CA1277600C - Tumor therapeutic agent and a process for its preparation - Google Patents
Tumor therapeutic agent and a process for its preparationInfo
- Publication number
- CA1277600C CA1277600C CA000490026A CA490026A CA1277600C CA 1277600 C CA1277600 C CA 1277600C CA 000490026 A CA000490026 A CA 000490026A CA 490026 A CA490026 A CA 490026A CA 1277600 C CA1277600 C CA 1277600C
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- Prior art keywords
- antigen
- molecular weight
- approximately
- therapeutic agent
- determinable
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Expired - Lifetime
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Classifications
-
- 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
-
- 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
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- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Veterinary Medicine (AREA)
- Chemical & Material Sciences (AREA)
- Immunology (AREA)
- Medicinal Chemistry (AREA)
- Pharmacology & Pharmacy (AREA)
- Epidemiology (AREA)
- Animal Behavior & Ethology (AREA)
- General Health & Medical Sciences (AREA)
- Public Health (AREA)
- Oncology (AREA)
- Microbiology (AREA)
- Mycology (AREA)
- Engineering & Computer Science (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Gastroenterology & Hepatology (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)
- Peptides Or Proteins (AREA)
- Pharmaceuticals Containing Other Organic And Inorganic Compounds (AREA)
- Diaphragms For Electromechanical Transducers (AREA)
- Measuring Pulse, Heart Rate, Blood Pressure Or Blood Flow (AREA)
- Window Of Vehicle (AREA)
- Saccharide Compounds (AREA)
Abstract
ABSTRACT OF THE INVENTION
A process for the preparation of a therapeutic agent from tumor cells for the therapy of tumorous diseases is described.
This entails either human tumor cells which carry antigens which are bound by the monoclonal antibodies which recognize an antigen (Ag1) having a molecular weight of approximately 33 KD + 3, or an antigen (Ag2) having a molecular weight of approximately 134 KD + 3, or an antigen (Ag3) having a molecular weight of approximately 80 KD + 3, or an antigen (Ag4) having a molecular weight of approximately 55 KD + 3, or an antigen (Ag5) having a molecular weight of approximately 60 KD + 3, or an antigen (Ag6) having a molecular weight of approximately 54 KD + 3, or an antigen (Ag7) having a molecular weight of approximately 260 KD + 3, or an antigen (Ag8) having a non-determinable molecular weight, or an antigen (Ag9) having a non-determinable molec-ular weight, or an antigen (Ag10) having a molecular weight of approximately 143 KD + 3 and 119 KD + 3, or an antigen (Ag11) having a molecular weight of approximately 178 KD + 3, or an antigen (Ag12) having a non-determinable molecular weight, or an antigen (Ag13) having a molecular weight of approximately 34 KD + 3, or an antigen (Ag14) having a molec-ular weight of approximately 195 KD + 3, or an antigen (Ag15) having a molecular weight of approximately 44 KD + 7, or an antigen (Ag16) having a molecular weight of approximately 43 KD + 3, or an antigen (Ag17) having a molecular weight of approximately 130 KD + 3, being dried or stabilized by chemical treatment, or antigens being isolated from human tumor cells using these monoclonal antibodies, and being processed to a therapeutic agent, neuraminidase being added where appropriate.
A process for the preparation of a therapeutic agent from tumor cells for the therapy of tumorous diseases is described.
This entails either human tumor cells which carry antigens which are bound by the monoclonal antibodies which recognize an antigen (Ag1) having a molecular weight of approximately 33 KD + 3, or an antigen (Ag2) having a molecular weight of approximately 134 KD + 3, or an antigen (Ag3) having a molecular weight of approximately 80 KD + 3, or an antigen (Ag4) having a molecular weight of approximately 55 KD + 3, or an antigen (Ag5) having a molecular weight of approximately 60 KD + 3, or an antigen (Ag6) having a molecular weight of approximately 54 KD + 3, or an antigen (Ag7) having a molecular weight of approximately 260 KD + 3, or an antigen (Ag8) having a non-determinable molecular weight, or an antigen (Ag9) having a non-determinable molec-ular weight, or an antigen (Ag10) having a molecular weight of approximately 143 KD + 3 and 119 KD + 3, or an antigen (Ag11) having a molecular weight of approximately 178 KD + 3, or an antigen (Ag12) having a non-determinable molecular weight, or an antigen (Ag13) having a molecular weight of approximately 34 KD + 3, or an antigen (Ag14) having a molec-ular weight of approximately 195 KD + 3, or an antigen (Ag15) having a molecular weight of approximately 44 KD + 7, or an antigen (Ag16) having a molecular weight of approximately 43 KD + 3, or an antigen (Ag17) having a molecular weight of approximately 130 KD + 3, being dried or stabilized by chemical treatment, or antigens being isolated from human tumor cells using these monoclonal antibodies, and being processed to a therapeutic agent, neuraminidase being added where appropriate.
Description
~27~i0~
The invention relates to a process for the prepar~
ation of a therapeutic agent from tumor cells for the therapy of tumorous diseases, and ~o a therapeutic agent of this type.
S It ;s known, for example from "Mechanisms of Tumor }mmuni~yf" &ree ~t al.~ e~s., John W;~ey and Sons, ~.Y., 197~, pa~e 156, that attempts have already ~een made to treat tumorous diseases by inoculat;on with tumor ce~ls ~h;ch have been mod;f;ed by freez;ng and thaw;ng, freeze-dry;ng, pressure or homogenization Subcellular fractions or cell extracts have also been used for th;s purposeO
However, as yet no vacc;ne aga;nst a tumorous d;sease has been d;sclosed.
We have foundO surprisingly,-that cells, which have been freeze-dr;ed or treated ~ith an aldehyde, from human tumors or from cell aggregates obta;ned from the ~atter, wh;ch carry ant;gens wh;ch are bound by ~he monoclonal ant;-bod;es descr;bed ;n ou~ co-pending Cana~ian Patent A~plication Serial Number 460,725, filed August 10, 1984, can be used as a therapeutic agent for t~e treatment of t~orous diseases~
Thus the invention relates to a therapeutic agent ~or the ereatment of a tumorous d;sease, contain;ng human cells ~hich have been dr;ed or stab;l;zed by a chemical trea~Ment and ~hich carry ant;gens ~h;ch are bound by the monoclonal antibod;es described in the above-ident.ified Canadiar~ Patent application.
One advantage of a therapeut;c agent of th;s type compared w;th the use.of unmod;f;ed cells ;s that unmod;-f;ed cells are not stable, so that they have to be pre-pared fre~h each time Moreoever, for thi~ reason, they cannot be standard;zed The invent;on furthermore relates to a process for the preparat;on of a therapeutic agent for the treatment of a tumorous d;sease, which comprises the drying or sta-bilizat;on, by a chemical treatment, and the process;ng to a therapeutic agent, neuram;nidase being added where appropr;ate, of human tu~or cells which carry antigens wh;ch are bound by the monoclonal ant;bod;es described in the above-identified Canadian Patent Application.
The invention also relates to a process for the preparation of a therapeutic agent for the treatment of a tumorous disease, which comprises the isolation, by means of a monoclonal antibody described in the above-identified Canadian Patent Application, of an antigen from human cells and its processing to a therapeutic agent, neuraminidase being added where appropriate.
The possibility of potentiating an immune response by neuraminidase is disclosed in German Offenlegungsschrift 2,620,649.
A therapeutic agent is to be understood to be an agent which may be suitable both as a prophylactic and for the treatment of a manifest disease.
The monoclonal antibodies described in co-pending Canadian patent application serial number 46~,725 are those which recognize an antigen (Agl) having a molecular weight of approximately 33 KD + 3, or an an-tigen (Ag2) having a mol-ecular weight of approximately 134 KD + 3, or an antigen (Ag3) having a molecular weight of approximately 80 KD + 3, or an antigen (Ag4) having a molecular weight of approx-imately 55 KD + 3, or an antigen (Ag5) having a molecular weight of approxima-tely 60 KD + 3, or an antigen (Ag6) having a molecular weight of approximately 54 KD + 3, or an antigen (Ag7) having a molecular weight of approximately 260 KD + 3, or an antigen (Ag8) having a non-determinable molecular weight, or an antigen (Ag9) having a non-determinable molec-ular weight, or an antigen (AglO) having a molecular weight of approximately 143 KD + 3 ancl 119 KD + 3, or an antigen (Agll) having a molecular weight of approximately 178 KD + 3, or an antigen (Agl2) having a non-determinable molecular weight, or an antigen (Agl3) having a molecular weight of approximately 34 KD + 3, or an antigen (Agl4) having a molec-ular weight of approximately 195 KD ~ 3, or an antigen (Agl5) having a molecular weight of approximately 44 KD + 7, or an antigen (Agl6) having a molecular weight of approximately 43 ~2~
- 3a -KD + 3, or an antigen (Agl7) having a molecular weight of approximately 130 KD -~ 3.
The antigens 4, 8, 9 and 10 are mycoplasma antigens which are associated with the cell lines described in the table.
The immunogens used to induce these antibodies are human cell lines cultured in vitro, cell extracts or extracts of human tissues. Permanent human cell lines, in particular the CaLu-l, Chago, Oat 75, PaTu II and Bewo cell lines, are preferred. It is also possible to use Agl-Agl7 to induce Abl-Abl7.
Mammals, preferably mice, are immunized intra-peritoneally with 1 x 106-108 cells J but preferably 107 cells, of a cell line of this type (days 0-120, but pre-ferably on days 0 and 7) and, after 1-150 days, but prefer-ably on day 11, the spleen cells from such animals are fused with the X63 Ag 8653 cell line (The Journal of I~munology 173, 4, ~548-1550, 1979) (Nature 256, 495-497~ 1975).
The hybridomas resulting after 3 weeks are tested for antibodies of the desired specificity. In this case, a panel of 30 cell lines cultured in vitro, human peripheral blood cells and human bone marrow were tested for reactivity with the antibodies by means of indirect immunofluorescence (Behring Inst. Mitt. 59, 64-70, 1976) and cell-sorter analysis (Acta Cytol. 19, 374-377, 1975), (Proc. Natl. Acad.
Sci. USA 77/8, 4914-4917, 1980).
Surprisingly, among the hybridoma supernatants which were tested, there were some which contained antibodies with the interesting specificities described above and in Table I.
ation of a therapeutic agent from tumor cells for the therapy of tumorous diseases, and ~o a therapeutic agent of this type.
S It ;s known, for example from "Mechanisms of Tumor }mmuni~yf" &ree ~t al.~ e~s., John W;~ey and Sons, ~.Y., 197~, pa~e 156, that attempts have already ~een made to treat tumorous diseases by inoculat;on with tumor ce~ls ~h;ch have been mod;f;ed by freez;ng and thaw;ng, freeze-dry;ng, pressure or homogenization Subcellular fractions or cell extracts have also been used for th;s purposeO
However, as yet no vacc;ne aga;nst a tumorous d;sease has been d;sclosed.
We have foundO surprisingly,-that cells, which have been freeze-dr;ed or treated ~ith an aldehyde, from human tumors or from cell aggregates obta;ned from the ~atter, wh;ch carry ant;gens wh;ch are bound by ~he monoclonal ant;-bod;es descr;bed ;n ou~ co-pending Cana~ian Patent A~plication Serial Number 460,725, filed August 10, 1984, can be used as a therapeutic agent for t~e treatment of t~orous diseases~
Thus the invention relates to a therapeutic agent ~or the ereatment of a tumorous d;sease, contain;ng human cells ~hich have been dr;ed or stab;l;zed by a chemical trea~Ment and ~hich carry ant;gens ~h;ch are bound by the monoclonal antibod;es described in the above-ident.ified Canadiar~ Patent application.
One advantage of a therapeut;c agent of th;s type compared w;th the use.of unmod;f;ed cells ;s that unmod;-f;ed cells are not stable, so that they have to be pre-pared fre~h each time Moreoever, for thi~ reason, they cannot be standard;zed The invent;on furthermore relates to a process for the preparat;on of a therapeutic agent for the treatment of a tumorous d;sease, which comprises the drying or sta-bilizat;on, by a chemical treatment, and the process;ng to a therapeutic agent, neuram;nidase being added where appropr;ate, of human tu~or cells which carry antigens wh;ch are bound by the monoclonal ant;bod;es described in the above-identified Canadian Patent Application.
The invention also relates to a process for the preparation of a therapeutic agent for the treatment of a tumorous disease, which comprises the isolation, by means of a monoclonal antibody described in the above-identified Canadian Patent Application, of an antigen from human cells and its processing to a therapeutic agent, neuraminidase being added where appropriate.
The possibility of potentiating an immune response by neuraminidase is disclosed in German Offenlegungsschrift 2,620,649.
A therapeutic agent is to be understood to be an agent which may be suitable both as a prophylactic and for the treatment of a manifest disease.
The monoclonal antibodies described in co-pending Canadian patent application serial number 46~,725 are those which recognize an antigen (Agl) having a molecular weight of approximately 33 KD + 3, or an an-tigen (Ag2) having a mol-ecular weight of approximately 134 KD + 3, or an antigen (Ag3) having a molecular weight of approximately 80 KD + 3, or an antigen (Ag4) having a molecular weight of approx-imately 55 KD + 3, or an antigen (Ag5) having a molecular weight of approxima-tely 60 KD + 3, or an antigen (Ag6) having a molecular weight of approximately 54 KD + 3, or an antigen (Ag7) having a molecular weight of approximately 260 KD + 3, or an antigen (Ag8) having a non-determinable molecular weight, or an antigen (Ag9) having a non-determinable molec-ular weight, or an antigen (AglO) having a molecular weight of approximately 143 KD + 3 ancl 119 KD + 3, or an antigen (Agll) having a molecular weight of approximately 178 KD + 3, or an antigen (Agl2) having a non-determinable molecular weight, or an antigen (Agl3) having a molecular weight of approximately 34 KD + 3, or an antigen (Agl4) having a molec-ular weight of approximately 195 KD ~ 3, or an antigen (Agl5) having a molecular weight of approximately 44 KD + 7, or an antigen (Agl6) having a molecular weight of approximately 43 ~2~
- 3a -KD + 3, or an antigen (Agl7) having a molecular weight of approximately 130 KD -~ 3.
The antigens 4, 8, 9 and 10 are mycoplasma antigens which are associated with the cell lines described in the table.
The immunogens used to induce these antibodies are human cell lines cultured in vitro, cell extracts or extracts of human tissues. Permanent human cell lines, in particular the CaLu-l, Chago, Oat 75, PaTu II and Bewo cell lines, are preferred. It is also possible to use Agl-Agl7 to induce Abl-Abl7.
Mammals, preferably mice, are immunized intra-peritoneally with 1 x 106-108 cells J but preferably 107 cells, of a cell line of this type (days 0-120, but pre-ferably on days 0 and 7) and, after 1-150 days, but prefer-ably on day 11, the spleen cells from such animals are fused with the X63 Ag 8653 cell line (The Journal of I~munology 173, 4, ~548-1550, 1979) (Nature 256, 495-497~ 1975).
The hybridomas resulting after 3 weeks are tested for antibodies of the desired specificity. In this case, a panel of 30 cell lines cultured in vitro, human peripheral blood cells and human bone marrow were tested for reactivity with the antibodies by means of indirect immunofluorescence (Behring Inst. Mitt. 59, 64-70, 1976) and cell-sorter analysis (Acta Cytol. 19, 374-377, 1975), (Proc. Natl. Acad.
Sci. USA 77/8, 4914-4917, 1980).
Surprisingly, among the hybridoma supernatants which were tested, there were some which contained antibodies with the interesting specificities described above and in Table I.
3~ ~.277~0~
Table 1 Reactivity o~ A~ 1-17 with cells in vitro Cells tested 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 1b 17 Lun~ tumor celL lines 5 CaLu-1 + + ~ + ~
E-14 ~ - - + ~ ~ t -~ ~ ~ t 109/4 ~ +
~ 549 ~ + +
Oat 75 + + - ~ - + ~ + -SHP-77 + * ~ + ~ + 1 Chago -~ t -~ +
Bro-Ca-Hoff + + -Pancreas tun-or cell l;nes 15 Pa Tu II ~ + - ~ +
Pa Tu III
MIA-Pa-Ca 2 ~ + - - -Panc-1 * ~ - ~ +
Gynecologi-caL tu~nor cell Lincs Bew~ * l I
~eLa + + * -Pa-1 ~ ~ ~ +
- 3_ Cells tested 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 _, . ~ . .. .. _ .
Melanoma cell l;nes Mel-ULF + ~ + t - + - +
S Mel-RPMI ~ I ~ * - ~ - +
llel-51-2 +
Mel-21-C-48 + +
Unrelatc~
tumor cell 10 lines ~R-75-1 ~ * - -Mamr,la-Ca~12 ~ - - * ~ ~ -Colon-Ca-hx 15 Colon-lli + + - ~
HT~-10~0 ~ ~ _ Leiomyo- +
sarcoma Hyper- _ 20 nephroma TUW
Raj;
L)a~di 173~
Immunocytoma ~27~6~
~ 3d -Cells tested 1 2 3 4 5 6 7 ~ 9 10 11 1Z 13 14 15 16 17 Normal lymph-o;d cel~s Lymphocytes ~ ~ ~ ~ +
S Monocytes ~ * ~
Granulocytes ~ +
rythrocytes Rone m a r r o w _+ + - - +~
l~ormal hur,lan 10 f;broblasts LL-29 t 1~u-F i-Br ~2 Hu-Fi-Br 43 + ~ ~ - - - - - - - -Hu-Fi-Br 47 ~ ~ ~ ~ ~ ~ ~ ~ ~ ~
~5 ~lu-F;-Br 10 Hu F;-Mel-1 H u- F; -l~l a ~l -13 Animal cells ~le~ - + ~ - -Grcyhound R~t fibro- - - - - - - - - - - - ~ -~ asts Mouse f;br o~
2 ~ b l a 9 1: S
~ 3e -+ = sign;ficant positive reaction ;n an ;ndirect ;mmuno,luorescence assay, ;n radioimmunoassay and ;n cytofluorometr;c analys;s.
~ = ueakly positive reaction with part of the popula-t;on tested - ~ no s;gnif;cant reaction The cells ~hich can be used ~ith1n the scope of the ~nvent1On are obtained from tumors by kno~n cell culture processes~ Cells ~hich have been obtained from such cul-tures by mechanical or enzymatlc means and have~ where appropriate, been inactivated ~ith mitomycin C, are dried, preferabl~ freeze~dr;ed~ or treated ~;th an agent kno~n to those sk;lled in the art as a stabil1z1ng agent for organ;c tissue, preferably a monoaldehyde or d1aldehyde having I
lS to 6 carbon a~oms.
The agents which are particularly suitable for chemical stabiliza~1On and fixation include, in part~cular, bifunct1Onal compounds~ that is to say those ~hich con-tain t~o groups which ca~ react w1th functional groups on the biological materiaL - in other words car. "cross-link" ;t. Examples of these are dialdehydes, in particular aliphatic dialdehydes having 2-8 carbon atoms. Ho~ever, monoalkanals hav~ng 1-4 carbon atoms, such as formaldehyde, ~hich can undergo bifunctional reactions, as ~ell as bi-functional imino esters, such as suberim;date, isocyanates or lsothiocyanates, are also suitable for this purpose.
It is also possible ~or so-ralled tanning agents such as, for example, tann1c acld and its derivatives, or chro~1um ~alts, to be used as agents ~h1ch can stab1~1ze biolog1cal mater1al. Sulfosalicylic ac1d is also su1t-able.
~776~
In general, the cells are ;n the form of cell aggre-gates or single cells. $t ;s also poss;ble to use cell fragments or antigens isolated from the tu~or cells. Ant;-gens of th;s type can be a~tained from tumorous tissue S from pat;ents, as uell as from human tu~crs ~h~eh grow ;n immunef;cient an;mals, and can be used.
Defined ant;gens are obta;ned from the tumor cells or fragments thereof using the monoclonal antibod;es des~
rr;bed in co-pending Canadi~n Pat~en~ Application Serial Nwnber 460,725.
Examples of ant;gens of this type are CEA tcarc;no-embyron;c antlgen; J.exp. Med. (1965) 122, 467) NCA ~nor,-speclfic crossreacting an~;gen; J. ~mmun. (1973) III" 1926) ~hich can be isolated from the tumor cells by ~mmunoadsorp-t1On chromatography. For th;s purpose, the monoclonalantlbod;es described ;n Table I of German Offenlegungs-schr;ft 30416,774 are covalently bound as pur;fied prs-teins, to CNBr-activated sepharos~ 4~O and the antigens ~CEA, NCA) recognised by these monoclonal antib3dies, are ;solated from DE-TA colon carclnoma cell extract . A suit-able process ;s descr;bed in the Pharmacia book "Aff;n;ty Chromatography~ Principles and Methods"~ 12-t8 ~1979), sum~arized on page 15.
An ant;gen of th;s type obta;ned uC;ng monoclonal ant;bod;es can be used as a therapeut;c agent, for example as act;ve compound ;n vacc;nes aga;nst a disease wh;ch 1s caused by the tumor cells from ~h;ch the antigen ~as obtained.
qual;ty control of a mater;al ~hich is to be used as a vaccine is carr;ed ou~ by, for çxample~ typing w;th monoclonal antibodies, or by ~ractionation of the total cellular proteins using SDS-polyacrylamide gel electro~
phoresis or 1s~electric focusing ~1st dimension) combined ~ith SDS-polyacrylam;de gel electrophores;s (2nd d~men-sion3 followed by sta;ning of the gel ~s;lver sta;n).
The antigenic mater;al is preferably adm~nisteredir,tradermally, preferably by the checkerboard vacc;nation method ~Cancer Immunol. and Immunother. 6,47-58 t1979), ;n rart;cular page 4~) in comb;nation ~ith an adjuvant, in particular neuramin;dase (6erman Offenlegungsschr;ft 2,~20,649).
A varcine of th;s type is preferably used for cer-tain stages of colon carcinoma (Duke C) and for other tumors wh;ch carry anti~ens or ep;topes wh;ch are present in the 5 vaccine. Other tumors of th;s type are solid tumors, for example carc;nomas of the pancreas~ of ~he stomach, of the breast and of the lungs. The vaccine can be adm;nistered parenterally or orally. The ant;gens can be administered dissolved or suspended ;n phys;ological sal;ne, preferably intrader~ally in PBS.
Two tests were carr;ed out to assess the stab;lity of the antigenic compos;tion of the vaccine:
a) the Terasaki IIF Assay (ind;rect immunofluorescence using tumor cells ~hich grow in the welLs of the Terasaki microt;ter plate) ~;th monoclonal ant;bodies of various specificiti~s~ It ;s pcssible by mea ns of this ~est to measure the expression of membrane 3ntigens on in~act tumor cells, against which a number of moncclonal ant;bod;es are available tCancer Detec~;on and Prevent;on 6, 181-184, 1983). It was possible by this means to detect drastic changes to the D~TA cell ~embrane during cul~iYation;
b) solubil;zation of the total cellular proteins us;ng a detergen~ (Hybrndo~a 1, 413-421, 1982) follo~ed by S~S-polyacrylam;de gel electrophoresis combined ~;th a silver sta;n (Anal. 8;ochem. 105, 361-363,1980). The comb;nat;on of these techniques ensures that no s;gnificant changes in the total protain content of the DE-TA cell line have occurred.
The examples wh;ch follo~ illustrate the invention.
Example 1 The carc;noma cell line ~ X was cultivated ;n a cell culture ;n plastic bottles, growing as a monolayer in RPMI-1640 med;um ~Moore, G.E., Gerner, R.E., Franklin, H.A., Culture of normal human leukocytes, J.A.M.A. 199, 519-524 (1967)) with 10X fetal calf serum. The adheren~ cells growing ;n confluent cultures ~ere separated mechanically or using trypsin wh;ch ~as d;ssolved ;n RPMI-1640 med;um conta;n;ng no fetal calf serum, the collagenase was ;n-act;vated by add;tion of fetal calf serum d;ssolved ;n ! 6 -RPM1~1640, and then the ~ells were detached from the t;s-sue culture bottles and washed 3 times in phosphate-buffered sal;ne (PBS) at 37C.
About 107 cells, the major part of wh;ch as in the form of aggregates, were incubated at 37C for 1 hour in 1 ml of P3S ~h;ch contained 100 yg of m;tomyc;n C.
Then the cells thus inactivated ~ere ~ashed 3 ti0es ~;th P~S and a) washed 3 t;mes ;n 0.18 molar ammonium bicarbonate buffer wh;ch had been adjusted to pH 7.4 w;th acetic acid. A
cell sed;ment corresponding to 107 cells was taken up ;n 100 ~l of the same ammon;um bicarbonate buffer and frozen at -70C. The frozen mater;al ~as then freeze-dr;ed and stored ;n a small glass bottle, which was closed air-t;ght, in a refrigerator at ~4C. The cell material thus treated can, after havin~ been taken up in ~BS, be used for vaccina-tion of patients.
Alternatively b) incubated w;th 0.1X glutaraldehyde in PBS at ~4C for 5 minu~es, the excess glutaraldehyde be;ng removed by wash-ing 3 times ~;th PBS, and then incubated with 2X BSA
(~ovine Serum Albumin) at +4C for S ~inutes and washed 3 times in P~S. The cells thus treated can be stored a~
~4C and used for the vaccinat;on of pat;ents.
Or c) incubated in formalin according to Lilly ~enno Rome;s ~1968), page 65, section 266, Oldenburg Verlag, Munich) at 25C overnight~ shak;ng occasionally. The cells ~about 108) were centr;fuged w;th decantation of the supernat~ t (10 minutes at 800 x 9), and the cell sediment was suspen-ded ;n 7 ml of double-d;stilled water (~ 1st wash). Th;s washing process was repeated 4 times at intervals of 1 hour.
The cell sed;ment was then washed 3 t;mes, at ;ntervals of 1 hour, in 7 ml of 70X ethanol eash t;me. The cell sedi-ment was then washed 3 t;mes, at ;ntervaLs of 30 M;nutes,;n 7 ml of 80X ethanol each t;me~ The cell sed;ment was then washed 3 times, at ;ntervals of 30 minutes, in 7 ml of 96% ethanol each time. The cell sediment was then washed 3 t;mes, at intervals of 30 minutes, ;n 7 ml 1~
76~9 of 99X ethanol each timeO The cell sediment was then washed 3 t;mes, at intervals of 30 minutes, in 7 ml of sterile P~S each time, and was stored sterile at 4C.
The cell~ thus treated can be stored at 4C and used for the vacc;nat;on of pat;ents.
~e~
In order to isolate antigens from tumor cells by immunoadsorption chromatography, purified monoclonal anti-bodies which unambiguously react with antigens on the tumor cells which are to be used as vaccine are covalently bound to CN3r-act;vated sepharose 48. The process was that of the Pharmac;a book "Aff;nity Chromatography", Princ;ples and Methods, 12-18 ~1979), in particular page tS. The carr;er-bound monoclonal ant;bod;es were then ;ncubatecl with cell solubilizates at ~4C for 2 hours, shaking occasionally. The latter were obtained from cultured cells, which had been mechanically removed from the culture bot-tles~ by means of extract;on with lys;s buffer ~5 9/ l sodium deoxycholate, û.5 mmol/l PMSF - phenylmethylsulfonyl fluoride, PBS, pH 8O3~ as described in Hybridoma 1, 413-421 t1982), in particular on page 414.
The loaded carrier was centrifuged and suspended in lys;s buffer-SDS ~2a mM tris.HCl pH s.n, 1 mmoltl PMSF, 5 gll Nonidet P-40 ~~ octylphenyl ethylene oxide;
Fluka A6), 5 g/l sod;um deoxycholate~ 1 mmol/l ethylened;-am;netetraacetate and 1 g/l sodium dodecyl sulfate ~SDS)) for ~ashing.
Th;s washing process was repeated 3 times. The carrier was then washed twice ;n lysis buffer without the 30 addit;on of SDS and then washed once with a washing buffer ~2 M tris.HCl~ pH s.a, 10 mmol/l NaCl, 0.1 mmol/l EDTA and 0.5 g/l NP-40). The ant;gens thus purified were removed from the solid carrier either by heating at +95C
for 5 m;nutes or by incubat;on in 6 mol/l NH4SCN at +4C
for 30 m;nutes.
Table 1 Reactivity o~ A~ 1-17 with cells in vitro Cells tested 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 1b 17 Lun~ tumor celL lines 5 CaLu-1 + + ~ + ~
E-14 ~ - - + ~ ~ t -~ ~ ~ t 109/4 ~ +
~ 549 ~ + +
Oat 75 + + - ~ - + ~ + -SHP-77 + * ~ + ~ + 1 Chago -~ t -~ +
Bro-Ca-Hoff + + -Pancreas tun-or cell l;nes 15 Pa Tu II ~ + - ~ +
Pa Tu III
MIA-Pa-Ca 2 ~ + - - -Panc-1 * ~ - ~ +
Gynecologi-caL tu~nor cell Lincs Bew~ * l I
~eLa + + * -Pa-1 ~ ~ ~ +
- 3_ Cells tested 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 _, . ~ . .. .. _ .
Melanoma cell l;nes Mel-ULF + ~ + t - + - +
S Mel-RPMI ~ I ~ * - ~ - +
llel-51-2 +
Mel-21-C-48 + +
Unrelatc~
tumor cell 10 lines ~R-75-1 ~ * - -Mamr,la-Ca~12 ~ - - * ~ ~ -Colon-Ca-hx 15 Colon-lli + + - ~
HT~-10~0 ~ ~ _ Leiomyo- +
sarcoma Hyper- _ 20 nephroma TUW
Raj;
L)a~di 173~
Immunocytoma ~27~6~
~ 3d -Cells tested 1 2 3 4 5 6 7 ~ 9 10 11 1Z 13 14 15 16 17 Normal lymph-o;d cel~s Lymphocytes ~ ~ ~ ~ +
S Monocytes ~ * ~
Granulocytes ~ +
rythrocytes Rone m a r r o w _+ + - - +~
l~ormal hur,lan 10 f;broblasts LL-29 t 1~u-F i-Br ~2 Hu-Fi-Br 43 + ~ ~ - - - - - - - -Hu-Fi-Br 47 ~ ~ ~ ~ ~ ~ ~ ~ ~ ~
~5 ~lu-F;-Br 10 Hu F;-Mel-1 H u- F; -l~l a ~l -13 Animal cells ~le~ - + ~ - -Grcyhound R~t fibro- - - - - - - - - - - - ~ -~ asts Mouse f;br o~
2 ~ b l a 9 1: S
~ 3e -+ = sign;ficant positive reaction ;n an ;ndirect ;mmuno,luorescence assay, ;n radioimmunoassay and ;n cytofluorometr;c analys;s.
~ = ueakly positive reaction with part of the popula-t;on tested - ~ no s;gnif;cant reaction The cells ~hich can be used ~ith1n the scope of the ~nvent1On are obtained from tumors by kno~n cell culture processes~ Cells ~hich have been obtained from such cul-tures by mechanical or enzymatlc means and have~ where appropriate, been inactivated ~ith mitomycin C, are dried, preferabl~ freeze~dr;ed~ or treated ~;th an agent kno~n to those sk;lled in the art as a stabil1z1ng agent for organ;c tissue, preferably a monoaldehyde or d1aldehyde having I
lS to 6 carbon a~oms.
The agents which are particularly suitable for chemical stabiliza~1On and fixation include, in part~cular, bifunct1Onal compounds~ that is to say those ~hich con-tain t~o groups which ca~ react w1th functional groups on the biological materiaL - in other words car. "cross-link" ;t. Examples of these are dialdehydes, in particular aliphatic dialdehydes having 2-8 carbon atoms. Ho~ever, monoalkanals hav~ng 1-4 carbon atoms, such as formaldehyde, ~hich can undergo bifunctional reactions, as ~ell as bi-functional imino esters, such as suberim;date, isocyanates or lsothiocyanates, are also suitable for this purpose.
It is also possible ~or so-ralled tanning agents such as, for example, tann1c acld and its derivatives, or chro~1um ~alts, to be used as agents ~h1ch can stab1~1ze biolog1cal mater1al. Sulfosalicylic ac1d is also su1t-able.
~776~
In general, the cells are ;n the form of cell aggre-gates or single cells. $t ;s also poss;ble to use cell fragments or antigens isolated from the tu~or cells. Ant;-gens of th;s type can be a~tained from tumorous tissue S from pat;ents, as uell as from human tu~crs ~h~eh grow ;n immunef;cient an;mals, and can be used.
Defined ant;gens are obta;ned from the tumor cells or fragments thereof using the monoclonal antibod;es des~
rr;bed in co-pending Canadi~n Pat~en~ Application Serial Nwnber 460,725.
Examples of ant;gens of this type are CEA tcarc;no-embyron;c antlgen; J.exp. Med. (1965) 122, 467) NCA ~nor,-speclfic crossreacting an~;gen; J. ~mmun. (1973) III" 1926) ~hich can be isolated from the tumor cells by ~mmunoadsorp-t1On chromatography. For th;s purpose, the monoclonalantlbod;es described ;n Table I of German Offenlegungs-schr;ft 30416,774 are covalently bound as pur;fied prs-teins, to CNBr-activated sepharos~ 4~O and the antigens ~CEA, NCA) recognised by these monoclonal antib3dies, are ;solated from DE-TA colon carclnoma cell extract . A suit-able process ;s descr;bed in the Pharmacia book "Aff;n;ty Chromatography~ Principles and Methods"~ 12-t8 ~1979), sum~arized on page 15.
An ant;gen of th;s type obta;ned uC;ng monoclonal ant;bod;es can be used as a therapeut;c agent, for example as act;ve compound ;n vacc;nes aga;nst a disease wh;ch 1s caused by the tumor cells from ~h;ch the antigen ~as obtained.
qual;ty control of a mater;al ~hich is to be used as a vaccine is carr;ed ou~ by, for çxample~ typing w;th monoclonal antibodies, or by ~ractionation of the total cellular proteins using SDS-polyacrylamide gel electro~
phoresis or 1s~electric focusing ~1st dimension) combined ~ith SDS-polyacrylam;de gel electrophores;s (2nd d~men-sion3 followed by sta;ning of the gel ~s;lver sta;n).
The antigenic mater;al is preferably adm~nisteredir,tradermally, preferably by the checkerboard vacc;nation method ~Cancer Immunol. and Immunother. 6,47-58 t1979), ;n rart;cular page 4~) in comb;nation ~ith an adjuvant, in particular neuramin;dase (6erman Offenlegungsschr;ft 2,~20,649).
A varcine of th;s type is preferably used for cer-tain stages of colon carcinoma (Duke C) and for other tumors wh;ch carry anti~ens or ep;topes wh;ch are present in the 5 vaccine. Other tumors of th;s type are solid tumors, for example carc;nomas of the pancreas~ of ~he stomach, of the breast and of the lungs. The vaccine can be adm;nistered parenterally or orally. The ant;gens can be administered dissolved or suspended ;n phys;ological sal;ne, preferably intrader~ally in PBS.
Two tests were carr;ed out to assess the stab;lity of the antigenic compos;tion of the vaccine:
a) the Terasaki IIF Assay (ind;rect immunofluorescence using tumor cells ~hich grow in the welLs of the Terasaki microt;ter plate) ~;th monoclonal ant;bodies of various specificiti~s~ It ;s pcssible by mea ns of this ~est to measure the expression of membrane 3ntigens on in~act tumor cells, against which a number of moncclonal ant;bod;es are available tCancer Detec~;on and Prevent;on 6, 181-184, 1983). It was possible by this means to detect drastic changes to the D~TA cell ~embrane during cul~iYation;
b) solubil;zation of the total cellular proteins us;ng a detergen~ (Hybrndo~a 1, 413-421, 1982) follo~ed by S~S-polyacrylam;de gel electrophoresis combined ~;th a silver sta;n (Anal. 8;ochem. 105, 361-363,1980). The comb;nat;on of these techniques ensures that no s;gnificant changes in the total protain content of the DE-TA cell line have occurred.
The examples wh;ch follo~ illustrate the invention.
Example 1 The carc;noma cell line ~ X was cultivated ;n a cell culture ;n plastic bottles, growing as a monolayer in RPMI-1640 med;um ~Moore, G.E., Gerner, R.E., Franklin, H.A., Culture of normal human leukocytes, J.A.M.A. 199, 519-524 (1967)) with 10X fetal calf serum. The adheren~ cells growing ;n confluent cultures ~ere separated mechanically or using trypsin wh;ch ~as d;ssolved ;n RPMI-1640 med;um conta;n;ng no fetal calf serum, the collagenase was ;n-act;vated by add;tion of fetal calf serum d;ssolved ;n ! 6 -RPM1~1640, and then the ~ells were detached from the t;s-sue culture bottles and washed 3 times in phosphate-buffered sal;ne (PBS) at 37C.
About 107 cells, the major part of wh;ch as in the form of aggregates, were incubated at 37C for 1 hour in 1 ml of P3S ~h;ch contained 100 yg of m;tomyc;n C.
Then the cells thus inactivated ~ere ~ashed 3 ti0es ~;th P~S and a) washed 3 t;mes ;n 0.18 molar ammonium bicarbonate buffer wh;ch had been adjusted to pH 7.4 w;th acetic acid. A
cell sed;ment corresponding to 107 cells was taken up ;n 100 ~l of the same ammon;um bicarbonate buffer and frozen at -70C. The frozen mater;al ~as then freeze-dr;ed and stored ;n a small glass bottle, which was closed air-t;ght, in a refrigerator at ~4C. The cell material thus treated can, after havin~ been taken up in ~BS, be used for vaccina-tion of patients.
Alternatively b) incubated w;th 0.1X glutaraldehyde in PBS at ~4C for 5 minu~es, the excess glutaraldehyde be;ng removed by wash-ing 3 times ~;th PBS, and then incubated with 2X BSA
(~ovine Serum Albumin) at +4C for S ~inutes and washed 3 times in P~S. The cells thus treated can be stored a~
~4C and used for the vaccinat;on of pat;ents.
Or c) incubated in formalin according to Lilly ~enno Rome;s ~1968), page 65, section 266, Oldenburg Verlag, Munich) at 25C overnight~ shak;ng occasionally. The cells ~about 108) were centr;fuged w;th decantation of the supernat~ t (10 minutes at 800 x 9), and the cell sediment was suspen-ded ;n 7 ml of double-d;stilled water (~ 1st wash). Th;s washing process was repeated 4 times at intervals of 1 hour.
The cell sed;ment was then washed 3 t;mes, at ;ntervals of 1 hour, in 7 ml of 70X ethanol eash t;me. The cell sedi-ment was then washed 3 t;mes, at ;ntervaLs of 30 M;nutes,;n 7 ml of 80X ethanol each t;me~ The cell sed;ment was then washed 3 times, at ;ntervals of 30 minutes, in 7 ml of 96% ethanol each time. The cell sediment was then washed 3 t;mes, at intervals of 30 minutes, ;n 7 ml 1~
76~9 of 99X ethanol each timeO The cell sediment was then washed 3 t;mes, at intervals of 30 minutes, in 7 ml of sterile P~S each time, and was stored sterile at 4C.
The cell~ thus treated can be stored at 4C and used for the vacc;nat;on of pat;ents.
~e~
In order to isolate antigens from tumor cells by immunoadsorption chromatography, purified monoclonal anti-bodies which unambiguously react with antigens on the tumor cells which are to be used as vaccine are covalently bound to CN3r-act;vated sepharose 48. The process was that of the Pharmac;a book "Aff;nity Chromatography", Princ;ples and Methods, 12-18 ~1979), in particular page tS. The carr;er-bound monoclonal ant;bod;es were then ;ncubatecl with cell solubilizates at ~4C for 2 hours, shaking occasionally. The latter were obtained from cultured cells, which had been mechanically removed from the culture bot-tles~ by means of extract;on with lys;s buffer ~5 9/ l sodium deoxycholate, û.5 mmol/l PMSF - phenylmethylsulfonyl fluoride, PBS, pH 8O3~ as described in Hybridoma 1, 413-421 t1982), in particular on page 414.
The loaded carrier was centrifuged and suspended in lys;s buffer-SDS ~2a mM tris.HCl pH s.n, 1 mmoltl PMSF, 5 gll Nonidet P-40 ~~ octylphenyl ethylene oxide;
Fluka A6), 5 g/l sod;um deoxycholate~ 1 mmol/l ethylened;-am;netetraacetate and 1 g/l sodium dodecyl sulfate ~SDS)) for ~ashing.
Th;s washing process was repeated 3 times. The carrier was then washed twice ;n lysis buffer without the 30 addit;on of SDS and then washed once with a washing buffer ~2 M tris.HCl~ pH s.a, 10 mmol/l NaCl, 0.1 mmol/l EDTA and 0.5 g/l NP-40). The ant;gens thus purified were removed from the solid carrier either by heating at +95C
for 5 m;nutes or by incubat;on in 6 mol/l NH4SCN at +4C
for 30 m;nutes.
Claims (7)
1. A process for the preparation of a therapeutic agent for the treatment of a tumorous disease, which comprises the drying or stabilization by a chemical treatment, and the processing to therapeutic agent of human tumor cells which carry antigens which are bound by the monoclonal antibodies which recognize an antigen (Ag1) having a molecular weight of approximately 33 KD ? 3, or an antigen (Ag2) having a molecular weight of approximately 134 KD ? 3, or an antigen (Ag3) having a molecular weight of approximately 80 KD ? 3, or an antigen (Ag4) having a molecular weight of approximately 55 KD ? 3, or an antigen (Ag5) having a molecular weight of approximately 60 KD ? 3, or an antigen (Ag6) having a molecular weight of approximately 54 KD ? 3, or an antigen (Ag7) having a molecular weight of approximately 260 KD ? 3, or an antigen (Ag8) having a non-determinable molecular weight, or an antigen (Ag9) having a non-determinable molec-ular weight, or an antigen (Ag10) having a molecular weight of approximately 143 KD ? 3 and 119 KD ? 3, or an antigen (Ag11) having a molecular weight of approximately 178 KD ? 3, or an antigen (Ag12) having a non-determinable molecular weight, or an antigen (Ag13) having a molecular weight of approximately 34 KD ? 3, or an antigen (Ag14) having a molec-ular weight of approximately 195 KD ? 3, or an antigen (Ag15) _ having a molecular weight of approximately 44 KD ? 7, or an antigen (Ag16) having a molecular weight of approximately 43 KD ? 3, or an antigen (Ag17) having a molecular weight of approximately 130 KD ? 3.
2. A process for the preparation of a therapeutic agent for the treatment of a tumorous disease, which comprises the isolation, by means of a monoclonal antibody which recognizes an antigen (Ag1) having a molecular weight of approximately 33 KD ? 3, or an antigen (Ag2) having a molecular weight of approximately 134 KD ? 3, or an antigen (Ag3) having a molecular weight of approximately 80 KD ? 3, or an antigen (Ag4) having a molecular weight of approximately 55 KD ? 3, or an antigen (Ag5) having a molecular weight of approximately 60 KD ? 3, or an antigen (Ag6) having a molec-ular weight of approximately 54 KD ? 3, or an antigen (Ag7) having a molecular weight of approximately 260 KD ? 3, or an antigen (Ag8) having a non-determinable molecular weight, or an antigen (Ag9) having a non-determinable molecular weight, or an antigen (Ag10) having a molecular weight of approx-imately 143 KD ? 3 and 119 KD ? 3, or an antigen (Ag11) having a molecular weight of approximately 178 KD ? 3, or an antigen (Ag12) having a non-determinable molecular weight, or an antigen (Ag13) having a molecular weight of approximately 34 KD ? 3, or an antigen (Ag14) having a molecular weight of approximately 195 KD ? 3, or an antigen (Ag15) having a mol-ecular weight of approximately 44 KD ? 7, or an antigen (Ag16) having a molecular weight of approximately 43 KD ? 3, or an antigen (Ag17) having a molecular weight of approx-imately 130 KD ? 3, of an antigen from human cells and its processing to a therapeutic agent.
3. The process as claimed in claim 1 or 2 wherein neurominidase is added.
4. A therapeutic agent for the treatment of a tumorous disease, containing human cells which have been dried or stabilized by a chemical treatment and which carry antigens which are bound by the monoclonal antibodies described in claim 1.
5. A therapeutic agent for the treatment of a tumorous disease, containing an antigen isolated from human tumor cells using a monoclonal antibody described in claim 2.
6. A therapeutic agent as claimed in claim 4, which contains neuraminidase.
7. A therapeutic agent as claimed in claim 5, which contains neuraminidase.
Applications Claiming Priority (2)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| DEP3432714.2 | 1984-09-06 | ||
| DE19843432714 DE3432714A1 (en) | 1984-09-06 | 1984-09-06 | TUMOR THERAPEUTICS AND METHOD FOR THE PRODUCTION THEREOF |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CA1277600C true CA1277600C (en) | 1990-12-11 |
Family
ID=6244749
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CA000490026A Expired - Lifetime CA1277600C (en) | 1984-09-06 | 1985-09-05 | Tumor therapeutic agent and a process for its preparation |
Country Status (13)
| Country | Link |
|---|---|
| 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) |
| IE (1) | IE58638B1 (en) |
| IL (1) | IL76289A (en) |
| PT (1) | PT81093B (en) |
| ZA (1) | ZA856807B (en) |
Families Citing this family (12)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| 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 |
| US7070782B1 (en) | 1992-11-05 | 2006-07-04 | Sloan-Kettering Institute For Cancer Research | Prostate-specific membrane antigen |
| JP4204642B2 (en) * | 1992-11-05 | 2009-01-07 | スローン − ケタリング・インスティテュート・フォー・キャンサー・リサーチ | Prostate-specific membrane antigen |
| US7105159B1 (en) | 1992-11-05 | 2006-09-12 | Sloan-Kettering Institute For Cancer Research | Antibodies to prostate-specific membrane antigen |
| US6953668B1 (en) | 1992-11-05 | 2005-10-11 | 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 |
| DE19506483A1 (en) * | 1995-02-24 | 1996-08-29 | Jens Dr Dr Atzpodien | Antitumour vaccines |
| DE69635801T2 (en) | 1995-02-24 | 2006-11-02 | Sloan-Kettering Institute For Cancer Research | PROSTATE-SPECIFIC MEMBRANES ANTIGEN AND ITS APPLICATIONS |
| US20050215472A1 (en) | 2001-10-23 | 2005-09-29 | Psma Development Company, Llc | PSMA formulations and uses thereof |
| WO2003034903A2 (en) | 2001-10-23 | 2003-05-01 | Psma Development Company, L.L.C. | 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 |
Family Cites Families (8)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| 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 |
-
1984
- 1984-09-06 DE DE19843432714 patent/DE3432714A1/en not_active Withdrawn
-
1985
- 1985-08-27 DE DE8585110757T patent/DE3583265D1/en not_active Expired - Lifetime
- 1985-08-27 EP EP85110757A patent/EP0173951B1/en not_active Expired - Lifetime
- 1985-08-27 AT AT85110757T patent/ATE64531T1/en not_active IP Right Cessation
- 1985-09-03 DK DK402485A patent/DK172253B1/en not_active IP Right Cessation
- 1985-09-04 IL IL76289A patent/IL76289A/en not_active IP Right Cessation
- 1985-09-04 FI FI853389A patent/FI88360C/en not_active IP Right Cessation
- 1985-09-04 GR GR852149A patent/GR852149B/el unknown
- 1985-09-04 ES ES546701A patent/ES8704082A1/en not_active Expired
- 1985-09-05 ZA ZA856807A patent/ZA856807B/en unknown
- 1985-09-05 PT PT81093A patent/PT81093B/en not_active IP Right Cessation
- 1985-09-05 IE IE219185A patent/IE58638B1/en not_active IP Right Cessation
- 1985-09-05 CA CA000490026A patent/CA1277600C/en not_active Expired - Lifetime
- 1985-09-05 JP JP60195000A patent/JPS6168427A/en active Pending
Also Published As
| Publication number | Publication date |
|---|---|
| IE58638B1 (en) | 1993-10-20 |
| DK402485A (en) | 1986-03-07 |
| DE3432714A1 (en) | 1986-04-24 |
| DK402485D0 (en) | 1985-09-03 |
| IE852191L (en) | 1986-03-06 |
| ES8704082A1 (en) | 1987-03-16 |
| ES546701A0 (en) | 1987-03-16 |
| FI88360B (en) | 1993-01-29 |
| EP0173951A3 (en) | 1988-06-08 |
| FI853389A0 (en) | 1985-09-04 |
| EP0173951B1 (en) | 1991-06-19 |
| ZA856807B (en) | 1986-04-30 |
| GR852149B (en) | 1986-01-03 |
| IL76289A0 (en) | 1986-01-31 |
| PT81093A (en) | 1985-10-01 |
| PT81093B (en) | 1988-07-01 |
| JPS6168427A (en) | 1986-04-08 |
| ATE64531T1 (en) | 1991-07-15 |
| IL76289A (en) | 1992-01-15 |
| FI88360C (en) | 1993-05-10 |
| DE3583265D1 (en) | 1991-07-25 |
| FI853389L (en) | 1986-03-07 |
| DK172253B1 (en) | 1998-02-09 |
| EP0173951A2 (en) | 1986-03-12 |
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