Implantable agarose-collagen beads containing cells which produce a diffusible biological product, and uses thereof

territorially invention

The present invention relates to the encapsulation of cells in agarose and collagen, followed by coating with agarose, therapeutic methods which cell materials use, and the production thereof.

background and prior art

The encapsulation of various biological materials in biologically compatible materials is a technique while some time has been used, albeit with limited success. examples for the prior art are US Patent No. 5,227,298 (Weber et al.). 5,053,332 (Cook et al.); 4,997,443 (Walthall, et al.); 4,971,83 (Larsson et al.); 4,902,295 (Walthall, et al.); 4,798,786 (Tice, et al.); 4,673,566 (Gooser et al.); 4,647,536 (Mosbach et al.); 4,409,331 (Lim); 4,392,909 (Lim); 4,352,883 (Lim) and 4,663,286 (Tsang, et al.). Also of interest is <patcit><text>US 5,643,569</text></patcit> (Jain et al.), filed June 7, 1995, and WO-A-95/19430, Jain et al., discussed in some detail encapsulating secretory cells in various bio-compatible Materials. As discussed therein, secretory cells are cells Biological products secrete. Generally possess secretory Cells at least some properties of endocrine cells, and can generally as equivalent to be cells which are endocrine nature, treated. The also pending Application discusses z. B. the encapsulation of insulin-producing Cells, preferably in the form of islands, in agarose-collagen beads, which are coated with agarose. The resulting products are useful in the treatment of conditions in which an animal requires an insulin therapy, such as about diabetes.

The application of Jain et al. discussed some detail earlier Approaches, which have been made in the art in transplantation therapy are. These are summarized as herein.

There are five main approaches known to the transplanted To protect tissue from the immune response of the host. All involve attempts to isolate the transplanted tissue from the host immune system. The Immune isolation techniques used to date include: extravascular diffusion chambers, intravascular Diffusion chambers, intravascular, Ultrafiltration chambers, microencapsulation and macroencapsulation. All However, these techniques have due to one or more of the following fails problems: a fibrotic reaction of the host against the implanted material, instability of the implanted material, limited diffusion of nutrients across semi-permeable Membranes, permeability of secretion-promoting Agents and product, and method of diffusion delay through semi-permeable Membrane barriers.

For example, in 1978 by Lim a microencapsulation for enclosing viable cells, tissues and other labile membranes within a semi-permeable membrane developed (Lim, Research report to Damon Corporation (1978)). Lim used microcapsules of alginate and poly-L-lysine to encapsulate islets of Langerhans. 1980 was the first successful in vivo application of this new technology published in Diabetes Research (Lim et al, Science 210:. 908 (1980)). microencapsulated The implantation of these Langerhans Islands led mean that in diabetic animals a normal blood glucose level was maintained long. Other researchers, these experiments repeated, however, found that alginate, a tissue reaction provoked and confirmed the results of Lim et al. not reproduce (Lamberti et al, Applied Biochemistry and Biotechnology. 10: 101 (1984); Dupuy et al., J. Biomed. . Material and Res 2-2: 1061 (1988); . Weber et al, Transplantation 49: 396 (1990); and Doon-Shiong et Al., Transplantation Proceedings 22: 754 (1990)). It is now assumed that the water solubility these polymers for the limited stability and biocompatibility these microcapsules is responsible in vivo (Dupuy et al., supra, Weber et al., Supra, Doon-Shiong, above, and Smidsrod, Faraday Discussion of Chemical Society 57: 263 (1974)).

used Recently Iwata et al. (.. Iwata et al, Jour Biomedical Material and Res. 26: 967 (1992)) agarose microencapsulation of allogeneic pancreatic islets and found out that they as a medium for the production of microspheres could be used. In their study, 1500-2000 islets were microencapsulated individually in 5% agarose and implanted in Streptozotocininduzierte diabetic mice. The implant survived a long time, and the recipients maintained a normal Blood glucose content indefinitely.

However, their method has a number of on disadvantages. It is cumbersome and inaccurate. For example remain many beads partially coated and several hundred beads of empty agarose educated. is therefore additional Time required to encapsulated islands of blank beads separate. About that addition, most of the implanted microbeads gather in the pelvic cavity, and it is a great Number of islands in full coated individual beads required to reach a normal blood glucose level. About that addition, the transplanted beads are difficult as&lt;?page 3?&gt;derzugewinnen, tend to be fragile, and set slightly to the minor damage Islands free.

A Makroeinkapselungsverfahren is was also tested. Macro capsules from various different Materials such as poly-2-hydroxyethyl methacrylate, Polyvinylchloride-c-acrylic acid and cellulose acetate were for prepared the immune isolation of islets of Langerhans (See Altman et al, Diabetes 35: 625 (1986);. Altman et al, Transplantation.: American Society of Artificial Internal Organs 30: 382 (1984); Ronel et al., Jour. Biomedical Material Research 17: 855 (1983); Klomp et al., Jour. Biomedical Material Research 17: 865-871 (1983)). In all these studies, only a transitory normalization was the achieved blood glucose level.

Archer et al., Journal of Surgical Research 28: 77 to (1980), used acrylic copolymer hollow fibers temporarily rejection of islet xenografts to prevent. They reported that dispersed neonatal pancreas transplants from mouse in hollow fibers after transplantation into diabetic hamsters long time survived. Recently confirmed Lacy et al, Science 254:. 1782-1784 (1991), their results, but found that the normal blood glucose level a temporary Phase. They found that if the islands in the fiber are injected, they aggregate within the hollow fiber, with resultant Necrosis in the central region of the island masses. The central necrosis concluded a longer Service life of the graft. used to solve this problem alginate to disperse the islands in the fiber. This experiment However repeated not comprehensive. Therefore, the function the membrane as a transplant medium for islands in humans questionable.

There was thus a demand, the achieve transplantation of secretory cells, and in particular the survival allografts and xenografts of pancreatic Islands without the use of chronic immunosuppressive agents.

In the work discussed above Jain et al. reported the inventors, that the encapsulant of secretory cells in a hydrophilic gel material to a hydro performs functional, non-immunogenic material transplanted into animals and can be long periods can be stored. The encapsulation of the secretory cells provided a more effective and manageable technique for transplanting secretory cells. The encapsulation technique was described suitable for the encapsulation of other biological materials, such as enzymes, microorganisms, trophic agents, including recombinantly produced trophic agents, cytotoxic agents and chemotherapeutic Agents. It has been discussed that the encapsulated biological Agents for the treatment of conditions are suitable, which are known to the biological agent appeal.

The application does not discuss in any way the incorporation of cells which produce biological materials, which can diffuse, the latter in a therapeutic context are useful. It herein is a difference made between secretory cells, and cells that produce biological materials which diffuse can. The former refer, according to the examples in the application of Jain, generally to products such as Hormones, cell signaling substances, etc., which are generally considered biological "messengers" considered. In contrast, denote biological materials that diffuse can, presented MHC materials such as Peptides, cell expression regulators such as suppressors, promoters, Inductors, and so on. This difference is in the field be recognized oncology, z. B. with the following discussion.

Extensive studies in cancer research who works with heterogeneous cell extracts, and various cellular components includes. about the use of monoclonal antibodies has relevant technology, with cancer identified associated antigens, eg. as GM2, TF, STn, MUC-1, and various epitopes derived therefrom. The current Theory postulates that epitopes different from these tumor markers are derived, non-covalently complexed with MHC molecules, thereby of specific cytolytic T cells, a type Agro is formed. This Mechanism is the various mechanisms on the biological Reaction against viral infections are involved, not dissimilar. See in this respect . Van der Bruggen et al, Science 254: 1643-1647 (1991); Boon et al., 5,405,940, and Boon et al., 5,342,774.

Additional research, parallel to work on the identification of the so-called Krebsepitope, has to be the regulation of cancer proliferation concentrated, such as via suppression or general biomodulation. See, for. Example, Mitchell, J. Clin. Pharmacol. 32: 2-9 (1992); Maclean et al., Can. J. Oncol. 4: 249-254 (1994). The goal all these different approaches anticancer one, is the modification of the host immune response, produce so a certain improvement in the condition of the patient.

The key to all these approaches the activity of one or more biological products, which diffuse can, and which cooperate with other materials to the immune response to modulate. For example, disclose Boon et al. and van der Bruggen et al. small peptide molecules. Mitchell discusses larger molecules z. B. act as suppressors.

One problem with all therapeutic approaches, using these materials whose delivery is in a safe, effective form. This is not easily achieved. It was now surprisingly found that the techniques of Jain et al., which in the development treatments for states, &lt;?page 4?&gt;Which need products from secretory cells, were so helpful, now in another can be used areas.

How this is accomplished is the subject of the invention, the detailed Description follows.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

example 1

This example, and those which follow, using RENCA cells. These are spontaneous Nierenadenokarzinomzellen from BALB / C mice, widely available and are held both in vitro and in vivo in culture. See Franco et al., Cytokine Induced Tumor Immunogenecity, 181-193 (1994).

Samples from frozen RENCA cells were incubated at 37 ° C thawed and then placed in tissue culture flasks containing Dulbecco's Modified Medium (D-MEM) containing with 10% bovine serum, penicillin (100 u / ml) and Streptomycin (50 ug / ml) added had been, is being obtained, which is hereinafter referred to herein as "complete medium".

Cells were grown to confluence left and then trypsinized, followed by washing with Hank's Balanced Salt Solution and then with the above complete medium.

In order to determine if the RENCA cells efficiently produce tumors were two BALB / C mice 10⁶ these cells injected intraperitoneally. The mice were about a period of 3-4 Weeks observed. From a clinical standpoint it appeared while the first two weeks healthy and exhibited normal activity. After that the clinical manifestations of cancer became evident. One mouse died after 23 days and the second after 25 days. After the death, the mice investigated, and there were found numerous tumors of various sizes. Some of the tumors also showed bleeding.

A sample of a tumor, comprising one of the mice had been removed, was used for latter histological examination in 10% formalin.

example 2

After it was shown that grew the RENCA cells in vivo, studies were carried out to determine whether these cells grow in beads according to the invention.

RENCA cells were grown to confluence grown as described above, treated with trypsin and washed, also as described above. Thereafter Samples of 60000-90000 cells produced. cells were then centrifuged at 750 rpm, and fluid was removed. The cells were then placed in solutions 1% incomplete suspended developed collagen in phosphate buffered saline at a pH of 6.5.

A solution of 1% agarose low viscosity was in minimal essential medium (MEM, minimal essential medium) prepared at 60 ° C held and then 100 .mu.l thereof to the above-described suspension of RENCA cells and incomplete developed collagen added. The materials were then promptly as one great Drop in sterile mineral oil transferred at room temperature. The mixture formed a single, smooth semi-solid bead. This procedure was repeated a number of beads produce.

After one minute the beads were As described above, transferred to complete medium at 37 ° C. The beads were then washed three times in minimal essential medium containing the above listed Containing antibiotics, washed. The beads were then overnight at 37 ° C in a humidified atmosphere of air and 5% CO₂ incubated. After Incubation, the now solid beads transferred to a sterile spoon, of 1 ml containing 5% agarose in minimal essential medium. The beads were 2-3 times in the solution rolled around it uniformly to coat them with agarose. Before the agarose was, were the beads in mineral oil transferred to obtain a smooth outer surface. After 60 seconds, the beads were five times with complete medium at 37 ° C washed by the oil to remove. It follows overnight incubation (37 ° C, humidified the atmosphere of air, 5% CO₂).

These RENCA containing beads were used in subsequent experiments.

example 3

Before conducting in vivo studies it was necessary to determine if the RENCA cells in the beads, were manufactured in the manner described above, grow.

For this, beads prepared as described in Example 2 discussed in the medium described in Example 2 while a period of three weeks under the listed conditions incubated. Three of the beads were then cut into small pieces cut and cultured in standard culture flasks, by direct Contact with both the flask and to the culture medium.

&lt;?page 5?&gt;

Observation of these cultures showed that the cells grew and formed standard RENCA colonies. This showed that the cells in the beads viable remained.

example 4

Thereafter, in vivo experiments executed. In these experiments, the beads were incubated for seven days at 37 ° C. After received Mice Bead transplants. To this was added in four mice each made a mid-line incision was performed intraperitoneally. Three beads, each of which contained 60,000 RENCA cells were transplanted. The incisions were then closed (two layer closure), using an absorbable suture. The four mice (BALB / C) were normal male mice between 24-26 Grams weighed and appeared to be healthy. There were two sets of controls performed. At the first set received two mice three beads that no RENCA cells contained, and the second, two mice were completely untreated.

Three weeks after the implantation received all mice intraperitoneal injections of 10⁶ RENCA cells. Eighteen days later died a control mouse. All remaining mice were sacrificed and examined.

Control mice showed numerous tumors, while the mice, which the implants in beads encapsulated cells had received across the entire abdominal cavity just random Nodes showed.

These encouraging results laid the planning of the experiments, which indicated the following example are close.

example 5

In these experiments, it was established Cancer simulated by RENCA cells under each kidney capsule of six BALB / C mice were injected. Fifteen days later the mice were divided into two groups. received The three mice in the first group three spheres as described above in Example 4. Fig. The second group (the control group) received beads, no RENCA cells contained.

After 4-5 days appeared Mice received RENCA cell containing implants were lethargic and their fur had become spiky, while the control group vigorously remained without a change in the condition of the coat.

Ten days after implantation (25 days after injection of RENCA cells), however, the control mice were lazy and reported inflated bellies on. One of the three control mice died on day fourteen after transplantation of the beads. This was followed by sacrifice of the mice.

showed the body cavities of the control mice copious bleeding, with numerous tumors via the alimentary canal, liver, the Stomach and lungs. The entire abdominal cavity was due by lush tumor growth no longer perceive. The mice, the beads had received with encapsulated RENCA cells, however showed no bleeding and only a few nodes on the diet cankers. On Comparison of test and control groups showed that in the Test group nodes had not evolved.

example 6

inoculated growth of free RENCA cells is inhibited when used in combination with encapsulated RENCA cells are incubated. There was another set of experiments executed to determine whether this effect is observed with other cells can be.

An adenocarcinoma cell line, ie MMT (mouse mammary tumor) was obtained from the American Type Culture Collection receive. Encapsulated MMT cells were prepared as described above, prepared with MMT cells, beads with 120,000 or 240,000 Cells per bead were produced. After preparation of the beads, they were used to determine whether they inhibit proliferation of RENCA cells in vitro would. In exact words of two petri dishes with six wells were prepared by inoculation with 1 × 10⁴ RENCA Cells per well in 4 ml of medium. In each dish served three Wells as a control, and three as test. One of the three control wells in each dish received a bead. Each of the other wells received either two or three empty Beads. The second well was similar treated, the depressions one, two or three beads, containing 120,000 or 240,000 MMT cells received. The wells were one week at 37 ° C incubated, after which RENCA cells treated with trypsin, washed and using a counting chamber counted were. The results are as follows:

&lt;?page 6?&gt;

<img img-content="tb" img-format="tif" he="52" wi="163" file="00110001.tif" />

example 7

Following the results of Example 6 The same experiments conducted using 1 x 10⁴ MMT cells rather than RENCA cells. The Experiment was carried out exactly the same as Example 6. FIG. The Results are reported below.

<img img-content="tb" img-format="tif" he="37" wi="160" file="00110002.tif" />

These results encouraged a in vivo experiment. This is illustrated in Example 8. FIG.

example 8

RENCA cells, as in the above used examples, are renal cancer cells. To the general Effectiveness of the invention more fully to show work were using a different Type of cancer cells carried. In exact words adenocarcinoma cells were used.

A mouse mammary tumor cell line (MMT) was obtained from the American Type Culture Collection. Under use the protocols given above were prepared implants 120,000 cells per bead and 240,000 cells per bead contained.

The experimental model used was the mouse model, supra. Twenty-two mice were divided into groups of 4, 9 and 9 divided. The first group, that is, the controls were further divided into three groups of two, one and one. The first Subgroup received implants of one bead containing no cells. One mouse received two empty beads and one received three empty beads.

In the experimental group A (9 animals) containing beads 120,000 cells, while in group B, the beads 240,000 cells contained. Within groups "A" and "B" gave it three sub-groups, each of which contained three mice. The sub-groups received one, two or three beads with MMT cells.

Twenty-one days after implantation received all animals injections of 40,000 RENCA cells. Directly after injection, the mice were lethargic, with spiky hair. This held about five days the data by which normal behavior was observed.

After twenty days designated control mice bloated bellies and extremely spiky hair on. One control mouse died 25 days after injection, while the remaining control mice located appeared as the final stage. All mice were sacrificed and the Tumor development was observed. These observations are below recorded:

&lt;?page 7?&gt;

<img img-content="tb" img-format="tif" he="96" wi="135" file="00130001.tif" />

These results indicate that of eighteen mice tested showed thirteen no disease. Of the mice in Group (A) had a Mouse a few nodes and another mouse showed a few tumors on. A mouse, the two beads had received, had a few tumors.

In group B had a mouse that a bead had received, and a mouse that had received two beads, a few tumors that with intestines were tangled. One of the mice, the three spheres had received, had developed a large solid tumor and was apparently very sick.

Nevertheless, the results showed total that the encapsulated mouse breast cancer cells tumor formation inhibited.

example 9

As suggested above, the Practice of the invention for the production of any material or factor, the or inhibits tumor cell proliferation and / or prevented. This was examined in the following experiment on.

Additional beads were prepared as described above in Example 2 except that incomplete developed Collagen is not recorded. These beads are therefore agarose / agarose beads. RENCA cells, as described above, were placed in these beads installed, in turn, as described above.

Two sets of three plates with the Six wells were thereafter as the control and experimental groups used. In the control group the wells with 4 ml RPMI complete medium (10% fetal calf serum and 11 ml / l of penicillin) filled. Each control group was then inoculated with 10,000 RENCA cells.

In the experimental group that was RPMI Complete medium conditioned, by adding material obtained immune isolated by incubating 10, RENCA containing Beads (120,000 Cells per bead) in a 35 × 100 mm Petri dish containing 50 ml RPMI complete medium. After five days of incubation Medium was collected from these shells, and 4 ml of which was added to each test well. These wells were then inoculated with 10,000 RENCA cells.

All plates (both control as also attempts) were five Days at 37 ° C incubated. After the incubation period, the cells were trypsinized treated, washed, and using a hemocytometer tallied. The cells of each well in the dishes were after trypsin pooled and counted, the result was as follows.

&lt;?page 8?&gt;

<img img-content="tb" img-format="tif" he="28" wi="152" file="00140001.tif" />

These results indicate that the Cells when in z. B. the beads of the invention are restricted, producing some factor, which leads to a suppression of tumor cell proliferation. This Restriktionsinhibitionsfaktor is of de n cells because of their Inclusion in the beads produces and differs from other materials such as a Kontaktinhibitionsfaktor, which are produced when cells contact each other.

example 10

The experiment above showed that the growth of RENCA cells in conditioned medium for about the half the growth of the cells in control medium was. specified herein Experiments investigated whether the growth inhibiting factor by Freezing of the conditioned medium would remain active.

Conditioned medium was RENCA produced by five-day incubation 10 immune-isolated, RENCA containing beads. Incubation was in 35 × 100 mm Petri dishes containing 50 ml RPMI complete medium at 37 ° C. After Incubation was collected, the medium and stored at -20 ° C. conditioned Medium was prepared by incubating immunoisolated cells, the MMT (mouse mammary tumor) cells contained. The beads contained 240,000 cells per bead, all other conditions being equal.

Frozen medium was thawed at 37 ° C and then used in the subsequent tests. Three bowls Six wells were for used each treatment, ie, (1) RPMI control medium, (2) frozen conditioned RENCA medium and (3) frozen conditioned MMT medium. A total of 4 ml of medium was pipetted into each well. All wells were then inoculated with 10,000 RENCA cells and five Days at 37 ° C incubated. After incubation, two sample cups were from each well taken, treated with trypsin, pooled and transferred to a hemocytometer tallied. After eight days, the remaining three cups each well tested in the same manner.

follow the results:

<img img-content="tb" img-format="tif" he="46" wi="153" file="00160001.tif" />

If these results with those of Example 6, above, compared, it will be appreciated that while frozen / thawed conditioned RENCA medium growth is not inhibited to the same extent as not frozen medium (compare Examples 6 and 7), it, nevertheless, inhibited the growth. Frozen conditioned Medium using MMT cells inhibited the growth even stronger as non-frozen conditioned medium MMT. These results show that eighteen mice tested, thirteen no disease showed. Of Mice in group (A) had a mouse a few nodes and another Mouse showed a few tumors.

The foregoing describes the preparation of of implantable beads, containing one or more types of cells which a produce a biological product that can diffuse as that term is defined herein. The biological product, which diffuse can, is one which has an effect on the animal, in which comprises beads is implanted. Preferably, this effect is immunomodulation, such as the stimulation of an immune response or the suppression a reaction. In the case of cancer, the biological product, can diffuse the, for example, a peptide to be that on with MHC molecules Cancer cells complexed in a subject, whereby a CTL response caused contrast &lt;?page 9?&gt;is that in turn to a reduction Tumor burden results in the subject. The product, diffuse the can also be a suppressor of tumor growth may be. In connection with this form of therapy, it is possible, although not necessarily preferably, the implanted beads in one or in the vicinity an identified tumor position.

"Biological Product, can diffuse the " as used herein refers to materials such as proteins, Glycoproteins, lipoproteins, carbohydrates, lipids, glycolipids, and Peptides. In more specific words, materials such as antibodies, cytokines, Hormones, enzymes and so on by way of example, but by no means the only type of materials received. Excludes the well known "end products" of cellular processes, such as about CO₂ and H₂O.

As the experiments show, the implantable beads also be used prophylactically. It is well known that at least a portion of the population of cancer patients for recurrence of state vulnerable is. The experiments described herein that the implants can prevent the occurrence or reoccurrence of cancer, via the biological effect, which can diffuse the product, exerts on the system of the organism.

The discussion of the invention has on in vivo approaches concentrated. However, it should also be understood that there are many in vitro approaches for the Invention are, some of which are discussed hereinafter. For example, it is well known that many cells which desired products produce, when cultured in vitro the presence of feeder cells need. With such feeder cells there are always problems. You can grow faster than the desirable Cells, a de facto "strangulation" of interest Materials results. Furthermore, it may be a problem with various toxic Products, which are produced by the layers of feeder cells. The implantable beads the invention act almost as cellular incubators, while the built-cells protect, while they allow, that the products, which can diffuse, in a culture medium Moving inside, where they can be collected for. example.

As stated above, requires the Preparation of the implantable beads first suspending the cells in solution, preferably aqueous in a solution of collagen. Preferably, the collagen is incomplete entwickelts Collagen in a solution from about 0.5 to about 2%. Dependent on the type of cells used, the number of cells in the solution at a given time, and thus the number of cells in a bead, vary. Preferably, from about 10,000 to about 200,000 cells per bead used more preferably from about 30,000 to about 100,000. Most preferably, be about 40,000 to about 60,000 cells used.

After suspending the cells in the collagen solution A agarose added. Preferably, this agarose solution is in a range of about 0.5% to about 5%, preferably by 1%. In this dropwise addition Mixture onto or into inert materials, such as TEFLON^® or mineral oil forms a bead. This beads is semi-fixed. The semi-solid beads is then transferred to a sterile medium, preferably one, containing antibiotics, washed, and incubated to polymerize collagen. polymerization of collagen is a well studied phenomenon, and the conditions, under which it takes place, herein do not clearly outlined will.

After the solidification of the bead It is then coated with agarose, preferably by a agarose is rolled. A preferred way to accomplish this is to cause a easier with TEFLON^®-coated spoon An agarose, preferably 5% to 10%, contains.

The above discussion regarding biological Products which can diffuse should not be considered limited are designed to wild type materials. One can, for example, as easily transformed or transfected host cells installing, such as eukaryotic cells (eg. B. 293 cells, CHO cells, COS Cells), or even prokaryotic cells (eg. E. coli), the treated were to produce heterogeneous protein, or modified via z. B. homologous recombination to increased Amounts of desired biological products to produce. Other materials, such as Hybridomas can also be used, wherein said biological product, to diffuse the can, a monoclonal antibody is.