Classifications

• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
  • A61K35/12—Materials from mammals; Compositions comprising non-specified tissues or cells; Compositions comprising non-embryonic stem cells; Genetically modified cells
  • A61K35/26—Lymph; Lymph nodes; Thymus; Spleen; Splenocytes; Thymocytes
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
  • A61K35/12—Materials from mammals; Compositions comprising non-specified tissues or cells; Compositions comprising non-embryonic stem cells; Genetically modified cells
  • A61K35/28—Bone marrow; Haematopoietic stem cells; Mesenchymal stem cells of any origin, e.g. adipose-derived stem cells

Definitions

• the fractionation is effected by means of certain agglutinins adapted to bind selectively to terminal galactose residues on cell surfaces.
• the substances used are of the type of galactose and fucose binding lectins, and among preferred leetins there may be mentioned soybean agglutinin, peanut agglutinin.
• stem cells when different cell types are present, only stem cells interact with lectins of this type, while no interaction takes place with nature T-cells. This brings about an aggregation of the stem cells, the aggregates are easily separated from the other cells and the aggregates can be subsequently broken up to yield cells which can be injected.
• Mouse bone marrow and spleen cells were fractionate by means of agglutinins of the type defined above.
• a test for spleen colony forming .units in the isolated fractions showed that the hemopoietic stem cells are agglutinated by such lectins.
• Specific substances used were soybean and peanut agglutinin.
• the capacity of the agglutininated fractions to reconstitute lethally irradiated allogeneic mice was investigated.
• the cell fractions thus obtained are substantially depleted of graft-versus-host (GVH) reaction, and thus it is possible to overcome difficulties encountered hitherto when it was tried to graft allogeneic hemopoiet cell transplants to irradiated mammals or to severely immune deficient patients.
• the difficulties are due to the presence of cells which cause a GVH reaction, and these are substantially removed from the cell fractions obtained according to the process of the present invention.
• Peanut agglutinin is a lectin specific for D- galactosyl-(1-3)-N-acetyl-D-galactosamine while soybean agglutinin (SBA) is a lectin specific for terminal N-acetyl-D-galactosamine and D-galactose. It seems that receptors for such lectins, and specifically from PNA or SBA are present on the surface of henopoietic stem cells, whereas in most cells of the T-type the PNA and SBA receptors are masked.
• the agglutinated cells are the ones which can be used subsequently for the treatment, the resulting fraction being substantially devoid of GVH reaction.
• the following Example is intended to illustrate the invention and is not to be construed in a limitative manner.
• mice Female (Balb/c x C57BL/6)F 1 hybrids and female SWR mice were used as recipients.
• Donor animals were Balb/c and SWR female mice. The recipients were 8-12 weeks old and the donors' age was 6-8 weeks. All mice were raised at the Weizmann Institute Animal Center. Irradiation. Mice were exposed to a single dose 60 of 900 R from a Gamma beam 150A, Co source, produced by Atomic Energy of Canada, with F.S.D. of 75 cm and 65 R/min dose rate.
• Bone marrow was obtained from the femurs and suspended in phosphate buffered saline (PBS). Spleen suspension was prepared by straining the tissue in PBS solution through nylon gauze. The cells were then washed twice in PBS and resuspended in the same buffer at the required final concentration. Nucleated cells were counted in Turk's solution. Spleen colony forming units (CFU-S) assay. Details of the procedure have been previously described. Bone marrow and spleen suspensions were suspended in PBS at various concentrations (see Results) and injected into the caudal vein of irradiated recipients. Each experimental group consisted of 8 SWR mice.
• PBS phosphate buffered saline
• PNA and SBA were purified by affinity chromatography on a column of Sepharose-N- ( ⁇ -amino- caproyl) -B-D-galactopyranosylamine. Separation of CFU-S enriched fractions by agglutination with SBA and PNA:
• Fractionation of splenocytes by SBA was carried out as follows: The splenocyte suspension (2 x 10 8 cells in 0.5 ml PBS) was incubated in polystyrene tubes (17 x 100 mm) with SBA (0.5 ml, 2 mg/ml) for 5 minutes at room temperature. The cells were then gently layered with Pasteur pipette on top of a solution of bovine serum albumin (5% w/v in PBS, 40 ml) in a 50 ml conical glass tube. After 15 minutes at room temperature, most of the agglutinated cells sedimented, whereas the unagglutinated cells remained on the surface of the bovine serum albumin solution.
• the bottom and top fractions were removed separately by Pasteur pipettes and transferred to 15 ml conical plastic tubes.
• the cells were then suspended in D-galactose (0.2 M) in PBS. After 10 minutes at room temperature, the cells were collected by centrifugation (200 x g, 5 min) and washed twice with the D-galactose solution. Finally the cells were washed twice with PBS. About 6 x 10 7 cells were obtained from both agglutinated and unagglutinated fractions.
• the agglutinated fraction is enriched 2.8 fold in hemopoietic activity relative to the unagglutinated fraction.
• a similar fractionation of bone marrow cells resulted in total agglutination (more than 90% of the cells were recovered in the bottom fraction) thus strengthening our assumption that the SBA receptor is present on hemopoietic cells as well as on B cells.
• Staining of bone marrow and spleen cells with PNA conjugated to fluorescein isothiocyanate revealed receptors for the lectin on only a small portion of the cells (10-20%).
• Fractionation of bone marrow or spleen cells by PNA could be achieved, however, when rabbit erythrocytes, which carry the PNA receptors were added to the incubation mixture to form mixed aggregates of nucleated mouse spleen or bone marrow cells with rabbit erythrocytes. These aggregates were large enough to be separated from the unagglutinated fraction.
• the spleen or bone marrow cells in PBS (3 x 10 8 cells/ 0.5 ml) were incubated in polystyrene tubes (17 x 100 mm) with PNA (0.3 ml, 1 -mg/ml) for 5 min at room temperature and then rabbit erythrocytes (1.8 x 10 9 cells in 0.3 ml
• FIG. 1 shows the cumulative mortality of irradiated (BALB/c x C57BL/6)F 1 mice after transplantation with splenocytes (10 7 cells per animal) from BALB/c mice starting with 15 mice in each group.
• Grafts unfractionated splenocytes , splenocytes agglutinated by SBA; , splenocytes unagglutinated by SBA; , control without graft.
• Fig. 2 shows the cumulative mortality of irradiated (BALB/c x C57BL/6)F 1 mice after transplantation with splenocytes (10 7 cells per animal) from SWR mice, starting with 15 mice in each group.
• Grafts unfractionated splenocytes; , splenocytes sequentially agglutinated by SBA and PNA; , splenocytes unagglutinated by SBA; , control without graft. Grafting lethally irradiated allogeneic mice with unfractionated splenocytes or with cells unagglutinated by SBA resulted in high mortality (13/15 and 15/15 respectively) within the first 30 days. The two mice surviving the first four weeks were suffering from wasting disease (delayed GVH reaction) and died within the second month after transplantation.
• Spleen histology and bone marrow differential count revealed typical GVH symptoms damage to the white pulp of the (myelo-, erthro- and thrombopoiesis) in the spleen of these mice was normal or slightly hyperplastic, indicating that complete hemopoiesis had occurred and that the high mortality rate among these mice was due to acute GVH reaction and not to deficiency of hemopoietic stem cells.
• mice grafted with the twice agglutinated fraction only one out of fifteen died (Fig. 2). The remaining mice have already survived more than six months.
• Spleen histology (Figs. 3 and 4) and bone marrow differential count when taken four and five weeks after transplantation in a parallel experiment, revealed that in reconstituted mice compared with intact animals, both spleen and bone marrow were completely restored.
• Typical spleen histology of ⁇ BALB/c x C57BL/6)F 1 mice irradiated by 900 R and grafted with splenocytes (10 7 cells per animal) of SWR donors is shown in Figs. 3 and 4. The specimen was taken from mice sacrificed 4 weeks after transplantation.
• Mouse spleen cells can serve as a good model for allogeneic bone marrow transplantations in humans, since both mouse splenocytes and human bone marrow aspirates cause an early type of GVH reaction which is fatal in most cases, whereas mouse bone marrow cells may cause only a mild type of delayed GVH reaction.

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• 1978
  • 1978-06-15 IL IL7854911A patent/IL54911A0/xx unknown
• 1979
  • 1979-06-14 WO PCT/US1979/000432 patent/WO1980000058A1/en unknown
  • 1979-06-14 JP JP50106379A patent/JPS55500688A/ja active Pending
• 1980
  • 1980-01-29 EP EP79900747A patent/EP0016790A1/en not_active Withdrawn

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