well-known in the art, for example see Sternberger, L.A. et al, J. Histochem. Cytochem. 18:315 (1970), Bayer, E.A. et al, Meth. Enzym. 62:308 (1979), Engval, E. et al,
  • the agents of the present invention can be used to: a) assay for the presence of an IRBC in vivo as well as in vitro; and b) localize the presence of an IRBC to a specific location in vivo.
  • the labeled agents of the present invention can readily incorporate into any of the currently available in vivo or in vitro assay formats such as an ELISA assay, a latex agglutination assay, and magnetic resonance imaging.
  • the agents of the present invention can be used to: a) purify an IRBC from a population containing non-infected cells; and b) be used in the assay formats described above.
  • An IRBC can be purified from a population of cells using affinity chromatography. Specifically, an infected cell expressing either the ICAM-
  • CD36 binding site can be isolated from a mixture of cells by passing the cells over a column which contains an immobilized agent capable of binding the ICAM-1 or CD36 binding site present on the infected cell.
  • the intercellular adhesion molecule-1 (ICAM-1, CD54) has been implicated as a cytoadhesion receptor for Plasmodium falciparum-infected erythrocytes. Wild type and mutant ICAM-1 expressed in COS cells were examined for binding to laboratory-adapted and naturally-acquired malaria-infected erythrocytes. Domain deletion, human-mouse chimeric ICAM-1 molecules, and amino acid substitution mutants localized the primary binding site for parasitized erythrocytes to the first NH 2 -terminal immunoglobulin-like domain of ICAM-1. The ICAM-1 binding sites are distinct from those recognized by LFA-1, Mac-1, and the human major-type rhinoviruses.
  • Oligonucleotide-directed mutagenesis (Kunkel, T.A., Proc. Natl. Acad Sci USA 52:488-492 (1985)) was used to generate IC .M-1 deletion, chimeric, and amino acid substitution mutants as described (Staunton et al, Cell (52:243-254 (1990)). Transfection of COS Cells
  • COS cells at 50% confluency were tiansfected by the DEAE- dextran method using vector alone or vector containing wild-type or mutant ICAM-1 cDNA.
  • COS cells were harvested 72 hours after transfection and the efficiency of transfection of ICAM-1 constructs was analyzed by indirect immunofluorescence and flow cytometry using anti- human ICAM-1 MAbs CL203 (Maio et al, J. Immunol 245:181-185 (1989)) (a gift of Dr. S. Ferrone), and RR1/1 (Dustin et al, J. Immunol 257:245-254 (1986)); and anti-murine MAb YNl/1 (Takei, F., J. Immunol
  • ICAM-1 peptides Pro ⁇ -Thr 23 and overlapping hexapeptides spanning residues Gln l -Thr 23 were synthesized on an Applied Biosystems peptide synthesizer.
  • Transfected COS cells in RPMI 1640 plus 10% fetal bovine serum were reseeded (2.5 - 4xl0 4 /well) 24-48 hours prior to assay into 24-well tissue culture plates at 37 ⁇ C in 5% CO,.
  • Malaria-infected erythrocytes 400 ul/well; 2% hematocrit; 20-35% parasitemia
  • Unattached erythrocytes were removed by rinsing the wells with RPMI 1640.
  • the anti-ICAM-1 MAbs CL203 or RR1/1 were added to each well. After 45 minutes incubation at room temperature, the wells were washed twice with RPMI 1640, and the cells were fixed with an ice-cold acetone-methanol (50% v/v) mixture for one minute. Cells were rinsed with PBS and colloidal gold-labelled anti- mouse antibody (Amersham, Arlington, IL) was added to each well for 30 minutes, followed by three washes with phosphate-buffered saline.
  • IRBC binding to ICAM-1-coated or CD36-coated surfaces was performed as follows. Soluble ICAM-1 (lOug/ml) (Marlin et al, Nature
  • ICAM-1 peptides were preincubated for 30 minutes with the IRBC prior to addition to receptor-coated plates. The number of IRBC bound per mm 2 surface area was quantitated by light microscopy.
  • a 1.3kb fragment containing the ⁇ l hinge, C ⁇ and C j3 sequence was generated by PCR from a plasmid containing the human gene
  • the fragment contains a 5' Hindlll site, a translational stop codon following the codon for F185, the 5' donor splice that follows the ⁇ l C H I exon, and a 3' Xhol site. This fragment was subcloned into Hindlll and Xhol sites of pCDGl to produce pCDG185Gl.
  • Culture supematants of COS cells transfected with pCDG185Gl contained approximately 0.5 ⁇ g/ml ICAM-1-IgGl chimera (F185G1) as determined by ELISA on day 3 post transfection.
  • F185G1 was purified from culture media of transfected COS cells by ICAM-1 mAB (R6.5)-Sepharose and protein A-Sepharose chromatography. Figures 4a and b.
  • Tandon et al reviewed Tandon et al 's information that the mature sequence began with the second translated amino acid and contained a hydrophobic stretch near the N-terminus, but stated that "It is not clear whether the single Arg residue preceding the hydrophobic region would be sufficient to allow amino-terminal membrane anchoring.” Based on the prior art of Tandon et al and their own findings, Oquendo et al believed that this would not be a stop transfer sequence because they include the N-te ⁇ ninal hydrophobic region as part of the extracellular domain.
  • CD36 might be membrane bound, rather than folded up with the extracellular domain, and made constructs deleted in the N-terminal as well as C-te ⁇ nigal hydrophobic regions. It was impossible to predict the best place to truncate the CD36 molecule relative to the hydrophobic regions, because often "connector" segments are present between the globular extracellular domain and membrane spanning segments.
  • CD36 is deleted for hydrophobic regions, residue 6-28, and 439-465. In one aspect of this embodiment residues 1-5 and 466-471 are also deleted.
  • constructs were made so that a signal sequence was spliced to amino acids D30, Q34, Q40, G46, or F50 of CD36.
  • the CD36 sequence continued to amino acid N416, F429, V433, G435, or L439 of CD36, and was then spliced to the hinge, CH2, and CH3 exons of human IgGl heavy chain.
  • Constructs were transfected into COS cells. Expression was measured by ELISA using protein A as capture reagent and CD36 MAb as detector reagent or with protein A Sepharose.
  • chimeric constructs including the following amino acid segments of CD36: D30F429, Q34L439, and D30N416. This suggests that preferred embodiments contain a portion of CD36 beginning with amino acids 30- 34, and ending with amino acids 416 to 439.
  • CD36-IgGl chimeras were constructed by ligating CD36 PCR fragments lacking the transmembrane domain sequences to the expression vectors CDIG1 and CDBG1.
  • CDIG1 and CDBG1 were derived by inserting an Ig and a ⁇ 2 microglobin signal peptide sequence and stuffer sequence into the HindlH XhoI sites of CDG1 respectively.
  • the signal peptide sequence and the 5' UT sequence of ICAM-1 were generated by PCR using a long antisence primer containing the signal peptide sequence and 24bp of ICAM-1 5'UT.
  • the vectors were digested with EC0R47III(CDIG1) os KASI(CDBGl) and Xhol and then purified via electrophoresis in a low melt agarose gel.
  • the KasI site was blunt ended with Klenow prior to Xhol digestion.
  • the blunt enjged vectors terminate with the codon for the -1 position of the signal peptides.
  • CD36 PCR fragments with a 5' blunt end terminating with the amino terminal codons, and a Xhol site and donor splice sequence were ligated to CDIG1 and CDBG1.
  • the resulting chimeras were expressed in COS cells as described earlier.
  • the culture supematants were assayed in an ELISA assay for reactivity to a CD36 mAb and an anti-human IgG Horseradish peroxidase (HRP) conjugate.
  • HRP horseradish peroxidase
  • Soluble ICAM-1 truncated before the hydrophobic transmembrane region was purified from the supematants of transfected CHO cells (Marlin et al, Nature 344:10-12 (1990)) or baculovirus-vecto ⁇ infected insect cells
  • ICAM-1 Intraerythrocytic P. falciparum parasites selected in vitro to bind to purified ICAM-1 (ItG-ICAM) (Ockenhouse et al, J. Infec. Dis. 164:163- 169 (1991)) were maintained in synchronous continuous culture and used in adhesion assays at the trophozoite/schizont stage of development
  • ItG-ICAM Intraerythrocytic P. falciparum parasites
  • IRBC ICAM-1-coated plates (40-50% parasitemia, 1% hematocrit) for one hour at room temperature.
  • ItG- ICAM IRBC were incubated in solution with increasing concentrations of F185G1 chimera, sICAM-1/CHO, or normal human IgG for 30 minutes prior to addition to plates coated with sICAM-1/CHO (10 ⁇ g/ml).
  • sICAM-1 and F185G1 were determined with a capture ELISA assay (Marlin et al, Nature 344:10-12 (1990)), using sICAM-1/CHO as a standard, Figure 5.
  • F185G1 for SKW-3 cell binding, F185G1 at the concentration indicated was absorbed to 96-well microtiter plates which had previously been coated with protein A (20 ⁇ g/ml) and blocked with 1% BSA-PBS.
  • SKW-3 cells in binding buffer (RPMI 10% FBS/20mM HEPES) were treated with or without 100 ng ml PMA for 15 minutes at 37°C and then labeled with 2',7'-bis(2-carboxyethyl)-(5 and 6)-carboxyfluorecein acetomethyl ester
  • sICAM-1 10 ⁇ g/ml, 2 hours, 37°C
  • BSA-PBS 1% BSA-PBS
  • PMA treated SKW-3 (10-cells) were incubated for 30 minutes in 50 ⁇ l of binding buffer, with or without F18561 or mAb TS1/18 to the LFA-1 ⁇ subunit (1:100 ascites) and then added directly to sICAM- 1 coated wells. Binding was for 1 hour at 37°C. Unbound cells were removed by inverting microtiter plates in a tank of PBS/lmm Mg++/.5mM Ca++/0. 1% BSA for 45 minutes.
  • Bound cells were quantitated on a fluorescence concentration analyzer (Pandex). Percent bound ( ⁇ SD) was calculated by subtracting background binding to wells that were not coated with ICAM-1 from binding to ICAM-1 coated wells, divided by input fluorescence x 100.
  • IRBC IRBC (5 X 10 ⁇ per 100 ⁇ l) selected in vitro for binding to ICAM-1 (ItG-ICAM) or CD36 (ItG-CD36) were incubated with F185G1 chimera or normal human IgG (20 ⁇ g/ml final concentration) for 30 minutes prior to addition to monolayers of adherent freshly isolated human monocytes. After two hours incubation at 37°C, unattached red blood cells were removed by washing coverslips three times with RPMI 1640. In order to avoid quantitating IRBC attached to the phagocyte surface but not internalized, coverslips were rinsed in hypotonic 0.85% NH 4 C1 to lyse attached IRBC.
  • IRBC from individuals with uncomplicated malaria, CY25, or complicated severe cerebral malaria, G15 were cultured in vitro for 24 hours to allow intiaerythrocytic parasite maturation to the trophozoite stage of development
  • These infected eiythrocytes bound to COS cells expressing wild-type and domain deleted ICAM-1 (Table 1).
  • ICAM-1 molecules were constructed from cDNAs containing a conserved Bgl II restriction site at amino acid residue 168 of the human sequence (Staunton et al, Cell 61:243-254 (1990). Human domains Dl and D2 (hmICAM-1) or murine domains Dl and D2 (mhICAM-1) were recombined with domains D3-D5 of the other species. The chimeric cDNAs were expressed in COS cells and IRBC binding determined. The efficiency of expression was determined using two MAbs to human ICAM-1, RR/1 and CL203, and MAb YNl/1 (Horley et al, EMBO J.
  • IRBC IRBC binds to hmICAM-1 but not mhICAM-1 (Fig. 1), thus the first 168 residues of human ICAM-1 are sufficient to support binding of an IRBC counter-receptor.
  • Amino acid substitution mutants of ICAM-1 have profound effects on LFA-1, Mac-1, and human rhinovirus binding. Similarly, the adhesion of IRBC to single and multiple amino acid substitution mutants was examined. Amino acid substitutions in Dl and D2 are denoted by one- letter code for the wild-type sequence followed by a slash and the one letter code for the mutant sequence (Table 2). The efficiency of mutant
  • ICAM-1 expression on COS cells was determined using MAb CL203 by immunocytofluorimetiy and in adhesion assays by immunogold silver staining.
  • Mab CL203 which recognizes an epitope located within the D4 region had no effect on IRBC binding.
  • the amino acid substitution mutants, D60S/KL and R13G/EA, which conformationally disrupt the secondary structure of domains 1 and 2 also abrogate IRBC adhesion (Table 2).
  • ⁇ - strands A, B, E, D form one sheet while C, F, G strands fashion the opposing sheet
  • the contact site for Plasmodium falciparum-infected erythrocytes is predicted to be localized in domain 1 to a loop between ⁇ strands A and B and extend into ⁇ strand B. This contact site is distinct from the binding sites for LFA-1 and HRV (Fig. 2).
  • ICAM-1 amino add substitution mutants were generated by oligonucleotide- directed mutagenesis (Staunton et al, Cell (52:243-254 (1990)). Wild-type (wt) residues precede the slash and are followed by the substitution residues in the mutant.
  • IRBC adhesion to COS cells expressing mutant ICAM-1 was assessed by concurrent monoclonal antibody CL203 staining and IRBC adhesion and expressed as the mean percentage ⁇ standard deviation (sd) binding of IRBC to wild-type" ICAM-1 transfected cells. The values for LFA-1 binding and HRV14 binding to the new mutants generated for these studies are shown in the columns within the table. * Amino acid substitution mutants with decreased binding as previously published (Staunton et al, Cell 62:243-254 (1990)).
  • Chimeric proteins consisting of soluble ICAM-1 or soluble CD36 and an Antibody Fragment
  • CD36 Other immunoadhesin based on CD36 (CDBG1) have been rationally designed or generated and expressed as described earlier. These include Q40G435, G46G435, Q34V433, Q40V433, F50V433, D30L 39,
  • CD36 immunoadhesins can have its collagen binding ability deleted using the methods described earlier.
  • T-lymphoblastoid cells SKW-3
  • F185G1 T-lymphoblastoid cells
  • PMA-induced activation of LFA-1 Fig. 2c
  • ICAM-1 immunoadhesin was chosen for the immunoadhesin because this subclass is the most effective in triggering antibody-dependent cellular cytotoxicity (Riechmann et al, Nature 552:323-327 (1988)) and binds avidly to all three classes of Fc ⁇ receptor
  • CD36-binding IRBC incubated in the presence or absence of F185G1 chimera were not phagocytosed.
  • the F185Gl-treated internalized IRBC are quickly degraded and residual parasite-derived hemozoin pigment observed intracellularly (Fig. 4b,c).
  • CD36-binding IRBC attach to CD36 on the surface .of monocytes but are not phagocytized through this receptor (Fig 4a).
  • the rosetting of ItG-CD36 IRBC with monocytes was blocked completely by the anti-CD36 monoclonal antibody OKM5 (data not shown).
  • the ICAM-1-binding IRBC are not resetted or phagocytosed in the absence of F185G1 (Fig. 4d).
  • ICAM-1 immunoadhesin and a CD36 immunoadhesin that is effective against P. falciparum parasitized erythrocytes but does not block lymphocytic binding to ICAM-1.
  • Sequestration of P. falciparum IRBC plays a pivotal role in the pathology of malaria, probably by triggering a cascade of deleterious events including local anoxia, induction of toxic inflammatory mediators, edema and tissue damage. Sequestration in the brain leads to the most fatal form of the disease, cerebral malaria (World Health Organization Malaria Action Programme, Trans. R Soc. Trop. Med Hyg. 80 Suppl.:3-50 (1986)). lmmunoadhesins mimicking P.
  • falciparum sequestration receptors can be therapeutically effective through two distinct mechanisms. First they should reverse sequestration; a combination of adhesins, including ICAM-1 and CD36 immunoadhesin, may be required for maximal effectiveness. Reversal of sequestration is predicted to alleviate much of the associated pathology and especially mortality resulting from cerebral malaria or placental insufficiency. Second, immunoadhesins can sensitize parasitized eiythrocytes for recognition and elimination by the immune system, as exemplified here by monocyte phagocytosis and destruction mediated by an lCAM-1 immunoadhesin.
  • ADDRESSEE Sterne, Kessler, Goldstein & Fox

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Abstract

Structure d'immunoadhésines de CD36 et leur utilisation pour tuer de manière sélective les érythrocytes infectés par Plasmodium falciparum. Les immunoadhésines contiennent le CD36 ou des fragments de CD36 ayant subi une délétion dans une ou plusieurs des régions des restes 1-5, 6-28, 439-465 et 466-471.
PCT/US1992/008482 1991-10-03 1992-10-05 IMMUNOADHESINES DE CD36 ET LEUR UTILISATION POUR TUER DE MANIERE SELECTIVE LES ERYTHROCYTES INFECTES PAR $i(PLASMODIUM FALCIPARUM) WO1993006848A1 (fr)

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Cited By (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP0578342A3 (en) * 1992-05-14 1994-06-29 Grace W R & Co Purification and characterization of a csvtcg-specific tumor cell adhesion receptor
WO2001002005A3 (fr) * 1999-06-30 2001-07-05 Isis Innovation Induction de la tolerance immunitaire
WO2001036675A3 (fr) * 1999-11-19 2002-05-02 Isis Innovation Polymorphismes cd36
US8921534B2 (en) 2001-12-12 2014-12-30 Sanofi Pasteur Limited Enhancement of the immune response using CD36-binding domain
US20150044207A1 (en) * 2003-05-06 2015-02-12 Biogen Idec Hemophilia Inc. Clotting Factor-Fc Chimeric Proteins to Treat Hemophilia
WO2015085311A1 (fr) * 2013-12-07 2015-06-11 Case Western Reserve University Compositions et méthodes de traitement de thrombose

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* Cited by examiner, † Cited by third party
Title
CELL, Volume 58, issued 14 July 1989, P. OQUENDO et al., "CD36 Mediates Cytoadherence of Plasmodium Falciparum Parasitized Erythrocytes", pages 95-101. *
JOURNAL OF CLINICAL INVESTIGATION, Volume 84, issued, September 1989, J.W. BARNWELL et al., "A Human 88-kD Membrane Glycoprotein (CD36) Functions as a Receptor for Cytoadherence on Plasmodium Falciparum-Infected Erythrocytes", pages 765-722. *
NATURE, Volume 332, issued 24 March 1988, L. REICHMANN et al., "Reshaping Human Antibodies for Therapy", pages 323-327. *
NATURE, Volume 339, issued 04 May 1989, A. TRAUNECKER et al., "Highly Efficient Neutralization of HIV with Recombinant CD4-Immunoglobulin Molecules", pages 68-70. *
PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCE U.S.A., Volume 88, issued April 1989, C.F. OCKENHOUSE et al., "Sequestrin, A CD36 Recognition Protein on Plasmodium Falciparum Malaria-Infected Erythrocytes Identified by Anti-Idiotype Antibodies", pages 3175-3179. *
SCIENCE, Volume 238, issued 20 November 1987, E.S. VITTETTA et al., "Redesigning Nature's Poisons to Create Anti-Tumor Reagents", pages 1098-1104. *
SCIENCE, Volume 243, issued 17 March 1989, C.F. OCKENHOUSE et al., "Identification of a Platelet Membrane Glycoprotein as a Falciparum Malaria Sequestration Receptor", pages 1469-1471. *
THE JOURNAL OF BIOLOGICAL CHEMISTRY, Volume 264, No. 13, issued 05 May 1989, N.N. TANDON et al., "Isolation and Characterization of Platelet Glycoprotein IV (CD36)", pages 7570-7575. *

Cited By (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP0578342A3 (en) * 1992-05-14 1994-06-29 Grace W R & Co Purification and characterization of a csvtcg-specific tumor cell adhesion receptor
WO2001002005A3 (fr) * 1999-06-30 2001-07-05 Isis Innovation Induction de la tolerance immunitaire
WO2001036675A3 (fr) * 1999-11-19 2002-05-02 Isis Innovation Polymorphismes cd36
US8921534B2 (en) 2001-12-12 2014-12-30 Sanofi Pasteur Limited Enhancement of the immune response using CD36-binding domain
US20150044207A1 (en) * 2003-05-06 2015-02-12 Biogen Idec Hemophilia Inc. Clotting Factor-Fc Chimeric Proteins to Treat Hemophilia
WO2015085311A1 (fr) * 2013-12-07 2015-06-11 Case Western Reserve University Compositions et méthodes de traitement de thrombose

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