CA1139219A - Medicinal preparation for treating carcinomas and process of producing the preparation - Google Patents

Abstract

Abstract:
The invention relates to treatment of carcinomas with cytostatic agents. The method involves admnistering to the patient specially treated carcinomas cells until the tolerance of the patient to the cytostatic agent is improved, and then administering the cytostatic agent. The special treatment of the cells involves culturing carcinoma cells isolated from the patient, subjecting portions of the cultured cells to different cytostatic agents and observing the visible morphology damage of each portion, selecting the most effective cytostatic agent and treating the remainder of the cultured cells with it until 30 to 70% of the cells remain intact but partially weakened, removing the cytostatic agent by washing and then suspending the cells in a physiologically acceptable fluid to form a pharmaceutical preparation. The medical treatment outlined above employing such pharmaceutical preparation allows tumor cells to be treated without undue harm to the patient.

Description

MEDICINAI, PRE~R~TION ~O~ ~R~TI~G C~RCINOMAS
~ND PROCESS OF PROD~CIN(, l'~ll. PREPAI~ATIbN
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BACKGROUND OF T~-7E INVF.MTI~N

The treatment of carcinomas with cytostatic agents has been ~,nown for a considerable time. Almost all cytostatic ac,~ents have the disadvarltage that they are highly toxic and yenerally speaking also adversely affect the growth of healthy cells. Further, it has also been found that the known cytostatic agents are not equally active on all kinds of carcinomas. Even di~ferent f~ J
patients having similar carcinomas often exhibit different responses to one and the sa~e cytostatic agent. In order to select the optimum cytostatic agent in such cases and proceed with a selective treatment with the agent thus determined, Limburg and Krahe have suggested testing tis-sue cultures taken from the involved carcinoma for sensi-tivity to various cytostatic agents to establish an onco-biogram. 5ee Deutsche Medizinsche Wochenschrift, Vol. 89 (1964), page l933.
While the above-mentioned procedure affords the advantage that the involved organ or or~ans of the patient are not subjected to the toxic action of cytostatic agent.s which are not effective on the specific ~ype of tumor, the patient is still directly affected by a cytostatic agent.
This exposure has proven to be deleterious particularly when high dosages are administered over a prolonge~1 period of time.

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It is an object of the present invention to provide a medicinal preparation which is intended for the treatment of carcinomas and which is suitable either for use alone, or jointly with a cytostatic agent, which agent is specifically effective on the type of tumor involved. The medicinal prepa-ration should be satisfactorily tolerated by the patient.
According to one aspect of the invention there is provided a method for isolating, culturing and growing selected carci-noma cells comprising the steps of: (1) providing a source of carcinoma cells; (2) culturing and incubating said cells in a nutrient growth media under cell growth conditions to produce a cell culture; (3) subjecting a plurality of pre-determined portions of the cell culture of step (2) to a plurality of cytostatic agents and observing the visible morphological damage in each portion; (4) selecting that cytostatic agent that exhibits visible morphological damage to said susceptible cells; (5) treating the remaining portion of the cell culture of step (2) with the cytostatic agent of step (4) and incubating such that from about 30 to 70~ of said cells remain intact under microscopic examination and that said cells are not completely killed but only partially weakened; (6) washing the weakened cells of step (5) with a physiologically acceptable solution to remove substantially completely all of said cytostatic agent from said weakened cells; and thereafter (7) suspending the cells thus treated in a physiologically acceptable fluid for a pharmaceutically 1~3~2~

acceptable preparation.
According to another aspect of the invention there is provided a method for producing a suspension of weakened selected carcinoma cells that are susceptible to the cyto-static activity of a selected cytostatic agent comprisingtreating a cell culture of carcinoma cells with a cycostatic agent to which said cells are sensitive and incubating for a period of time until microscopic examination indicates that only about 30 to about 70~ of the thus treated cells are intact, and thereafter washing the thus weakened cells with a physiologically acceptable solution to remove said cytostatic agent.
The objects of the invention are accomplished as will be apparent from the following description, examples and appended claims.
DETAILED DESCRIPTION OF THE INVENTION
Carcinoma cells are used as a starting material in the process according to the present invention and can be re-covered in a conventional manner. For instance, the cancer cells can be released by a treatment with trypsin from surgically explanted tumor tissue; see Limburg et al.., Deutsche Medizinische Wochenschrift, Vol. 89 (1964), page 1938.
A preferred procedure of recovering carcinoma cells is explained as follows:
At the beginning of the procedure, any desired malignant tissue of a tumor is excised under sterile conditions during - 2a -~139Z~

a surgical operation. It is then treated with a trypsin solution and subsequently cultured on mica flakes in ~eighton tubes. The tissue which has been excised is mechanically macerated disintegrated under sterile conditions to form a tissue pulp. Care should be taken to use only firm cancer tissue which is structurally - 2b -~L~ 39Zl~
intact and free from suhstantial admixtures of blood or dead cells. ~ solution of 0.25~ tryp~in in a phosphate-buffered physiologica~l salt solution (~BS), which contains 100 units/ml penicillin and 0.1 mg/ml streptomycin, for sterilization, is added to that pulp, which is subsequent-ly stirred at 37C. For certain firmer tissues of harder consistency, the concentration of the trypsin solution ma~
be increased up to 2%. ~fter a~out 20 minutes the re-leased individual cells, without tissue portions, to the extent possible, are poured into a second sterile flask.
To interrupt the continued action of trypsin on the cell suspension which has been poured into the second sterile flask, the suspension is cooled to ~4C. by means o~ an ice bath. This sequence of stirring and pouring is re-peated until there is no more solid tissue in the flaskused for the treatment with trypsin. When the trypsin treatment has been completed, the cooled cell suspension is coarsely filtered, preferably through a layer of gauze that is sterile but has not been treated with any chemi-cals. The filtrate is centrifuged at about 800 to about1000 revolutions per minute and the separated cell sedi-ment isolated from the filtrate.
The trypsin treatment may be omitted with carci-noma cells that are already available in the form of indi-vidual cells, such as ascites carcinoma cells and leukemiacells.
The cancer cells are next grown to form a cell culture or cell turf according to known procedures, for example as in the Limburg et al article in Deutsche r~ed-izinische Wochensch~ift,mentioned above. The cells arecultured, and the following is an illustration:

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The c~ll s~iment is dilute~ with a liquid nu-trient to provide a concentration of from 2 to 4 million viable cells per milliliter and is equally distributed among about 30 tubes after it has been thoroughly pipet-ted. One drop is placed on a slide so that the extent ofdilution can be ascertained. The dilution is satisfactory when about 20 cells can be seen in the field of view under a magnification power of about 125. For a tube test, 1 milliliter of liquid is filled into each Leightor) tube.
10 Preferably, the liquid nutrient employed has the following composition:

% By Volume Tissue culture medium (TCM) according to Morgan, Morton, Parker (available commercially from DIFCO) 25.0 Hanks solution (~1% of a phenol red solution prepared q.s.ing 0.5 g phenol red up to 20 ml with N/20 NaOH) 55.0 Vitamin MEM solution (10'~) 5-L-glutamin (10% concentration)5.0 Calf serum 10.0 100 units of penicillin per milliliter and 0.1 mg of streptomycin per milliliter are added to the nu-'~ trient to ensure its sterility.
The cell suspension is incubated in the incuba~
tor at 37C. The cell metabolism is indicated by the change of the nutrient color from red to ycllow. 'I'he nu-trient is continually renewed whenever the metabolism has been taken place. The growth of the culture is examined and monitored under a chemist's microscope with reflected light. Depending on the nature of the starting material, the cultures have different growth characteristics. For example, cells from an endometrial or ovarian carcinoma -~ can grow to form a good cell monolayer within 48 hours Cells from other malignant growths, such as cells from squamous cell carcinomas, take up to 120 hours, depending on the degree to which they have matured.
The test of the cell cultures for sensitivity to various cytostatic agents is also conducted according to methods, for instance, as described by Limburg et al., Deutsche Medizinische Wochenschrift, mentioned above. An illustrative procedure is as follows:
Only well grown cell cultures are tested. De-i.,, ,~
pending on the quantity of the culture material which isavailable, the culture can be tested for sensitivity to any given cytostatic agent. A single culture can be tested for sensitivity of from 10 to 20 chemotherapeutic agents at the same time, which agents are added to respec-tive tubes in a weight-adequate concentration. Each agent used for sensitivity testing is used in the same concen-tration in which it is used in man for continuous or in-termittent therapy; as a guide see the values stated in Table 1 that follows. The various cytostatic agents are prepared in a pharmaceutical form and added to the respec-tive test tubes. Continuous monitorinq using a chemist' 9 microscope will reveal visable morphological darnage in most cases after 24 hours and, where antimetabolites are used, after 48 hours.
In the test in vitro, the following concentra~
tions suitable for therapy are used. These doses are cal-culated for recomrnended therapy concentrations for humans recommended by th~ man~facturers, relate~ to an averagebody wei~ht of 60 kg or to square meters of body surface and approximately 5000 ml blood v~lu~e, as the case may be.

Cytostatic Agent Concentration (Jug/ml) Cyclophosphamide (Endoxan) 33 Triethylene thiophosphoramide (Thio-Tepa*) 0.42 Podophyllic acid-2-ethylhydrazide (Proresid*) 33 Vincristin 0.017 Actinomycin D 0.05 Adriblastin 5-Fluoruracil 16.7 Amethopterin (Methotrexate) 2 Using this procedure, the dilution of any other cytostatic agent can easily be calculated.
IN VITRO SENSITIVITY TESTING

_ The sensitivity or resistance of a culture deri-ved from cancerous human tissue is determined by a semi-quantitative method. A culture which is completely resis-tant will correspond entirely with that in the control tube. Partial activity is indicated by a partial dissolu-tiOIl of about 50% of the cells in the field of view. ~igh sensitivity will be revealed by the fact that the field of view is free from viable cells and contains only a few shrunken cells, which have been killed. Only such a re-sult is regarded as revealing full sensitivity. On anaverage, I have found that at least two cultures treated * Trade Mark ~ iL39Z~
with different cytostatic atlt?rlts prove to he sensitive.
Only these cultures are to be used for the cell therapy which will be explained hereinafter. Such tests have been conducted in more than 3000 individual cases and have given the unique result that cells from eàch of several tumors from similar sites and/or of similar histology, but from different patients, may exhibit greatly different respollses to the same cytostatic agent, and further that cells from metastases may differ in response to cells from the primary tumor.
~he test of carcinoma cell cultures for sensiti-vity to various cytostatic a~ents may be carried out in J vivo in the abdomen of rats.
IN VIVO SENSITIVITY TESTING
The cytostatic sensitivity test is carried out in vivo in diffusion chambers placed into the abdomen of rats when less than 20 g of tumor material are available.
The cell suspension is prepared in the same manner as in the in vitro method. The diffusion chamber consists of two filters, which are placed into two Teflon rings, which are connected together by screws. The filters act as a semipermeable membrane. The pore size is selected so that ,J
liquid, electrolyte and proteins can permeate throuqh the membrane whereas cells such as leucocytes, macropha~Jes and the like cannot. A typical pore size for the filters is about 0.25 microns. This chamber is filled with 0.3 ml of prepared cell suspension.
Imrnediately after their preparation, the cham-bers are implanted into the peritoneal cavity of inbred strain BD2 rats. Two chambers are usetl for each animal.
When the chambers have been implanted~ the peritoneum and the abdominal wall are ~closcc~ ~ ~sing this method, 100~ of the tumor cells retained on the membrane filters are grown.
On the fifth day after the imulantation of the diffusion chambers into the abdomina of the rats, various cytostatic agents are repeatedly injected into the peri-oneal cavities of respective rats. Just as in the in vitro method, the concentration employed is the same as that used in human therapy. Forty-eight hours after the last injection, the rats are sacrificed and the diffusion chambers removed. The mernbrane filters are isolated and fixed, and the cells are strained accordiny to Papanico-J laou's method. The cytotoxic activity is determined in accordance with the above desribed morpholoyic criteria as in the in vitro test.
CELL WEAKENING TREATMENT
When the cells have been tested for sensitivity to various cytostatic agents as described above, untreated carcinoma cells having the same origin as the previously tested cells are found to have been weakened by the treat-ment and procedures accordin~ to the present invention.
In order to conduct the process accordiny to my invention, it is essential that when the optimum cytosta-tic agent for a given cell culture has been ascertained, the cells are treated with the selected ayent so that they are not completely killed but only partly weakened. To that end, care must be taken that the time of incubation in the presence of the cytostatic ayent is not too lony.
As would be expected, the time for which a culture is incubated will vary with different cell cultures and with the activity of the cytostatic agent. The progressive weakening can be watche~ un~er a microscope having a mag-nification of about 125. This can be performed by a skil-led technician without difficulty. The incubation is pre-ferably continued until a microscopic examination reveals that only about 30 to about 70~, and preferably only about 40 to about 60%, of the cells appear to be intact. Incu-bation times of about 4 to about 15 hours, and particular-ly of about 6 to about 12 hours, have generally proved ap-propriate. The incubation i.s preferably effected at a temperature o~ about 37C.
For the weakening treatment, each cytostatic agent is used in the same concentration as in J the above-described test.
The cell weakening treatment is preferably car-ried out as follows. Cells which have been recovered by treatment with trypsin, as described above and which have not been treated with a cytostatic agent, and are suspen-ded in a nutrient media, placed into Roux bottles and heated in an incubator to 37C. The particular cytostatic

2~ agent which has given optimum results in the above-men-tioned test is placed into the bottles in a concentration ;! ~J as indicated in Table 1.
When the cells have been suitahly weak.ened to the extent desired, the culture is then thoroughly washed to remove the cytostatic agent. This step serves to at-tenuate the action of the cytostatic agent on the cells and mainly to prevent a continucd reaction of the cyto.sta-tiC agent with the cells or possibly killing the cells. A
phosphate-buffered saline solution is preferred as a wash-ing fluid although other suitable physiologically compat-ible solutions may also be used.

ILIh~ c~lls w~ich hav~ ~us ~eerl ~eakerled 'co the ex-- tent desired, are celltrifuged at about ~OO to about lOO0 r.p.m.
ror 1O minutes and preferably suspended in a physiologic-ally acceptable fluid, such as physiological salt solution or calf serum. The concentration of the thus-treated cells in the resulting pharmaceutical preparations ready `
for administration after dil~tion is preferably about 106 cells per milliliter.
Pharmaceutical pr~ara~ions made according to the invention are preferably prepared from tunnor cells taken from the specific patient to be treated. This method will ensure a particularly high tolerance and optimum activity.
It is also recogni2ed that there may be cases in which it is not possible to make the preparation from cells taken from the patient to be treated for various reasons, such as there is insufficient time available or because persons skilled in the preparation and testing of cultures grown from carcinoma cells and equipment required for such work are not available. In such cases, preparations made from cells taken from the carcinomas of other patients may be used. It is, of course, most preferable to treat a patient with preparations made from cells of carcinomas which are similar to the carcinoma to be treated. For instance, patients suffering from an ovarian carcinoma will be treated with preparations made from cells taken fronn ovarian carcinomas of other patients.It is also possible to make mixed preparations, which contain cells from dif-ferent kinds of carcinomas, such as weaXened cells from mammary and ovary carcinomas.
The pharmaceucical preparations accordin~ to the present invention are stable in suspension for about 4 ~13~9 ~139Z~
wee~s or so at a storage temperature of about -~4C. ~o increase the shelf life of the preparations, the pharma-ceutical prep~ations may be quick frozen to temperatures as low as -80. When deeply frozen the preparations are stable for about one year. For this purpose, they are preferably sealed and paekaged together with a slight ex cess of nutrient solution. The pharrnaceutical prepara-tions can also be stored for a prolonc3ed time in a lyo-philized state.
Pharmaeeutical preparations according to the present invention ean be administered throuc,3h any conve-'~ J nient and effective route such as by subcutaneous or in-tramuseular injeetion or by intravenous infusion. Admin-istration by subcutaneous or intramuscular in~ection is preferred.
In one preferred treatment method about 1 ml of a eell suspension aecording to the invention diluted to 10 times its volume and containing about 106 cells are ad-ministered in each sinr,31e treatment. The treatment is suitably repeated 5 to 6 tim~s in intervals of about 48 hours. It has been found that treatment with the pharma-.~
ceutical preparations according to the present invention will result in a greatly improved tolerance of the patient to the corresponding cytostatic chernical agents. For thiC;
reason, it is highly desirable to treat a patient with cy-tostatic agents alone in a conventional manrler according to known procedures for about 8 days after a sequence c~
treatments with the preparations according to the present invention. Further details of this aspect will become apparent from the following clinical stu~ies which serve to further illustrate and explain the invention.

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EXAMPLE

The following rea9ents ancl solutions were used:

Washing Solution -- this was prepared by placing 80 ml solution A, 100 ml solution B, 100 ml solution C and 5 ml of an antibiotic solution, into a container and bi-distilled water was added to make l,000 ml of solution.

Solution A -- was prepared by dissolving the following in 400 ml bidistilled water:

NaCl 40.0 g KCl 1.0 g N ~PO4.2H2O 5~75 9 KH2P04 1. O g Solution B -- was prepared by dissolving in 500 ml bidistilled water:
CaC12 0.5 9 Solution C -- was prepared by dissolving in 500 ml bidistilled water:

MgC12 . 6H20 Solutions A, B and C were sterilized in an autoclave and stored at 4C.
Antibiotic Solution Penicillin (Grunenthal, Stolberg) 6 mil:Lion I.[J.
Streptomycin (Specia, Paris) 1 g were dissolved in 300 ml bidistilled water and stored at -20C.

Trypsin Solution -- was prepared in a water bath at 3 7 C . ~139219 Trypsin powder (Serva-Heidelberg) 25 g Hanks (BSS) solution (see below) 475 ml The solution was filtered under sterile conditions and then filled in small bottles and stored at -20C. ~or the trypsin treatment this solution was adjusted to the de-10 sired concentration with washing solution.
Behring IV Nutrient Hanks solution (55 ml stock solu-.~ tion A + 55 ml stock solution B
-to which was added bidistilled water 1100 ml) 1100 ml Tissue culture medium solution500 ml Calf serum 200 ml Vitamin solution 100 ml Glutamin solution 100 ml Antibiotic solution 10 ml The mixture was filtered under sterile condi-tions and was then bottled and stored at -20C. Before t'l :~
use, the nutrient was adjusted to a pH value in the range of 6.8 to 7.2 with sodium hydrogen carbonate solution (22 g NaHCO3 and 478 ml bidistilled water).
Hanks Stock Solution A -- the following ingre-dients were dissolved in 250 ml of bidistilled water and stored at 4C:

NaCl 40.0 g KCl 2. 0 g ~ g2 MgSO4.7H2O
Mgcl2 6H20 CaC12 0,7 g Hanks Stock Solution B -- the following mat--erials were dissolved in 240 ml bidistilled water and stored at 4C:
Na2HP042~120 KH2PO~ 0.3 9 Glucose 5.0 g Phenol red solution (1%) 10.0 ml Phenol Red Solution Phenol red 0.5 9 1/20 N NaOH 15 ml Phenol red was dissolved in the cold in the sodium hy-droxide solution within~about 30 minutes and the solution was then filled up to a volume of 50 ml with bidistilled water. The solution wa.s stored at 4C.
TCM So_ution TCM powder (according to Morgan, Morton and Parker, Difco Labora-tories, Detroit, ~.S.) 5.5 g Hanks Stock Solution A 25 ml Hanks Stock Solution B 25 ml Bidistilled water was added to the two stock solutions to make 500 ml. The powder was dissolved in the resulting solution.

Glutamin Solution Glutamin powder (Difco ~139219 Laboratories) 10.0 g Hanks Stock Solution ~ 6 ml Hanks Stock Solution B 6 ml Bidistilled water was added to the stock solutions at a volume of 100 ml. The glutamin powder was dissolved in the resulting solution.
Vitamin Solution Vitamin (MEM 100 x, Boehringer, Mannheim) 100 ml Hanks Stock Solution A 50 ml ' j Hanks Stock Solution B 50 ml Bidistilled water was added to the stock solutions to a volume of 900 ml and the vitamins were added. The result-ing solution was then filtered under sterile conditions, filled into small bottles of 100 ml and stored at -2noc.
Calf Serum Fetal calf serum (Boehringer, Mannheim) was stored at -20C. and before use was thawed in a water bath at 37C~

. ,f Tissue (50 g) excised from an ovarian carcinoma of a 46 year old patient in stage T4N5Mlc were manually com-minuted together with 700 ml of trypsin solution dilutedto a concentration of 0.5% and placed into a lO00 ml flask. The mixture was agitated for 10 minutes with a magnetic stirrer. The supernatant liquor was then ~oured into an ice-cooled flask of the same capacity. The se-quence of agitating the tissue-trypsin mixture and of pouring the supernatant liquor was repeated four tim~s The ~ourth repetition resulted in a clear supernatant liquor. The combined supernatant liquors and the re~ain-ing tissue were then filtered through sterile gauze. The filtrate was centrifuged at 800 to 1000 r.p.m. for 10 minutes. Washing solution (100 ml) was poured onto the sediment. The resulting mixture was re centrifuged and spun down.
The resulting sediment was thoroughly pipetted with 5 ml nutrient and was then filled with nutrient to a total volume of about 400 ml. After this dilution, the examination with a Zeiss inverted microscope having a maq-nification of 125 revealed about 20 to 25 cells in the field of view. The dilute cell suspension was distributed among a series of test tubes, 1 ml per tube, and to Roux bottles, 100 ml per bottle. The growth and metabolism of the cells were observed and recorded daily for six days.
As soon as the nutrient solution exhibited a color change from red to yellow, the nutrient solution was renewed.
Good cell growth was observed when the cell suspension had been incubated at 37C. in an incubator for 6 days.
The cell cultures in the tubes were subjected to the in vitro test explained above with the cytostatic agents listed in Table 1 in the concentrations stated there. After 24 hours the morphological damage was deter-mined under a microscope. The cell suspension proved sen-sitive to the following cytostatic a~ents: A~riblastin, Vincristin and Proresid. All of the remaining listed cytostatic agents, 6 in this case, were ineffective.

Four Roux bottles which contained the cell sus-pension obtained by the above method were then prepared.
Adriblastin, Vincristin and Proresid were used to treat the contents of respective bottles in the concentrations stated in Table 1. The cell suspension in the remaining bottle was not treated and served as a contrpl sample.
After the cytostatic agent had been allowed to act on the cells for 12 hours, the samples were examined under a microscope and only 60% of the cells were found to be in-tact. The cells were then separated by centrifugation at800 to 1000 r.p.m. for 10 minutes. 10 ml of fresh nu-trient were poured onto the sediment. The weakened cells thus obtained were suspended in the fresh nutrient in a concentration of 106 cells per ml. The suspension was stored at 4C.
Test In Vitro 1 ml of an untreated cell suspension, containing 106 cells, was placed into each of a plurality of test tubes. These cells had been obtained by the above-des-cribed treatment of the carcinoma tissue with trypsin buthad not been treated with a cytostatic agent. For six days, 0.3 ml of one of the preparations obtained as des-cribed above and consisting of cells that had been weak-ened with Adriblastin, Vincristin and Proresid, respect-ively, was added daily to each tube. During this test,the tubes were kept in an incubator a~: a temperature of 37C. After 7 to 9 days, about 80 to 90% of the untreated cells in each tube had been destroyed by tlle preparation according to the present invention. A.s the cytostatic agents had been thoroughly washed out of the preparations, ~13~Z~9 it was observed that tl-i.s is the result oE an intercell-ular reaction.
Clinical Treatment Based on the results of the above experiments as -*
: i~
well as additional experiments of like character and after ~
,~.
corresponding experiments on strains of cells from animal tumors, the preparations according to the present inven-tion were used for clinical therapy. With one exception, all patients suffered from cancer in terminal stages and in spite of surgical operations, irradiation treatments and/or cytostatic treatments were in stages T 4 N3-5 Mlc in accordance with the classification introduced by the ~nion International contre le Cancer. The average sur-vival time of such patients is less than 60 days. The preparations according to the invention were used mainly for an adjuvant immune therapy in an attempt to strengthen the immune system as a preliminary to a cytostatic treat-ment with the corresponding cytostatic agents. In all 22 patients were treated, who had all been fully informed of their state and the intended course of treatment. Four-teen of the patients suffered from malignant ovarian tumors ;~J (one of them from mesosigmoid tumor as a recurrent growth),four ~om mammary carcinomas, one from a pancreas carcinoma, one from a retothelial sarcoma, one rom a cecum carcinoma and one from a carcinoma of the corpus uteri.
Cancerous tissues were excisec] from the patients by surgical operations and were treated in accordance with the above-described procedures to formulate the prepara-tions according to the invention. On alternate days, the 113~Zl~
- lR -individually prepared cell suspension in a total of ~ to 8 10-ml portions was administered to each patient by intra-- gluteal injection. Each 10-ml portion contained 106 cells, that is l ml of the preparation in a nutrient solu-tion and 9 ml physiological salt solution or calf serum. b~'`
In some cases, 1 ml Lidocaine solution (xyloc~ine) in a con-centration of 0.5% was administered with each 10-ml por-tion, if desired. In the first sequence, the number of injections depended on the number of cells which were available and in most cases this amounted to 5 to 8 injec-tions in intervals of 43 hours. After a waiting time of 8 to 20 days (with patients in extre~e condition the waiting J time is at the upper limit), the cytostatic treatment was carried out in accordance with the individual test result, in most cases in accordance with the following schedule:
l mg Vincristin by intravenous injection; 6 hours later Adriblastin by intravenous injection in an amount of 40 mg per m2 of body surface area; 48 hours later Cyclophos-phamide or Proresid in an amount of 25 mg per kg body weight, dissolved in the same concentration in 500 ml of physiological salt solution. Owing to the toxicity of J Adriblastin, an interval of 3 weeks was left between two cytostatic treatment sequences, until a total of 550 mg of ~hat agent had been administered. In case of resistclrlce after test result, a change was made to the other cytost~tic agents, such as 5-FU, methotrexate Leukoverin and the like, in weekly intervals.
This treatment resulted in a subjective and objec-tive improvement of all patients. Tllere were no loccll or general secondary effects. Although thc cell suspension 1~L3S'~1~

had to be assumecl to be toxic, its injection did not re-local or genel~al sult in/reddening or swelling. A sensation of pain or similar response to the reaction was not observed. The improvement wa~ so lasting that in all cases the normally dangerous cytostatic therapy could be started after an interval of 20 days, on a maximum.
Four of the twenty-two patients mentioned above were not given a subsequent cytostatic thcrapy because they refused it or their tumc~r.s werc apparerltly t.oo advan-ced. Nevertheless, even these patients had an unusually long survival time. For instance, a woman of 60 who had a mammary t~mor larger than a fist and bone metastases and J also proven brain metastases was subjectetl to a simple ablatio mammae and given a subseqùent treatment with the preparation accordiny to the invention. Without additio-nal therapy she had a survival time of 270 days.
Five patients were also treated with heterolo-gous carcinoma cell suspensions and showed a very good tolerance.
The average survival time of all patients is presently 350 days, which is almost six times the previous average. Five patients were still alive after 245 to 665 days. Of these, three have had no recurrent yrowtils ant]
two have recurrent growths which are amenable to treat-ment. All patients treated are in a 9Ood general state.
The result of the treatment with the prepara-tions according to the invention on two of the twenty-two patients mentioned above an~ on an atlditional patient will now be described.

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1) A patient of ~6 was admitted to the hospi-tal when she suffered from mammary ~morson both sides that had existed for two years, and large abdominal tumors.
The patient was in an extremely poor general state. After a general pre-treatment, her two breasts, which had been extensively invaded by cancer, as well as the associated lymph nodes at the axillae, were removed in two sur~ical operations in June 1978. Fourteen days later, her uterus and the two ovar~n ~mors were removed. The remaining can-cerous tissue in her omentum anc3 on her peritoneum, on lymph nodes and at other sites could not be surgically rè
moved. The patient was not irradiated but was given 12 J injections with the preparation according to the invention made fr~m pretreated carcinoma cells from her own body (prepared in accordance with the above example) and was subsequently given the combined chemotherapy. I'he patient recovered completely during the immune therapy, and subse-quently showed a high tolerance to the cytostatic agents.
After a total of 12 months had passed, the patient reports that she feels excellent. She is entirely capable of per-forming her work and has no complaints at all. Her clini-9 cal examination does not reveal any recurrent growth, nei-ther abdominally nor in the chest region. At the begin-niny of the treatment, she was in a tumor stage ~:r4 N5 Mlc.
2) A patient of 32 had a large ~bdominal tumor when she was admitted to the hospital. Iler uterus ancl the two adnexes had been removed in another hospita] one year previous for a treatment of a fairly large ovarian cancer.
She had also received cytostatic treatment for the pre-vious incident. After relapse, a mesosigma tumor of 1~3g;~1g almost the size of c ~ ~ was excised, which had developed as a recurrent growth due to the previous ovarian carcinoma. It was impossible to excise the mesosigma tumor entirely in healthy tissue. She had also metastases at the lymph nodes on the pelvic wall. The patient was given a post-surgical adjuvant immune therapy consisting of 12 injections of preparations produced by a treatment of cells from her own tumors. Because the patient subse-quently moved away, ~he could not be given a subsequent cytostatic treatment. The patient returned several times for subsequent examination. Eleven months from the begin-ning of the immune therapy she is in an excellent state without any clinical indication of recurrent growths. The entire abdomen is free from tumors.

3) A patient of 41, who has not been included in the above-described group because she is in the Tl stage, came to the hospital for admittance because she had a small, possibly cancerous node in the left mammary gland. The result of mammography, thermography and punc-ture cytology were positive on the left and doubtful re-garding another small node on the right tPapanicolaou 3).
0 ; The histology of tumorous tissue excised on the left breast revealed a scirrhus mammary carcinoma (~fl Nx Mo) about 2 cm in diameter. This patient's case is reported separately because she was the only early-stage case. The patient agreed to a simple mastectomy of the left breast but refused additional measures particularly on the right breast. She also refused cytostatic or other subsequent treatments but agreed to a cell treatment. According to the present invention, she was given a heterologous cell ~1392~9 treatment with mammary carcinoma cells which had been obtained from another patient and treated accor ~ g to the procedures of the present invention. The cell treatment was performed in accordance with the routine described above comprising 6 intramuscular injections of 10 ml cell suspension containing 106 cells each on ~lternate days.
Twelve months after the treatment the patient has no in-dication of recurrent growth. The suspecte~3 t~mor of the right breast has completely disappeare~.
It is apparent from the previous investigations that it was not possible to administer a sufficiently large dosage in most cases owing to a lack of material.
It would be desirable to give every patient suffering from a carcinoma, even if it is in an initial stage, a therapy consisting of a sequence of injections five or six times every 48 hours within one month, then in each of three alternate months, then in corresponding months of four successive half-years and finally in corresponding months of several successive years. Such treatmellts can be car-ried out only with heterologous cancer cells but theseshould be taken from similar organs, if possible, in case no autologous cells are available. ~he heterologous can-cer cells may be mixed in a ratio of 1:1 with lyophilized cancer cells, which need not be pretreated.

1~L3~9 l~ile not wishing to ~e bound by any particular theory or mode of operation, it appears that the process of my invention and the products so produced are effective in the treatment of carcinoma on the following: Based on S the assumption that malignant tissue growth starts on the base of an autonomous, progressive and overshooting proli-feration of body-specific cells, this method of cancer treatment was developed. The principle of my method appears, in larye part, to be an amelioration of the immu-nological starting condition of the patient before the cy-tostatic therapy itself by repeated injections of ultrafil-trates of body-specific cytostatically pretreated carcinoma ; cells, as hereinabove described.

Claims (11)

C L A I M S

1. A method for isolating, culturing and growing selected carcinoma cells comprising the steps of:
(1) providing a source of carcinoma cells;
(2) culturing and incubating said cells in a nutrient growth media under cell growth conditions to produce a cell culture;
(3) subjecting a plurality of pre-determined portions of the cell culture of step (2) to a plurality of cytostatic agents and observing the visible morphological damage in each portion;
(4) selecting that cytostatic agent that exhibits visible morphological damage to said susceptible cells;
(5) treating the remaining portion of the cell culture of step (2) with the cytostatic agent of step (4) and incubating such that from about 30 to 70 % of said cells remain intact under microscopic examination and that said cells are not completely killed but only partially weakened;
(6) washing the weakened cells of step (5) with a physiologically acceptable solution to remove substantially completely all of said cytostatic agent from said weakened cells; and thereafter (7) suspending the cells thus treated in a physiologically acceptable fluid for a pharmaceuti-cally acceptable preparation.

2. The method according to claim 1 wherein the incubation of step (5) is conducted for a period of about 4 to 15 hours.

3. The method according to claim 2 wherein the incubation of step (5) is conducted for a period of about 6 to about 12 hours.

4. The method according to claim 1 wherein the incubation of step (5) is conducted at a temperature of about 37°C.

5. The method according to claim 1 wherein the incubation of step (5) is conducted such that from about 40 to about 60% of said cells remain intact.

6. A method for producing a suspension of weakened selected carcinoma cells that are susceptible to the cytostatic activity of a selected cytostatic agent compris-ing treating a cell culture of carcinoma cells with a cytostatic agent to which said cells are sensitive and incubating for a period of time until microscopic examination indicates that only about 30 to about 70% of the thus treated cells are intact, and thereafter washing the thus weakened cells with a physiologically acceptable solution to remove said cytostatic agent.

7. A method according to claim 1 wherein by step (1) a source of individual ascites carcinoma cells or leukemia cells is provided.

8. The method of claim 6 including the additional step of freezing the resulting solution of weakened cells.

9. The method of claim 6 including the additional step of lyophilizing the resulting solution of weakened cells.

10. The method of claim 6 including freezing a portion of the resulting solution of weakened cells and lyophilizing a portion of the resulting solution of weakened cells, main-taining the frozen and lyophilized portions under storage conditions and thereafter thawing the frozen portion and mixing same with an equal amount of lyophilized cells.

11. The method according to claim 1 wherein the weakened cells of step (5) are separated by centrifuge at a speed of about 800 to about 1,000 r.p.m. and thereafter washed according to step (6).