FI88360B - FOERFARANDE FOER FRAMSTAELLNING AV TUMOERLAEKEMEDEL - Google Patents
FOERFARANDE FOER FRAMSTAELLNING AV TUMOERLAEKEMEDEL Download PDFInfo
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- FI88360B FI88360B FI853389A FI853389A FI88360B FI 88360 B FI88360 B FI 88360B FI 853389 A FI853389 A FI 853389A FI 853389 A FI853389 A FI 853389A FI 88360 B FI88360 B FI 88360B
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- mak
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- 239000000427 antigen Substances 0.000 claims abstract description 22
- 102000036639 antigens Human genes 0.000 claims abstract description 22
- 108091007433 antigens Proteins 0.000 claims abstract description 22
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- 238000000034 method Methods 0.000 claims abstract description 14
- 239000003814 drug Substances 0.000 claims abstract description 12
- 229940124597 therapeutic agent Drugs 0.000 claims abstract description 12
- 102000005348 Neuraminidase Human genes 0.000 claims abstract description 7
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- 101100182914 Caenorhabditis elegans mak-1 gene Proteins 0.000 claims 4
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- LOKCTEFSRHRXRJ-UHFFFAOYSA-I dipotassium trisodium dihydrogen phosphate hydrogen phosphate dichloride Chemical compound P(=O)(O)(O)[O-].[K+].P(=O)(O)([O-])[O-].[Na+].[Na+].[Cl-].[K+].[Cl-].[Na+] LOKCTEFSRHRXRJ-UHFFFAOYSA-I 0.000 description 10
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- ATDGTVJJHBUTRL-UHFFFAOYSA-N cyanogen bromide Chemical compound BrC#N ATDGTVJJHBUTRL-UHFFFAOYSA-N 0.000 description 2
- 229960003964 deoxycholic acid Drugs 0.000 description 2
- KXGVEGMKQFWNSR-LLQZFEROSA-N deoxycholic acid Chemical compound C([C@H]1CC2)[C@H](O)CC[C@]1(C)[C@@H]1[C@@H]2[C@@H]2CC[C@H]([C@@H](CCC(O)=O)C)[C@@]2(C)[C@@H](O)C1 KXGVEGMKQFWNSR-LLQZFEROSA-N 0.000 description 2
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- WSFSSNUMVMOOMR-NJFSPNSNSA-N methanone Chemical compound O=[14CH2] WSFSSNUMVMOOMR-NJFSPNSNSA-N 0.000 description 1
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Classifications
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K39/00—Medicinal preparations containing antigens or antibodies
- A61K39/0005—Vertebrate antigens
- A61K39/0011—Cancer antigens
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K38/00—Medicinal preparations containing peptides
- A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- A61K38/43—Enzymes; Proenzymes; Derivatives thereof
- A61K38/46—Hydrolases (3)
- A61K38/47—Hydrolases (3) acting on glycosyl compounds (3.2), e.g. cellulases, lactases
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- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Epidemiology (AREA)
- Chemical & Material Sciences (AREA)
- Immunology (AREA)
- Medicinal Chemistry (AREA)
- Pharmacology & Pharmacy (AREA)
- Animal Behavior & Ethology (AREA)
- General Health & Medical Sciences (AREA)
- Public Health (AREA)
- Veterinary Medicine (AREA)
- Microbiology (AREA)
- Mycology (AREA)
- Oncology (AREA)
- Gastroenterology & Hepatology (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Engineering & Computer Science (AREA)
- Medicines Containing Antibodies Or Antigens For Use As Internal Diagnostic Agents (AREA)
- Medicines Containing Material From Animals Or Micro-Organisms (AREA)
- Preparation Of Compounds By Using Micro-Organisms (AREA)
- Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)
- Diaphragms For Electromechanical Transducers (AREA)
- Window Of Vehicle (AREA)
- Measuring Pulse, Heart Rate, Blood Pressure Or Blood Flow (AREA)
- Pharmaceuticals Containing Other Organic And Inorganic Compounds (AREA)
- Peptides Or Proteins (AREA)
- Saccharide Compounds (AREA)
Abstract
Description
1 883601 88360
Menetelmä kasvainlääkkeen valmistamiseksiA method of making an antitumor drug
Keksintö koskee menetelmää terapeuttisen aineen valmistamiseksi kasvainsoluista kasvainsairauksien hoi-5 toon.The invention relates to a method of preparing a therapeutic agent from tumor cells for the treatment of tumor diseases.
Esimerkiksi teoksen Mechanisms of Tumor Immunity, toim. Gree et ai., John Wiley & Sons, N.Y., 1977, s. 196, perusteella on tunnettua, että kasvainsairauksia on jo yritetty hoitaa rokottamalla kasvainsoluilla, joita oli 10 muunnettu jäädyttämällä ja sulattamalla, lyofilisoimalla, paineen avulla tai homogenisoimalla. Myös subsellulaarisia fraktioita tai solu-uutteita on käytetty tähän tarkoitukseen. Tähän mennessä kasvainsairautta vastaan ei kuitenkaan tunneta mitään rokotetta.For example, Mechanisms of Tumor Immunity, ed. It is known from Gree et al., John Wiley & Sons, N.Y., 1977, p. 196, that attempts have already been made to treat tumor diseases by vaccinating with tumor cells modified by freezing and thawing, lyophilization, pressure or homogenization. Subcellular fractions or cell extracts have also been used for this purpose. To date, however, no vaccine is known against tumor disease.
15 Nyt on yllättäen todettu, että ihmisen kasvaimista peräisin olevia, lyofilisoituja tai aldehydillä käsiteltyjä soluja tai sellaisista saatuja soluaggregaatteja, jotka kantavat antigeenejä, joita DE-hakemusjulkaisussa 33 29 184 kuvatut monoklonaaliset vasta-aineet sitovat, 20 voidaan käyttää terapeuttisena aineena kasvainsairauksien hoidossa.It has now surprisingly been found that lyophilized or aldehyde-treated cells derived from human tumors or cell aggregates derived therefrom which carry antigens bound by the monoclonal antibodies described in DE 33 33 184 can be used as a therapeutic agent in the treatment of tumor diseases.
Keksinnön kohteena on siten menetelmä terapeuttisen aineen valmistamiseksi kasvainsairauden hoitoon, joka aine sisältää kuivattuja tai kemiallisella käsittelyllä stabi-25 loituja ihmisen soluja, jotka kantavat antigeenejä, joita ' ‘ DE-hakemusjulkaisussa 33 29 184 kuvatut monoklonaaliset : vasta-aineet sitovat. Sellaisen terapeuttisen aineen etuna natiivisten kasvainsolujen käyttöön verrattuna on se, että natiiviset solut eivät ole stabiileja, joten ne on valmis-: 30 tettava aina juuri ennen käyttöä. Sen vuoksi niitä ei voi- da standardoidakaan.The invention thus relates to a process for the preparation of a therapeutic agent for the treatment of a tumor disease, which comprises dried or chemically stabilized human cells carrying antigens which are bound by the monoclonal antibodies described in DE '33 33 184. The advantage of such a therapeutic agent over the use of native tumor cells is that the native cells are not stable, so they must always be prepared just before use. Therefore, they cannot be standardized.
Keksinnön mukaiselle menetelmälle kasvainsairauden hoitoon soveltuvan terapeuttisen aineen valmistamiseksi on tunnusomaista se, mitä patenttivaatimuksessa 1 esitetään.The method according to the invention for the preparation of a therapeutic agent suitable for the treatment of a tumor disease is characterized by what is stated in claim 1.
: 35 Ihmisen kasvainsolut, jotka kantavat antigeenejä, joita 2 88360 DE-hakemusjulkaisussa 33 29 184 kuvatut monoklonaaliset vasta-aineet sitovat, kuivataan tai stabiloidaan aldehydi-käsittelyllä ja viimeistellään terapeuttiseksi aineeksi, jolloin mahdollisesti lisätään neuraminidaasia.: 35 Human tumor cells carrying antigens that are bound by the monoclonal antibodies described in DE 2 88360 DE 33 29 184 are dried or stabilized by aldehyde treatment and finalized as a therapeutic agent, possibly with the addition of neuraminidase.
5 Lisäksi esitetään menetelmä kasvainsairauden hoi toon soveltuvan terapeuttisen aineen valmistamiseksi, jolloin ihmisen kasvainsoluista eristetään DE-hakemusjulkaisussa 33 29 184 kuvatun monoklonaalisen vasta-aineen avulla antigeeni ja se viimeistellään terapeuttiseksi aineek-10 si, jolloin mahdollisesti lisätään neuraminidaasia.In addition, there is provided a method of preparing a therapeutic agent suitable for the treatment of a tumor disease, comprising isolating an antigen from human tumor cells using a monoclonal antibody described in DE 33 29 184 and finalizing it as a therapeutic agent, optionally adding neuraminidase.
Mahdollisuus vahvistaa immuunivastetta neuramini-daasilla on DE-hakemusjulkaisun 26 20 649 perusteella tunnettu .The possibility of enhancing the immune response with neuraminidase is known from DE-A-26 20 649.
Terapeuttisella aineella tarkoitetaan ainetta, joka 15 voi olla sekä profylaktinen että ilmenneen sairauden hoitoon soveltuva.By therapeutic agent is meant an agent that may be both prophylactic and suitable for the treatment of an established disease.
Keksinnön puitteissa käyttökelpoisia soluja saadaan kasvaimista tunnetuilla soluviljelymenetelmillä. Mekaanisesti tai entsymaattisesti sellaisista viljelmistä saadut, 20 mahdollisesti mitomysiini C:llä inaktivoidut solut kuivataan, edullisesti lyofilisoidaan, tai käsitellään aldehy-dillä, edullisesti 1-6 hiiliatomia sisältävällä mono- tai dialdehydillä.Cells useful in the context of the invention are obtained from tumors by known cell culture methods. Mechanically or enzymatically, cells obtained from such cultures, optionally inactivated by mitomycin C, are dried, preferably lyophilized, or treated with an aldehyde, preferably a mono- or dialdehyde having 1 to 6 carbon atoms.
Kemialliseen stabilointiin ja fiksaatioon erityi-25 sen hyvin soveltuviin aineisiin kuuluvat bifunktionaali-set yhdisteet, siis sellaiset yhdisteet, jotka sisältävät kaksi sellaista ryhmää, jotka kykenevät reagoimaan biologisen materiaalin funktionaalisten ryhmien kanssa, ts. "silloittamaan" sen. Niitä ovat esimerkiksi dialdehydit, ·: 30 erityisesti alifaattiset 2-8 hiiliatomia sisältävät dialdehydit. Lisäksi soveltuvia ovat kuitenkin myös 1-4 hiili-atomia sisältävät monoalkanaalit, kuten formaldehydi, joka kykenee reagoimaan bifunktionaalisesti.Particularly suitable substances for chemical stabilization and fixation include bifunctional compounds, i.e. compounds containing two groups capable of reacting with the functional groups of the biological material, i.e. "crosslinking" it. These are, for example, dialdehydes, in particular aliphatic dialdehydes having 2 to 8 carbon atoms. However, monoalkanals having 1 to 4 carbon atoms, such as formaldehyde, which is capable of reacting bifunctionally, are also suitable.
Solut esiintyvät yleensä solurykelminä tai yksit-; 35 täisinä soluina. Myös kasvainsoluista eristettyjä anti- 3 88360 geenejä tai solunkappaleita voidaan käyttää. Sellaisia antigeenejä voidaan saada potilaiden kasvainkudoksista, kuten ihmisen kasvaimista, jotka kasvavat vajavaisen immuniteetin omaavissa eläimissä.Cells usually occur in cell clusters or singly; 35 as whole cells. Anti-3,88360 genes or cell fragments isolated from tumor cells can also be used. Such antigens can be obtained from tumor tissues of patients, such as human tumors, that grow in immunocompromised animals.
5 Määriteltyjä antigeenejä saadaan kasvainsoluista tai niiden kappaleista DE-hakemusjulkaisussa 33 29 184 kuvattujen monoklonaalisten vasta-aineiden avulla.The specified antigens are obtained from tumor cells or fragments thereof by means of the monoclonal antibodies described in DE-A-33 29 184.
Esimerkkejä sellaisista antigeeneistä ovat CEA (carcinoembryonic antigen; J. Exp. Med. 122 (1965) 467) ja 10 NCA (non-specific crossreacting antigen; J. Immun. III (1973) 1926), Jotka voidaan eristää kasvainsoluista immuu-niaffiniteettikromatografian avulla. Tätä varten DE-hake-musjulkaisun 34 16 774 taulukossa I esitetyt monoklonaaliset vasta-aineet sidotaan puhdistettuina proteiineina ko-15 valenttisesti CNBr-aktivoituun sefaroosi 4B:hen, ja näiden monoklonaalisten vasta-aineiden tunnistamat antigeenit (CEA, NCA) eristetään DE-TA-paksusuolisyöpäsolu-uutteista. Erästä sopivaa menetelmää on kuvattu Pharmacia-teoksessa Affinity Chromatography, Principles and Methods, 1979, 12-20 18; tiivistelmä on esitetty sivulla 15.Examples of such antigens are CEA (carcinoembryonic antigen; J. Exp. Med. 122 (1965) 467) and 10 NCA (non-specific crossreacting antigen; J. Immun. III (1973) 1926), which can be isolated from tumor cells by immunoaffinity chromatography. . To this end, the monoclonal antibodies shown in Table I of DE-A-34 16 774 are bound as purified proteins covalently to CNBr-activated sepharose 4B, and the antigens recognized by these monoclonal antibodies (CEA, NCA) are isolated from DE-TA. colon cancer cell extracts. A suitable method is described in Pharmacia, Affinity Chromatography, Principles and Methods, 1979, 12-20 18; a summary is presented on page 15.
Sellaista monoklonaalisen vasta-aineen avulla saatua antigeeniä voidaan käyttää terapeuttisena aineena, .·.·. esimerkiksi vaikuttavana aineena rokotteissa niiden kas- vainsolujen, joista antigeeni saatiin, aiheuttamaa sai-! 25 rautta vastaan.An antigen obtained with a monoclonal antibody can be used as a therapeutic agent,. for example, as an active ingredient in vaccines caused by the tumor cells from which the antigen was obtained! 25 against iron.
‘ ; Rokotteena käytettävän materiaalin laadunvalvonta ·;* suoritetaan esimerkiksi monoklonaalisilla vasta-aineilla toteuttavan vakioinnin avulla tai erottamalla kaikki solu-proteiinit SDS-polyakryyliamidigeelielektroforeesin tai : 30 isoelektrisen fokusoinnin (1. dimensio), joka on yhdistetty SDS-polyakryyliamidigeelielektroforeesiin (2. dimensio), avulla ja värjäämällä sen jälkeen geeli (hopeavär-‘ : jäys).‘; Quality control of the material to be used as a vaccine is performed, for example, by conditioning with monoclonal antibodies or by separating all cellular proteins by SDS-polyacrylamide gel electrophoresis or by: isoelectric focusing (1st dimension) combined with SDS-polyacrylamide gel electrophoresis then staining the gel (silver color).
Antigeenimateriaalia käytetään edullisesti yhdessä 35 apuaineen, erityisesti neuraminidaasin (DE-hakemusjulkaisu 4 88360 26 20 649), kanssa intradermaalisesti ja edullisesti sakki lautarokotusmenettelyä (Cancer Immunol, and Immunother. 6 (1979) 47-58, erityisesti s. 48) noudattaen. Sellaista rokotetta käytetään edullisesti määrätyissä vaiheissa ole-5 via paksusuolensyöpiä (Duke C) vastaan sekä muita sellaisia kasvaimia vastaan, jotka kantavat rokotteessa olevia antigeenejä tai epitooppeja. Sellaisia muita kasvaimia ovat kiinteät tuumorit, esimerkiksi haimasyöpä, mahasyövät, rintasyövät ja keuhkosyövät. Rokotetta voidaan antaa 10 parenteraalisesti tai suun kautta. Antigeenejä voidaan käyttää fysiologiseen suolaliuokseen liuotettuina tai sus-pendoituina, edullisesti intradermaalisesti PBS:ssä.The antigenic material is preferably used in combination with 35 excipients, in particular neuraminidase (DE-A-4 88360 26 20 649), intradermally and preferably in accordance with the sac board vaccination procedure (Cancer Immunol, and Immunother. 6 (1979) 47-58, in particular p. 48). Such a vaccine is preferably used against colon cancers (Duke C) at certain stages, as well as against other tumors that carry antigens or epitopes in the vaccine. Such other tumors include solid tumors such as pancreatic cancer, gastric cancers, breast cancers, and lung cancers. The vaccine can be administered parenterally or orally. Antigens can be used dissolved or suspended in physiological saline, preferably intradermally in PBS.
Rokotteen antigeenikoostumuksen stabiilisuuden arvioimiseksi suoritettiin kaksi testiä: 15 a) Terasaki-lIF-koe (epäsuora immunofluoresenssiTo assess the stability of the antigenic composition of the vaccine, two tests were performed: a) Terasaki-IFF assay (indirect immunofluorescence
Terasaki-mikromaljan syvennyksissä kasvavia tuumorisoluja käyttäen) monoklonaalisilla vasta-aineilla, joiden spesifisyys vaihteli. Tämän kokeen avulla on mahdollista mitata membraaniantigeenien, joita vastaan on käytettävissä usei-20 ta monoklonaalisia vasta-aineita, esiintyminen intaktien tuumorisolujen pinnalla [Cancer Detection and Prevention 6 (1983) 181-184]. Näin oli mahdollista todeta voimakkaat muutokset DE-TA-solumembraanissa viljelyn aikana.In wells of a Terasaki microplate using growing tumor cells) with monoclonal antibodies of varying specificity. This experiment makes it possible to measure the presence of membrane antigens against which several monoclonal antibodies are available on the surface of intact tumor cells [Cancer Detection and Prevention 6 (1983) 181-184]. Thus, it was possible to detect strong changes in the DE-TA cell membrane during culture.
b) Kaikkien soluproteiinien saattaminen liukene-25 viksi detergentin avulla (Hybridoma 1 (1982) 413-421) ja sen jälkeinen SDS-polyakryyliamidigeelielektroforeesi yhdistettynä hopeavärjäykseen [Anal. Biochcm. 105 (1980) 361-363]. Näiden menetelmien yhdistäminen takaa sen, että DE-TA-solulinjan kokonaisproteiinisisällössä ei ole tapah-30 tunut merkittäviä muutoksia.b) Dissolution of all cellular proteins with detergent (Hybridoma 1 (1982) 413-421) and subsequent SDS-polyacrylamide gel electrophoresis combined with silver staining [Anal. Biochem. 105 (1980) 361-363]. The combination of these methods ensures that there are no significant changes in the total protein content of the DE-TA cell line.
Seuraavat esimerkit valaisevat keksintöä.The following examples illustrate the invention.
Esimerkki 1Example 1
Karsinoomasolulinjaa BW X (Wienin syöpäsolututki-muskeskus) viljeltiin soluviljelmässä yksisolukerroksena : 35 muovipulloissa RPMI-1640-väliaineessa [Moore, G.E., 5 88360The carcinoma cell line BW X (Vienna Cancer Cell Research Center) was cultured in cell culture as a monolayer: 35 in plastic flasks in RPMI-1640 medium [Moore, G.E., 5,88360
Gerner, R.E., Franklin, H.A., Culture of Normal Human Leucocytes, J.A.M.A. 199 (1967) 519-524], joka sisälsi 10 % fetaalisen vasikan seerumia. Yhteensulautuneiden vilje-mien kiinni toisissaan kasvavat solut irrotettiin mekaani-5 sesti tai trypsiinillä, joka oli liuotettu fetaalisen vasikan seerumia sisältämättömään RPMI-1640-väliaineeseen, kollagenaasi inaktivoitiin lisäämällä RPMI-1640:een liuotettua fetaalisen vasikan seerumia, ja sen jälkeen solut poistettiin kudosviljelypulloista ja pestiin kolmesti fos-10 faattipuskuroidulla keittosuolaliuoksella (PBS), jonka lämpötila oli 37 °C.Gerner, R.E., Franklin, H.A., Culture of Normal Human Leucocytes, J.A.M.A. 199 (1967) 519-524] containing 10% fetal calf serum. Cells growing from fused cultures were mechanically detached or trypsinized in RPMI-1640 medium without fetal calf serum, collagenase was inactivated by adding fetal calf serum dissolved in RPMI-1640, and then the cells were removed from the tissue culture. three times with phosphate-buffered saline (PBS) at 37 ° C.
Soluja, joiden määrä oli noin 107 ja jotka esiintyivät suurimmaksi osaksi rykelminä, inkuboitiin 1 tunnin ajan 37 °C:ssa 1 ml:ssa PBS:ä, joka sisälsi 100 pg miιοί 5 mysiini C:tä. Näin inaktivoidut solut pestiin sen jälkeen kolmesti PBS:llä ja (a) kolmesti 0,18 M ammoniumvetykarbo-naattipuskurilla, jonka pH oli säädetty etikkahapolla arvoon 7,4. 107 solua vastaava solusedimentti sekoitettiin 100 μΐ:aan samaa ammoniumvetykarbonaattipuskuria ja jäädy-20 tettiin -70 °C:ssa. Tämän jälkeen jäädytetty materiaali kylmäkuivattiin ja säilytettiin jääkaapissa +4 °C:ssa pie-nessä ilmatiiviisti suljetussa lasipullossa. Tällä tavalla käsiteltyä solumateriaalia voidaan antaa rokotteena poti-laalle PBS:ään sekoitettuna.Cells numbering about 107, most of which were present in clusters, were incubated for 1 hour at 37 ° C in 1 ml of PBS containing 100 μg of myicin C. The cells thus inactivated were then washed three times with PBS and (a) three times with 0.18 M ammonium hydrogen carbonate buffer adjusted to pH 7.4 with acetic acid. The cell sediment corresponding to 107 cells was mixed with 100 μΐ of the same ammonium bicarbonate buffer and frozen at -70 ° C. The frozen material was then lyophilized and stored in a refrigerator at + 4 ° C in a small hermetically sealed glass bottle. The cellular material treated in this manner can be administered as a vaccine to a patient mixed with PBS.
I. I 25 Vaihtoehtoisesti (b) soluja inkuboitiin 5 minuut tia +4 °C:ssa PBSrssä, joka sisälsi 0,1 % glutaraldehydiä, "** ’ ylimääräinen glutaraldehydi poistettiin kolmesti suori tetulla PBS-pesulla, jonka jälkeen soluja inkuboitiin 2-%:iseen BSA:n (bovine serum albumin) kanssa 5 minuuttia *.*·! 30 +4 °C:ssa ja ne pestiin kolmesti PBStllä. Näin käsiteltyjä : ; soluja voidaan säilyttää +4 °C:ssa ja antaa rokotteena po- ’ : tilaalle.I. I 25 Alternatively (b) the cells were incubated for 5 minutes at + 4 ° C in PBS containing 0.1% glutaraldehyde, "** 'excess glutaraldehyde was removed three times by washing with PBS, after which the cells were incubated with 2-% with BSA (bovine Serum albumin) for 5 minutes at * + 1 ° C and washed three times with PBSt. The cells thus treated can be stored at + 4 ° C and administered as a vaccine. : to replace.
; Vaihtoehtoisesti (c) soluja inkuboitiin yön yli formaliinissa 25 °C:ssa Lillyn [Benno Romeis, Oldenburg 35 Verlag MUnchen, (1968), luku 266, s. 65] menettelyä nou- 6 88360 dattaen ja aikä ajoin ravistaen. Solut (noin 108) sentrifu-goitiin (10 minuuttia keskipakokiihtyvyydellä 800 g), su-pernatantti dekantoitiin, ja solusedimentti suspendoitiin 7 ml:aan kahdesti tislattua vettä (* yksi pesu). Tämä pe-5 sutoimenpide toistettiin neljästi 1 tunnin välein. Tämän jälkeen solusedimentti pestiin kolmesti 1 tunnin välein 70-%:isella etanolilla kunkin erän ollessa 7 ml. Seuraa-vaksi solusedimentti pestiin kolmesti 30 minuutin välein 80-%:isella etanolilla kunkin erän ollessa 7 ml. Seuraa-10 vaksi solusedimentti pestiin kolmesti 30 minuutin välein 96-%:isella etanolilla kunkin erän ollessa 7 ml. Seuraa-vaksi solusedimentti pestiin kolmesti 30 minuutin välein 99-%:isella etanolilla kunkin erän ollessa 7 ml. Tämän jälkeen solusedimentti pestiin 30 minuutin välein kolmella 15 7 ml:n erällä steriiliä PBS:ä ja säilytettiin steriilisti 4 °C:ssa. Tällä tavalla käsiteltyjä soluja voidaan varastoida 4 °C:ssa ja antaa rokotteena potilaalle.; Alternatively (c), the cells were incubated overnight in formalin at 25 ° C according to the procedure of Lilly [Benno Romeis, Oldenburg 35 Verlag MUnchen, (1968), Chapter 266, p. 65] and shaking from time to time. The cells (about 108) were centrifuged (10 minutes at 800 g centrifugal acceleration), the supernatant was decanted, and the cell sediment was suspended in 7 ml of doubly distilled water (* one wash). This Fri-5 procedure was repeated Four times every 1 hour. The cell pellet was then washed three times every 1 hour with 70% ethanol in 7 ml portions. Next, the cell sediment was washed three times every 30 minutes with 80% ethanol, each batch being 7 ml. For the next 10 hours, the cell sediment was washed three times every 30 minutes with 96% ethanol with 7 ml each. Next, the cell sediment was washed three times every 30 minutes with 99% ethanol, each batch being 7 ml. The cell sediment was then washed every 30 minutes with three 7 ml aliquots of sterile PBS and stored sterile at 4 ° C. Cells treated in this manner can be stored at 4 ° C and administered to a patient as a vaccine.
Esimerkki 2Example 2
Antigeenien eristämiseksi kasvainsoluista immuuni-20 affiniteettikromatografian avulla puhdistetut monoklonaaliset vasta-aineet, jotka reagoivat spesifisesti rokotteena käytettävien kasvainsolujen pinnalla olevien antigeenien kanssa, sidottiin kovalenttisesti CNBr-aktivoi-tuun sefaroosi 4B:hen. Tämä suoritettiin Pharmacia-teoksen 25 Affinity Chromatography, Principles and Methods, 1979, s. 12-18, erityisesti s. 15, mukaisesti. Sen jälkeen kantajaan sidottuja monoklonaalisia vasta-aineita inkuboitiin solusolubilisaattien kanssa 2 tuntia +4 °C:ssa aika ajoin ravistaen. Nämä oli saatu hajotuspuskuriuutolla [5 g/1 : 30 natriumdeoksikolaattia, 0,5 mmol/1 PMSF:ä (* fenyylimetyy-lisulfonyylifluoridi), PBS, pH 8,3] mekaanisesti viljely-pulloista poistetuista viljelmäsoluista, kuten artikkelissa Hybridoma 1 (1982) 413-421, erityisesti sivulla 414 on kuvattu.To isolate antigens from tumor cells, monoclonal antibodies purified by immuno-affinity chromatography that specifically reacted with antigens on the surface of tumor cells used as vaccines were covalently bound to CNBr-activated sepharose 4B. This was performed according to Pharmacia's 25 Affinity Chromatography, Principles and Methods, 1979, pp. 12-18, especially p. 15. The carrier-bound monoclonal antibodies were then incubated with cell solubilizers for 2 hours at + 4 ° C with occasional shaking. These were obtained by lysis buffer extraction [5 g / 1: 30 sodium deoxycholate, 0.5 mmol / l PMSF (* phenylmethylsulfonyl fluoride), PBS, pH 8.3] from culture cells mechanically removed from culture flasks as described in Hybridoma 1 (1982). 413-421, especially on page 414.
35 Kuormitettu kantaja sentrifugoitiin ja suspendoiThe loaded carrier was centrifuged and suspended
tiin pesun suorittamiseksi hajotuspuskuri SDS:ään [20 mMwas performed in lysis buffer in SDS [20 mM
7 883607 88360
Tris-HCl, pH 8,0, 0 5 g/1 Nonidet P-40:ä (= oktyylifenyy- lietyleenioksidi; Fluka AG), 5 g/1 natriumdeoksikolaat-tla, 1 mmol/1 etyleenidiaminotetra-asetaattia, 1 g/1 nat-riumdodekyylisulfaattia (SDS)]. Tämä pesutoimenpide tois-5 tettlin kolmesti. Seuraavaksi kantaja pestiin kahdesti hajotuspuskurilla, johon ei ollut lisätty SDS:ä, ja sen jälkeen kerran pesupuskurilla (2 M Tris-HCl, pH 8,0, 10 mmol/1 NaCl:ä, 0,1 mmol/1 EDTA:a, 0,5 g/1 NP-40:ä). Näin puhdistetut antigeenit irrotettiin kiinteästä kantajasta 10 joko +95 °C:ssa suoritetulla 5 minuutin kiehauttamisella tai +4 °C:ssa 6 M NH4SCN:ssä toteutetulla 30 minuutin inku-boinnilla.Tris-HCl, pH 8.0, 0.5 g / l Nonidet P-40 (= octylphenylethylene oxide; Fluka AG), 5 g / l sodium deoxycholate, 1 mmol / l ethylenediaminotetraacetate, 1 g / l sodium dodecyl sulfate (SDS)]. This wash another-5 teglin three times. The support was then washed twice with lysis buffer without SDS and then once with wash buffer (2 M Tris-HCl, pH 8.0, 10 mmol / l NaCl, 0.1 mmol / l EDTA, 0 , 5 g / l NP-40). The antigens thus purified were detached from the solid support 10 either by boiling for 5 minutes at + 95 ° C or by incubation for 30 minutes in 6 M NH 4 SCN at + 4 ° C.
> f «> f «
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| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| DE19843432714 DE3432714A1 (en) | 1984-09-06 | 1984-09-06 | TUMOR THERAPEUTICS AND METHOD FOR THE PRODUCTION THEREOF |
| DE3432714 | 1984-09-06 |
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| FI853389A0 FI853389A0 (en) | 1985-09-04 |
| FI853389L FI853389L (en) | 1986-03-07 |
| FI88360B true FI88360B (en) | 1993-01-29 |
| FI88360C FI88360C (en) | 1993-05-10 |
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| EP (1) | EP0173951B1 (en) |
| JP (1) | JPS6168427A (en) |
| AT (1) | ATE64531T1 (en) |
| CA (1) | CA1277600C (en) |
| DE (2) | DE3432714A1 (en) |
| DK (1) | DK172253B1 (en) |
| ES (1) | ES8704082A1 (en) |
| FI (1) | FI88360C (en) |
| GR (1) | GR852149B (en) |
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| DE3806565A1 (en) * | 1988-03-01 | 1989-09-14 | Deutsches Krebsforsch | VIRUS-MODIFIED TUMOR VACCINES FOR THE IMMUNOTHERAPY OF TUMOR METAL KEYS |
| AT398900B (en) * | 1991-02-12 | 1995-02-27 | Pekar Rudolf Dr | Vaccine for immunotherapy |
| US6953668B1 (en) | 1992-11-05 | 2005-10-11 | Sloan-Kettering Institute For Cancer Research | Prostate-specific membrane antigen |
| CA2147499C (en) * | 1992-11-05 | 2010-10-19 | Ron S. Israeli | Prostate-specific membrane antigen |
| US7105159B1 (en) | 1992-11-05 | 2006-09-12 | Sloan-Kettering Institute For Cancer Research | Antibodies to prostate-specific membrane antigen |
| US7070782B1 (en) | 1992-11-05 | 2006-07-04 | Sloan-Kettering Institute For Cancer Research | Prostate-specific membrane antigen |
| US6569432B1 (en) | 1995-02-24 | 2003-05-27 | Sloan-Kettering Institute For Cancer Research | Prostate-specific membrane antigen and uses thereof |
| DE69635801T2 (en) | 1995-02-24 | 2006-11-02 | Sloan-Kettering Institute For Cancer Research | PROSTATE-SPECIFIC MEMBRANES ANTIGEN AND ITS APPLICATIONS |
| DE19506483A1 (en) * | 1995-02-24 | 1996-08-29 | Jens Dr Dr Atzpodien | Antitumour vaccines |
| US20050215472A1 (en) | 2001-10-23 | 2005-09-29 | Psma Development Company, Llc | PSMA formulations and uses thereof |
| JP5355839B2 (en) | 2001-10-23 | 2013-11-27 | ピーエスエムエー デベロプメント カンパニー， エル．エル．シー． | PSMA antibodies and protein multimers |
| CN1315536C (en) * | 2002-09-13 | 2007-05-16 | 李进 | Novel vaccine of tumor antigen, its preparation method and vaccine composition |
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| US4152410A (en) * | 1975-09-03 | 1979-05-01 | Eisai Co., Ltd. | Diagnosis reagent for neoplasm and method for diagnosis of neoplasm |
| DE2643215A1 (en) * | 1976-09-25 | 1978-04-06 | Bayer Ag | CANCEROSTATICALLY EFFECTIVE PREPARATIONS FROM TUMOR CELLS AND A METHOD FOR THEIR PRODUCTION |
| ZA776822B (en) * | 1976-11-24 | 1979-06-27 | D Mccollester | Vaccine and method for immunotherapy of neoplastic disease |
| DE2918927A1 (en) * | 1979-05-10 | 1980-11-20 | Hans Prof Dr Limburg | MEDICINAL PRODUCT FOR TREATING CARCINOMAS AND METHOD FOR THE PRODUCTION THEREOF |
| US4446122A (en) * | 1979-12-28 | 1984-05-01 | Research Corporation | Purified human prostate antigen |
| US4468457A (en) * | 1981-06-01 | 1984-08-28 | David M. Goldenberg | Method for producing a CSAp tryptic peptide and anti-CSAp antibodies |
| DE3329184A1 (en) * | 1983-08-12 | 1985-02-21 | Behringwerke Ag, 3550 Marburg | MONOCLONAL ANTIBODIES WITH SPECIFICITY FOR MEMBRANE-ASSOCIATED ANTIGENS |
| DE3416774A1 (en) * | 1984-05-07 | 1985-11-14 | Behringwerke Ag, 3550 Marburg | MONOCLONAL ANTIBODIES, METHOD FOR THE PRODUCTION THEREOF AND THEIR USE |
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- 1984-09-06 DE DE19843432714 patent/DE3432714A1/en not_active Withdrawn
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- 1985-08-27 AT AT85110757T patent/ATE64531T1/en not_active IP Right Cessation
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- 1985-09-04 GR GR852149A patent/GR852149B/el unknown
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| Publication number | Publication date |
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| FI853389A0 (en) | 1985-09-04 |
| ES546701A0 (en) | 1987-03-16 |
| IL76289A0 (en) | 1986-01-31 |
| ES8704082A1 (en) | 1987-03-16 |
| PT81093B (en) | 1988-07-01 |
| GR852149B (en) | 1986-01-03 |
| DK402485A (en) | 1986-03-07 |
| DE3583265D1 (en) | 1991-07-25 |
| JPS6168427A (en) | 1986-04-08 |
| EP0173951B1 (en) | 1991-06-19 |
| PT81093A (en) | 1985-10-01 |
| FI88360C (en) | 1993-05-10 |
| FI853389L (en) | 1986-03-07 |
| IE58638B1 (en) | 1993-10-20 |
| CA1277600C (en) | 1990-12-11 |
| IL76289A (en) | 1992-01-15 |
| EP0173951A3 (en) | 1988-06-08 |
| ZA856807B (en) | 1986-04-30 |
| DE3432714A1 (en) | 1986-04-24 |
| EP0173951A2 (en) | 1986-03-12 |
| DK172253B1 (en) | 1998-02-09 |
| ATE64531T1 (en) | 1991-07-15 |
| DK402485D0 (en) | 1985-09-03 |
| IE852191L (en) | 1986-03-06 |
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