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WO1993006850A1 - Erythrocytes infectes par plasmodium falciparum et lies a l'icam-1 et au cd36 - Google Patents

Erythrocytes infectes par plasmodium falciparum et lies a l'icam-1 et au cd36 Download PDF

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Publication number
WO1993006850A1
WO1993006850A1 PCT/US1992/008484 US9208484W WO9306850A1 WO 1993006850 A1 WO1993006850 A1 WO 1993006850A1 US 9208484 W US9208484 W US 9208484W WO 9306850 A1 WO9306850 A1 WO 9306850A1
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Prior art keywords
icam
irbc
binding
binding site
antibody
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PCT/US1992/008484
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English (en)
Inventor
Donald E. Staunton
Timothy A. Springer
Christian F. Ockenhouse
Original Assignee
The Center For Blood Research
The Government Of The United States, As Represented By The Secretary Of The Army
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Application filed by The Center For Blood Research, The Government Of The United States, As Represented By The Secretary Of The Army filed Critical The Center For Blood Research
Publication of WO1993006850A1 publication Critical patent/WO1993006850A1/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 erythrocytes (IRBC).
  • the present invention additionally relates to molecules capable of binding to the IRBC binding site on ICAM-1 or on CD36.
  • 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.
  • Erythrocytes infected with the human malaria parasite Plasmodium falciparum, adhere to vascular post-capillary endothelium, and the sequestration of the- malaria-infected erythrocytes (IRBC) is a primary event responsible for the clinical complications of severe and cerebral malaria. While immature ring stage parasitized erythrocytes circulate unobstructed throughout the vasculature, adhesion of mature fntraerythrocytic stages of the parasite to endothelium averts splenic clearance of IRBC and allows parasite maturation in a microenvironment of low oxygen tension.
  • ICM-1 intercellular adhesion molecule-1
  • CD54 Bosset et al, Nature (Lor L) 342:57-59 (1989)
  • CD36 GPIV
  • 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.
  • 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 (Barnwell et al, /. Clin. Invest. 84:765-172 (1989); Aikawa et al, Am. J. Trop. Med. Hyg. 43:30 (1990)).
  • MAb monoclonal antibody
  • ICAM-1 a member of the immunoglobulin-like superfamily, is a monomeric unpaired 90-115 M, glycoprotem 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.) 331:624-627 (1988)).
  • ICAM-1 is a ligand for the leukocyte integrins, lymphocyte function antigen-1 (LFA-1; CDlla/CD18) (Rothlein et al, J. Immunol 237:1270-1274 (1986); Marlin et al, CeU 52:813-819 (1987)) and
  • Mac-1 (CDllb/CD18) (Diamond et al,J. Cell Biol 222 219-3139 (1990); Smith et al, J. Clin. Invest 53:2008-2017 (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), the etiologic agent of the common cold (Staunton et al, Cell 56:849-853
  • red blood cells infected with mature intracellular forms of the malaria parasite bind to a region located within the amino-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 65:71-81 (1992)).
  • ICAM-1 has a restricted distribution in vivo, and its expression is regulated by LPS and the cytokines TNF, L-l ⁇ , and interferon-gamma
  • TNF up regulate the surface expression of ICAM-1 and support adhesion of malaria-infected erythrocytes (Berendt et al, Nature (Lond.) 341:57-59 (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 erythrocytes 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 erythrocytes 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 erythrocytes from naturally-acquired infections bind only to CD36 in vitro, a smaller subpopulation of parasitized erythrocytes 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 of 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-iDfected erythrocytes.
  • An IRBC binds to the first NH 2 -terminal 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, Iyse, and kill sequestered intraerythrocytic parasites in cases of severe and complicated ⁇ / ⁇ ro 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 uninfected 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. By inhibiting the binding of an IRBC to a non-infected cell 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; and (d) 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, fused to an immunoglobulin or a fragment thereof.
  • ICAM-1 fusion protein herein designated F185G1
  • F185G1 consists of soluble-ICAM-1 fused to the hinge region and constant domains CH2 and CH3 of human IgGl heavy chain. Fusion proteins of this nature have been demonstrated to stimulate phagocytosis of an IRBC when bound to the IRBC's surface.
  • 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, fused to an immunoglobulin or a fragment thereof.
  • hmICAM-1 human ICAM-1, domains 1 and 2; murine ICAM-1, domains 3-5
  • mhICAM-1 human ICAM-1, domains 1 and 2; murine ICAM-1, domains 3-5
  • results represent the mean of three determinations ⁇ standard deviation.
  • ICAM-1 ICAM-1, murine ICAM-1, and human ICAM-2. Amino acid substitution mutations within human ICAM-1 affecting binding of Plasmodium falciparum IRBC (Pf), LFA-1 (L), and HRV (R) are indicated by the solid line. The alignment of sequences by predicted secondary structure is indicated by ⁇ -strands A-G.
  • 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-te ⁇ ninus. 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.
  • ItG-CD36 IRBC open symbols
  • sICAM-1 or synthetic peptides at concentrations indicated were incubated on plates previously coated with 10 ug ml ICAM-1 or 1 ug/ml CD36, respectively. Binding of IRBC to adhesion receptors were determined and the results represent the mean per cent binding compared to control samples incubated in PBS alone. Control binding of ItG-ICAM-1 IRBC to purified ICAM-1 is 1578 ⁇ 225 IRBC/mm 2 and binding of G-CD36 to purified CD36 is 860 ⁇ 108 IRBC/mm 2 .
  • FIG. 1 A. Schematic diagram of the F185G1 expression construct (pCDF185Gl) and the F185G1 immunoadhesion.
  • Phagocytosis of Plasmodium falc ⁇ arum-ioiected erythrocytes by human monocytes Phagocytosis of Plasmodium falc ⁇ arum-ioiected erythrocytes by human monocytes.
  • CD36-binding IRBC preincubated with F185G1 chimera 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.
  • 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-infected erythrocytes is the first NH 2 -terminal domain between residues Gh '-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.
  • the present invention provides agents and methods for the freatment 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
  • 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, or a fragment thereof.
  • These 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
  • a “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 IRBC binding activity and are soluble are especially preferred. Soluble fragments of CD36 or ICAM-1 can be rationally designed by one skilled in the art. 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. As used herein, 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 cytotcsriciry (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-forming 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
  • an antibody-derivatized agent 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. 266:1351-1361 (1987) for a review of effector functions); however, it is preferable to employ 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.
  • 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 the 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 CD36 and then proteolytically mapping, using agents such as trypsin, the cross-linked protein to identify the collagen linked fragment
  • linker scanning mutagenesis can be employed to optimize the directed nature of the mutagenesis.
  • 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 ch ⁇ nerization 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
  • 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 at, Exp. Cell Res. 275:109-124 (1988), Campbell, A.M., Monoclonal Antibody Technology: Laboratory Techniques in Biochemistry and Molecular Biology, Elsevier Science
  • 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 heterologous protein (such as globulin or /3-galactosidase) or through the inclusion of an adjuvant during immunization.
  • a heterologous protein such as globulin or /3-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.
  • 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 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 one skilled in the art 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 &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)). Based on the present disclosure a 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 of an agent 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.
  • 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 covalentiy liked to a toxin such as ricin.
  • a toxin such as ricin.
  • an IRBC can be preferentially killed by utilizing a mammal's natural defense systems.
  • an IRBC with an antibody-derivatized agent which is capable of selectively binding the IRBC, the constant regions of the antibody moiety of the antibody- derivative agent will stimulate biological activities such as phagocytosis,
  • CDC and ADC
  • an agent includes the F185G1 chimeric antibody which consist of the hinge region and constant domains CH2 and CH3 of the human IgGl heavy chain covalentiy linked to a soluble derivative of ICAM-1.
  • F185G1 chimeric antibody which consist of the hinge region and constant domains CH2 and CH3 of the human IgGl heavy chain covalentiy linked to a soluble derivative of ICAM-1.
  • 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 mvention 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 Pharmaceutical 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 FTTC 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.
  • 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 -te ⁇ ninal 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.
  • the addition of overlapping synthetic peptides encompassing the binding site on ICAM-1 inhibited malaria-infected erythrocyte adhesion to recombinant soluble ICAM-1-coated surfaces.
  • Oligonucleotide-directed mutagenesis (Kunkel, T.A., Proc. Natl. Acad. Sci USA 52:488-492 (1985)) was used to generate ICAM-1 deletion, chimeric, and amino acid substitution mutants as described (Staunton et al, Cell 62:243-254 (1990)). Transfection of COS Cells
  • COS cells at 50% confluency were transfected 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 243:181-185 (1989)) (a gift of Dr. S. Ferrone), and RR1/1 (Dustin et al, J. Immunol 237:245-254 (1986)); and anti-murine MAb YNl/1 (Takei, F., /. Immunol
  • ICAM-1 peptides Pro 12 -Thr 23 and overlapping hexapeptides spanning residues Gln ⁇ 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 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.
  • IRBC binding to ICAM-1-coated or CD36-coated surfaces was performed as follows. Soluble ICAM-1 (lOug/ml) (Marlin et al, Nature (Lond.) 344:70-72 (1990)) or CD36 (1 ug ⁇ nl) (Tandon et al, J. Biol Chem.
  • 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 13kb fragment containing the ⁇ l hinge, 0 ⁇ 2 and C ⁇ sequence was generated by PCR from a plasmid containing the human gene
  • F185G1 was subcloned into Hindm and Xhol sites of pCDGl to produce pCDG185Gl.
  • Culture supernatants 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.
  • Soluble ICAM-1 truncated before the hydrophobic transmembrane region was purified from the supernatants of transfected CHO cells (Marlin et al, Nature 344:70-72 (1990)) or baculovirus-vector infected insect cells (Diamond et al, Cell 65:961-971 (1991)).
  • ICAM-1 was adsorbed (20 ⁇ l aliquots) to plastic bacteriological plates (Falcon 1007) overnight at4°C.
  • F185G1 IAM-1-IgGl chimera
  • ICAM-1-coated plates 40-50% parasitemia, 1% hematocrit
  • 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). Erythrocytes not attached to the sICAM-1-coated surface were removed by gentle rinsing of the plates. Cells were fixed with 2% glutaraldehyde and stained with Giemsa. The number of malaria-infected erythrocytes bound per mm 2 surface are represents the mean of three separate determinations. The concentrations of sICAM-1 and F185G1 was determined with a capture ELISA assay (Marlin et al, Nature 344:70-72
  • 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 ⁇ nl) and blocked with 1% BSA-PBS.
  • SKW-3 cells in binding buffer RPMI/10% FBS 20mM HEPES
  • binding buffer RPMI/10% FBS 20mM HEPES
  • 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 X 10 6 per 100 ⁇ l
  • ICAM-1 ItG-ICAM
  • CD36 ItG-CD36
  • F185G1 chimera F185G1 chimera
  • normal human IgG 20 ⁇ g/ml final concentration
  • IRBC from individuals with uncomplicated malaria, CY25, or complicated severe cerebral malaria, G15 were cultured in vitro for 24 hours to allow intraerythrocytic parasite maturation to the frophozoite stage of development These infected erythrocytes bound to COS cells expressing wild-type and domain deleted ICAM-1 (Table 1).
  • Example 2
  • Human and murine mutant chimeric 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 immunocytofiuorimetry 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-mf cted 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).
  • a linear peptide Pro ⁇ -Thr 23 and the hexapeptide GSVLVT inhibited IRBC binding in a dose-dependent manner with 50% inhibition at approximately 0.125 and 03mM, respectively (Fig. 3b).
  • the inhibitory effect of these peptides was three orders of magnitude less than that observed using sICAM-1 as the inhibitor of IRBC binding (Fig. 3b).
  • the inhibition by the ICAM-1 peptides was specific for ICAM-1-binding infected erythrocytes, since parasitized red cells which bind to an alternative sequestration receptor, CD36, were not inhibited from binding to immobilized CD36 (Fig. 3b).
  • ICAM-1 amino acid substitution mutants were generated by oligonucleotide- d ⁇ rected mutagenesis (Staunton et al, Cell 62:243-254 (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 61:243-254 (1990)).
  • the immunoadhesin did not bind uninfected erythrocytes nor erythrocytes infected with malaria parasites which bind to an alternative endothelial receptor, CD36 (data not shown).
  • the ICAM-1 immunoadhesin is a more effective inhibitor of IRBC adhesion to ICAM-1-coated surface than sICAM-1 (Fig. 2b). Fifty percent inhibition of IRBC binding is achieved with approximately 8 fold less F185G1 than sICAM-1. Enhanced binding may reflect the multivalent nature of F185G1.
  • T-lymphoblastoid cells SKW-3 cells adhere to F185G1 on a solid substrate and binding is enhanced by PMA- induced activation of LFA-1 (Fig. 2c). Concentrations of soluble F185G1 completely block IRBC binding do not inhibit LFA-1 dependent SKW-3 binding to sICAM-1 coated surfaces (Fig. 2d). In addition binding of soluble F185G1 to lymphoblastoid cells with or without PMA treatment can not be detected by indirect immunofluorescence (data not presented). Hence the avidity of F185G1 is higher for the receptor on IRBC than for
  • 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
  • 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 RG-CD36 IRBC with monocytes was blocked completely by the anti-CD36 monoclonal antibody OKM5 (data not shown).
  • the ICAM-1-binding IRBC are not rosetted or phagocytosed in the absence of F185G1 (Fig. 4d).
  • 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 fo ⁇ n of the disease, cerebral malaria (World Health Organization Malaria Action Programme, Trans. R Soc. Trop. Med. Hyg. 80 Sup ⁇ l.:3-50 (1986)). lmmunoadhesins mimicking P. falciparum sequestration receptors can be therapeutically effective through two distinct mechanisms.
  • immunoadhesins can sensitize parasitized erythrocytes for recognition and elimination by the immune system, as exemplified here by monocyte phagocytosis and destruction mediated by an ICAM-l immunoadhesin. Release from sequestration is not necessarily required for this effector mechanism, as it could presumably be mediated by monocytes and granulocytes at sites of sequestration in post capillary venules.
  • a side benefit of clearance of parasites by phagocytes is that it boosts host humoral and cellular immunity to P. falciparum. Cytoadherence receptor binding must be conserved and thus pathogen strain variation, which is extensive for P. falciparum, would not be an effective mechanism for evasion of this therapy.
  • ADDRESSEE Sterne, Kessler, Goldstein & Fox
  • Gin Val Ser lie His Pro Arg Glu Ala Phe Leu Pro Gin Gly Gly Ser 1 5 10 15
  • MOLECULE TYPE DNA
  • SEQUENCE DESCRIPTION SEQ ID NO:10: TTTCTCGAGG GTGTCTGCTG GAAGCAGGCT CAG 33

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Abstract

On a découvert la séquence d'acides aminés du site de liaison sur la molécule d'adhésion intracellulaire-1 (ICAM-1) des érythrocytes infectés par le paludisme (IRBC). On a prévu des procédés utilisant cette séquence dans le traitement et le diagnostic du paludisme. En outre, on a prévu des agents aptes à inhiber la liaison d'un IRBC à l'endothélium, ainsi que des agents aptes à stimuler la phagocytose d'un IRBC.
PCT/US1992/008484 1991-10-03 1992-10-05 Erythrocytes infectes par plasmodium falciparum et lies a l'icam-1 et au cd36 WO1993006850A1 (fr)

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WO2004009810A3 (fr) * 2002-07-18 2004-04-29 Glaxo Group Ltd Modeles animaux
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Cited By (8)

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US7132395B1 (en) 1988-09-01 2006-11-07 Bayer Pharmaceuticals Corporation Antiviral methods using human rhinovirus receptor (ICAM-1)
US6107461A (en) * 1990-07-20 2000-08-22 Bayer Corporation Multimeric forms of human rhinovirus receptor and fragments thereof, and method of use
US6130202A (en) * 1990-07-20 2000-10-10 Bayer Corporation Antiviral methods
US5953908A (en) * 1994-10-07 1999-09-21 Appleby; Anthony John Method and apparatus for heating a catalytic converter to reduce emissions
US5845485A (en) * 1996-07-16 1998-12-08 Lynntech, Inc. Method and apparatus for injecting hydrogen into a catalytic converter
US6036827A (en) * 1997-06-27 2000-03-14 Lynntech, Inc. Electrolyzer
WO2002024307A3 (fr) * 2000-09-15 2003-10-16 Allied Therapeutics Ltd Reduction de la quantite de cellules presentes dans un echantillon
WO2004009810A3 (fr) * 2002-07-18 2004-04-29 Glaxo Group Ltd Modeles animaux

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