EP0906444A1 - Cellules dendritiques humaines genetiquement transfectees, leur production et leur utilisation, de preference comme vaccins - Google Patents

Cellules dendritiques humaines genetiquement transfectees, leur production et leur utilisation, de preference comme vaccins

Info

Publication number
EP0906444A1
EP0906444A1 EP97922844A EP97922844A EP0906444A1 EP 0906444 A1 EP0906444 A1 EP 0906444A1 EP 97922844 A EP97922844 A EP 97922844A EP 97922844 A EP97922844 A EP 97922844A EP 0906444 A1 EP0906444 A1 EP 0906444A1
Authority
EP
European Patent Office
Prior art keywords
dendritic cells
human
gene
mucin
cells
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.)
Withdrawn
Application number
EP97922844A
Other languages
German (de)
English (en)
Inventor
Gabriele Pecher
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Individual
Original Assignee
Individual
Priority date (The priority date 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 date listed.)
Filing date
Publication date
Priority claimed from DE19617837A external-priority patent/DE19617837A1/de
Priority claimed from DE1996117846 external-priority patent/DE19617846A1/de
Application filed by Individual filed Critical Individual
Publication of EP0906444A1 publication Critical patent/EP0906444A1/fr
Withdrawn legal-status Critical Current

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Classifications

    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
    • C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
    • C07K14/4701—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals not used
    • C07K14/4727—Mucins, e.g. human intestinal mucin
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K40/00—Cellular immunotherapy
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K40/00—Cellular immunotherapy
    • A61K40/10—Cellular immunotherapy characterised by the cell type used
    • A61K40/19—Dendritic cells
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K40/00—Cellular immunotherapy
    • A61K40/20—Cellular immunotherapy characterised by the effect or the function of the cells
    • A61K40/24—Antigen-presenting cells [APC]
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K40/00—Cellular immunotherapy
    • A61K40/40—Cellular immunotherapy characterised by antigens that are targeted or presented by cells of the immune system
    • A61K40/41—Vertebrate antigens
    • A61K40/42—Cancer antigens
    • A61K40/4256—Tumor associated carbohydrates
    • A61K40/4257—Mucins, e.g. MUC-1
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K2239/00—Indexing codes associated with cellular immunotherapy of group A61K40/00
    • A61K2239/46—Indexing codes associated with cellular immunotherapy of group A61K40/00 characterised by the cancer treated
    • 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
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K48/00—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy

Definitions

  • the invention relates to gene-transfected human dendritic cells.
  • Gene-transfected dendritic cells can be used in basic research as well as in the construction of vaccines, e.g. B. tumor vaccines, find application. An efficient gene transfer to human dendritic cells has not been described so far.
  • the invention further relates to a method for producing these cells and their use, preferably as a vaccine.
  • the glycoprotein mucin encoded by the MUC1 gene, is expressed on the surface of pancreatic, Mamraa, colon, Parotis and ovarian carcinomas as well as on the corresponding healthy cells.
  • Muzin encoded by MUC1 consists of two thirds of 20 to 100 "tandem nucleotide repeats".
  • a "tandem nucleotide repeat” consists of 60 nucleotides that encode a polypeptide of 20 amino acids (see Fig. 2).
  • peptide epitopes are exposed on tumor cells and can be recognized as foreign by the immune system, in particular T cells (these peptide epitopes are "hidden” by healthy carbohydrates on cells and therefore dissolve normal cells no immune response). These mucin epitopes are suitable for stimulating the immune system to protect the body against a tumor.
  • Electroporation was used as the transfection method, a method which is not always successful and in which a large number of cells die.
  • EBV-immortalized B cells have been used as immune cells and transfected with MUCl vectors and used to stimulate the immune system (described in Jerome KR, N. Domenech, and OJ Finn. 1993. Tumor-specific cytotoxic T cell clones from patients with breast and pancreatic adenocarcinoma recognize EBV-immortalized B cells tranfected with polymorphyc epitheal mucin complementary DNA. J. of Immunol. 151: 1654-1662 and in Pecher G. and Finn O. j. 1996. Induction of cellular immunity in chimpanzees to tumor-associated antigen mucin by vaccination with MUC1 cDNA-transfected EBV-immortalized autologous B-cells.
  • EBV Epstein-Barr virus
  • the aim of the invention is to provide gene-transfected human dendritic cells.
  • These cells are also to be genetically engineered to develop a vaccine which specifically stimulates the immune system against tumor cells already present in the body and which is intended to reduce or eliminate the tumor.
  • "professional” immune cells for the expression of tumor-associated epitopes are to be used to construct a vaccine.
  • Certain "professional” immune cells, in contrast to tumor cells, express the costimulatory ligands necessary for optimal T cell activation, such as CD80 and CD86.
  • the essential feature of the manufacturing process is the transfection of the foreign gene into the dendrial cells using liposomes.
  • the method according to the invention is efficient, simple to carry out, safe to use and, in comparison to, for example, retroviral gene transfer, inexpensive.
  • the vaccine consists of human autologous dendritic cells which are transfected with a partial sequence of the human mucin MUCl gene, which contains several "tandem repeat nucleotide sequences" from the MUCl (FIG. 2), by means of lipofectin using the plasmid, and which are by Treatment with the glycosylation inhibitor phenyl-N-acetyl- ⁇ -D-galactosaminide express tumor-associated epitopes.
  • the MUCl transfected cells are treated with the glycosylation inhibitor phenyl-N-acetyl- ⁇ -D-galactosaminide for 24 to 36 hours so that the immunogenic, tumor-associated mucin epitopes are formed.
  • the Expression can be checked by a FACS analysis using mucin-epitope-specific antibodies.
  • the invention also relates to the vector pCMV / MUCl according to FIG. 1 for transfection of the dendritic cells, which consists of the following essential components:
  • CMV cytomegalovirus
  • the vaccine according to the invention has the following advantages / innovations compared to previous tumor vaccines:
  • the vaccine does not contain tumor cells, but a clearly defined antigen (MUCl).
  • Immune cells used to construct the vaccine are dendritic cells.
  • dendritic cells do not produce any immunosuppressive substances such as interleukin 10.
  • Human dendritic cells are produced from peripheral blood of patients or healthy people using interleukin 4 and granulocyte macrophage colony stimulating factor. This is a simple and easy to practice procedure.
  • the transfection method is lipofection. A high gene transfer rate in dendritic cells is achieved. The method is easy to carry out and reproducible.
  • PCMV / MUCl according to Fig.l is used as the vector for gene transfer.
  • the corresponding mucin cDNA was cloned into the vector under the immediate early promoter of CMV.
  • the vector contains no cDNA for resistance to antibiotics or the like. The vector thus fulfills high requirements Safety requirements for human use.
  • Another advantage of the vaccine according to the invention is that the recognition of the mucin-peptide epitopes by cytotoxic T cells does not follow the previously known classic way of recognizing short peptide epitopes in connection with the HLA complex.
  • the mucin-peptide epitopes are recognized by the T cells without "help" from the HLA complex.
  • This peculiarity in the detection of tumor-associated mucin epitopes can be explained by the above-mentioned special "tandem repeat" structure of the molecule and the high density of the antigen on the presenting cell.
  • the repeated repetition of the immunogenic peptide-epitope motif leads to an activation of the T cells by "crosslinking" the T cell receptor without the HLA complex having to be present.
  • peptide epitopes are exposed on tumor cells that can be recognized as foreign by the immune system.
  • the activation of the immune system triggered by this is not sufficient in tumor patients to eliminate the tumor because (due to the lack of expression of CD80 and CD86 on tumor cells) there is no costimulation of T cells.
  • the vaccine according to the invention triggers an efficient, tumor-specific immune response, which is based on the activation of mucine epitope-specific, cytotoxic T cells. These T cells lead to downsizing or elimination of the tumor cells.
  • dendritic cells are transfected with MUCl (copies of the tandem nucleotide sequence of MUCl cloned into the vector) and, if appropriate, treated with the glycosylation inhibitor phenyl-N-acetyl- ⁇ -D-galactosaminide, these express the tumor-associated epitopes.
  • MUCl copies of the tandem nucleotide sequence of MUCl cloned into the vector
  • Dendritic cells are isolated from human peripheral blood and cultured. On day 4 of the culture of the dendritic cells, the dendritic cells are transfected. For this a vector is used which contains CMV as a promoter for the foreign gene MUCl. 15 ⁇ l lipofectin (Fig. 3) is used for 750,000 dendritic cells. The successful transfection of the foreign gene MUCl is detected by means of FACS analysis with the monoclonal antibody HMFG-2 against mucin epitopes. After transfection, 12% of the dendritic cells show an expression of mucin epitopes. By using a glycosylation inhibitor (Gl), mucine epitopes can be detected on the surface of 48% of the dendritic cells. This marks the successful gene transfer. Even without using the glycosylation inhibitor, there are already sufficient immunogenic mucin epitopes on the surface.
  • Gl glycosylation inhibitor
  • Mock (vector without foreign gene) transfected cells express mucin epitopes to a maximum of 2% (see Fig. 4)
  • Lymphocytes are obtained from human peripheral blood by Ficoll gradient centrifugation and kept in culture. Dendritic cells are selected by the addition of interleukin 4 and granulocyte-macrophage colony stimulating factor and by adherence to platik. The dendritic cells are transfected with the MUCl vector using liposomes. Mucin expression is checked using Western blot methods and FACS analysis with monoclonal mucin antibodies. Phenyl-N-acetyl- ⁇ -D-galactosarainide (concentration 5 mM) is added to the culture medium of the transfected cells for 36 hours. The expression of the tumor-associated mucin-peptide epitopes generated in this way persists for 72 hours and is checked with monoclonal mucin-peptide antibodies by means of FACS analysis. The vaccine is applied to the patient within these 72 hours.
  • the vaccine can be used for therapy in patients with mucin (MUCl) -expressing tumors.
  • MUCl mucin
  • the treatment of breast, pancreas, ovarian, colon and parotid tumors is preferred.
  • This vaccine can also be used in healthy people to prevent a tumor expressing mucine epitopes.

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  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • General Health & Medical Sciences (AREA)
  • Epidemiology (AREA)
  • Animal Behavior & Ethology (AREA)
  • Public Health (AREA)
  • Veterinary Medicine (AREA)
  • Chemical & Material Sciences (AREA)
  • Organic Chemistry (AREA)
  • Zoology (AREA)
  • Gastroenterology & Hepatology (AREA)
  • Toxicology (AREA)
  • Biochemistry (AREA)
  • Biophysics (AREA)
  • Genetics & Genomics (AREA)
  • Medicinal Chemistry (AREA)
  • Molecular Biology (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Medicines Containing Antibodies Or Antigens For Use As Internal Diagnostic Agents (AREA)
  • Micro-Organisms Or Cultivation Processes Thereof (AREA)

Abstract

L'invention concerne des cellules dendritiques humaines génétiquement transfectées, leur production et leur utilisation, de préférence comme vaccins. Ces cellules ont des applications dans l'industrie pharmaceutique et en médecine. Ces cellules dendritiques sont produites par transfection avec un gène étranger au moyen de liposomes, de préférence au moyen de lipofectine, qui est une préparation de liposomes. Le vaccin décrit est constitué de cellules dendritiques autologues humaines transfectées par une séquence partielle du gène humain de mucine-MUC1 qui contient plusieurs séquences de nucléotides de MUC1 répétées en tandem, des épitopes associés à des tumeurs étant créés dans ces cellules, de préférence sur leur surface, au moyen d'un inhibiteur de la glycosylation.
EP97922844A 1996-04-19 1997-04-14 Cellules dendritiques humaines genetiquement transfectees, leur production et leur utilisation, de preference comme vaccins Withdrawn EP0906444A1 (fr)

Applications Claiming Priority (5)

Application Number Priority Date Filing Date Title
DE19617846 1996-04-19
DE19617837A DE19617837A1 (de) 1996-04-19 1996-04-19 Dendritische Zellen transfiziert mit Muzin-cDNA als Vakzine gegen humane Tumorerkrankungen
DE19617837 1996-04-19
DE1996117846 DE19617846A1 (de) 1996-04-19 1996-04-19 Verfahren zur Gentransfektion von humanen dendritischen Zellen
PCT/DE1997/000772 WO1997040182A1 (fr) 1996-04-19 1997-04-14 Cellules dendritiques humaines genetiquement transfectees, leur production et leur utilisation, de preference comme vaccins

Publications (1)

Publication Number Publication Date
EP0906444A1 true EP0906444A1 (fr) 1999-04-07

Family

ID=26025378

Family Applications (1)

Application Number Title Priority Date Filing Date
EP97922844A Withdrawn EP0906444A1 (fr) 1996-04-19 1997-04-14 Cellules dendritiques humaines genetiquement transfectees, leur production et leur utilisation, de preference comme vaccins

Country Status (2)

Country Link
EP (1) EP0906444A1 (fr)
WO (1) WO1997040182A1 (fr)

Families Citing this family (12)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5834015A (en) * 1996-09-11 1998-11-10 Albany Medical College Protein-lipid vesicles and autogenous vaccine comprising the same
CA2282300C (fr) 1997-02-24 2011-08-02 Therion Biologics Corporation Poxvirus recombine destine a une immunisation contre l'antigene associe aux tumeurs muc1
US5965381A (en) * 1998-03-06 1999-10-12 Ludwig Institute For Cancer Research Delivery of proteins into eukaryotic cells with recombinant yersinia
EP1068296B1 (fr) 1998-03-31 2011-08-10 Geron Corporation Compositions permettant de faire apparaitre une reponse immunitaire a un antigene de telomerase
US7402307B2 (en) 1998-03-31 2008-07-22 Geron Corporation Method for identifying and killing cancer cells
DE10048710A1 (de) * 1999-09-27 2001-10-04 Gabriele Pecher Pharmazeutische Zusammensetzung zur Behandlung und Prophylaxe von humanen Tumoren, die das Tumorantigen Muzin und/oder das Carcinoembryonale Antigen (CEA) exprimieren und ihre Verwendung
DK1530628T3 (da) 2002-08-16 2011-01-10 Glycotope Gmbh Fremgangsmåde til fremstilling af temperaturinducerede tumorcellelysater til anvendelse som immunogene forbindelser
SI1654353T1 (sl) 2003-08-18 2013-09-30 Glycotope Gmbh Tumorski celični liniji NM-F9 (DSM ACC2606) in NM-D4 (DSM ACC2605), njihove uporabe
EP3539981A1 (fr) 2006-09-10 2019-09-18 Glycotope GmbH Utilisation de cellules humaines d'origine leucémique myéloïde pour l'expression d'anticorps
PL1920781T3 (pl) 2006-11-10 2015-06-30 Glycotope Gmbh Kompozycje zawierające core-1-dodatnie mikroorganizmy i ich zastosowanie w leczeniu lub profilaktyce nowotworów
ES2686313T3 (es) 2011-08-22 2018-10-17 Glycotope Gmbh Microorganismos que portan un antígeno tumoral
SG11202010496WA (en) 2018-05-18 2020-12-30 Daiichi Sankyo Co Ltd Anti-muc1 antibody-drug conjugate

Family Cites Families (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
AU6504596A (en) * 1995-07-21 1997-02-18 Rhone-Poulenc Rorer Pharmaceuticals Inc. Adeno-associated viral liposomes and their use in transfecting dendritic cells to stimulate specific immunity

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
See references of WO9740182A1 *

Also Published As

Publication number Publication date
WO1997040182A1 (fr) 1997-10-30

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