AU3420689A - Method and means for immuno-stimulating blood treatment with a mitogen - Google Patents

Description

Method and means for immunostimulating blood treatment with mitogen.

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.

Background of the invention The human (or animal) body's immune defence system against foreign "invaders" is very complex and many questions still remain to be answered. For example, many of the factors which cause such diseases as cancers, rheumatism and the like are completely unknown. On the other hand, the knowledge about what happens immunologically - i.e. how the immune system of the body acts in order to take care of pathological cells - has been clarified to a greater extent. In particular, the dis¬ covery of the endogenous substanses called lymphokins, especi¬ ally interferons and interleukins, their structure and their important functions for the immune defense system represents the start of industrial scale production of biotechnological recombinates, and great investments have since then been made in extensive clinical programs.

After more than a decade's experience of using such recom¬ binates, it is generally recognized that such substanses, when produced by genetic engineering techniques, have not given the expected results. In particular, the healing effect obtained usually is no better than for other types of therapy, whereas the side effects can be rather serious.

It is also known that many substances, so-called mitogens and/or antigens, stimulate the production of tumor killing substances such as TNF, lymphokines, e.g. gamma-interferon and interleukines such as interleukin II (IL-II), which - as indi¬ cated above - are considered to have a very great potential for the prophylaxis and treatment of many different ( "interferon- sensitive") diseases such as various types of tumours.

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. pn the one hand that whole blood was used instead of only selected blood com¬ ponents - as had been common previously - for the in vitro incubation with the mitogen, and on the other hand that the sample _r after having been charged with interferon by incubation with the mitogen and separation of the latter, was re-adminis- tered to the very same patient, from which the sample had been taken. In other words, the treatment was not only species- specific but individual-specific ("autologous") . This procedure has many advantages, such as avoiding the risk of complications and side effects because of differences in the composition of the blood when the donor and the receiver are different in¬ dividuals. 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.

As a result of the discourageing experiences of the above mentioned use of biotechnologically produced substances, Prof. Rosenberg et al isolated, using a very complex technique, a selection of important white blood cells from a patient suffe¬ ring from cancer, and then stimulated the selected white blood cells with the substances (Interleukin 2) produced by the above mentioned recombinates (see e.g. Rosenberg M, et al, "Ly pho- kine-activated killer cells, lysis of fresh syngeneic natural killer resistant murine tumour cells by lymphocytes cultured in interleukin 2", Cancer Res., 44: 1946-1953, 1984, which is incorporated herein by reference) . Many research teams all over the world are now trying to improve this technique (called LAK cell therapy), epecially in order to reduce the undesired side effects .

Although the so-called autologeous blood treatment has proven to be advantageous in several respects, there is a continuing need of further improvements. It is, for example, desirable to find better ways and means for stimulating the patient's blood cells so as to make the procedure practically and economically useful outside research laboratories.

Further examples of background art are found in EP-A2-147689, EP-A2-107119, EP-A2-79221, and US-A-4551435. 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.

Summary of the invention 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) . For example, there are strong indications that about 70% of the human population carries antibodies against the mitogen SEA (Staph. enterotoxin A), and also 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. In accordance with this first aspect of the invention, 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.

As used in this specification and the appended claims, the ex¬ pression "cell-containing blood samples" and the like 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. The expression "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). hen using 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. In accordance with this second aspect of the invention 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. This separation method offers several essential advantages compared to the separation methods disclosed in the above mentioned PCT application.

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.

According to a still further aspect of the invention it has been found that, thanks to said pre-separation of anti-mitogen antibodies, it is not always necessary to keep on stimulating the blood cells until interferons, interleukins and/or other lymphokines have been produced in vitro, but a proper choice of the mitogen and the conditions for its reaction with the blood cells can make it sufficient to just "prime" the blood cells as regards their immune-stimulating capability during the in vitro process, so that they are ready to start production of the above mentioned defense substances when returned to the optimal medium of growth, viz. the donor's own blood system. Thus, the term "in vitro stimulation" (and similar expressions) as used in the present specification and claims 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). In the latter case, the in vitro stimulation may be very short, e.g. from a few seconds to some five or ten minutes. Mitogens having a strong specificity for MHC II are especially useful in this embodiment. Such a short time outside the donor's = recei¬ ver's body represents a further important advantage of then invention. Description of preferred embodiments

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) . In a particularly interesting embodiment 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. In other words, when using such fragments of SEA as the mitogen, it may not be absolutely necessary to separate the mitogen from the stimulated blood before re-administering the same to the pati¬ ent. Such fragments of SEA (called F_j_ and F3) have been desc¬ ribed by Noskova V P et al in Int.J.Biochem, Vol 16, No.2, p.201-6, 1984, and by Ezepchuk Yu. V. et al in Int. J. Biochem. Vol 18, No. 5, p. 485-88, 1986, theses two publications being included herein by reference.

However, mitogens are usually very toxic to the patient. Therefore, it is usually most essential to remove the mitogen completely from the blood cells before re-administering the same to the patient. In accordance with a preferred embodiment of this aspect of the invention, 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. In an especially preferred embodiment 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. Thus, 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. As an alternative, 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. In 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.

Description of the 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). 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. The latter are preferably con¬ siderably larger than the blood cells, for example about 200 μm compared to about 7 μm 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. In the bag 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. If 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 Fτ_, F3), the preparation will now be- administered to the patient (full lines). In the opposite case 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-

.MITOGEN. 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.

Figure 2 illustrates an alternative embodiment of the inven¬ tion, wherein a εingel blood bag can be used instead of the several bags used in the embodiment of Figure 1. As illustrated in position A_. in Figure 2, 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 β illustrates the subsequent addition of an additional amount of carrier, sufficient for adsorbing any excess of mitogen in the BLOOD BAG. For the described preferred embodiment, the carrier in step ^~) is avidin-carrier, the avidin part of which will adsorb the biotin part of SEA-biotin rapidly and strongly. Finally, 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.

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. When practising this embodiment 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.

The advantageous results obtained when working in accordance with the invention will be further illustrated in the following tests on rabbits. io

Results of SEA stimulation of whole blood of rabbits bearing VX2 carcinoma.

At Mater al?

1. Animal: White rabbits (male), B.W. 2.2-3.0 kg. 2. Tumor: VX2 carcinoma (virus cell line).

3. Preparation of VX2 carcinoma-bearing rabbits.

- inject cell suspension (50 μl, 5xl0⁶ cells), directly to the external left lobe of the liver. Methods 1. 3 ml whole blood is taken out from the VX2 bearing rab¬ bits 2 weeks after inoculation in a sterile tube containing heparin.

². SEA-Sepharoεe^R acrobeads (100 ml) or Sepharoεe^R-avidin- biotin-SEA are added to the sterile tube. The tube is shaken carefully at 4°C for 30 minutes.

3. The mixture from (2) is put in a column K9 containing Net (80 m) and then the column is washed out by 1 ml saline. The final volume is 4 ml.

4. 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.

5. The whole blood from (4) is injected back to the same rabbit, with and without removal of SEA or fragments of SEA.

6. The procedure is repeated every 3 days. Procedure:

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. Experiments. The experiments have been performed with the following groups: 1. Control group without any kind of treatment, only saline injection.

2. Treatment with Adriamycin.

3. Removal of SEA - antibody from the blood samples with SEA-Sepharose^R and then stimulation of the whole blood with SEA and injection of the blood without removal of the SEA.

4. As number 3, but removal of SEA with anti-SEA-Sepharose^R after the stimulation.

5. Removal of the SEA-antibody from the blood samples with SEA-biotin-avidin-Sepharose^R beads and then stimulation of the whole blood with SEA-biotin and, after stimula¬ tion, removal of the SEA-biotin by coupling the same to avidin-Sepharose^R beads, and separation of the blood before re-injection to the rabbits. 6. Stimulation of the blood samples with SEA-biotin, SEA, SEA fragments without removal of blocking SEA-antibo- dies.

Results:

1: The rabbits in the control and Adriamycin groups showed no reduction of the size of the tumor (see Figure 4).

2: The rabbits in the group without removal of blocking antibodies to SEA did not show any reduction of the size of the tumor (see Figure 2).

3: The rabbits with removal of the blocking antibodies and then treatment with SEA, SEA-biotin or fragments of SEA showed a good reduction of the size of the tumor and increased survival (see Figures 2 and 3).

4: Histopathological examination of the rabbits treated by removal of antibodies and then stimulation as in (3) above showed that the tumor was necrotic and, surrounding the tumor, macrophage-like cells could be visulized.

Rabbits in the control group and the rabbits without removal of blocking SEA-antibodies showed no necrotic areas and no macrophage-like cells could be detected.

Preliminary study of dosage variations. In a preliminary study VX2 tumor cells are implanted into three groups of rabbits (three animals in each group) as desc¬ ribed above. A control group received no treatment, a first group treated with SEA according to the invention received a dosage of 1 ng/ml blood and a second group 10 ng/ml blood. The treatment started one week after the implantation, and the rabbits were treated twice per week for four weeks and then sacrificed. The size and visual appearence of the tumor could be studied. The group receiving 10 ng/ml had tumors of about the same size and the same appearence as the control group (very little spontaneous necrosis in the middle of the tumor). The group receiving only 1 ng/ml showed considerably reduced tumor sizes and strongly necrotic tumors. These preliminary results intimate that the best effect of the invention might be obtained in a dosage interval which is neither too low nor too high.

Claims (10)

ι3C AIMS

1. A method of preparing an immuno-stimulating preparation by incubating a cell-containing blood sample, especially a cell-containing whole blood sample, taken from a specific individual, with a mitogen in vitro so as to produce a blood preparation designed to be re-administered to the same indivi¬ dual, said method comprising the further step of removing anti¬ bodies against said mitogen from said blood sample before carrying out said incubation with said mitogen in vitro.

2. A method according to claim 1, wherein said anti-bodies against said mitogen are removed from said blood sample by contacting the blood sample with the corresponding mitogen or its active derivatives or active immobilized forms of the mitogen.

3. A method according to claim 1 or 2, wherein said mitogen has a strong specificity for MHC II on accessory cells such as monocytes and/or B cells.

4. A method according to any one of claims 1 to 3, wherein said mitogen is selected from the group comprising SEA, SEB and fragments thereof, in particular fragments having mitogenic activity and being substantially non-toxic to the human body.

5. A method according to claim 4, wherein said mitogen has mitogenic activity while being sustantially non-toxic to the human body, and wherein said blood preparation to be re-ad- ministered contains said mitogen.

6. A method according to any one of the preceding claims, wherein said in vitro incubation of said blood sample comprises in vitro priming of the blood cells for subsequent production of tumor killing substances in vivo.

7. A method of separating a mitogen from a cell-containing blood sample, which has been incubated with said mitogen, comprising the steps of contacting said mitogen-containing blood sample with anti-bodies which are specific for said mitogen so as to bind said mitogen to said anti-bodies, and separating said anti-bodies with attached mitogen from said blood sample.

8. A method according to claim 5, wherein said anti-bodies are immobilized to a carrier having a substantially larger particle size than said blood sample.

9. A blood treatment kit for carrying out the method accor¬ ding to any one of the preceeding claims, comprising 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 separating said mitoge¬ nic agent from said blood sample, so that the treated blood sample leaving said outlet substantially lacks mitogenic ac- tivity.

10. A method according to any one of claims 1 to 6, compris¬ ing the further step of re-administering said blood preparation to the individual from which the blood sample was taken.