WO1990012632A1 - Procede servant a enlever la proteine c-reactive et les anticorps anti-phosphorylcholines dans des fluides biologiques - Google Patents

Procede servant a enlever la proteine c-reactive et les anticorps anti-phosphorylcholines dans des fluides biologiques Download PDF

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WO1990012632A1
WO1990012632A1 PCT/US1990/001873 US9001873W WO9012632A1 WO 1990012632 A1 WO1990012632 A1 WO 1990012632A1 US 9001873 W US9001873 W US 9001873W WO 9012632 A1 WO9012632 A1 WO 9012632A1
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blood
plasma
reactive protein
antibodies
phosphorylcholine
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PCT/US1990/001873
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Igal Kedar
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The United States Of America, Represented By The Secretary, United States Department Of Commerce
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61M—DEVICES FOR INTRODUCING MEDIA INTO, OR ONTO, THE BODY; DEVICES FOR TRANSDUCING BODY MEDIA OR FOR TAKING MEDIA FROM THE BODY; DEVICES FOR PRODUCING OR ENDING SLEEP OR STUPOR
    • A61M1/00—Suction or pumping devices for medical purposes; Devices for carrying-off, for treatment of, or for carrying-over, body-liquids; Drainage systems
    • A61M1/36—Other treatment of blood in a by-pass of the natural circulatory system, e.g. temperature adaptation, irradiation ; Extra-corporeal blood circuits
    • A61M1/3679—Other treatment of blood in a by-pass of the natural circulatory system, e.g. temperature adaptation, irradiation ; Extra-corporeal blood circuits by absorption
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
    • B01J20/00—Solid sorbent compositions or filter aid compositions; Sorbents for chromatography; Processes for preparing, regenerating or reactivating thereof
    • B01J20/02—Solid sorbent compositions or filter aid compositions; Sorbents for chromatography; Processes for preparing, regenerating or reactivating thereof comprising inorganic material
    • B01J20/10—Solid sorbent compositions or filter aid compositions; Sorbents for chromatography; Processes for preparing, regenerating or reactivating thereof comprising inorganic material comprising silica or silicate
    • B01J20/103—Solid sorbent compositions or filter aid compositions; Sorbents for chromatography; Processes for preparing, regenerating or reactivating thereof comprising inorganic material comprising silica or silicate comprising silica
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
    • B01J20/00—Solid sorbent compositions or filter aid compositions; Sorbents for chromatography; Processes for preparing, regenerating or reactivating thereof
    • B01J20/28—Solid sorbent compositions or filter aid compositions; Sorbents for chromatography; Processes for preparing, regenerating or reactivating thereof characterised by their form or physical properties
    • B01J20/28002—Solid sorbent compositions or filter aid compositions; Sorbents for chromatography; Processes for preparing, regenerating or reactivating thereof characterised by their form or physical properties characterised by their physical properties
    • B01J20/28004—Sorbent size or size distribution, e.g. particle size
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
    • B01J20/00—Solid sorbent compositions or filter aid compositions; Sorbents for chromatography; Processes for preparing, regenerating or reactivating thereof
    • B01J20/30—Processes for preparing, regenerating, or reactivating
    • B01J20/3092—Packing of a container, e.g. packing a cartridge or column
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
    • B01J20/00—Solid sorbent compositions or filter aid compositions; Sorbents for chromatography; Processes for preparing, regenerating or reactivating thereof
    • B01J20/30—Processes for preparing, regenerating, or reactivating
    • B01J20/32—Impregnating or coating ; Solid sorbent compositions obtained from processes involving impregnating or coating
    • B01J20/3202—Impregnating or coating ; Solid sorbent compositions obtained from processes involving impregnating or coating characterised by the carrier, support or substrate used for impregnation or coating
    • B01J20/3204—Inorganic carriers, supports or substrates
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
    • B01J20/00—Solid sorbent compositions or filter aid compositions; Sorbents for chromatography; Processes for preparing, regenerating or reactivating thereof
    • B01J20/30—Processes for preparing, regenerating, or reactivating
    • B01J20/32—Impregnating or coating ; Solid sorbent compositions obtained from processes involving impregnating or coating
    • B01J20/3202—Impregnating or coating ; Solid sorbent compositions obtained from processes involving impregnating or coating characterised by the carrier, support or substrate used for impregnation or coating
    • B01J20/3206—Organic carriers, supports or substrates
    • B01J20/3208—Polymeric carriers, supports or substrates
    • B01J20/3212—Polymeric carriers, supports or substrates consisting of a polymer obtained by reactions otherwise than involving only carbon to carbon unsaturated bonds
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
    • B01J20/00—Solid sorbent compositions or filter aid compositions; Sorbents for chromatography; Processes for preparing, regenerating or reactivating thereof
    • B01J20/30—Processes for preparing, regenerating, or reactivating
    • B01J20/32—Impregnating or coating ; Solid sorbent compositions obtained from processes involving impregnating or coating
    • B01J20/3231—Impregnating or coating ; Solid sorbent compositions obtained from processes involving impregnating or coating characterised by the coating or impregnating layer
    • B01J20/3242—Layers with a functional group, e.g. an affinity material, a ligand, a reactant or a complexing group
    • B01J20/3244—Non-macromolecular compounds
    • B01J20/3246—Non-macromolecular compounds having a well defined chemical structure
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
    • B01J20/00—Solid sorbent compositions or filter aid compositions; Sorbents for chromatography; Processes for preparing, regenerating or reactivating thereof
    • B01J20/30—Processes for preparing, regenerating, or reactivating
    • B01J20/32—Impregnating or coating ; Solid sorbent compositions obtained from processes involving impregnating or coating
    • B01J20/3231—Impregnating or coating ; Solid sorbent compositions obtained from processes involving impregnating or coating characterised by the coating or impregnating layer
    • B01J20/3242—Layers with a functional group, e.g. an affinity material, a ligand, a reactant or a complexing group
    • B01J20/3244—Non-macromolecular compounds
    • B01J20/3246—Non-macromolecular compounds having a well defined chemical structure
    • B01J20/3248—Non-macromolecular compounds having a well defined chemical structure the functional group or the linking, spacer or anchoring group as a whole comprising at least one type of heteroatom selected from a nitrogen, oxygen or sulfur, these atoms not being part of the carrier as such
    • B01J20/3251—Non-macromolecular compounds having a well defined chemical structure the functional group or the linking, spacer or anchoring group as a whole comprising at least one type of heteroatom selected from a nitrogen, oxygen or sulfur, these atoms not being part of the carrier as such comprising at least two different types of heteroatoms selected from nitrogen, oxygen or sulphur
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
    • B01J20/00—Solid sorbent compositions or filter aid compositions; Sorbents for chromatography; Processes for preparing, regenerating or reactivating thereof
    • B01J20/30—Processes for preparing, regenerating, or reactivating
    • B01J20/32—Impregnating or coating ; Solid sorbent compositions obtained from processes involving impregnating or coating
    • B01J20/3231—Impregnating or coating ; Solid sorbent compositions obtained from processes involving impregnating or coating characterised by the coating or impregnating layer
    • B01J20/3242—Layers with a functional group, e.g. an affinity material, a ligand, a reactant or a complexing group
    • B01J20/3244—Non-macromolecular compounds
    • B01J20/3246—Non-macromolecular compounds having a well defined chemical structure
    • B01J20/3248—Non-macromolecular compounds having a well defined chemical structure the functional group or the linking, spacer or anchoring group as a whole comprising at least one type of heteroatom selected from a nitrogen, oxygen or sulfur, these atoms not being part of the carrier as such
    • B01J20/3253—Non-macromolecular compounds having a well defined chemical structure the functional group or the linking, spacer or anchoring group as a whole comprising at least one type of heteroatom selected from a nitrogen, oxygen or sulfur, these atoms not being part of the carrier as such comprising a cyclic structure not containing any of the heteroatoms nitrogen, oxygen or sulfur, e.g. aromatic structures
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/44—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material not provided for elsewhere, e.g. haptens, metals, DNA, RNA, amino acids
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
    • B01J2220/00—Aspects relating to sorbent materials
    • B01J2220/50—Aspects relating to the use of sorbent or filter aid materials
    • B01J2220/58—Use in a single column

Definitions

  • the present invention relates to a method for removing C-reactive protein and antiphosphorylcholine antibodies from biological fluids to improve the cellular immune responses thereof, and specifically to a method for removing C-reactive protein and antiphosphorylcholine antibodies from the circulation of patients with cancer by conducting extracorporeal perfusion of a patient's blood plasma through a phosphorylcholine-matrix adsorp ⁇ tion device so as to improve the patient's cellular immune responses against the cancer.
  • the method of the present invention may be employed alone or in combination with other cancer treating modalities, such as inter- leukin 2 or other cytokines.
  • effector cells to kill tumor cells when placed in direct contact with the tumor cells in vivo could reflect the presence of host effector which interferes with tumor cells recognition or killing by the adoptively transferred effector cells and the host defense cells present in the body.
  • a number of experi ⁇ mental studies have supported the idea that tumor cells themselves produce or elicit the production by the host of "tumor protecting agents" or “blocking” or “sup ⁇ pressor” factors.
  • the present invention further provides a method and system for employing the same, which comprises pumping blood from a patient through a cell separate which separates the cells into blood cells and plasma, passing the blood plasma through a device, such as a column, which contains adsorbent matrix material that includes phosphorylcholine for removing C-reactive protein and antiphosphorylcholine antibodies, and recombining the plasma and cells before returning the same to the patient.
  • a device such as a column, which contains adsorbent matrix material that includes phosphorylcholine for removing C-reactive protein and antiphosphorylcholine antibodies
  • Figure 1 is a schematic suggestion for C- reactive protein involvement in the macrophage recogni ⁇ tion and killing of tumor cells;
  • Figure 2 is a diagrammatic representation of a system for the extracorporeal treatnent of blood which may be used to carry out the method of the present inven ⁇ tion;
  • Figure 3 is a graph indicating the influence of C-reactive protein when it is combined with IL 2- stimulated human serum or ascites fluid with regard to rosette formation;
  • Figure 4 is a graph indicating the influence of the antibody to C-reactive protein when it is added to IL 2-stimulated human serum with regard to rosette forma ⁇ tion; and Figures 5(A) and 5(B) are graphs indicating the percent lysis of tumor cells in various mixtures includ ⁇ ing IL 2-stimulated human serum, IL 2-stimulated ascites fluid, and C-reactive protein before and after the mix-
  • C-reactive protein is expressed on the membrarie of human macrophages and that the presence of interleukin 2 (IL 2) in human plasma induces a signi ⁇ ficant increase in C-reactive protein (CRP) expression on macrophage membrane, and further that the presence of either soluble CRP, or antiphosphorlcholine (anti-PC) antibodies in a killing mixture of macrophages and target tumor eells almost completely blocks the killing capacity of the effector cells, even after activation with IL2.
  • IL 2 interleukin 2
  • anti-PC antiphosphorlcholine
  • CRP-adsorbent device such as a colunm, having an appropriate adsorbent matrix contained therein in accord- ance with the present invention.
  • a CRP-adsorbent material therein for the extracorporeal treatment of a biological fluid, such as blood plasma, in order to remove CRP and anti-PC antibodies therefrom.
  • the treatment may be conducted by continuously removing a patient's blook separating the blood cells therefrom, treating the separated plasma in the CRP-adsorbent column or device so as to remove the CRP and anti-PC, and mixing and returning the treated plasma and blood cells directly to the patient.
  • the blood cells may be directly rein- fused into the patient.
  • the separated plasma may be collected, treated in the CRP-adsorbent column, again collected, and then returned to the patient as early as possible.
  • the CRP-adsorbent material contained in the column which is used in the method of the present inven ⁇ tion comprises phosphorylcholine (PC) or PC derivatives bonded to a matrix so as to maximize the activity of the PC or PC derivative and the binding capacity of the column or device, while minimizing leakage of the PC and the PC derivatives, as well as other substances, from the column during use.
  • PC phosphorylcholine
  • an effective amount of PC or PC derivatives is used in preparing a column.
  • a few milligrams of PC per gram of matrix mater- ial may be used.
  • One example of a non-limiting range is from about 0.6 to 1.6 mg of PC or PC derivative per gram of column matrix material.
  • the PC or PC derivatives are cross linked to amino groups of a formatted silicon matrix so as to be capable of removing CRP and anti-PC antibodies to improve cellular immune responses against cancer.
  • PC and PC derivatives useful in the method of the present invention include all PC derivatives that sufficiently bind CRP and anti-PC antibodies.
  • PC derivatives include PC esters, such as p- nitrophenyl-6-(O-phosphorylcholine)hydroxy hexanoate.
  • the matrix contained in the extracorporeal device or column used in the method of the present inven ⁇ tion may be formed from any material suitable for carry ⁇ ing the PC and PC derivatives, such as silicon, Agarose, Sepharose, acryloid beads, other suitable polymeric sub- stances a ⁇ i matrixes, and solid-phase silica.
  • the solid- phase silica matrix may comprise virtually any form of particulate silica including amorphous silicas, such as colloidal silica, silica gels, precipitated silicas, and fumed or pyrogenic silicas; microcrystalline silicas such as diatomites; and crystalline silicas such as quartz.
  • the silica should have a particle size in the range from about 45 to 120 mesh, usually in the range from about 45 to 60 ⁇ es .
  • Other materials useful for forming a matrix as disclosed in U.S. Patent 4,681,870 may also be used in the device or column employed in the method of the present invention.
  • U.S. Patent 4,681,870 is herein incorporated by reference.
  • the PC and/or PC derivatives are bound to the device or column matrix in a suitable manner so as to retain the ability of the PC and PC derivatives to remove CRP and anti-PC from the biologiccal fluid passed over the column matrix.
  • the PC or PC derivatives may be cross linked to amino groups of a formatted silicon matrix.
  • Other methods for binding the PC or PC derivatives to the matrix material may be used such as those applicable methods disclosed by U.S. Patent 4,681,870.
  • a system for conducting the extra ⁇ corporeal treatment method of the present invention includes a column or device 10 which is connected to a cell separator 20.
  • the column or device 10 may be sterilized, for example with a gas sterilant such as ethylene oxide, and either used immediately or sealed and stored for later use. Prior to use, the column or device 10 may be washed with normal saline followed by a wash with normal saline containing any other suitable prepara ⁇ tory ingredients. However, no calcium ion chelating agents should be introduced.
  • the column or device 10 is then connected to the cell separator 20 to receive separated plasma there ⁇ from.
  • the cell separator 20 may be continuous flow cell separator, such as an IBM Model 2997, available from IBM, or may comprise a semi-permeable membrane which allows passage of the plasma and blood proteins, but prevents passage of the cellular elements of the blood.
  • a semi-permeable membrane which allows passage of the plasma and blood proteins, but prevents passage of the cellular elements of the blood.
  • a blood pump 22 is used to pass the blood through the membrane. Suitable blood pumps include a tube and a peristalic pump wherein the blood is isolated from the pumping machinery to prevent contamina ⁇ tion.
  • the blood passes through the cell separator 20 at a rate which may be in the range of from about 10 to 20 ml/min. typically until a total desired volume of blood has been passed.
  • the blood cells are mixed with the plasma passing through the treatment column or device 10, and the recombined blood returned to the patient.
  • a microfilter 24 may be provided at the outlet of the treatment column or device 10 to prevent passage of macroscopic particles which might be lost from the column or device 10.
  • the ascitic cells were then isolated. Freshly ⁇ obtained ascite specimens were centrifuged in 250 ml sterile polypropylene containers at 1200 rpm for 10 minutes at 4°C. The cell-free ascitic fluid was harvested and kept at 4°C until used. The cell pellet was washed three times in phosphate-buffered saline (PBS) . supplemented with 0.4% sodium citrate. After the third wash, the cells were -resuspended in a small volume of cold RPMI 1640 and layered over Ficoll.
  • PBS phosphate-buffered saline
  • the interphase cell layer was collected, washed twice in - RPMI 1640, and resuspended at 10 8 cells/ ml in RPMI 1640 witii 2% pooled human heat-inactivated AB serum, 50 yg/ml gentamicin, and 2mM glutamine. The cells were then kept at 4°C until use.
  • Tumor cytotoxicity was quantified based on the following procedures. Ficoll-enriched, washed ascite cells were incubated under a variety of experimental conditions at . 37°C in humidified air with 5% C0 2 with gentle agitation at a concentration of 3-4 x 10° cells/ml. Controlled incubations were performed at 4°C. Samples of 100 microliters (304 x 10 5 cells ) were withdrawn from the culture at various time points and slides " were made by spinning the sample in a cytocentri- fuge, fixation in methanol, and staining with Giemsa. A total of 10 low-power (25x) fields were examined (2 slides, 5 fields each).
  • a total number of tumor cells in these fields were enumerated and the fraction of cells forming rosettes (at least 4 cells directly attached to the tumor cell) was determined.
  • a semi-quantitive assessment of the degree of rosette formation was formu- lated based on the percentage of tumor cells forming rosettes and the number of host effector cells surround ⁇ ing each tumor cell. The criteria were as follows: 4+ - all tumor cells form rosettes, all rosettes com ⁇ prised of at least 8 cells;
  • 1+ 20-80% of tumor cells form rosettes, ⁇ 20% of rosettes comprised of at least 8 cells, 80% or more hav- ing 4-7 cells;
  • Cytotoxicity was determined by harvesting cells cultured under various experimental conditions for 24 hours and making Giesma-stained cytocentrifuge prepara ⁇ tions, as noted above for assessment of rosette forma ⁇ tion. Surviving tumor cells were identified morpho ⁇ logically and counted in 10 low-power fields (2 slides, 5 fields each) . Duplicate samples were evaluated for each culture condition. Parallel cultures were incubated at 4°C for the same time period and were considered negative controls. Cytotoxicity was calculated as follows: Cytotoxicity - (1 - (avq. number of surviving tumor cells at 37°C)) x 100 (avg. number of surviving tumor cells at 4°C)
  • C-reactive protein was isolated and purified from malignant ascites by a modification of the procedure described by Volanakis et al, "C-reactive Protein: Puri- fication by Affinity Chromatography and Physiochemical Characterization," J. Immunol. Meth. (1978), 23:285; and Oliveira et al, "comparative Studies on the Binding Properties of Human and Rabbit C-reactive Protein,” J. Immunol. Meth. (1980), 124:1396.
  • the C-reactive protein so obtained formed a single band on polyacrylamide gel electrophoresis, binded to antibodies to C-reactive protein, and was stored at -70°C at 1.5 mg/ l in PBS.
  • C-reactive protein was biotinylated according to the procedure of Bayer et al, "The Avidin-Biotin Complex in Affinity Cytochemistry," Meth. Enz mol. (1979), 62:308, and was stored at 4°C in a 1 mg/ml solution. Unlabelled (lot 25962) and fluorescein isothiocyanated (FITC- labelled) (lot 12217) F(ab') 2 fragments of sheep anti- human C-reactive protein antibodies were purchased from Cappel (Melvern, PA), dialyzed against PBS and sterilized by filtration before use. Murine monoclonal IgM anti- bodies 17/207 (obtained from Dr. J.
  • Kenny, PRI, NCI-FCRF Kenny, PRI, NCI-FCRF
  • HPCM2 obtained from Dr. P. Gearhart, Johns Hopkins University
  • the anti-phosphoryl- choline antibodies were biotinylated by the method of Bayer et al, supra ⁇ Recombinant human IL 2 was obtained from the Cetus Corp. (Emeryville, CA) .
  • Cytospin slides that had been fixed in cold acetone (minus 20°C) for 45 minutes were coated with FITC- labelled F(ab') 2 fragments of antibodies to C-reactive protein, biotinylated antiphosphorylcholine antibodies, or biotinylated C-reactive proteins for 90 minutes at room temperature * Biotinylated antibodies were developed with a secjDnd incubation of 60 minutes with FITC-labelled avidin.
  • the slides were washed in PBS buffer containing 0.2%. albumin, ,100 mg/L Ca ++ , 100 mg/L mg ++ , and 0.01% sodium azide, and the slides were counterstained with 0.5% Evans blue dye. The slides were then examined with a fluorescence microscope.
  • a phosphorylcholine affinity column was prepared using 50 mg of P-nitrophenyl-6-(0- phosphocholine)hydroxy hexanoate, prepared by "molecular probes" in accordance with the procedure of T.F. Spande, J. Organic Che . (1980) 45: 3081, which was dissolved in 0.2 ml of dry acetonitrile and the resulting solution then added to 60 i ml of a 1:1 suspension of -aminohexyl agarose (Sigma) in rJoorate buffer saline (BBS), at pH 8.4. This mixture was stirred gently overnight at 4°C and washed with 1 liter of BBS. The washed PC-matrix was packed in a glass column 2 cm in diameter and about 15 cm in height. The column was then used to remove CRP and anti-PC anti- bodies from human malignant ascites. Results:
  • the malignant ascites were examined under the microscope after Giemsa staining, and abundant malignant cells, lymphocytes, macrophages, neutrophils, and a few mesothelial cells were observed. There were very few tumor cells interacting with the host defense cells present in the ascitic fluid, regardless of the tumor cell type or the number and type of host cells present. Cells from 8 of the 9 patients were incubated for one hour at 37°C either in autologous cell-free ascitic fluid or an RPMI 1640 with 2% human AB serum. Spontaneous rosette formation in the ascites averaged a score of 0.49 +/- 0.06. However in serum-containing media, the value was 1.86 +/- 0.16.
  • the rosette formation in the serum- containing medium was significantly higher than that in the ascites.
  • Spontaneous rosette formation in the ascites was not different from the levels seen in the serum- containing medium incubated at 4°C.
  • Rosette formation was significantly increased by incubating the ascitic cells for one hour at 37°C in serum-containing media supple ⁇ mented with 500 U/ml recombinant human IL 2.
  • the effect of added IL 2 on rosette formation in the ascitic fluid was considerably less dramatic, though still significantly increased over spontaneous rosette formation in the ascitic fluid.
  • the ascitic fluid significantly inhibited both the spontaneous and the IL 2-induced rosette formation among tumor and host defense cells.
  • Ascitic cells were then incubated for one hour at 37°C in autologous ascites and the autologous ascites depleted of C-reactive protein by passage over a column of immobilized phosphorylcholine.
  • IL 2-stimulated rosette formation was 3 times higher in ascites from which C-reactive protein had been removed and was similar to the IL 2-stimulated rosette score observed in the serum.
  • purified C-reactive pro ⁇ tein was added to the ascitic cell cultures after these cultures had been depleted of C-reactive protein.
  • purified C-reactive protein inhibits IL 2-stimulated rosette formation in either ascites or in a serum-containing medium although not completely to the levej. of the pre-column ascites.
  • the C-reactive protein is apparently capable of blocking the interaction between host defense cells and malignant cells in the ascitic fluid. If C-reactive protein is the effector's targeting ⁇ device, antibodies to C-reactive protein should also block rosette formation.
  • the JL 2-stimulated formation of tumor rosettes was signi ⁇ ficantly inhibited by antibodies to C-reactive proteins.
  • the antibodies completely blocked the stimulatory effects of the IL2, reducing IL 2-stimulated rosette formation to the level of spontaneous rosette formation in the serum.
  • cytocentrifuge preparations of ascitic cells were stained with FITC-conjugated F(ab') 2 fragments of antibodies to C- reactive protein.
  • the macrophages expressed abundant cell surface molecules recognized by the antibodies to C- reactive protein.
  • ascitic cells were cultured under a variety of conditions for 24 hours and the number of surviving tumor cells was determined by counting the cells on a Giemsa-stained cytocentrifuge preparation. Tumor cells death was considered to be at the baseline level (i.e., 0% killing) in cultures incubated at 4°C, i.e., conditions that interfere with rosette formation.
  • the formula for calculating cyto- toxicity as described above incorporates this assump ⁇ tion.
  • the lytic effect on tumor cells incubated for 24 hours precisely paralleled the propensity to form rosettes.
  • Figures 5(A) and 5(B) are graphic representations of the results of photomicrographs at 25 x of Giemsa-stained ascitic cells cultured under a variety of conditions for eight different patients, each patient being represented by a different symbol. These symbols include a circle, an hourglass shape, a triangle directed upwards, a triangle directed downwards, a star, a diamond shape, a blank square, and a square having two crossed diagonal lines. Both killing and rosette formation occur in serum but not in ascites. Addition of IL 2 enhances both killing and rosette forma ⁇ tion in serum but the effect is substantially less impera- tive in ascites.
  • IL 2-stimulated rosette formation and killing reached a level comparable to that seen in serum when C-reactive protein was first removed from the ascites by passage over a phosphorylcholine column.
  • Addition of either purified C-reactive protein or antibody to C- reactive protein inhibits both rosette formation and killing in either serum or ascites.
  • a correlation coeffi ⁇ cient for rosette formation and cytotoxicity was determined to be 0.85 (p ⁇ 0.05). Thus, rosette formation is a reasonably accurate surrogate measure for tumor cytotoxicity.

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Abstract

Un procédé servant à traiter le cancer par la réalisation extracorporelle d'une perfusion de plasma sanguin d'un malade du cancer au moyen d'un dispositif, tel qu'une colonne (10), contenant une matière matricielle absorbante comportant de la phosphrylcholine (PC) ou des dérivés de PC afin d'enlever la protéine C-réactive et les anticorps anti-PC de façon à améliorer les réponses immunisantes cellulaires du malade contre le cancer. Le procédé peut être utilisé comme seul traitement ou en combinaison avec une autre modalité de traitement du cancer telle que le traitement par l'IL-2 ou d'autres cytokines.
PCT/US1990/001873 1989-04-19 1990-04-06 Procede servant a enlever la proteine c-reactive et les anticorps anti-phosphorylcholines dans des fluides biologiques WO1990012632A1 (fr)

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Cited By (12)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP0787500A1 (fr) * 1996-02-06 1997-08-06 BELLCO S.p.A. Procede et dispositif de seperation de toxines en particulier de cytokines
WO2003019135A2 (fr) * 2001-08-23 2003-03-06 Bio-Rad Laboratories, Inc. Temoin de reference pour test de proteine c-reactive a sensibilite elevee
WO2004076486A1 (fr) * 2003-02-27 2004-09-10 Theravision Gmbh Methode de diminution des niveaux de proteine c-reactive
WO2005100405A3 (fr) * 2004-04-15 2006-04-13 Athera Biotechnologies Ab Nouvelle composition
DE102005061715A1 (de) * 2005-12-22 2007-06-28 Biobarries Gmbh Prozess zur Entfernung von C-reactivem Protein aus biologischen Flüssigkeiten durch Apherese
US20100285044A1 (en) * 1998-05-22 2010-11-11 Lentz M Rigdon Method and compositions for treatment of cancers
WO2015193504A1 (fr) * 2014-06-19 2015-12-23 Pentracor Gmbh Matériau de séparation comprenant des dérivés phosphorylcholine
EP3020726A1 (fr) 2014-11-12 2016-05-18 Pentracor GmbH Utilisation d'une solution de citrate destiné au nettoyage chromatographique d'affinés de CRP au moyen de phosphocholine et leurs dérivés
US9796786B2 (en) 2011-08-09 2017-10-24 Athera Biotechnologies Ab Antibodies binding to phosphorylcholine (PC) and/or PC conjugates
US9803028B2 (en) 2011-08-09 2017-10-31 Athera Biotechnologies Ab Antibodies against phosphorylcholine
EP3607978A1 (fr) 2018-08-06 2020-02-12 Pentracor GmbH Régénération simplifiée de colonnes d'aphérèse
US11896969B2 (en) 2019-09-05 2024-02-13 Bio-Rad Laboratories, Inc. Anionic exchange-hydrophobic mixed mode chromatography resins

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US4203893A (en) * 1977-09-07 1980-05-20 Institut National De La Recherche Agronomique Coupling products of cytidine-diphosphocholine and amino-compounds for pharmaceutical use
US4384954A (en) * 1980-04-16 1983-05-24 Kuraray Co., Ltd. Column for adsorption of blood proteins
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US4681870A (en) * 1985-01-11 1987-07-21 Imre Corporation Protein A-silica immunoadsorbent and process for its production
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US4203893A (en) * 1977-09-07 1980-05-20 Institut National De La Recherche Agronomique Coupling products of cytidine-diphosphocholine and amino-compounds for pharmaceutical use
US4384954A (en) * 1980-04-16 1983-05-24 Kuraray Co., Ltd. Column for adsorption of blood proteins
US4472303A (en) * 1981-07-10 1984-09-18 Kuraray Co., Ltd. Blood purification method
US4681870A (en) * 1985-01-11 1987-07-21 Imre Corporation Protein A-silica immunoadsorbent and process for its production
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Cited By (29)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP0958839A2 (fr) * 1996-02-06 1999-11-24 BELLCO S.p.A. Dispositif de séparation de toxines en particulier de cytokines
EP0958839A3 (fr) * 1996-02-06 1999-12-15 BELLCO S.p.A. Dispositif de séparation de toxines en particulier de cytokines
EP0787500A1 (fr) * 1996-02-06 1997-08-06 BELLCO S.p.A. Procede et dispositif de seperation de toxines en particulier de cytokines
US20100285044A1 (en) * 1998-05-22 2010-11-11 Lentz M Rigdon Method and compositions for treatment of cancers
WO2003019135A2 (fr) * 2001-08-23 2003-03-06 Bio-Rad Laboratories, Inc. Temoin de reference pour test de proteine c-reactive a sensibilite elevee
US6548646B1 (en) * 2001-08-23 2003-04-15 Bio-Rad Laboratories, Inc. Reference control for high-sensitivity C-reactive protein testing
WO2003019135A3 (fr) * 2001-08-23 2003-11-20 Bio Rad Laboratories Temoin de reference pour test de proteine c-reactive a sensibilite elevee
AU2002326403B2 (en) * 2001-08-23 2006-08-24 Bio-Rad Laboratories, Inc. Reference control for high-sensitivity C-reactive protein testing
JP2007523837A (ja) * 2003-02-27 2007-08-23 テラヴィジョン ゲゼルシャフト ミット ベシュレンクテル ハフツング Ｃ反応性タンパク質のレベルを低減するための方法
WO2004076486A1 (fr) * 2003-02-27 2004-09-10 Theravision Gmbh Methode de diminution des niveaux de proteine c-reactive
US8012483B2 (en) 2004-04-15 2011-09-06 Athera Biotechnologies Ab Phosphorylcholine conjugates and corresponding antibodies
EP1797893A3 (fr) * 2004-04-15 2007-06-27 Athera Biotechnologies Ab Conjugués de la phosphorylcholine et anticorps correspondants
WO2005100405A3 (fr) * 2004-04-15 2006-04-13 Athera Biotechnologies Ab Nouvelle composition
US10222382B2 (en) 2004-04-15 2019-03-05 Athera Biotechnologies Ab Phosphorylcholine conjugates and corresponding antibodies
WO2007076844A1 (fr) * 2005-12-22 2007-07-12 Beta V3 Gmbh Utilisation d'une matrice pour eliminer la proteine c reactive de liquides biologiques
DE102005061715A1 (de) * 2005-12-22 2007-06-28 Biobarries Gmbh Prozess zur Entfernung von C-reactivem Protein aus biologischen Flüssigkeiten durch Apherese
US9803028B2 (en) 2011-08-09 2017-10-31 Athera Biotechnologies Ab Antibodies against phosphorylcholine
US9796786B2 (en) 2011-08-09 2017-10-24 Athera Biotechnologies Ab Antibodies binding to phosphorylcholine (PC) and/or PC conjugates
JP2017526732A (ja) * 2014-06-19 2017-09-14 ペントラコール ゲーエムベーハー ホスホリルコリン誘導体を含む分離物質
CN106459108A (zh) * 2014-06-19 2017-02-22 彭特科尔有限公司 包含磷酰胆碱衍生物的分离材料
EP2957563A1 (fr) 2014-06-19 2015-12-23 Pentracor GmbH Matériau de séparation comprenant des dérivés de la phosphorylcholine
RU2666357C2 (ru) * 2014-06-19 2018-09-07 Пентракор Гмбх Сепарационный материал, включающий производные фосфорилхолина
WO2015193504A1 (fr) * 2014-06-19 2015-12-23 Pentracor Gmbh Matériau de séparation comprenant des dérivés phosphorylcholine
CN106459108B (zh) * 2014-06-19 2019-10-15 彭特科尔有限公司 包含磷酰胆碱衍生物的分离材料
EP3020726A1 (fr) 2014-11-12 2016-05-18 Pentracor GmbH Utilisation d'une solution de citrate destiné au nettoyage chromatographique d'affinés de CRP au moyen de phosphocholine et leurs dérivés
US9962628B2 (en) 2014-11-12 2018-05-08 Pentracor Gmbh Use of citrate solution for affinity chromatographic purification of CRP using phosphocholine and derivatives thereof
EP3607978A1 (fr) 2018-08-06 2020-02-12 Pentracor GmbH Régénération simplifiée de colonnes d'aphérèse
WO2020030532A1 (fr) 2018-08-06 2020-02-13 Pentracor Gmbh Régénération simplifiée de colonnes d'aphérèse
US11896969B2 (en) 2019-09-05 2024-02-13 Bio-Rad Laboratories, Inc. Anionic exchange-hydrophobic mixed mode chromatography resins

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