Classifications

• • B—PERFORMING OPERATIONS; TRANSPORTING
  • B29—WORKING OF PLASTICS; WORKING OF SUBSTANCES IN A PLASTIC STATE IN GENERAL
  • B29C—SHAPING OR JOINING OF PLASTICS; SHAPING OF MATERIAL IN A PLASTIC STATE, NOT OTHERWISE PROVIDED FOR; AFTER-TREATMENT OF THE SHAPED PRODUCTS, e.g. REPAIRING
  • B29C45/00—Injection moulding, i.e. forcing the required volume of moulding material through a nozzle into a closed mould; Apparatus therefor
  • B29C45/17—Component parts, details or accessories; Auxiliary operations
  • B29C45/64—Mould opening, closing or clamping devices
  • B29C45/66—Mould opening, closing or clamping devices mechanical
• • 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
  • A61K35/14—Blood; Artificial blood
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P37/00—Drugs for immunological or allergic disorders
• • B—PERFORMING OPERATIONS; TRANSPORTING
  • B29—WORKING OF PLASTICS; WORKING OF SUBSTANCES IN A PLASTIC STATE IN GENERAL
  • B29C—SHAPING OR JOINING OF PLASTICS; SHAPING OF MATERIAL IN A PLASTIC STATE, NOT OTHERWISE PROVIDED FOR; AFTER-TREATMENT OF THE SHAPED PRODUCTS, e.g. REPAIRING
  • B29C45/00—Injection moulding, i.e. forcing the required volume of moulding material through a nozzle into a closed mould; Apparatus therefor
  • B29C45/17—Component parts, details or accessories; Auxiliary operations
  • B29C45/64—Mould opening, closing or clamping devices
  • B29C45/66—Mould opening, closing or clamping devices mechanical
  • B29C2045/667—Cam drive for mould closing or clamping

Definitions

• the present invention relates to methods and means for the in vitro treatment of whole blood taken from an individual (human or animal) for the purpose of providing an im uno-stimulated blood product designed for re-admininstration to the very same same individual, from which the whole blood sample was taken, for prophylactic or therapeutic purposes.
• TNF tumor killing substances
• lymphokines e.g. gamma-interferon and interleukines
• interleukin II IL-II
• IL-II interleukin II
• My PCT application PCT/SE84/00222 discloses a completely new approach to the problem of preparing and using preparations for the treatment of diseases which are sensitive to gamma-inter- feron and/or interleukins.
• the invention described in said publication was based on the per se well known fact that mito- gens can induce production of i.a. gamma-interferon in vitro. This approach involved two critical steps, viz.
• Another important advantage of the autologeous procedure is that the stimulated cells are the patient's own cells, with the result that the stimulation will produce an optimal mixture of tumor killing substances. Furthermore, the procedure is rather simple, rapid, inexpensive and eliminates or considerably reduces the instability problems of gamma- interferon preparations. It also was most unexpected that the autologeous treatment could result in such a high yield of interferon production, since it was previously known that sick patients, such as tumour patients, have a reduced capability of producing interferon.
• the first-men- tioned paper generally relates to the stimulation of purified leucocytes by contacting the leucocytes with an insoluble, antitumor i munocyte-inducing material capable of binding to T- cells, said material comprising a ligand which is covalently bound to an insoluble carrier.
• the stimulation may be carried out after immunosuppressor cells (suppressor T-cells, macro- phages) have been removed.
• EP-A2-107119 discloses a specific blood-treating material having antitumor effect and consisting of a lipopolysaccharide derived from the cell walls of Gram-negative bacteria and immobilized by an insoluble carrier containing an a ino group or a carboxyl group. This modification of the lipopolysaccha ⁇ ride is said to reduce the lethal toxicity of the lipopoly ⁇ saccharide.
• EP-A2-79221 i.a. discloses the use of immobilized Protein A for removing immuno complexes (immunoglobulines) from a pati ⁇ ent's blood plasma and then providing for specific tumor anti ⁇ body generation. Also US-A-4551435 is concerned with the remo ⁇ val of immuno complexes from the blood.
• the present invention relates to further developments of the basic concept of the autologous stimulation as disclosed in the above mentioned PCT application, i.e. the procedure com ⁇ prising the steps of taking a blood sample from an specific individual, incubating the sample in vitro with a suitable mitogen, and re-administering the incubated blood sample, or parts thereof, to the very same individual.
• a first aspect of the invention is based on the insight that a very great proportion of the human population is carrying antibodies against one or more immune-stimulating substances, or mitogens (as herein defined) .
• mitogens as herein defined
• mitogen SEA Staleukin-associated plasminogen SEA
• anti-bodies against other mitogens are common.
• Such anti- bodies completely destroy, or at least severely inhibit, the cell stimulating capability of the corresponding mitogen.
• it is sugg ⁇ ested to remove the antibodies against the mitogen in question from the cell-containing blood sample before stimulating the sample in vitro with the respective mitogen.
• cell-containing blood samples primarily means whole blood, but the expression may also include any blood fraction or blood component containing cells which can be stimulated by a mitogen, in particular containing monocytes and/or B cells.
• mitogen is to be understood in a broad sence, and it is intended to include any substance or agent, or parts or fragments thereof (including mitogenically active components prepared enzymatically or by recombinant technology), which are capable of stimulating the immune-defen ⁇ se of living blood cells when brought into contact with such cells (such agents may in certain publications be called “mito ⁇ gens", “antigens”, “immuno-stim lants”, “immuno-inducers”, or the like) .
• the present invention has shown that the above mentioned pre- separation of anti-mitogen antibodies dramatically reduces the amount of mitogen which is required for efficiently stimulating the blood cells (if at all possible in the presence of anti- mitogen antibodies).
• SEA as the mitogen, it may as an example be mentioned that only a few molecules of SEA per blood cell may be sufficient for triggering an efficient cell stimulation when the preparation has been made substantially free from anti-SEA in accordance with the invention.
• a second aspect of the invention relates to a new method of separating mitogens from the incubated cell-containing blood sample.
• the separation is carried out by contacting the mitogen-con- taining sample with anti-bodies which are specific for the mitogen used for the stimulation of the sample, said anti- bodies being bonded to a carrier which can be separated from the sample, with the mitogen attached to the corresponding anti-mitogen of the carrier.
• a further aspect of the invention relates to a kit for carry ⁇ ing out either or both of the above mentioned methods, said kit including at least one blood container having a blood sample inlet and a blood sample outlet, separation means provided in said outlet, and means for contacting said blood sample with at least one agent having mitogenic activity in said at least one blood container, said separation means being capable of separa ⁇ ting said mitogenic agent from said blood sample, so that the stimulated blood sample leaving said outlet is substantially free from mitogenic activity.
• in vitro stimulation should be interpreted to include not only actual production of tumor killing substan- ces (as defined above) in vitro, but also the in vitro priming of the blood cells for subsequent production of said substan ⁇ ces in vivo (after the re-administration to the patient).
• the presently most preferred mitogen is SEA which, as already mentioned, is a tremendously potent mitogen in the absence of anti-SEA antibodies, but it is within the scope of the inven- tion possible to use any other immuno-stimulating mitogen, including mitogenically active fragments of SEA, SEB, and the like, in particular mitogens having, like SEA, a strong speci ⁇ ficity for MHC II, especially on accessory cells such as mono ⁇ cytes and/or B cells (MHC — Major Histoco patibility Complex) .
• the invention makes use of either or both of two mitogenically active fragments of SEA, which seem to be non-toxic to the human body.
• the mitogen is labelled with a substance capable of binding to the mitogen (e.g. by a covalent bond) without substantially destroying the mitogenic activity of the mitogen, the labelled mitogen then being removed from the blood cells by means of an agent capable of strongly bind- ing to said labelling substance.
• the labelled mitogen is biotinylated (labelled with biotin), especially biotinylated SEA or active fragments there ⁇ of (SEA-B).
• Avidin which is known to have an extremely high affinity for biotin also when immobilized on a carrier, is the preferred agent for removing the biotinylated mitogen from the stimulated blood sample.
• a biotinylated mitogen such as SEA-B can be removed quantitavely by absorption on avidin immobilized on a suitable carrier such as a polysaccharide type carrier, e.g. an agarose carrier.
• the carrier usually has the form of beads or similar particles, which can be removed from the treated blood sample any convenient separation technique such as filtering, chromatographic separation, and the like.
• the inner walls of e.g. a blood bag might serve as the avidin-carrier.
• the presently preferred procedure for removing the anti-mito ⁇ gen before the incubation with the mitogen is to immobilize anti-mitogen binding substance on a solid carrier, which has a greater diameter than the blood cells and therefore can be conveniently separated by means of a conventional filter.
• the carrier may e.g. consist of polysaccharides, for example agaro- se, which are commersially available.
• the cell-containing blood starting material is preferably heparinized whole blood. Both the anti-mitogen elimination and the mitogen stimulation are preferably carried out batchwise as a suspension under (comparatively cautious) agitation.
• the treatment kit according to the invention it is pre ⁇ sently preferred to use, as the containers, bags of substan- tially the same type as is used in conventional blood separa ⁇ tion systems and the like. Examples of two different embodiment of the kit are illustrated in the enclosed drawings.
• Figure 1 is a diagrammatic representation of a first embodi- ment of a blood bag system for use according to the invention.
• Figure 2 is a diagrammatic representation of a second embodi ⁇ ment of a blood bag system for use according to the invention.
• Figure 3 is a diagram illustrating the development of the tumor size of implanted VX2 carcinoma in untreated rabbits.
• Figure 4 is a diagram illustrating the tumor development for rabbits subjected to different kinds of treatment.
• Figure 5 is a diagram comparing animals treated according to the invention with a control group as regards survival.
• Figure 1 of the drawing illustrates how whole blood is taken from a patient and collected in, for example, a conventional heparinized blood bag (WHOLE BLOOD).
• WHOLE BLOOD heparinized blood bag
• the latter bag is in turn connected - or connectable - to a second blood bag (IMMOBIL. MITOGEN), containing anti-mitogen binding particles, preferab ⁇ ly the mitogen (e.g. SEA) which is specific for the anti-mito- gen (e.g. anti-SEA) to be removed from the blood sample, said specific mitogen preferably being immobilized on a suitable carrier such as agarose beads.
• SEA anti-mito- gen
• the latter are preferably con ⁇ siderably larger than the blood cells, for example about 200 ⁇ m compared to about 7 ⁇ m for white blood cells.
• the proportions in this bag can suitably be of the order of one part of beads per nine parts of whole blood. It is usually sufficient to mix the contents of the bag for about half an hour in order to bind the anti-mitogen to the beads. As mentioned above, the duration of the treatment can be considerably shorter, in particular when the purpose is to only prime the blood cells for suse- quent production if tumor killing substances in vivo, as ex ⁇ plained above.
• the suspension is then squeezed into the next bag (MITOGEN) through a filter, through which the blood cells/whole blood but not the anti-mitogen binding beads can pass.
• the cell stimulation/lymphokin production is carried out by mixing- /agitation for a suitable period of time, usually from about one hour up to a few days, depending on the components used, but the duration of the stimulation can be much shorter, as explained above.
• the mitogen used is not considered to be harmful to the patient (e.g. as in the case of mitogenic but non-toxic SEA-fragments F ⁇ _, F3), the preparation will now be- administered to the patient (full lines).
• the mitogen may be removed, e.g. by passing the preparation into a further bag (IMMOBIL. ANTI-MITOGEN), wherein the mitogen may be collected by anti-mitogen immobilized on a suitable carrier similar to the process carried out in the bag IMMOBIL-
• the carrier/beads are collected by a second, filter and the stimulated blood preparation is then re-administered to the patient, as illustrated by the dashed lines in Figure 1.
• FIG. 2 illustrates an alternative embodiment of the inven ⁇ tion, wherein a ⁇ ingel blood bag can be used instead of the several bags used in the embodiment of Figure 1.
• a pre-determined amount of a whole blood sample is taken from a patient and passed into a treat ⁇ ment bag designated BLOOD BAG.
• the blood sample can be taken into the BLOOD BAG either directly or via a separate bag (WHOLE BLOOD).
• the illustrated BLOOD BAG is shown having a blood inlet and a blood outlet, the latter being provided with a separation means such as a filter F.
• Position ⁇ illustrates the step of intruducing into the the BLOOD BAG, as an anti-body binding agent, a pre-determined amount of the corresponding mitcgen immobilized on a carrier.
• a presently preferred mitogen-carrier combination is SEA-biotin-avidin-polysaccharide-type carrier.
• Position £ illustrates the subsequent stimulation of the blood sample from Position £ by adding a suitable form of the mitogen to the BLOOD BAG (still containing the SEA-carrier matrix with attached antibodies removed from the blood during step ⁇ ) .
• the presently preferred mitogen for this embodiment is biotiny- lized SEA (SEA-biotin) .
• the anti-mitogen-free blood sample is incubated with the mitogen added in step £ for a time suitable for obtaining the desired stimulation/priming of the blood cells.
• Position ⁇ illustrates the subsequent addition of an additional amount of carrier, sufficient for adsorbing any excess of mitogen in the BLOOD BAG.
• the carrier in step ⁇ ) is avidin-carrier, the avidin part of which will adsorb the biotin part of SEA-biotin rapidly and strongly.
• the blood sample from step ⁇ is taken out from the BLOOD BAG through filter F for re-administration to the patient, the filter F being chosen such that the above mentioned carriers will remain in the BLOOD BAG.
• FIG. 2 A simpler procedure, which might be useful in certain situa- tions, is illustrated by the dashed line in Figure 2.
• This embodiment only includes treatment step £, after which the treated blood sample will be re-administered to the patient via filter F (as in step ⁇ ⁇ ) .
• This embodiment may be used with carrier-mitogen complexes which retain a useful mitogenic activity despite the coupling to the carrier.
• the carrier-immobilized mitogen should be added in an amount sufficient both for removing the anti-mitogen anti-bodies contained in the blood sample, and for providing the desired stimulation of the blood cells.
• Animal White rabbits (male), B.W. 2.2-3.0 kg.
• Tumor VX2 carcinoma (virus cell line).
• SEA-Sepharo ⁇ e R acrobeads 100 ml
• Sepharo ⁇ e R -avidin- biotin-SEA are added to the sterile tube. The tube is shaken carefully at 4°C for 30 minutes.
• SEA SEA-biotin or fragments of SEA or SEA-fragments- biotin (1 and 10 ng/ml whole blood) is added to the whole blood from (3), and the samples are incubated for 6 hours at 37°C.
• VX2 tumor cells are implanted into one of the 5 lobes in the liver of male rabbits. After 2 weeks the tumor has grown to a size sufficient for being measured without any risk of measure ⁇ ment errors ( Figure 1).
• the experiments were divided into two different pars, viz. part 1 for judgement of the size of the tumor, and part 2 with prolonged treatment but only histopatho- logical examination of the tumor, depending on the high death rate of the rabbits in the control group ( see Figure 3).
• the rabbits in the control group are normally dieing after 4-6 weeks because of pulmonary metastatis. Part 1
• a first treatment is given 2 weeks after the implantation of the tumor cells, a second treatment after another 3 days and so on.
• the rabbits are sacrified after 2 weeks of treatment, and the total weight of the liver is measured, as is the size of the tumor.
• the reason for killing the animals after 4 (2+2) weeks is that the VX2 cell line used is so malignant that further waiting would result in the entire liver being occupied by tumors (making evaluation impossible).
• a first tretment is given 2 weeks after the implantation of the tumor cells, a second treatment after 3 days and so on.
• the rabbits in the treatment group are sacrified after 4 weeks of treatment.
• the rabbits in the control group are all dead at week 6. In this group only survival and histopatology could be performed.
• the experiments have been performed with the following groups: 1. Control group without any kind of treatment, only saline injection.

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• 1988
  • 1988-04-15 SE SE8801426A patent/SE8801426D0/xx unknown
• 1989
  • 1989-04-14 WO PCT/SE1989/000208 patent/WO1989009619A1/en not_active Application Discontinuation
  • 1989-04-14 EP EP19890904646 patent/EP0406317A1/en not_active Withdrawn
  • 1989-04-14 AU AU34206/89A patent/AU632999B2/en not_active Ceased
  • 1989-04-14 JP JP1504245A patent/JPH03503640A/ja active Pending
  • 1989-04-14 KR KR1019890702371A patent/KR900700131A/ko not_active Application Discontinuation

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