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WO1993004696A1 - Procede et composition servant a detruire les parasites transmettant le paludisme - Google Patents

Procede et composition servant a detruire les parasites transmettant le paludisme Download PDF

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Publication number
WO1993004696A1
WO1993004696A1 PCT/US1992/007380 US9207380W WO9304696A1 WO 1993004696 A1 WO1993004696 A1 WO 1993004696A1 US 9207380 W US9207380 W US 9207380W WO 9304696 A1 WO9304696 A1 WO 9304696A1
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WO
WIPO (PCT)
Prior art keywords
parasite
malaria
antibody
parasitophorous
duct
Prior art date
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PCT/US1992/007380
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English (en)
Inventor
Theodore F. Taraschi
Bruno Pouvelle
Original Assignee
Thomas Jefferson University
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
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Publication date
Application filed by Thomas Jefferson University filed Critical Thomas Jefferson University
Publication of WO1993004696A1 publication Critical patent/WO1993004696A1/fr

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Classifications

    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/20—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans from protozoa
    • C07K16/205—Plasmodium
    • 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
    • C07K14/44—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from protozoa
    • C07K14/445—Plasmodium
    • 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

  • Malaria is a serious parasitic disease spread by the bite of Anopheles mosquitoes.
  • the World Health Organization recognizes malaria as the world's major primary health problem, causing more morbidity and mortality than any other disease.
  • WHO World Health Organization
  • Mosquitoes have developed resistance to insecticides and the parasites have developed resistance to drugs.
  • the organisms responsible for malaria are parasitic protozoans of the genus Plasmodium .
  • the species that cause the disease in humans are P. falciparum, P. vivax, P. ovale, and P. malar iae, each producing a different form of malaria.
  • P. falciparum Prevalent throughout the tropics, the global incidence of malaria is now approaching 500 million cases annually, with P. falciparum responsible for nearly half of all cases.
  • malaria has been virtually eliminated in the United States, Canada,
  • Malaria parasites spend part of their life cycle in the Anopheles mosquitoes and part in humans.
  • Mosquitoes become infected with malaria parasites by • ingesting blood containing the sexual forms of the protozoa.
  • plasmodia are transmitted by bites of the mosquito whose saliva carries the spore of the parasite.
  • an infectious spore merozoite
  • the parasite enters the blood stream and infects red blood cells (erythrocytes) .
  • P. falciparum which causes the most severe type of malaria, infects all ages of red blood cells (other species attack only young or old cells) and therefore, affects a greater proportion of the blood cells.
  • a composition and method for killing blood-stage malaria parasites by selective delivery of parasiticidal agents, including antibodies and antibody-toxin conjugates, are provided.
  • This novel approach to treating malaria is made possible by the inventors' discovery of a parasitophorous duct which directly exposes the intraerythrocytic parasite to the serum during the 48 hour blood-stage life cycle.
  • selected chemotherapeutic agents or antibody raised by vaccination would be required in relatively small amounts.
  • the parasite would be vulnerable to treatment for a relatively long period of time (days) .
  • the parasitemia % of erythrocytes infected with malaria
  • Figure 1 is a graph showing the typical time course for accumulation of rhodamine-labeled dextran (10K) in the parasite of trophozoite-stage infections.
  • Figure 2 is a graph showing the accumulation of rhodamine-dextran in the parasite of trophozoite-stage infections as a function of dextran concentration.
  • IgG antibodies and crisis-form factor from human immune serum have been found to retard the in vitro growth of P. falciparum.
  • the parasitophorous duct may provide the pathway for these molecules to reach the intracellular parasite.
  • surface antigens on trophozoites and schizonts a parasite developed from a trophozoite which undergoes multiple fission to form erozoites
  • the parasitophorous duct which we have discovered will provide the means through which potentially lethal antibodies or antibody-toxin recombinants (immunotoxins) can be delivered specifically to the intraerythrocytic parasite.
  • the parasitophorous duct can be used to (1) deliver monoclonal or polyclonal antibodies to inhibit parasite growth and/or agglutinate segmented schizonts, preventing reinvasion by binding to specific antigens on the parasite plasma membrane; (2) selectively deliver immunotoxins to kill trophozoite or schizont stage parasites; (3) inhibit parasite growth by blocking the entrance to the duct.
  • a blood- stage malaria vaccine will be developed wherein vaccination will be achieved either by immunization with isolated, prematurely lysed parasites (which would no longer be infectious) , killed parasites or purified parasite surface antigens from ring, trophozoite or schizont-stage infections.
  • isolated, prematurely lysed parasites which would no longer be infectious
  • killed parasites or purified parasite surface antigens from ring, trophozoite or schizont-stage infections.
  • parasite vacuolar membranes Irrespective of the phospholipid headgroup, tubular extensions of membrane appeared to bridge the erythrocyte and parasite vacuolar membranes. These structures, termed by us “parasitophorous ducts" appear to interconnect the parasitophorous vacuole and the external medium to provide a path by which macromolecules in the serum can passively diffuse to the parasite.
  • trophozoite-stage parasites incubated in vitro with a rhodamine-goat anti-mouse IgG 2a antibody at 37'C are heavily labeled, but are devoid of any antibody labeling at ⁇ C.
  • the kinetics of uptake of rhodamine-dextran (10K) into the parasite at 37°C were shown to be characteristic of endocytosis. Accumulation of rhodamine-dextran was inhibited by depletion of ATP.
  • rhodamine-dextran The accumulation of rhodamine-dextran in the parasite of trophozoite-stage cells was also measured as a function of rhodamine-dextran concentration, and a linear relationship, typical of fluid- phase endocytosis, was observed.
  • the parasite was found to be heavily labeled compared with the plasma membrane of the erythrocyte due to the density and proximity of labeled intraparasitic membranes and organelles.
  • Tubular membranes which we refer to as parasitophorous ducts, were seen interconnecting the parasite vacuolar and erythrocyte membranes.
  • Cells were also incubated at 1 hour at 37 ⁇ C with carboxylate-modified fluorescent latex beads, diluted 200 times with PBS which contained 1% bovine serum albumin and 3% of 40K dextran. Ducts projecting from the parasitophorous vacuolar membrane
  • Example 2 Accumulation of Rhodamine-Labeled Dextran in the Parasite of Trophozoite Stage Infections
  • Cells were incubated in tissue culture with rhodamine-dextran (1 mg/ l) at 37°C. For each time point, cells were taken from culture, washed 4 times at 4°C to remove external rhodamine-dextran, and the fluorescence intensity in the parasite compartment quantified by CFIM. The results are shown in Figure 1. Each fluorescence value is the average from 70-100 infected cells. Fluorescence values were obtained at fixed photomultiplier tube gains and expressed in arbitrary units. To deplete ATP (0) , infected cells were incubated in glucose-free tissue culture medium, supplemented with 50 mM deoxy-glucose and 5 mM NaN 3 , for 30 minutes at 37°C.
  • Example 3 Labeling of Parasites with Monoclonal Antibodies
  • IA2 An IgG monoclonal antibody, IA2, specific for the parasite surface antigen found during all stages of infection was purified from Ascites fluid. An aliquot of the antibody was labeled with rhodamine (Rh) .
  • Rh-IA2 was characterized and did not contain any unassociated rhodamine or contaminating proteins.
  • Rh-IA2 When the Rh-IA2 was incubated at 4"C with trophozoite-stage infected red blood cells, labeling of the parasitic plasma membrane was apparent. When the temperature was raised to 37"C, it appeared that internal structures in the parasite also became labeled. This is important because the immunotoxin must be endocytosed by the parasite to be efficacious. Controls done with unlabeled IA2 indicate that the addition of Rh does not alter the binding properties of the antibody.
  • Rh-IA2 Trophozoite-stage infected red blood cells will be incubated with Rh-IA2 at 4 C (to prevent parasite uptake by fluid-phase endocytosis) with serial dilutions of the antibody to determine the minimum amount of antibody necessary for visualization by CFIM. Having determined this, we will examine the ability of Rh-IA2 to label parasites in vitro at different developmental stages. Infected cells will be incubated at 4'C with Rh-IA2 and examined by CFIM for reactivity. The presence of the antigen will be confirmed by Western blotting.
  • Rh-IA2 To identify the membranes that Rh-IA2 is associated with, cells will be fixed, sectioned and reacted with a gold-conjugated goat anti-mouse IgG and examined by electron microscopy. When the incubation is done at 4'C, only the parasite plasma membrane should be labeled. If the incubation is done at 4'C, the cells are washed and the temperature raised to 37"C, IA2 should also be associated with vesicles/organelles in the parasite cytosol if it has been endocytosed.
  • Example 4 Determination of Antibody Toxicity
  • Infected red blood cells will also be examined by CFIM. At each new invasion, the parasitemia of the culture containing the antibody will be measured and compared to controls. Cell killing will be expressed as % dead infected cells versus time (hours) . If cell killing is achieved, serial dilutions of antibody will be incubated with infected red blood cells to determine the LD 50 (amount of antibody necessary to kill 50% of the infected cells) . Our preliminary results indicate that a significant amount of parasite killing occurs during the trophozoite stage of the life cycle.
  • this experiment will distinguish between direct killing of the parasite (by examination of cell viability as a function of stage) and blocking of re-invasion by agglutination (which- should be obvious in segmented schizonts) of released merozoites.
  • Immunotoxins are cytotoxic agents prepared by linking cell-reactive antibodies to potent toxins or their A chains.
  • Ricin a disulfide-bonded heterodimeric glycoprotein-consisting of a B chain (which binds galactose-containing molecules on cell surfaces) and an A chain (which kills cells by enzymatically inactivating the 60 S ribosomal unit) is the most widely used toxin.
  • B chain which binds galactose-containing molecules on cell surfaces
  • a chain which kills cells by enzymatically inactivating the 60 S ribosomal unit
  • ricin-antibody conjugates In order for ricin-antibody conjugates to be lethal, the complex must be endocytosed, the antibody-ricin link broken, and free ricin released to the cytosol.
  • the procedures for conjugating ricin A chain to monoclonal are well known in the art.
  • a ricin A- IA2 immunotoxin will be prepared.
  • the toxicity of ricin A plus B chain, ricin A chain, and ricin A-IA2 will be established in our cell system as described in previous examples. We expect that ricin will kill the parasite since we have shown that the parasite actively endocytoses.
  • Cultures will be incubated for two cycles with ricin A plus B, ricin A, and ricin in combination with a selected antibody in the culture medium. As a control, identical cultures without the toxins will also be maintained. New aliquots of toxins and ricin-antibody will be added to the culture when the medium is changed. Ricin A chain agglutinates erythrocytes at concentrations above 20 micrograms/ml. This is well above the concentrations (10 ⁇ -10 " M) where ricin A-toxins have been shown to be toxic. Throughout the life cycle, an aliquot of infected cells will be removed and viability will be assayed using a viability assay (Molecular Probes, Inc., Eugene, Oregon).
  • Parasite stage will be determined by Giemsa-stained blood smears. At each new invasion, the parasitemia of the culture containing the antibody will be measured and compared to controls. Cell killing will be expressed as % dead infected cells versus time (hours) . If cell killing is achieved, serial dilutions of ricin-antibody will be incubated with infected red blood cells to determine the LD 50 .

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  • Health & Medical Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Organic Chemistry (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Medicinal Chemistry (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Biophysics (AREA)
  • General Health & Medical Sciences (AREA)
  • Genetics & Genomics (AREA)
  • Tropical Medicine & Parasitology (AREA)
  • Molecular Biology (AREA)
  • Biochemistry (AREA)
  • Immunology (AREA)
  • Toxicology (AREA)
  • Zoology (AREA)
  • Gastroenterology & Hepatology (AREA)
  • Medicines Containing Antibodies Or Antigens For Use As Internal Diagnostic Agents (AREA)
  • Pharmaceuticals Containing Other Organic And Inorganic Compounds (AREA)

Abstract

Composition et procédé servant à détruire les parasites transmettant le paludisme et consistant à administrer sélectivement des agents parasiticides, comprenant des anticorps ainsi que des conjugués anticorps-toxine.
PCT/US1992/007380 1991-08-30 1992-08-28 Procede et composition servant a detruire les parasites transmettant le paludisme WO1993004696A1 (fr)

Applications Claiming Priority (2)

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US75237291A 1991-08-30 1991-08-30
US752,372 1991-08-30

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Cited By (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1996009398A1 (fr) * 1994-09-19 1996-03-28 Rijkslandbouwuniversiteit Wageningen Produits d'assemblage geniques codant des agents de protection des cultures, plantes transformees renfermant et exprimant de tels produits d'assemblage, et procedes de lutte contre les agents pathogenes et organismes pesteux des cultures
US7431936B2 (en) * 2000-05-31 2008-10-07 Institut Pasteur Proteins involved in cytoadhesion of plasmodium falciparum ring-stage-infected erythrocytes, antibodies which bind to the proteins, and methods for detecting infection, stage of infection and vaccines for protecting against infection
EP2990059A1 (fr) * 2014-08-26 2016-03-02 Fraunhofer-Gesellschaft zur Förderung der angewandten Forschung e.V. Complexes anti-parasitaires
US10577412B2 (en) 2015-04-12 2020-03-03 Fraunhofer-Gesellschaft Zur Foerderung Der Angewandten Forschung E.V. Anti-plasmodium parasite antibodies

Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US4867973A (en) * 1984-08-31 1989-09-19 Cytogen Corporation Antibody-therapeutic agent conjugates

Patent Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US4867973A (en) * 1984-08-31 1989-09-19 Cytogen Corporation Antibody-therapeutic agent conjugates

Non-Patent Citations (2)

* Cited by examiner, † Cited by third party
Title
NATURE, Vol. 289, 22 January 1981, PERRIN et al., "Inhibition of P. Falciparum Growth in Human Erythrocytes by Monoclonal Antibodies", pages 301-303. *
SCIENCE, Vol. 252, issued 21 June 1991, WALDMANN, "Monoclonal Antibodies in Diagnosis and Therapy", pages 1657-1662. *

Cited By (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1996009398A1 (fr) * 1994-09-19 1996-03-28 Rijkslandbouwuniversiteit Wageningen Produits d'assemblage geniques codant des agents de protection des cultures, plantes transformees renfermant et exprimant de tels produits d'assemblage, et procedes de lutte contre les agents pathogenes et organismes pesteux des cultures
US7431936B2 (en) * 2000-05-31 2008-10-07 Institut Pasteur Proteins involved in cytoadhesion of plasmodium falciparum ring-stage-infected erythrocytes, antibodies which bind to the proteins, and methods for detecting infection, stage of infection and vaccines for protecting against infection
EP2990059A1 (fr) * 2014-08-26 2016-03-02 Fraunhofer-Gesellschaft zur Förderung der angewandten Forschung e.V. Complexes anti-parasitaires
WO2016030292A1 (fr) * 2014-08-26 2016-03-03 Fraunhofer-Gesellschaft zur Förderung der angewandten Forschung e.V. Complexes anti-parasitaires
US10344082B2 (en) 2014-08-26 2019-07-09 Fraunhofer-Gesellschaft Zur Foerderung Der Angewandten Forschung E.V. Anti-parasitic complexes
US10577412B2 (en) 2015-04-12 2020-03-03 Fraunhofer-Gesellschaft Zur Foerderung Der Angewandten Forschung E.V. Anti-plasmodium parasite antibodies

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