CN1291105A - Hapten-modified tumor cell membranes and preparation and method for using it - Google Patents
Hapten-modified tumor cell membranes and preparation and method for using it Download PDFInfo
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Classifications
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P35/00—Antineoplastic agents
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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
- A61K39/385—Haptens or antigens, bound to carriers
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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
- A61K39/001102—Receptors, cell surface antigens or cell surface determinants
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P43/00—Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
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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
- A61K2039/60—Medicinal preparations containing antigens or antibodies characteristics by the carrier linked to the antigen
- A61K2039/6012—Haptens, e.g. di- or trinitrophenyl (DNP, TNP)
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- Engineering & Computer Science (AREA)
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- Medicines Containing Antibodies Or Antigens For Use As Internal Diagnostic Agents (AREA)
- Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)
Abstract
The present invention is directed to isolated tumor cell membranes, compositions thereof, methods of making the membranes and compositions, and methods of treating cancer. The compositions of the present invention include a composition prepared from a tumor cell which is hapten modified. The tumor cell membranes and compositions of the invention have the properties, when administered to a mammal suffering from a malignant tumor of the same type as said tumor cell, of eliciting T lymphocytes that infiltrate the tumor of the mammal, of eliciting an inflammatory immune response against the tumor of the mammal, and of eliciting a delayed-type hypersensitivity response to the tumor of the mammal. The membranes and compositions of the invention also stimulate T cells in vitro. The methods of the present invention are directed to treating cancer comprising administering a therapeutically effective amount of a tumor cell membrane. The present invention is also directed to a method of making a hapten-modified tumor cell membrane and dosage forms containing the membranes.
Description
The list of references of government concerned's fund
The present invention described herein carries out in the permission of National Institutes of Health-fund CA-39248 of National Cancer Institute or in the work under authorizing.U.S. government can have some right of the present invention.
Background of invention
Nineteen sixties is set up such theory, and tumor cell has non-existent specific antigen (TSA) on normal cell, can make the individual tumor of repelling to these antigenic immunne response.Propose afterwards, the new immunologic determinants of introducing can strengthen the immunne response to TSA on cell.Mitchison, implantation method (Transplant.Proc.), 1970,2,92.This " auxiliary determinant " may be hapten, protein, virus coat antigen, transplantation antigen or heterogenous cell antigen, and it can be introduced in the tumor cell group.Then these injection cells can be tolerated in the individuality of growth of tumour cell of unmodified to expection.Clinically, wish to take place therefore the reaction of the TSA that follows to be strengthened, and make the tumor cell of otherwise tolerance obtain destroying assisting the immunoreation of determinant.Mitchison with above, has also proposed several binding modes of auxiliary determinant, comprises 1) cell of unmodified only is attenuated, and its speed of growth is slowed down, and perhaps its sensitivity to immune attack strengthens; 2) auxiliary determinant only provides the point of attack, and the cell that therefore makes modification can be that immunne response at TSA is killed and wounded; 3) auxiliary determinant has assosting effect, is being used for the especially location in lymph node, the immune correct position of health as binding antibody or promotion cell.
People such as Fujiwara, Journal of Immunology, 1984:132,1571 expressions, the tumor cell of some haptenization, promptly with the bonded tumor cell of hapten trinitrobenzene (TNP), can in the Mus system, induce systematicness immunity to the tumor cell of unmodified, as long as this mice at first under the situation of the special suppressor T cell of no hapten to this hapten sensitization.In untreated receptor animal, fully also stoped growth of tumor specifically by the splenocyte of treatment mice.People such as Flood, Journal of Immunology, 1987,138,3573 show, with " degeneration " tumor of the bonded ultraviolet induction of TNP in addition mice immunized can repel TNP bonded " carrying out property " tumor, this is a non-immunity.And, the attack of tumor that these mices tolerate subsequently unconjugated " carrying out property ".In another experimental system, people such as Fujiwara, Journal of Immunology, 1984,133,510 proofs, the mice with trinitro-chlorobenzene (TNCB) sensitization after the cyclophosphamide pretreatment can be cured the tumor of big (10mm) by the original position hapten of tumor cell is handled; Subsequently, these animals tolerate the attack of unconjugated tumor cell specifically.
People's such as Fujiwara instruction is owing to comprising that following several reasons is different from the present invention: the cell that uses in the compositions of A.Fujiwara derives from inductive transplantation Mus tumor, rather than derives from spontaneous human tumor; The compositions of B.Fujiwara is used for immunoprophylaxis, and the present invention uses immunization therapy; The compositions of C.Fujiwara is used as topical therapeutic, and compositions of the present invention is used by the whole body inoculation; The compositions of D.Fujiwara does not cause tumour regression, and compositions of the present invention is treated at quite a few at least and caused among the patient and degenerate and/or make prolonged survival period; Use tumor cell with E.Fujiwara, and the present invention instructs using of tumor cell membrane.
Proved recently exist can with the T cell of organizing cross reaction of unmodified.Weltzien and colleague show, the restricted T cell clone of I class MHC that produces from the mice of the homology lymphocyte immunity modified with TNP can be replied MHC " self " peptide relevant, that TNP modifies.Ortmann, people such as B., Journal of Immunology, 1992,148,1445.In addition, clear and definite, the lymphocyte immunity mice of modifying with TNP causes and can modify the development that cell has the splenic t-cell of cell-cytotoxic reaction in the secondary breeder reaction of external demonstration with to TNP.Shearer, G.M., European Journal of Immunology, 1974,4,527.Cause by the autogenous cell immunity of modifying with DNP or TNP, be quite significant with the lymphocytic potential of the autogenous cell of replying unmodified, because this may be relevant with two kinds of clinical problems: 1) drug-induced autoimmune disease, 2) cancer immunotherapy.For the former, as hapten, it combines formation with normal tissue protein can be by the immune complex of T cell recognition with oral medicine in suggestion once.Tsutsui, people such as H., Journal of Immunology, 1992,149,706.Subsequently, can develop into autoimmune disease, for example, systemic lupus erythematosus (sle), even after the medicine that cancellation is disliked, still continue.This will mean can with the lymphocytic final generation of the T that organizes cross reaction of unmodified.
The common trait of these experiments is to use hapten sensitization under the environment of not inducing the inhibition cell.Cyclophosphamide is pretreated, the splenocyte of the mice of TNCB sensitization shows radiation resistance " enhanced miscellaneous function ", that is, they strengthen the Cytotoxic external generation of anti-TNP specifically.And in case these enhanced miscellaneous functions are activated from the body lymphocyte by external contact TNP is bonded, then they also can strengthen the cytotoxicity (people such as Fujiwara, 1984) that irrelevant antigen is comprised tumor antigen.People such as Flood (1987) with above, show that this enhanced auxiliary activity is by having Lyt
-1
+, Lyt
-2
-, L3T4
+, I
-J
+The T cell of phenotype mediates, and to propose these cells are the anti-cells that suppress, a kind of immunomodulating T cell of newtype.
The immunization therapy of melanoma patient is shown, with elementary antigen keyhole relative hemocyanin sensitization administered with high dose (1000mg/M before three days
2) or low dosage (300mg/M
2) cyclophosphamide, significantly strengthened acquisition (people such as Berd, cancer research (Cancer Res.), 1982,42,4862 to this antigenic delayed hypersensitivity; Cancer research, 1984,44,1275).The low-dose cyclophosphamide pretreatment is replied from the autologous melanoma vaccine injection metastatic melanoma patient, produces from the delayed hypersensitivity of autologous melanoma cell (people such as Berd, cancer research, 1986,46,2572; Cancer research, 1988,6,335).Cyclophosphamide is used reduction (people such as Berd, cancer research, 1984,44,5439 that cause the non-specific T inhibit feature of peripheral blood lymphocyte; Cancer research, 1987,47,3317), this may be since consumption poor CD4+, CD45R+ suppress inducing T cell people such as (, cancer research, 1988,48,1671) Berd.As if the antitumor action of this immunization therapy scheme is begun to use vaccine and forms limits (people such as Berd to the long interval between the delayed hypersensitivity of tumor cell, american cancer EASD journal (Amer.Assoc.Cancer Res.), 1988,29,408 (#1626)).Therefore, still need to improve the therapeutic efficiency of this vaccine so that it has more immunogenicity.
Most of tumour immunity scholars approve of now, and the infiltration of the leukocyte of T lymphocyte-be responsible for tumour immunity-in tumor mass is the prerequisite that immune system is destroyed tumor.Therefore, many attention have concentrated on known " TIL " treatment of being started as Stephen doctor Rosenberg of NCI.Doctor Rosenberg and other people extract a small amount of T lymphocyte from human metastatic carcinoma, the natural existence of these T lymphocytes is by increasing its quantity greatly with the lymphocytic a kind of somatomedin-In vitro culture of interleukin II (IL2)-T.People such as Topalian, Journal of Clinical Oncology (J.Clin.Oncol.), 1988,6,839.Yet this treatment also is not highly effective, because the ability of the T cell of injection only limits to " recurrence " to tumor locus.
In big quantity research, in treatment, utilized high concentration IL2 induction of lymphocyte to become ability (people such as Lotze, biologically magazine (J.Biol.Response), 1982,3,475 of non-specific cell toxicity killer cell; People such as West, New England Journal of Medicine (New Engl.J.Med.), 1987,316,898).Yet, because the strong toxicity of high dose intravenous IL2 makes this method have limitation.A small amount of attention is invested following phenomenon, quite the IL2 of low concentration can pass through the inducing antigen activating T cell expansion and as immunological adjuvant (people such as Talmadge, cancer research, 1987,47,5725; People such as Meuer, lancet (Lancet), 1989,1,15).Therefore, still need to understand and make great efforts the purposes of exploitation IL2 as immunological adjuvant.
It is believed that human melanoma is expressed the unique surface antigen that can be discerned by the T lymphocyte.Old, L.J., cancer research, 1981,41,361; Van der Bruggen, people such as P., science, 1991,254,1643; Mukherji, people such as B., Journal of Immunology, 1986,136,1888; And Anichini, people such as A., Journal of Immunology, 1989,142,3692.Yet, induce difficulty to these antigenic effective T cell-mediated reactions to limit immunotherapy before the inventor carries out work in vivo.
As if proposed several models and explained what is a toleration to the human tumor related antigen.Comprise:
1) specific for tumour antigen of downward modulation initial stage antitumor reaction suppresses cell.People such as Mukherji, together above; Berendt, M.J. and R.J.North., The Journal of Experimental Medicine (J.Exp.Med.), 1980,151,69.
2) human tumor cell can not cause t helper cell or provide costimulatory signal for these T cells.Fearon, people such as E.R., cell (Cell), 1990,60,397; Townsend, S.E. and J.P.Allison, science, 1993,259,368; With
3) surface expression that reduces on tumor cell of main histocompatibility product, this has limited it by the identification of T cell.Ruiter, D.J., carcinobiology seminar (Seminars in CancerBiology), 1991,2,35.Going back neither one in these hypothesis confirms in clinical system.
No matter whether these explanations are true, and still continuing need be to more effective treatment of multiple malignant tumor.
For acute myelogenous leukemia (AML), the treatment of AML is divided into process after one or two initial stage induction period and the several alleviation, be also referred to as and consolidate chemotherapy.Initial stage induces chemotherapy can induce completely in the patient of 55-88% according to used scheme and replys.Yet, the great majority recurrence among these patients, the AML patient of long-term (5 years+) survival only is 20-30%.Add in therapeutic scheme that between the catabasis first time high dose chemotherapy and bone marrow transplantation (BMT) result can cause the improvement of appropriateness.For example, the patient who stands allogeneic BMT has the growth of 5-10% on 5 annual survival rates.Yet the strict criterion of acceptability of BMT (for example availability of age, HLA coupling donor) has seriously limited medicable patient's quantity.In case AML patient's recurrence have only 30% chance realization alleviation for the second time, and few patient can become at last anosis.The form of therapy of recurrence is comprised and realizes alleviating used similar scheme (inductive treatment is the chemotherapy of consolidating of several courses of treatment subsequently) for the first time, although also can use the single medicament of high dose and BMT people such as () Keating.
The experience prompting of bone marrow transplantation aspect, immunologic rejection may work in the control of disease.Graft versus host disease (GVHD) and recurrence are two main causes with the death of BMT treatment.If weak GVHD takes place, then the danger of recurrence reduces people such as () Horowitz.Therefore the lymphocyte transplanted of supposition once can immunologic rejection host leukaemia (reaction of graft leukemia, GVL).T cell responses to specific leukemia cell antigen can mediate this GVL reaction, although immunogenicity human leukemia antigen does not obtain proof (like this equally for melanoma) as yet.Known person AML cell strong expression I class and II class major histocompatibility complex (MHC) antigen (people such as Ashman; People such as Andreasen), this is the prerequisite of inducing the t cell responses of CD8-and CD4-mediation respectively.Yet, at the success of inducing as yet of leukaemia's t cell responses.
There are several immunological methods once to be used for the treatment (people such as Foon of acute leukemia; Caron and Scheinberg).These methods are divided into non-specific method, as the methanol extracting residue of bacillus calmette-guerin vaccine (BCG), interleukin II, levamisole, tubercule bacillus; With specific method, as monoclonal antibody and vaccine (leukaemia of collection, cell-free extract and cultured cells).Great majority in these researchs carry out in the patient of having alleviated, and wherein immunization therapy can successfully be used to control remaining disease.
Later stage nineteen sixties and nineteen seventies are early stage, and the R.Powles seminar of England St.Barthlomew hospital has carried out a series of research (Powles, 1974 of using vaccine therapy AML patient after the alleviation of chemotherapy-induced; People such as Powles, 1977).They use allogeneic AML cell, with BCG as adjuvant.Having carried out test several times, all is small sample capacity (N=10-15).Compare with independent chemotherapy, use the combination of chemotherapy and immunotherapy that survival period is prolonged to some extent, and the survival of not having a recurrence does not prolong.Do not observe serious toxicity; Do not see the autoimmune toxicity of normal marrow (for example, to).In retrospect, these tests have many technical problems: 1) use allochthonous rather than from the leukaemia of body; 2) leukaemia's dosage is excessive (up to 10 in the vaccine
9Cell/dosage); 3) BCG dosage is high, and BCG uses by the position of time and leukaemia's vaccine and separates; 4) when accepting cytotoxic drug, the patient uses vaccine (keep or consolidate chemotherapy).
The limited successful immunochemistry basis of above-mentioned treatment is still waiting thinking, but is checking several hypothesis.Kim and Jang (1992) suggestion may not be because the shortage of T cell all the components to the shortage of the t cell responses of defined epitope, but owing to produce the difficulty of defined epitope.People such as Martin (1993) exist by supposition because lower and can escape the autoreactive T cell that thymus is selected to " self " peptide affinity, have explained their result.
The routine of treatment human cancer is attempted as yet not success.The development of the immunity of using the mediation of inducing cell reliably of the compositions that exemplifies as mentioned above is shown in delayed hypersensitivity (DTH), T cellular infiltration and inflammatory immunne response.
Therefore, no matter the trial quantity of the multiple theory of reporting in treatment of cancer that proposes based on immunization how, still need a kind of compositions, it can cause the T lymphocyte that soaks into tumor, causes inflammatory immunne response to tumor, and cause delayed hypersensitivity to tumor after being applied to mammal.The applicant is surprised to find now, and use isolating film from homology or allogeneic tumor cell has the characteristic of these hope.
Summary of the invention
The present invention relates to a kind of isolating tumor cell membrane, a kind ofly contain this tumor cell membrane of compositions, separation and preparation of this film and contain the method for compositions of this film and they are in purposes external and the treatment cancer.The tumor cell membrane of available hapten transformation preferably may homology or allochthonous tumor cell plasma membrane.Homologous tumor cell membrane is from body.Cancer to be treated comprises cancer and non-solid tumor, comprises leukemia (as acute myelogenous leukemia), lymphoma, multiple myeloma, ovarian cancer, colon cancer, rectal cancer, colorectal carcinoma, melanoma, breast carcinoma, pulmonary carcinoma, renal carcinoma and carcinoma of prostate.
On the one hand, the present invention relates to a kind of mammal (preferably being the people) tumor cell membrane of isolating hapten transformation.This hapten can be selected from: dinitro benzene, trinitrobenzene, N-iodoacetyl-N '-(5-sulfo group-1-naphthyl) ethylenediamine, trinitro-benzene-sulfonic acid, Fluorescein isothiocyanate, arsenic acid PhNCS, trinitro-benzene-sulfonic acid, Phosphorylcholine, sulfanilic acid, arsanilic acid and dinitro benzene-S-mustard and combination thereof.
On the other hand, the present invention relates to a kind of compositions that contains the mammalian tumor cell film of hapten transformation separately or contain the mammalian tumor cell of hapten transformation simultaneously.
Again on the other hand, the invention provides a kind of vaccine combination that contains mammal (preferably people) tumor cell membrane for the treatment of effective dose, be used for to the administration of suffering from the malignant tumor of this tumor cell membrane same type.
Another aspect of the present invention, said composition contains a kind of adjuvant, as endotoxin and cytokine such as interleukin II, interleukin-4, IFN-(IFN-γ), interleukin 12, interleukin 15 and the GM-CSF of bacillus calmette-guerin vaccine, QS-21, detoxification.
Film of the present invention and compositions (when to suffering from mammal (preferably people) with the tumor cell same type malignant tumor of separating this film when using) have one of following character at least: (ⅰ) cause the T lymphocyte that soaks into the mammal tumor of being treated, (ⅱ) initiation is to the inflammatory immunne response of this mammal tumor, and (ⅲ) initiation is to the delayed hypersensitivity of this mammal tumor.Film of the present invention and compositions also have the character at stimulated in vitro T cell.
Again on the other hand, the present invention relates to a kind of treatment method for cancer, comprise to mammal (preferably people) and use a kind of compositions that contains the human tumor cells film of the hapten transformation for the treatment of effective dose that wherein this mammal suffers from the malignant tumor with this tumor cell membrane same type.
In addition, the present invention relates to a kind of method, comprise and cause T lymphocyte with the character of soaking into this mammal (preferably people) tumor and randomly, measure the T lymphocyte that soaks into this mammal tumor.The invention further relates to a kind of method, comprise the inflammatory immunne response of initiation to this mammal tumor, randomly, measure this inflammatory immunne response, perhaps relate to a kind of method, comprise that initiation to the delayed hypersensitivity of this mammal tumor and randomly, measures this delayed hypersensitivity.The present invention also relates to a kind of method at stimulated in vitro T cell.
Of the present invention more on the other hand, the present invention relates to a kind of method for preparing the tumor cell membrane of hapten transformation.Detailed Description Of The Invention
All be incorporated herein by reference at this all patents, patent application and list of references of quoting.If contradiction is as the criterion with present disclosure.
The present invention relates to a kind of isolating modification and tumor cell membrane unmodified, a kind of new compositions that contains this film, separate and this film of preparation and contain the method for compositions of this film and use film of the present invention and method for compositions.
Film of the present invention and compositions can be used for treating preferably people's cancer of mammal, comprise transfer and preinvasive cancer, solid tumor and non-solid tumor, as rectal cancer, colorectal carcinoma, melanoma, breast carcinoma, pulmonary carcinoma, renal carcinoma and carcinoma of prostate.I, II, III or IV phase cancer can be treated with film, the compositions and methods of the invention that separate to modify, preferably III phase and IV phase, III phase more preferably again.The mammal that suffers from the metastatic carcinoma of aforementioned type, particularly people can be treated with film of the present invention, compositions and method.In one embodiment, treat the member of domestic animal such as cat, dog, horse and Bovidae with the present invention.
Film of the present invention and compositions also can be used for causing the T lymphocyte with the character of soaking into mammal tumor, initiation is to the inflammatory immunne response of mammal tumor, initiation is to the delayed hypersensitivity of mammal tumor, and/or at stimulated in vitro T lymphocyte.
Should be appreciated that relevant any disclosure of using isolating tumor cell is equally applicable to the application of tumor cell membrane in this description, or be applicable to the combination of tumor cell and tumor cell membrane.Tumor cell membrane and compositions thereof
The tumor cell membrane of isolating modification of the present invention is by preferably people's tumor cell preparation of mammal.In one embodiment of the invention, separating tumor cell film from the tumor of cat, dog, horse or bovid.
For the present invention, comprise complete and disruptive tumor cell in the definition of tumor cell.The tumor cell of diffusion barrier can be the cell that living cells, attenuation cell or quilt are killed and wounded.Thereby can use among the present invention and after the patient is used, not grow and divide the tumor cell that keeps non-growth conditions basically.If the patient used separately or combine with isolating tumor cell membrane use, then these cells are preferred.Should be appreciated that " cell of non-growth conditions " meaning be nondividing work in vivo, attenuation or killed and wounded, complete or disruptive (perhaps have simultaneously complete sum disruptive) cell.Suspend the conventional method of cell of non-growth conditions for known to the technical staff, and can be used for the present invention.For example, irradiating cell can not be grown and divide it before use.Tumor cell can used the back growth to stop cell with for example 2500R irradiation.In addition, also can from can grow in vivo and splitted tumor cell the separating tumor cell film.Preferably, in this case, the tumor cell membrane product is not polluted by splitted tumor cell in vivo.
The tumor cell membrance separation from the tumor cell of the cancer same type of desire treatment.For example, be used for the treatment of the membrance separation of ovarian cancer from ovarian cancer cell.Preferably, tumor cell derives from the same patient of desire treatment.Tumor cell preferably homologous (for example from body) with the patient, but also can be allochthonous.For being defined as " homologous ", tumor cell does not need that (promptly 100%) hereditism is last identical fully with treatment patient's tumor cell or non-tumor somatic cell.MHC molecular genetic homogeneity between (the separation membrane) tumor cell and the patient is normally sufficient.In addition, between the antigen that exists on as specific antigen on the membrane derived tumor cell and patient tumors cell genetic identity can be arranged.Genetic identity can be measured according to method well known in the art.Homologous tumor cell looks like also a kind of genetically engineered (for example utilizing recombinant DNA technology) and makes its identical cell on hereditism aspect the specific antigen on the specific MHC molecule of for example patient and/or the patient's cancerous cell.Also can use to derive from animal mutually of the same race and the last different tumor cell of hereditism,, prepare tumor cell membrane of the present invention as variant cell of the same race.Tumor cell can be but be not limited to from biopsy specimen or from tissue culture isolated cells.From homogeneous variant cell and the isolating film of stem cell also within the scope of the invention.
Tumor cell membrane can comprise all cells film, as adventitia, nuclear membrane, mitochondrial membrane, tonoplast, endoplasmic reticulum, Golgi complex film and lysosome membrane.In one embodiment of the invention, surpassing about 50% film is the tumor cell plasma membrane.Preferably, be made up of the tumor cell plasma membrane above about 60% film, it is preferred surpassing about 70%, the 80%th, in addition preferred, the 90%th, in addition preferred, the 95%th, in addition preferred, the 99%th, most preferred.
Preferably, isolating film is substantially free of nucleus and cell.For example, if about 2 * 10
8Contain in the membrane substance of cell equivalent (c.e.) and be less than about 100 cells and/or nucleus, then membrane product is for being substantially free of nucleus or cell.Cell equivalent be from shown in the quantity of isolating film the cell of quantity.Be substantially free of cell and/or nuclear isolating tumor cell membrane can contain lymphocyte and/or lymphocyte film.
Preferably, isolating tumor cell membrane is an epicyte, that is, and and the tumor cell plasma membrane.Membrane product of the present invention can contain complete adventitia or its part.The isolating film of the present invention that contains some adventitia includes at least a MHC molecular moiety and/or a kind of heatshock protein part of adventitia.The size of membrane-bound fragment is not important.
The allogeneic tumor cell membrane also can use with homology (for example from body) antigen-presenting cell in the method for the invention.This method allows to come immune patients with the tumor cell membrane that derives from non-patient self tumor.Isogeneic is delivery cell processing allogeneic film, makes patient's cell-mediated immune system to reply it.
For example, can use isolating tumor cell membrane of hapten transformation and tumor cell.The tumor cell membrane of these modifications (and tumor cell) has one of following character at least: (ⅰ) cause the T lymphocyte that soaks into the mammiferous tumor for the treatment of, (ⅱ) cause inflammatory immunne response, and (ⅲ) cause delayed hypersensitivity this mammal tumor to this mammal tumor.Tumor cell membrane of modifying and cell also have the character at stimulated in vitro T cell.
(modification or the unmodified) tumor cell membrane that relates in this description comprises arbitrary form that membrane product can be stored or use, and for example, is resuspended to film, the refrigerated or freeze dried film of film precipitation in the diluent.
Film of the present invention can use separately in the method for the invention, or includes but not limited to that with other chemical compound other compositions of the present invention uses.Therefore, tumor cell and tumor cell membrane can use separately or use jointly.For the present invention, use jointly and comprise simultaneously and continuous administration.In addition, tumor cell and tumor cell membrane can with other chemical compound, include but not limited to that cytokine such as interleukin II, interleukin-4, IFN-(IFN-γ), interleukin 12, interleukin 15 and GM-CSF use jointly.Tumor cell of the present invention and tumor cell membrane also can include but not limited to that chemotherapy, X-ray therapy, antibody therapy and antisense oligonucleotide treatment are used in combination with other cancer therapy.Yet, the invention has the advantages that it can be used alone as treatment of cancer, making to need other treatment.
Compositions of the present invention can contain isolating tumor cell membrane of the present invention (modification or unmodified) and a kind of pharmaceutically acceptable carrier or diluent, as but be not limited to Hanks solution, saline, phosphoric acid buffer saline solution, sucrose solution and water.Usually, according to the route of administration and the standard pharmacy choice of practice pharmaceutically acceptable carrier of expecting.The ratio of effective ingredient and carrier depends on chemical property, dissolubility and the stability of compositions and the dosage of estimating naturally, and can use the general knowledge optimization of this area.
In a preferred embodiment of the invention, compositions of the present invention is a kind of vaccine combination that contains the isolating modification tumor cell membrane of effective dose.For present disclosure, " effective dose " is the required amount of result that reaches hope.For example, in a kind of treatment method for cancer, " effective dose " meaning is the amount of isolating modification tumor cell membrane, this measurer has the character that causes one of following situation at least: (ⅰ) cause the T lymphocyte that soaks into tumor, (ⅱ) initiation is to the inflammatory reaction of tumor, (ⅲ) cause the delayed hypersensitivity of tumor and (ⅳ) tumour regression.Equally, in the method for stimulated in vitro T cell, " effective dose " is the amount that causes the film of T cytositimulation.
Vaccine combination can contain, for example, and every dosage at least 10
4C.e. isolating film, preferably at least 10
5C.e., most preferably at least 10
6C.e..The amount of the vaccine combination that dosage is in single administration to be used.In one embodiment, vaccine combination contains every dosage about 10
5-Yue 2.5 * 10
7C.e. film.More preferably about 5 * 10
6C.e..The used tumor cell of the present invention and the amount of tumor cell membrane depend on as chemical compound usually to the amount of the cancerous cell of the affinity of cancerous cell, existence and the factors such as dissolubility of compositions.Can set dosage according to patient's body weight, clinical condition.
Vaccine combination of the present invention can be packaged as the dosage form that is suitable for Intradermal, intravenous, intraperitoneal, intramuscular and subcutaneous administration.In addition, this dosage form also can contain separative tumor cell membrane, for example rebuilds with the suitable dilution agent when using.Hapten
Tumor cell of the present invention and tumor cell membrane can be used as tumor cell modification, unmodified and tumor cell membrane and use, or as modify, the tumor cell of unmodified and being used in combination of tumor cell membrane.For the present invention, modify and include but not limited to use hapten transformation.Separately induce immune response (but strengthen in conjunction with or the immunne response of another molecule of otherwise adhering to) arbitrary micromolecule can play haptenic effect.Usually, employed molecule should be less than about 1000mw.
Known multiple hapten in this area, for example: TNP (people such as Kempkes, Journal of Immunology 1991 147:2467); Phosphorylcholine (people such as Jang, European Journal of Immunology 1991 21:1303); Nickel (people such as Pistoor, experimental dermatology magazine (J.Invest.Dermatol.) 1995105:92); Arsenic acid (ester) (Nalefski and Rao, Journal of Immunology 1993 150:3806).
Usually, being applicable to that hapten of the present invention has can be in conjunction with the character of hydrophilic amino acid (for example lysine).Hapten can be by lysine epsilon-amino or-the COOH base combines with cell.In addition, also can use and to pass through the hapten of phenodiazine (diaza) coupling in conjunction with hydrophobic amino acid such as tyrosine and histidine.Be applicable to that haptenic example of the present invention comprises: dinitro benzene, trinitrobenzene, N-iodoacetyl-N '-(5-sulfo group-1-naphthyl) ethylenediamine, trinitro-benzene-sulfonic acid, Fluorescein isothiocyanate, arsenic acid PhNCS, trinitro-benzene-sulfonic acid, Phosphorylcholine, sulfanilic acid, arsanilic acid, dinitro benzene-S-mustard (Nahas and Leskowitz, cellular immunology (Cellular Immunol.) 198054:241) and combination thereof.In case learnt present disclosure, the technical staff just can select to be used for hapten of the present invention.For example, can test hapten with delayed hypersensitivity (DTH) testing routine.Adjuvant
In a preferred embodiment, tumor cell or tumor cell membrane are used with a kind of immunological adjuvant.This adjuvant has the character of enhancing to the immunne response of the tumor cell of hapten transformation and film.The representative example of adjuvant is a bacillus calmette-guerin vaccine, BCG, or synthetic adjuvant, comprise the homogenizing saponin from Quillaia saponaria (quillajasaponaria) skin purification, the QS-21 of Corynebacterium (Corynebacterium parvum), people such as McCune, cancer (Cancer) 1979 43:1619 generally are endotoxin and cytokine such as interleukin II, interleukin-4, IFN-(IFN-γ), interleukin 12, interleukin 15, GM-CSF and the combination thereof of saponin, detoxification.
Should be appreciated that and to be optimized adjuvant.In other words, the technical staff can utilize normal experiment to determine the best adjuvant that uses.The method for preparing tumor cell membrane of the present invention
The tumor cell that the present invention uses can be prepared as follows.As people such as Berd (1986) with above, people (1997), U.S. Patent number 5 such as Sato, 290,551 and U. S. application series number 08/203,004,08/479,016,08/899,905,08/942,794 or the described processing tumor of corresponding PCT application PCT/US96/09511, its each all this complete being incorporated herein by reference.In brief,,, use the agitator mechanical disintegration,, use mortar and pestle, be cut into small pieces with knife blade with the tweezers fluffing as with collagenase and the division of DNA enzyme enzymatic through the split extraction cell, or the like.For liquid tumors, can collect blood or bone marrow sample, and through density gradient centrifugation separating tumor cell.
Prepare tumor cell membrane by ruptured cell by tumor cell, for example, utilize hypotonic shock, mechanical disintegration and enzymatic division, and through the different cell component of centrifugalize.In brief, can use the following step: the cracking tumor cell, from cracked tumor cell, remove the tumor cell that nucleus obtains acellular nuclear, obtain not contain cell and nuclear purer basically film, this tumor cell membrane and hapten are done in order to obtain the tumor cell membrane of hapten transformation.Can carry out membrance separation according to people's such as Heike method.
In one embodiment of the invention, intact cell and nucleus can be removed by continuous centrifugal, up to be substantially free of nucleus and cell through microscopy.For example, cracked cell can be with low velocity, and 500-2000g is centrifugal about 5 minutes according to appointment.This separation method makes about 2 * 10
8Keep in the membrane substance of cell equivalent (c.e.) and be less than about 100 cells and/or nucleus.For example, can contain (postnuclear) supernatant behind the nuclear of film by the about 90 minutes Ultracentrifugation of about 100000g.Contain whole films in the precipitation.For example, film can be resuspended to about 8% sucrose, 5mM Tris, in the solution of pH7.6, and be frozen in approximately-80 ℃ until use.Can use arbitrary diluent, preferably as the diluent of stabilizing agent.In order to measure the quality of membrane product, can regularly cultivate (about 6 * 10
7C.e. film).Do not answer the cellulation colony, should detect yet less than cell or nucleus with optical microscope.
Can be undertaken by known method the modification of prepared cell or film with DNP or another kind of hapten, for example, the method of Miller and Claman, Journal of Immunology, 1976,117,1519, this complete quoting as a reference, this method comprises tumor cell or film and a kind of hapten incubation 30 minutes under aseptic condition, washs with Sterile Saline subsequently.With the monoclonal anti hapten antibody through flow cytometry susceptible of proof hapten transformation.
Splitted cell or isolating film can fresh use or for example the fridge of controlled speed or in liquid nitrogen refrigerated storage standby.Use immediately after in a single day cell and film melt.Preferably, melt behind cell or the film and use to the patient at once.For example, can melt cell or film the patient being made a skin test or treating the same day.Randomly, but washed cell or film, and randomly shine 2500R.Can wash once more, be suspended in then and do not contain in the phenol red Hanks balanced salt solution.
The allogeneic tumor cell membrane can prepare as mentioned above.Yet, before using to the experimenter, should with homology (for example from body) antigen-presenting cell incubation altogether.Isogeneic is delivery cell processing allogeneic film, makes patient's cell-mediated immune system to reply it.This method makes can come immune patients with the tumor cell membrane that derives from non-patient self tumor.A few hours arrive a period of time of a couple of days approximately approximately for allogeneic tumor cell membrane and antigen-presenting cell incubation.Wash the antigen-presenting cell of film pulse (cmembrane-pulsed) then, and to patient infusion.
Can prepare antigen-presenting cell with several different methods, comprise for example people such as Grabbe, 1995 and people such as Siena, 1995 method.In brief, for example from the patient who desires immunity, obtain blood by venipuncture.In addition, also can obtain bone marrow.In addition, also can obtain blood leucocyte by leukopheresis.Can from any of these sources, separate monocyte through gradient centrifugation.The monoclonal antibody of the further available antigens c D34 of leukocyte is through just selecting purification.
In tissue culture medium (TCM) (for example, being supplemented with the human serum or the autoserous RPMI-1640 of serum such as hyclone, merging), cultivate and enlarge the leukocyte of purification.In addition, also can use serum-free medium.For stimulator antigen is the growth of delivery cell, can in culture medium, add cytokine.Cytokine includes but not limited to granulocyte macrophage colony stimulating factor (GM-CSF), interleukin-4 (IL4), TNF (tumor necrosis factor), interleukin (IL3), FLT3 part and granulocyte colony-stimulating factor (G-CSF).
Separating also in cultivation, the antigen-presenting cell of amplification culture can be characterized by for example dendritic cell, mononuclear cell, macrophage and Langerhans cell.Use tumor cell membrane and method for compositions
The treatment method for cancer the present invention relates to a kind ofly treat diagnosis or suspect mammal people's the method preferably suffer from cancer, but comprises the tumor cell membrane of the hapten transformation of using a kind of pharmacy receiving amount, tumor cell or its combination of hapten transformation.Film and/or cell can mix with immunological adjuvant and/or pharmaceutically acceptable carrier.But before using said composition, can use the low-dose cyclophosphamide of pharmacy receiving amount or the chemotherapeutics of another kind of low dosage.But randomly can use the tumor cell or the tumor cell membrane of the non-haptenization of pharmacy receiving amount after the compositions of haptenization.Also can use the compositions of non-haptenization according to method of the present invention.
Can treat arbitrary malignant tumor according to the present invention, comprise and shifting and preinvasive cancer, solid tumor and non-solid tumor.Solid tumor comprises cancer, and non-solid tumor comprises hematologic malignancies.Cancer includes but not limited to adenocarcinoma and epithelial cancer.Hematologic malignancies comprises leukemia, lymphoma and multiple myeloma.Be can be with the limiting examples of the cancer of isolating modification tumor cell membrane treatment by method of the present invention below: ovarian cancer comprises that advanced ovarian cancer, leukemia include but not limited to acute myelogenous leukemia, colon cancer, comprises the colon cancer, rectal cancer, colorectal carcinoma, melanoma, breast carcinoma, pulmonary carcinoma, renal carcinoma and the carcinoma of prostate that are transferred to liver.Ovarian cancer may be adenocarcinoma or epithelial cancer.Colon and carcinoma of prostate are adenocarcinoma.Leukemia may derive from marrow sample bone marrow or lymph node.Leukemia may be acute, and the maturation that shows as the initial stage of development stops, and also may be chronic, shows as ripe lymphoid cell or myelocytic undue growth.Can treat I, II, III or IV phase cancer according to the present invention, preferably III and IV phase, III phase cancer more preferably again.
The mammal that suffers from the metastatic carcinoma of aforementioned type, particularly human, can treat with film of the present invention, compositions and method.In one embodiment of the invention, can treat domestic animal.
Before using vaccine combination of the present invention, can make the patient to being used for modifying the hapten immunity of tumor cell and film by it being imposed on skin.For example, can use dinitrofluorobenzene (DNFB).(for example, after about 2 weeks) can give a kind of tumor cell film composition of patient infusion subsequently.Said composition can be used (as injection again) at least three treatments altogether, preferably at least six treatments.In one embodiment, the sum of using (comprising initial application) can be 8 times, may be 10 times in another embodiment.Vaccination regimen can be designed by the attending doctor, to adapt to the state of an illness of particular patient.For example, can per 2 weeks, preferably implement vaccine injection weekly.Can use and once strengthen vaccine.Preferably, booster immunization once or twice.For example, can be after about 6 months of initial application or after about 1 year booster immunization.
Available medicine strengthens patient's immunne response.For example, can use cyclophosphamide (CY) before using at every turn.
Can use the present invention as the back of performing the operation in conventional treatment of cancer.For solid tumor, as ovarian cancer, can the best or suboptimum ground excision (debulking) tumor.Excision is meant among the feasible quilt of the tumor resection patient who treats and only leaves little tumor mass best.The excision of suboptimum ground is meant tumor resection and leaves bulk in the patient.For non-solid tumor, can collect suitable blood or bone marrow sample, and with known technical point in any list of references from cancerous cell.The tumor of excision or the tumor cell of collection can be used to prepare aforesaid tumor cell membrane.
Can use tumor cell membrane by arbitrary suitable way, comprise inoculation and injection, for example Intradermal, intravenous, intraperitoneal, intramuscular and subcutaneous injection.Each vaccine therapy can have a plurality of site of administration.For example, use at every turn can intradermal injection two preferably three adjacent regions use vaccine combination.In one embodiment of the invention, on upper arm or lower limb, use vaccine combination.
Use the usefulness that multiple biological respinse modifier can improve vaccine.These medicaments are replied by immune stimulatory directly or indirectly and are worked.Biological respinse modifier of the present invention includes but not limited to interleukin 12, interleukin 15 and IFN-.In one embodiment, after each vaccine injection, use IL12.Patient with inflammatory reaction is used IL12 can be made the T lymphopoiesis in the tumor mass and have more activity.The T cell number that increases and functionally cause the immune destruction of tumor and disappear.
The extensive work person has been developed and has been tested the human cancer vaccine.Although they can induce the weak immunity to patient's cancer sometimes, seldom can cause tumor regression or prolong the time-to-live.Use vaccine of the present invention and shockingly found the evidence of inflammatory reaction.Arrive the lymphocytic infiltration of T with microscopic examination.Therefore, this method that can strengthen inflammatory reaction and lymphocyte quantity has tangible progress in the art.Therefore, the present invention also provides the method that causes the T cell that has one of following character at least: (ⅰ) causes the T lymphocyte that soaks into the mammiferous tumor for the treatment of when using, (ⅱ) initiation is to the inflammatory immunne response of mammal tumor, and (ⅲ) initiation is to the delayed hypersensitivity of mammal tumor.
The method of stimulation T cell can be with isolating tumor cell membrane at stimulated in vitro T cell.Whether this test can be used for evaluation Example may success as the treatment of using the specific tumors film.The T cell that uses in this test can obtain according to following method, and this method is to describe to be used for the people, but can be applicable to any mammalian subject.
But use a kind of compositions of pharmacy receiving amount by the patient who diagnosis is suffered from certain types of cancer, wherein contain tumor cell, tumor cell membrane or its combination of hapten transformation, produce the T cell thus.Said composition randomly can contain a kind of immunological adjuvant and/or a kind of pharmaceutically acceptable carrier.Before using for the first time the tumor cell compositions, but randomly can use a kind of low-dose cyclophosphamide of pharmacy receiving amount or the chemotherapeutics of another kind of low dosage, as but be not limited to about 5-10mg/M
2Melphalan.But randomly can use a kind of vaccine combination of non-haptenization of pharmacy receiving amount after the compositions of haptenization, it contains film, tumor cell or its combination of non-haptenization.Can use the compositions of non-haptenization according to method of the present invention.
Can be from behind the autogenous cell of using hapten transformation or film, it being taken place to obtain peripheral blood lymphocyte (PBL) the patient of strong delayed allergy (DTH) reaction.The DTH reaction preferably has the diameter of about 10mm, more more preferably approximately greater than 10mm.Can set up T cell line by PBL by cancerous cell repetitive stimulation with hapten transformation.The T cell can separate with known method, preparation as single cell suspension, filter, monocytic eliminating and the separating of subgroup of expressing specific T cells receptor (TCR) type, method is to make this subgroup in the presence of TCR subtype sepcific antibody and/or increasing in the presence of the IL-2 and/or in the presence of superantigen.Purpose T cell can increase in vivo because they be from enrichment collect the infiltrate of tumor of purpose T cell.
Tumor cell of modifying and tumor cell membrane all have the character that stimulates the T cell.For the present invention, " stimulation " is meant that the propagation of inducing T cell and T cell produce in external cytokine.Film and tumor cell all have the ability that stimulates the T cell independently.Can detect and measure the propagation of T cell to the picked-up of modified nucleotide by the T cell, these modified nucleotides as but be not limited to
3The H thymidine,
125I UDR (iododeoxyuridine); And dyestuff, if can make the painted 3-of living cells (4,5-dimethylthiazole-2-yl)-2,5-diphenyl tetrazolium bromide (MTT).In addition, cytokine as but the generation that is not limited to IFN γ, tumor necrosis factor (TNF) and IL-2 can be used for showing the T cell proliferation.Production of cytokines should be higher than background level, common high 25 piks/ml, preferably high 100 piks/ml.
The T cell be the mediation two kinds of immunologic function-effector functions and regulatory function, secretory proteins (lymphokine) and kill and wound other cell (cytotoxicity)-lymphocyte.Effector function comprises reactivity), delayed hypersensitivity, allograft rejection, tumour immunity and graft versus host disease.Lymphokine produces and cytotoxicity is proved by T cytological effect thing function.The immunoglobulin that the regulatory function of T cell shows as cell-mediated Cytotoxic ability of other T of amplification and B cell produces.Regulatory function also needs the generation of lymphokine.T cell response induce stimulus object (as but be not limited to mitogen, antigen or agglutinin) produce IFN-(IFN γ).
In one embodiment of the invention, can followingly set up T cell line.PBL (1 * 10
6) and from the bonded B lymphoblast (1 * 10 of body DNP
5) in the flat culture plate in 24 holes, in lymphocytes culture medium, mix, cultivate after 7 days, and adding 100U/ml IL2 (Cetus Oncology, Emeryville, CA).The T cell culture that expands is stored in the culture medium that contains IL2, when needing it is separated, to keep in the 22mm diametric hole about 2 * 10
6The concentration of individual cell.Stimulated culture by adding again from the bonded B lymphoblast of body DNP in per 14 days.(Becton-Dickinson, San Jose CA) can determine phenotype by flow cytometry with monoclonal anti body surface (panel).CD8+ and separating by indirect elutriation of CD4+T cell finish, wherein, according to Wysoki, L.J. and V.L.Sato, institute of NAS newspaper, 1978,75,2844 method, the T cell attachment that makes anti-CD8 or anti-CD4 monoclonal antibody bag quilt with standard technique is on the plate of anti-immunoglobulin bag quilt, and this article is this complete quoting as a reference; Separate the cell that adheres to, and include but not limited to that with the stimulus object that DNP modifies the following stated, melanoma cells and β lymphoblast and IL2 expand.
For example,, obtain the T cell subsets of phenotype homogeneous, contain 2 * 10 in this culture medium by in the lymphocytes culture medium of round bottom microtitration plate, cultivating with the limiting dilution degree
5The allogeneic feeder cells of individual irradiation, 200 U/ml IL-2 and phytohemagglutinin.At replying the lymphoblastic multiplication capacity of B that DNP modifies, screening contains the hole of the lymphocyte colony of growth.Positive hole is expanded with IL2, and uses from per 14 days repetitive stimulations of the bonded B lymphoblast of body DNP.
Can be used as the effector lymphocyte and test peripheral blood lymphocyte (PBL).It is suspended in lymphocytes culture medium (RPMI-1640, the 10% people AB that merges
+Serum, insulin-transferrins-selenite media additive (Sigma Chemical Co.), 2mM L-glutaminate, 1% non essential amino acid, 25mM HEPES buffer, penicillin+streptomycin) in, and with 1 * 10
5The concentration of cells/well adds at the bottom of 96 hole circles in the microtitration plate.Also add irritation cell, comprising: 1) from body or allogeneic PBL, 2) by with Epstein-Barr virus transfection preparation from body or allogeneic B lymph matricyte system, 3) melanoma cells cultivated from body; By irradiation (5000R) deactivation.In the great majority experiment, effector: the stimulus object ratio is preferably 1: 1.Culture plate is at CO
237 ℃ of incubations are 5 days in the incubator; Use then
125(ICN Radiochemical, Costa Mesa CA) make these holes accept pulse 6 hours to the IUDR of I labelling, collect with automatic collecting device, and count with the γ calculating instrument.Calculate the meansigma methods of three repeating holes.Also detect the lymphproliferation response of the T cell of cultivating according to the method described above.
Obtain the also PBL of cold preservation from the patient when the maximum DTH that melts at the autogenous cell that DNP is modified is reactive, and the outer breeder reaction of detection bodies.Independent DNFB uses the cyclical effect cell that can not produce detectable amount.Anticipation reaction PBL (the 63rd day) after twice DNP-vaccine injection can detect, and will continue to detect during vaccine therapy, and this is based on the experience of the melanoma cells of former application DNP modification.
In order to detect production of cytokines, can be with 1 * 10 in the round bottom microtitration plate
5Cells/well adds the T cell.Add equal-energy stimulus (DNP modify from body B lymphoblast), incubation was collected supernatant after 18 hours.But ELISA kit measurement IFN-(Endogen, Boston, MA with the commodity acquisition; Sensitivity=5pg/ml).
In order to measure the MHC dependency of this reaction, before adding the effector lymphocyte, irritation cell can be the mhc class i (W6/32) of 10 μ g/ml or the precincubation of MHC II class (L243) monoclonal antibody 1 hour with concentration.Can detect the non-specific mouse immuning ball protein of same concentrations, as negative control.
The reactive CD8+T cell of DNP that obtains by a large amount of colonies of elutriation can by modify with DNP from body B lymphoblast repetitive stimulation, long-term (＞3 months) are incubated in the culture medium that contains IL-2; They keep stable phenotype, CD3+, CD8+.Article two, evidence shows that its reaction is that mhc class i is restrictive: 1) irritation cell and the precincubation of anti-I class framework antibody, IFN-capable of blocking produces, and can not block with anti-II antibody-like incubation, 2) the T cell can be replied the stimulus object of the allogeneic DNP modification of one or two HLA-A locus coupling, but can not reply the unmatched stimulus object of HLA-A.
For the IFN-of measuring the T cell produces, can from patient's blood, obtain lymphocyte.About 1,000,000 lymphocyte and mixing from the autologous melanoma cell membrane that DNP modifies are used for stimulating the T cell.Added the 100U/ml interleukin II in per 7 days.Expand the T cell by going down to posterity.Again stimulate the T cell with what DNP modified from the autologous melanoma cell membrane then.Obtain the T cell mass of enrichment, it can be to reacting from the autologous melanoma cell that DNP modifies.IFN-determination of yield according to the T cell stimulates.It has been generally acknowledged that it promptly is tangible that the IFN-that is higher than 15 piks/ml produces.
Further illustrate the present invention by the following example, its intention only is explanation, rather than desire is defined in these specific embodiments with the present invention.Embodiment 1 isolating melanoma film stimulates the T cells in vitro
After the foundation of T cell line is carried out the DNP-vaccine administration according to this method, from having the patient of strong delayed allergy (DTH) reaction, the autologous melanoma cell obtains peripheral blood lymphocyte (PBL) from what DNP-was modified.From blood, separate PBL by density gradient centrifugation, it is suspended in freezing culture medium, as contain among the RPMI-1640 of 2.5% human albumin and DMSO, freezing in the fridge of controlled speed, and be stored in the liquid nitrogen until using people such as (, 1995) Sato.
Set up T cell line from autologous melanoma cell (DNP-Mel) repetitive stimulation by these PBL by what modify, and keep people such as (, 1995) Sato with recombinant interleukin 2 (IL-2) with DNP-.
Melanoma cells as mentioned above, Enzymatic Extraction melanoma cells from the transfer piece that takes out by operation the same patient, and with preceding method cold preservation people such as (, 1997) Sato.Set up from autologous melanoma cell line by this melanoma cells suspension.In brief, enzyme process is isolated melanoma cells from shift piece, it is suspended in the tissue culture medium (TCM) (RPMI-1640 that contains hyclone or human serum), and is added in the tissue culturing plate.After a couple of days, remove the tumor cell that does not adhere to, and add fresh culture medium.After several weeks, the melanoma cells that adheres to begins fast breeding.When cell grows into the confluent cultures plate, by taking off cell, and add in the fresh tissue culturing plate and make cell separation with EDTA.
Method with Miller and Claman is modified this melanoma cell series cell with DNP.This comprises tumor cell and dinitrofluorobenzene (DNFB, Sigma Chemical Co.) incubation 30 minutes under aseptic condition, washes excessive DNFB off with Hanks solution subsequently.With the anti-DNP antibody of mouse monoclonal (SPE-7; Sigma Immunochemicals, St.Louis MO) confirms that through flow cytometry DNP modifies (100% cell demonstration is modified by DNP).
As a kind of selection method that is equipped with, can modify the melanoma cells of cold preservation as mentioned above with DNP, and need not set up the intermediate steps of cell line.
Cell membrane extracts with people's such as Heike method and extract cell membrane from the melanoma cells (DNP-Mel) that DNP modifies.In brief, by hypotonic shock in the 30mM of 5 times of volumes sodium bicarbonate buffer liquid, 1mM Phenylmethanesulfonyl fluoride (PMSF), and by Dounce homogenization (10-20 time), the cell that cracking DNP modifies.By removing remaining intact cell and nucleus in 5 minutes, do not contain nucleus and cell up to supernatant with the 1000g continuous centrifugal.Then, through 100, centrifugal 90 minutes precipitation membranes of 000g.With the precipitation in whole films with every milliliter 10
7Cell equivalent unit is (that is, from 10
7The film that extracts in the individual cell) be resuspended to 8% sucrose, 5mM Tris, among the pH7.6 ,-80 ℃ freezing until use down.
As a kind of selection method that is equipped with, with above-mentioned same procedure isolated cell film from the melanoma cells of unmodified.With film with different cell equivalent concentration (10
5-10
9Cell equivalent/ml) is suspended in and does not contain in the albuminous Hanks solution.Add DNFB then as mentioned above.Then, through 100, centrifugal 90 minutes precipitation membranes of 000g, and with twice of salt washing.
The cytokine that membrane product causes produces according to IFN-γ and produces, and measures the inductive t cell responses of melanoma film that DNP modifies.The T cell that will obtain from patient PBL is with 10
5The cells/well plating in culture plate at the bottom of 96 hole circles, every hole 100 μ l culture medium (adding the RPMI 1640 of 10% people AB serum, 2mM L-glutamic acid, 100mg/ml/100U/ml streptomycin/penicillin, 10mM HEPES, 1% non essential amino acid).In every hole, add not commensurability (about 10
5-Yue 10
8Cell equivalent) cell membrane, and adding other culture medium, to make the cumulative volume in every hole be 250 μ l.
Incubation is collected supernatant and is carried out IFN-γ mensuration after 18 hours.But ELISA test kit (Endogen, Boston, MA with the commodity acquisition; Sensitivity=5pg/ml) is measured the concentration of IFN-γ in the supernatant.
Produce (750pg/ml) with the IFN-γ that behind body DNP-Mel film incubation, detects tangible T cell.It is relevant that the IFN-γ of T cell produces the amount of the DNP-Mel film of incubation together.Do not cause significant reaction to the Mel film of unmodified.By setting up two kinds of T cell subbreed with positive panning technique enrichment CD4+ and CD8+T cell.Because IFN-γ produces, every kind of subbreed can react to the DNP-Mel film.The reaction of CD4+T subbreed and DNP-Mel film can be blocked by the antibody of MHC II class, and the reaction of CD8+ subbreed can be blocked (being respectively 73% and 80% blocking-up) by the antibody of mhc class i.
These results show that the tumor cell membrane of hapten transformation can be successfully used to inoculate the patient who needs oncotherapy.Embodiment 2: with the tumor cell membrane treatment III phase ovarian cancer of modifying
Can put into practice (extract (debulking) operation, follow) treatment patient at first by standard medical by chemotherapy.After treating that chemotherapy is finished, can use the vaccine of the ovarian cancer cell film that contains hapten dinitro benzene (DNP) modification and implement the treatment of 6 courses of treatment in week.Can use the cyclophosphamide of low dosage for the first time before the injection.After treating to finish the course of treatment, can detect patient's delayed hypersensitivity with cancer cell membrane unmodified that modify to DNP.With cold preservation extract from metastatic tumo(u)r and/or can carry out in vitro study from the isolating lymphocyte of peripheral blood.
For example, show after optionally connected patient who is subjected to surgical removal or the chemotherapy that the patient that tumor reduces treats.The tumor mass of downcutting from every patient enough obtains at least 100 * 10
6Individual tumor cell alive.This patient can accept chemotherapy, as carboplatin and paclitaxel, preferably tumor free clinically after chemotherapy is finished (that is, health check-up and CT research are normal, change of serum C A-125＜35IU/L).
The patient can not accept treatment of the present invention, and this is a foundation: for preparation vaccine and tuerculoderma tumor cell (＜100 * 10 in shortage
6Individual cell), be lower than 80 Karnovsky performance state, main region radiotherapy in 6 months in the past, the current of systemic corticosteroid used, hematocrit＜30% or WBC＜3000, age＜18, the activeness autoimmune disease, activeness severe infection, another kind of activeness malignant tumor, the evidence of hepatitis b virus infection (circulating antigen) or HIV (circulating antibody) maybe can not provide promise.
The patient is carried out the excision of tumor and the removal of metastasis.The patient who carries out removing in the best or suboptimum ground is qualified.Tumor tissues can be delivered to laboratory and processing to obtain film.This film can cold preservation and is stored in the liquid nitrogen.
Homologous and allochthonous tumor cell membrane all can prepare and use as described in this manual.
From performing the operation the back in six (6) weeks, the patient can begin chemotherapy, as application carboplatin or cisplatin+paclitaxel, according to following dosage: per 3 all carboplatin AUC 7.5mg/M
2Or cisplatin 75mg/M
2, per 3 all paclitaxel 175mg/M
2Intravenous was above 3 hours.Can implement six circulation chemotherapies.Also can implement any chemotherapy in addition.
After finishing around the chemotherapy approximately, can shift evaluation, comprise breast-abdomen-pelvis computer topography (CT) the patient.The patient who has only proof not have the recurrence cancer just is fit to select to carry out vaccine therapy.If CT research is negative for recurrence, then the patient of change of serum C A125 level rising is qualified.The tumor cell membrane treatment can implemented last at least 4 weeks of chemotherapy, begin after 12 weeks at the latest.
In the-7 days, available following material made a skin test to the patient: 1) DNP modify from body ovarian cancer cell or film, 2) diluent (the Hanks balanced salt solution that contains 0.1% human albumin) and 3) the PPD intermedium.Can measure the DTH reaction at the-5 days.At the 0th day, the patient can carry out 300mg/M
2The quick intravenous fluids of cyclophosphamide.After three days, but a kind of tumor cell film composition of their intradermal injection, and this can repeat weekly to continue for six (6) weeks.Vaccine may comprise the ovarian cancer cell film of modifying with the blended DNP of BCG.Can be in upper arm vaccinate.If excised left axillary gland for a certain reason, can use right arm.
After two weeks half of the 6th inoculation, can carry out clinical evaluation, comprise CBC, SMA-12, CA125 and chest X ray the patient.Can test that they modify the DTH:DNP of following material with unmodified from body cancerous cell, DNP autologous peripheral blood lymphocyte, diluent and PPD intermedium that modify and unmodified.Also can test tactiosensible property to dinitrofluorobenzene (DNFB).
Can in the time of 6th month, (when beginning, the vaccine plan count) the 7th (reinforcement) vaccine of patient that keeps not having recurrence.For every patient, the tumor cell membrane that can store at least one refrigerating bottle carries out 6th month booster injection.If estimate available cell number be not enough to carry out 6 times weekly vaccine add the reinforcement in June, then the beginning process of injection can be kept to 5 times weekly.Can after 1 year, use another time and strengthen vaccine, but but this is just in the cell time spent that capacity is arranged.Just before 1 year booster immunization, available autologous tumor cell or film make a skin test to the patient, determine whether to keep the immune level before it.The tumor cell membrane treatment melanoma that embodiment 3 usefulness are modified
Can handle tumor mass as previously mentioned.In brief, can be by dividing with collagenase and DNA enzyme enzymatic and extracting cell by mechanical disintegration.The isolated cell film is freezing in the fridge of controlled speed as described in this manual, and is stored in the liquid nitrogen and uses until needs.On treatment patient same day, melt, wash film, be resuspended to and do not contain in the phenol red Hanks balanced salt solution.Can carry out DNP according to the method for Miller and Claman (1976) modifies.This comprises tumor cell and dinitrofluorobenzene (DNFB) incubation 30 minutes under aseptic condition, washs with Sterile Saline subsequently.
This vaccine combination can contain minimum 2.5 * 10
6C.e. the melanoma cells film got rid of of trypan blue and the most how much 7.5 * 10
6C.e. melanoma cells film, it is suspended in the 0.2ml Hanks solution.Each vaccine injection can comprise three injections to adjacent regions.
Cryodesiccated material can be used 1ml sterilized water or phosphate buffered saline(PBS), and pH7.2 (PBS) rebuilds.Can in aseptic buffer salt solution, carry out suitable dilution.Drawing 0.1ml then before injection mixes with vaccine.For the first time and for the second time vaccine can mix with the Tice BCG (" Tice-1 ") of 0.1ml dilution in 1: 10.BCG is from Organon Teknika Corporation (Durham, NC) the Tice bacterial strain of Huo Deing (substrain of Institute Pasteur bacterial strain).Can mix with 0.1ml 1: 100 dilution (" Tice-3 ") with the 4th vaccine for the third time.The 5th time and the 6th time and strengthen vaccine and can mix with 0.1ml 1: 1000 dilution (" Tice-5 ").Ideal vaccine reaction is that little (＜5mm) the inflammatory pimple of center ulcer is just arranged.
Can make a skin test by injection 0.1ml substances in the forearm epithelium, and estimate 48 hours DTH by the average diameter of measuring scleroma.Can test following material: 1) 1 * 10
6Individual unmodified and with DNP modify from autologous melanoma cell membrane cell; Can use (TCM) tumor cell of enzymatic splitted (TCE) and mechanical disintegration; 2) 3 * 10
6Autologous peripheral blood lymphocyte individual unmodified and that modify with DNP; 3) Hanks solution; With 4) PPD intermedium intensity.By to upper arm outside of belly dermal administration 200 μ g DNFB and check that this zone at 48 hours scleroma circle, also can detect the tactiosensible property to DNFB.Can after six all vaccine administration processes, carry out a complete set of DTH test.Pretreatment DTH test only limits to melanoma cells film, PPD and the diluent that DNP modifies.This strategy is used for avoiding: the lymphocyte sensitization and 2 that the patient is modified DNP) tumor cell by the injection unmodified tolerates the patient.
When making a skin test, the blood that can collect all patients is used for separating and cold preservation of lymphocyte and serum at every turn.Can periodically detect: as by proliferation assay to from the replying of body cancerous cell, release of cytokines and cytotoxicity.
Can be before vaccine therapy begins the metastatic disease of assess patient.After treating that the vaccine therapy in 8 weeks finishes for the first time, every three months is once estimated.Can continue to estimate up to 1 year, the 3rd year per 4 months once, and per thereafter 6 months once.Each evaluation can check UP and conventional blood test (CBC, SMA-12 and CA125).Can be before using vaccine, (before the booster immunization) 6 months with carried out the CT of breast-abdomen-pelvis in 12 months, as clinical indication.Can determine survival rate that do not recur and total.All patients all can carry out at least 5 years or when death till.
Estimate that the patient can form the local response to BCG, comprise the tenderness pustule that discharge, 2-3 healed after the month, stay the cicatrix of similar variolation.When the patient formed the sensitivity of BCG, the intensity of these reactions may improve.In the patient of the vaccine that the inoculation hapten is handled not observe anaphylaxis, other allergia phenomenon and autoimmune.
The reaction of inoculation position can following classification: 0-is asymptomatic; 1-itches or is uncomfortable, but does not disturb arm motion or normal activity; 2-causes disturbing the arm motion but need not change the discomfort of normal activity; 3-causes mainly disturbing arm motion and needs to change the discomfort of normal activity; 4-causes carrying out with extremity the discomfort of normal activity.
Cyclophosphamide can be rebuild with sterilized water, and can use suitable dosage by quick intravenous fluids.Usually, about 1/3rd patient may be sick in the stomach after using low-dose cyclophosphamide, and about 10% may vomit.During this dosage leukopenia, alopecia and cystitis can not take place.Expect this scheme with lower nausea and vomiting incidence rate, use cyclophosphamide one time because may only combine with last vaccination.
Can after vaccinate, observe the patient.Allowing patient with unexpected symptom or sign get in touch the doctor estimates immediately.Cause the available acetyl aminophenol treatment of uncomfortable heating.The nauseating available oral prochlorperazine (prochlorperazine) that low-dose cyclophosphamide causes (prochlorperazine (Compazine)) treatment.If at inoculation position serious local response (＞5mm ulcer) takes place, then can reduce BCG dosage (on seeing) subsequently.
The patient who does not have recurrence during assessment in a year can accept last reinforcement vaccine injection.Can observe its state of an illness then and further do not treat.The patient who develops into transfer can no longer study, and treats (operation or chemotherapy usually) by clinical indication.
Can be used for also determining whether the DNP vaccine prolongs these patients' nothing recurrence and/or the effect research of total time-to-live.Can measure survival parameter (Ka-Mai (Kaplan-Meier) method).
Thomas Jefferson university, NIH and FDA are all observed about all regulations of promising to undertake.
We drew following result with the melanoma cells of hapten transformation to the research from the autologous melanoma vaccine that DNP modifies in the past: the patient (N=60) of treatment back 100% forms the positive DTH reaction (〉=5mm harden diameter) of the autologous tumor cell that DNP is modified, 85% formation strong positive reaction (〉=10mm harden diameter).That uses that DNP modifies has similar success from the expection of body cancer film vaccine, that is, the expection patient form to DNP that modify with the DTH from body cancer cell membrane unmodified.
From the narration of front as can be known, except that shown here and described, multiple modification of the present invention is obvious to those skilled in the art.These modifications also are within the scope of accessory claim book.
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U.S. Patent number 5,290,551
U.S. Patent Application Serial 08/203,004
U.S. Patent Application Serial 08/479,016
U.S. Patent Application Serial 08/899,905
U.S. Patent Application Serial 08/942,794
PCT?US?96／09511
Claims (52)
1. compositions that contains the human tumor cells film of hapten transformation.
2. the compositions of claim 1, the free tumor cell of wherein said tumor cell membrance separation from cancerous cell and non-solid tumor cell.
3. the compositions of claim 1, wherein said tumor cell membrane derives from the tumor that is selected from leukemia, lymphoma, multiple myeloma, ovarian cancer, colon cancer, rectal cancer, colorectal carcinoma, melanoma, breast carcinoma, pulmonary carcinoma, renal carcinoma and carcinoma of prostate.
4. the compositions of claim 3, wherein said leukemia is an acute myelogenous leukemia.
5. the compositions of claim 1, wherein said tumor cell membrane is the tumor cell membrane that is selected from homology tumor cell membrane and allogeneic tumor cell membrane.
6. the compositions of claim 5, wherein said homology tumor cell membrane is from body.
7. the compositions of claim 1, wherein said hapten is selected from: dinitro benzene, trinitrobenzene, N-iodoacetyl-N '-(5-sulfo group-1-naphthyl) ethylenediamine, trinitro-benzene-sulfonic acid, Fluorescein isothiocyanate, arsenic acid PhNCS, trinitro-benzene-sulfonic acid, sulfanilic acid, arsanilic acid, dinitro benzene-S-mustard and combination thereof.
8. the compositions of claim 1, it also contains adjuvant.
9. the compositions of claim 8, wherein said adjuvant is selected from the endotoxin and the cytokine of bacillus calmette-guerin vaccine, QS-21, detoxification.
10. the compositions of claim 1, it has when when suffering from people with the malignant tumor of described tumor cell membrane same type and use, and can cause the lymphocytic character of T of soaking into this patient's tumor.
11. the compositions of claim 1, it has when when suffering from people with the malignant tumor of described tumor cell membrane same type and use, and can cause the character to the inflammatory immunne response of this patient's tumor.
12. the compositions of claim 1, it has when when suffering from people with the malignant tumor of described tumor cell membrane same type and use, and can cause the character to the delayed hypersensitivity of this patient's tumor.
13. the compositions of claim 1, wherein said tumor cell membrane is allochthonous, and said composition further comprises a kind of antigen-presenting cell.
14. the compositions of claim 1, wherein said tumor cell membrane is allochthonous, and described antigen-presenting cell is homologous.
15. the compositions of claim 1, wherein said tumor cell membrane is allochthonous, and described antigen-presenting cell is from body.
16. a treatment method for cancer comprises the people is used a kind of compositions that contains the human tumor cells film of the hapten transformation for the treatment of effective dose that wherein this patient suffers from the malignant tumor with this tumor cell membrane same type.
17. the method for claim 16 comprises and causes the T lymphocyte that soaks into this patient's tumor and measure the T lymphocyte that soaks into this patient's tumor.
18. the method for claim 16 comprises initiation inflammatory immunne response and this inflammatory immunne response of mensuration to this patient's tumor.
19. the method for claim 16 comprises initiation delayed hypersensitivity and this delayed hypersensitivity of mensuration to this patient's tumor.
20. the method for claim 16, wherein said malignant tumor are selected from cancer and non-solid tumor.
21. the method for claim 16, wherein said malignant tumor is selected from leukemia, lymphoma, multiple myeloma, ovarian cancer, colon cancer, rectal cancer, colorectal carcinoma, melanoma, breast carcinoma, pulmonary carcinoma, renal carcinoma and carcinoma of prostate.
22. the method for claim 21, wherein said leukemia is an acute myelogenous leukemia.
23. the method for claim 16, wherein said tumor cell membrane are the tumor cell membranes that is selected from homology tumor cell membrane and allogeneic tumor cell membrane.
24. the method for claim 23, wherein said homology tumor cell membrane is from body.
25. the method for claim 16, wherein said hapten is selected from: dinitro benzene, trinitrobenzene, N-iodoacetyl-N '-(5-sulfo group-1-naphthyl) ethylenediamine, trinitro-benzene-sulfonic acid, Fluorescein isothiocyanate, arsenic acid PhNCS, trinitro-benzene-sulfonic acid, sulfanilic acid, arsanilic acid, dinitro benzene-S-mustard and combination thereof.
26. the method for claim 16, wherein said compositions also contains a kind of adjuvant.
27. the method for claim 26, wherein said adjuvant are selected from the endotoxin and the cytokine of bacillus calmette-guerin vaccine, QS-21, detoxification.
28. the method for claim 16, wherein said tumor cell membrane is allochthonous, and described compositions further contains a kind of antigen-presenting cell.
29. the method for claim 16, wherein said tumor cell membrane is allochthonous, and described antigen-presenting cell is homologous.
30. the method for claim 16, wherein said tumor cell membrane is allochthonous, and described antigen-presenting cell is from body.
31. one kind is caused the lymphocytic method of T of soaking into mammal tumor, comprise that to a kind of mammalian tumor cell film for the treatment of the hapten transformation of effective dose of this administration wherein this mammal suffers from a kind of and malignant tumor this tumor cell membrane same type.
32. the method for claim 31, wherein said mammal are people or the animal that is selected from dog, cat, cattle and equine.
33, a kind of method that causes the inflammatory immunne response of mammal tumor, comprise that to a kind of mammalian tumor cell film for the treatment of the hapten transformation of effective dose of this administration wherein this mammal suffers from a kind of and malignant tumor this tumor cell same type.
34. the method for claim 33, wherein said mammal are people or the animal that is selected from dog, cat, cattle and equine.
35. an initiation is to the method for the delayed hypersensitivity of mammal tumor, comprise that to a kind of mammalian tumor cell film for the treatment of the hapten transformation of effective dose of this administration wherein this mammal suffers from a kind of and malignant tumor this tumor cell membrane same type.
36. the method for claim 35, wherein said mammal are people or the animal that is selected from dog, cat, cattle and equine.
37. method for preparing the tumor cell membrane of hapten transformation, comprise that the cracking tumor cell obtains cracked tumor cell, from this cracked tumor cell, remove the tumor cell that nucleus obtains acellular nuclear, tumor cell by this acellular nuclear obtains acellular basically tumor cell membrane, and this tumor cell membrane is handled through a kind of hapten, obtain the tumor cell membrane of hapten transformation.
38. the method for claim 37, wherein said cracking are selected from hypotonic shock, mechanical disintegration and enzymatic division.
39. the method for claim 37, wherein said hapten is selected from: dinitro benzene, trinitrobenzene, N-iodoacetyl-N '-(5-sulfo group-1-naphthyl) ethylenediamine, trinitro-benzene-sulfonic acid, Fluorescein isothiocyanate, arsenic acid PhNCS, trinitro-benzene-sulfonic acid, sulfanilic acid, arsanilic acid, dinitro benzene-S-mustard and combination thereof.
40. the mammalian tumor cell film of an isolating hapten transformation.
41. the film of claim 40, wherein said mammal are people or the animal that is selected from dog, cat, cattle and equine.
42. the film of claim 41, wherein said mammal is the people.
43. the film of claim 42, wherein this membrance separation is from the tumor cell that is selected from cancerous cell and non-solid tumor cell.
44. the film of claim 42, wherein this is membrane derived in the tumor that is selected from leukemia, lymphoma, multiple myeloma, ovarian cancer, colon cancer, rectal cancer, colorectal carcinoma, melanoma, breast carcinoma, pulmonary carcinoma, renal carcinoma and carcinoma of prostate.
45. the film of claim 44, wherein said leukemia is an acute myelogenous leukemia.
46. the film of claim 40, it is selected from homology tumor cell membrane and allogeneic tumor cell membrane.
47. the film of claim 46, wherein said homology tumor cell membrane is from body.
48. the film of claim 40, wherein said hapten is selected from: dinitro benzene, trinitrobenzene, N-iodoacetyl-N '-(5-sulfo group-1-naphthyl) ethylenediamine, trinitro-benzene-sulfonic acid, Fluorescein isothiocyanate, arsenic acid PhNCS, trinitro-benzene-sulfonic acid, sulfanilic acid, arsanilic acid, dinitro benzene-S-mustard and combination thereof.
49. the film of claim 40, it has one of following character at least: (ⅰ) cause the T lymphocyte that soaks into mammal tumor, (ⅱ) initiation (ⅲ) causes to the delayed hypersensitivity of mammal tumor with (ⅳ) at stimulated in vitro T cell the inflammatory immunne response of mammal tumor.
50. the film of claim 40, wherein this film is a kind of epicyte.
51. the film of claim 40, wherein this film comprises the membrane portions that contains MHC molecule, heatshock protein or its combination.
52. compositions that comprises the film of claim 40.
Applications Claiming Priority (2)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| US2501298A | 1998-02-17 | 1998-02-17 | |
| US09/025,012 | 1998-02-17 |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN1291105A true CN1291105A (en) | 2001-04-11 |
Family
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Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN99803065A Pending CN1291105A (en) | 1998-02-17 | 1999-02-17 | Hapten-modified tumor cell membranes and preparation and method for using it |
Country Status (12)
| Country | Link |
|---|---|
| EP (1) | EP1054690A2 (en) |
| JP (1) | JP2002502880A (en) |
| KR (1) | KR20010041018A (en) |
| CN (1) | CN1291105A (en) |
| AR (1) | AR015237A1 (en) |
| AU (1) | AU2686999A (en) |
| BR (1) | BR9907912A (en) |
| CA (1) | CA2320969A1 (en) |
| IL (1) | IL137791A0 (en) |
| PL (1) | PL342856A1 (en) |
| WO (1) | WO1999040925A2 (en) |
| ZA (1) | ZA991245B (en) |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN115137814A (en) * | 2022-07-01 | 2022-10-04 | 可蓝赛生物医药(上海)有限公司 | Tumor vaccine adjuvant |
Families Citing this family (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CA2327339A1 (en) * | 1998-04-09 | 1999-10-21 | David Berd | A method of inducing an anti-tumor response against a lung metastasis in a melanoma patient |
| US6248585B1 (en) | 1998-11-19 | 2001-06-19 | Thomas Jefferson University | Compositions for preserving haptenized tumor cells for use in vaccines |
| JP2004507446A (en) * | 2000-02-04 | 2004-03-11 | トーマス・ジェファーソン・ユニバーシティ | Immunotherapy of low-dose haptenized tumor cells and tumor cell extracts |
| US7297330B2 (en) | 2000-02-04 | 2007-11-20 | Thomas Jefferson University | Low dose haptenized tumor cell and tumor cell extract immunotherapy |
| US7612251B2 (en) | 2000-09-26 | 2009-11-03 | Pioneer Hi-Bred International, Inc. | Nucleotide sequences mediating male fertility and method of using same |
| WO2003103389A2 (en) | 2002-06-10 | 2003-12-18 | Avax Technologies Inc. | Cryopreservation of haptenized tumor cells |
| US11214603B2 (en) | 2016-04-27 | 2022-01-04 | The Regents Of The University Of Michigan | C3D cellular and acellular vaccines for the prevention and treatment of cancer |
Family Cites Families (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US4086134A (en) * | 1973-09-18 | 1978-04-25 | University Of Glasgow | Method for preparation of vaccine against feline leukemia |
| CA2222135A1 (en) * | 1995-06-07 | 1996-12-19 | Thomas Jefferson University | Hapten modified tumor cell extract and methods of treating or screening for cancer |
-
1999
- 1999-02-17 EP EP99907137A patent/EP1054690A2/en not_active Withdrawn
- 1999-02-17 IL IL13779199A patent/IL137791A0/en unknown
- 1999-02-17 ZA ZA9901245A patent/ZA991245B/en unknown
- 1999-02-17 WO PCT/US1999/003536 patent/WO1999040925A2/en not_active Application Discontinuation
- 1999-02-17 AU AU26869/99A patent/AU2686999A/en not_active Abandoned
- 1999-02-17 CN CN99803065A patent/CN1291105A/en active Pending
- 1999-02-17 KR KR1020007009049A patent/KR20010041018A/en not_active Application Discontinuation
- 1999-02-17 JP JP2000531176A patent/JP2002502880A/en not_active Withdrawn
- 1999-02-17 CA CA002320969A patent/CA2320969A1/en not_active Abandoned
- 1999-02-17 BR BR9907912-7A patent/BR9907912A/en not_active Application Discontinuation
- 1999-02-17 PL PL99342856A patent/PL342856A1/en not_active Application Discontinuation
- 1999-02-23 AR ARP990100710A patent/AR015237A1/en unknown
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN115137814A (en) * | 2022-07-01 | 2022-10-04 | 可蓝赛生物医药(上海)有限公司 | Tumor vaccine adjuvant |
Also Published As
| Publication number | Publication date |
|---|---|
| BR9907912A (en) | 2000-10-24 |
| KR20010041018A (en) | 2001-05-15 |
| IL137791A0 (en) | 2001-10-31 |
| ZA991245B (en) | 1999-08-18 |
| WO1999040925A2 (en) | 1999-08-19 |
| PL342856A1 (en) | 2001-07-16 |
| AU2686999A (en) | 1999-08-30 |
| AR015237A1 (en) | 2001-04-18 |
| JP2002502880A (en) | 2002-01-29 |
| WO1999040925A3 (en) | 1999-10-21 |
| CA2320969A1 (en) | 1999-08-19 |
| EP1054690A2 (en) | 2000-11-29 |
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