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WO1997038109A1 - Rejet immunologique de cellules tumorales transfectees - Google Patents

Rejet immunologique de cellules tumorales transfectees Download PDF

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Publication number
WO1997038109A1
WO1997038109A1 PCT/AU1997/000214 AU9700214W WO9738109A1 WO 1997038109 A1 WO1997038109 A1 WO 1997038109A1 AU 9700214 W AU9700214 W AU 9700214W WO 9738109 A1 WO9738109 A1 WO 9738109A1
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WO
WIPO (PCT)
Prior art keywords
cells
gal
cell
transfected
galactosyltransferase
Prior art date
Application number
PCT/AU1997/000214
Other languages
English (en)
Inventor
David Anthony Power
Bruce Ernest Kemp
Original Assignee
St. Vincent Institute Of Medical Research
St. Vincent's Hospital
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
Application filed by St. Vincent Institute Of Medical Research, St. Vincent's Hospital filed Critical St. Vincent Institute Of Medical Research
Priority to AU21454/97A priority Critical patent/AU2145497A/en
Publication of WO1997038109A1 publication Critical patent/WO1997038109A1/fr

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/10—Transferases (2.)
    • C12N9/1048—Glycosyltransferases (2.4)
    • C12N9/1051—Hexosyltransferases (2.4.1)
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • 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 present invention relates to a method of inducing an immune response against human tumour cells.
  • the present invention also relates to methods of treatment of tumours in humans.
  • Various non-surgical methods of treating tumours in humans have been developed and trialed in recent years. Such methods include, for example, radiotherapy and/or chemotherapy; photoradiation therapy; thermotherapy, neutron therapy; the use of monoclonal antibodies for selectively identifying and destroying neoplastic cells; and the administration of recombinant hormones and cytokines such as ⁇ -interferon, ⁇ -interferon and interleukin-2.
  • a natural anti-Gal antibody is present in the serum of all humans. This anti-Gal antibody constitutes approximately 1% of circulating IgG in humans and interacts specifically with the Gal (1,3) Gal epitope (Galili et al (1993): Hamedeh et al (1992)).
  • porcine ⁇ (1,3) galactosyltransferase gene has been cloned and sequenced and is described in Strahan et al (1995) and PCT/AU94/00126. the entire disclosures of which are incorporated herein by reference.
  • Another immune response which leads to cell destruction occurs in the transfusion of blood into a recipient who has antibodies against the donor blood components.
  • An individual of a particular ABO blood group can recognise red cells carrying different blood group antigens and produce antibodies to them. Antibodies may be produced naturally, without immunisation with the foreign red cells. Table 1 indicates the genotypes and antigens of the ABO system. Most people naturally make antibodies to the antigens they lack.
  • the antigenic properties of these blood-group substances are determined by terminal sugars on an oligosaccharide chain built up of D- galactose, D-glucose, N-acetyl-D-glucosamine. N-acetyl-galactosamine and L- fucose. Briefly, the enzyme produced by the H transferase gene attaches a fucose residue (Fuc) to the terminal galactose (Gal) of the precursor oligosaccharide. Individuals possessing the A transferase gene (encoding ⁇ -
  • N-acetyl-D-galactosaminyltransferase attach N-acetyl galactosamine (NAGA) to this galactose residue while those with the B transferase gene (encoding ⁇ - D-galactosyltransferase) attach another galactose producing A and B antigens respectively.
  • the present inventors have found that human tumour cells stably transfected with a polynucleotide encoding porcine ⁇ (1,3) galactosyltransferase are more susceptible to lysis by human serum than non- transfected tumour cells.
  • tumour cells stably transfected with a polynucleotide encoding an appropriate glycosyltransferase involved in the formation of the A and/or B blood group antigens will also be more susceptible to lysis by human serum.
  • the present invention provides a method of inducing an immune response against a human tumour cell which method includes introducing into the tumour cell a polynucleotide encoding a glycosyltransferase such that the glycosyltransferase sequence is expressed in the tumour cell.
  • the present invention provides a method of treatment of tumours in humans which method includes introducing into the tumour cells a polynucleotide encoding a glycosyltransferase such that the glycosyltransferase sequence is expressed in the tumour cells.
  • the glycosyltransferase is ⁇ -1,3 galactosyltransferase.
  • the ⁇ -1.3 galactosyltransferase is a porcine ⁇ -1,3 galactosyltransferase.
  • the glycosyltransferase is selected from ⁇ -N-acetyl-D-galactosaminyltransferase and ⁇ -D-galactosyltransferase.
  • CD55 DAF - decay- accelerating factor
  • CD46 MCP - membrane cofactor protein
  • CD59 membrane inhibitor of reactive lysis
  • the method further includes treating the tumour cells with a suppressor of a natural inhibitor of complement activation.
  • Suppressors of these inhibitors include antibodies which bind to the inhibitory proteins.
  • the suppressor may, for example, be a monoclonal antibody which binds to the inhibitory protein CD55. CD46 or CD59.
  • the polynucleotide encoding the glycosyltransferase may be introduced into the tumour cell by any suitable method of gene delivery. Suitable methods of gene delivery may, for example, involve the use of viral vectors. Viruses can be modified to carry a desired gene and become vectors for gene delivery. International publication nos. WO 95/07994 and WO 94/29469 disclose vectors which have been adapted for delivery and expression of polynucleotides which encode immunogenic or therapeutic peptides. These vectors are particularly suitable for gene delivery to tumour cells.
  • a desired gene may be inserted into a bacterial plasmid along with promoter, enhancer and other sequences that enable the gene to be expressed in human cells.
  • the plasmid DNA can be incorporated into lipid vesicles (Liposomes including cationic lipids such as lipofectin) which then transfer the DNA into the target cell.
  • DNA can also be complexed with proteins that target the DNA to specific tissues in the same way as certain proteins are taken up (endocytosed) by specific cells.
  • International publication No. WO 94/23751 describes a nucleic acid transfer peptide for efficient delivery of nucleic acid to cells which is particularly suitable for use in gene therapy of tumours.
  • Another gene delivery technique involves "shooting" naked DNA on small gold beads into a cell using a "gun". It will be understood that the introduction of a polynucleotide encoding ⁇ (l,3) galactosyltransferase into a tumour cell according to the first aspect of the invention will result in the formation of the Gal (1,3) Gal epitope on the surface of the transfected cell.
  • Use of the Gal (1,3) Gal epitope provides advantages over the use of other epitopes (such as HLA-B7) in regard to the induction of an immune response in humans.
  • the first advantage resides in the fact that humans produce a natural antibody, anti- Gal, which interacts specifically with ⁇ -galactosyl epitopes and which constitutes up to 1% of circulating immunoglobulins in humans.
  • the Gal (1,3) Gal epitope is produced in abundance ( > 10 fi per cell) on cells of non-primate animals which naturally express the ⁇ (1,3) galactosyltransferase enzyme (Galili et al. (1988); Thall et al. (1991). Accordingly, human tumour cells expressing the Gal (1.3) Gal epitope are likely to be quickly recognised as foreign and strongly attacked by the immune system in a manner analogous to rejection of xenograft transplants.
  • Gal (1,3) Gal epitope causes cell death. It is possible, however, that the presence of the epitope induces antibody dependent cell-mediated cytotoxicity (ADCC). This mechanism involves binding of cells with cytotoxic potential to target cells coated with an antibody. ADCC generally causes rapid cell death and has been implicated in reported instances of cell destruction by anti-gal antibodies. For example, Galili (1993) demonstrated the destructive effect of anti-Gal on non primate cells in an in vitro ADCC assay.
  • Tumour cells expressing the A and/or B antigens are likely to be quickly recognised and attacked in humans who naturally produce the anti-A or anti-B antibodies.
  • the tumour cells are engineered to produce the antigen which is reactive with the naturally occurring antibodies in the individual.
  • a tumour in an individual of blood group A is preferably transfected with a polynucleotide encoding ⁇ -D-galactosyltransferase which gives rise to the B antigen.
  • individuals of the blood group phenotype AB do not naturally produce anti-A or anti-B antibodies. These individuals are therefore not ideal subjects in relation to this aspect of the invention.
  • individuals of the AB phenotype are relatively rare, occurring at a frequency of only around 4%
  • anti-Gal. anti-A and/or anti-B antibodies occur naturally in most individuals, it would be possible to boost levels of any one of these antibodies by immunisation with the corresponding antigen. Higher levels of the antibodies would presumably lead to a quicker and more effective attack on tumour cells transfected with either the u-1.3 galactosyltransferase, ⁇ -N- acetvl-D-galactosaminyltransferase and/or ⁇ -D-galactosyltransferase genes.
  • A431 is an epidermoid carcinoma cell line. It was grown in 10% fetal calf serum in D-MEM. Cells were passaged by trypsinisation with 0.1% trypsin/EDTA.
  • A431 cells were transfected using the DEAE-dextran method and transfected cells selected using media containing the antibiotic G418. After 2 weeks in culture, cells were subcloned and colonies selected according to the results of FACS analysis using FITC-labelled IB4 lectin (Sigma), which detects the gal ( ⁇ l,3) gal linkage.
  • a line (A431.gal) was derived after two rounds of cloning. 75% of cells from the line were positive in flow cytometric assay using FITC-conjugated IB4 lectin, when compared with non-transfected cells.
  • A431.gal or control non-transfected A431 cells were incubated for 30 mins at 23°C with 0-50% (v/v) normal human serum (NHS; blood group AB) and then washed xl in wash buffer. They were then analysed for (1) complement binding to the cell surface using a flow cytometric assay for C3c and C9, and (2) cell viability using (i) uptake of the vital dye calcein AM (Molecular Probes) by flow cytometry. and (ii) cell proliferation by incorporation of 3 H- thymidine.
  • NHS normal human serum
  • CAM is a nonfluorescent cell permeable substrate that is converted in the presence of intracellular esterase to intensely fluorescent calcein.
  • This assay was used to detect the number of live cells.
  • Calcein AM is a red fluorochrome which is a vital dye. so that only viable cells will take it up. The assay was performed using the manufacturer's instructions. Briefly, l ⁇ M calcein AM was added to 3xl0 5 cells in a volume of lOO ⁇ l for 20 mins at 23°C, washed and analysed on the flow cytometer.
  • A431.gal or A431 cells were grown in 24-well plates and then treated with 0-50% human AB serum for 30 mins at 23"C, the medium was changed and the cells then pulsed with I ⁇ Ci of 3 H-thymidine for 4 hours. Cells were harvested using NaOH and 50 ⁇ l added to liquid scintillant and counted in a beta counter.
  • A431 cells An initial step was assay of A431 cells for the presence of the membrane regulatory proteins CD55 (DAF), CD46 (MCP), and CD59 by flow cytometry. These proteins are found on many human cell types and are thought to act as natural inhibitors of complement activation and cell lysis. By FACS analysis, A431 cells were found to express high levels of all 3 of these proteins. This was important as future studies blocking the function of these inhibitory molecules could lead to increased susceptibility of the cells to lysis.
  • DAF membrane regulatory proteins
  • A431 cells did not possess detectable C3c or C9 on the cell surface.
  • Transfected A431.gal cells showed some binding of C3c with concentrations of NHS ⁇ 10%. However, even with very high serum concentration (up to 50%), C9 was not detected.
  • Non-transfected and transfected cells were placed in wells with NHS. Thymidine was added to the wells and after 4 hours cells were harvested and thymidine uptake was assessed. There was no significant difference in the rate of thymidine uptake between the transfected and non-transfected cells indicating negligible lysis of the transfected A431.gal cells.
  • A431 and A431.gal cells were incubated with varying concentration of NHS for 4 hours and then labelled with CAM. No detectable cell death occurred and there was no difference between transfected and non-transfected cells.
  • FACs were performed (as described in Example 1) on the MDA-gal clones, non-transfected MDA cells, A431-gal cells and non-transfected A431 cells to determine the levels of gal expression.
  • Gal clones were treated with FITC-labeled IB4 lectin, binding to the gal epitope. Fluorescence was measured using FACs.
  • MDA-gal clone L was chosen.
  • A431-gal cells showed a more depressed level of gal expression compared to MDA-gal I, ⁇ . or E with greater spread or variation within the population.
  • FACs was performed on the MDA and A431 cells to determine the presence and levels of membrane regulatory proteins CD55 (DAF).
  • CD55 CD46
  • MCP membrane regulatory proteins
  • CD59 thought to act as natural inhibitors of complement activation and cell lysis.
  • Both the MDA and A431 cell lines showed high levels of all three. CD55 and CD46 expression was the highest and lowest respectively. Levels of expression in both the MDA and A431 cells were very similar.
  • Cytotoxicity assays were done on the MDA and A431 cell lines, using the lactic dehydrogenase (LDH) release method. Cells were transfected with either galactosyltransferase or control construct. Two lines were used for MDA (one high and one low level expressor). and one line for A431.
  • LDH lactic dehydrogenase
  • LDH assays were performed. There was greater lysis in the cells transfected with galactosyltransferase (12-18% and 36-47% for the two transfected lines), compared with the control (3-8%).

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Abstract

La présente invention concerne un procédé d'induction d'une réponse immune dirigée contre des cellules tumorales humaines, lequel peut être utile dans le traitement de tumeurs chez l'homme. Ce procédé consiste à transfecter les cellules tumorales à l'aide d'un polynucléotide codant une glycosyltransférase, laquelle peut être choisie parmi une α-1,3 galactosyltransférase, une α-N-acétyl-D-galactosaminyltransférase et une α-D-galactosyltransférase.
PCT/AU1997/000214 1996-04-03 1997-04-03 Rejet immunologique de cellules tumorales transfectees WO1997038109A1 (fr)

Priority Applications (1)

Application Number Priority Date Filing Date Title
AU21454/97A AU2145497A (en) 1996-04-03 1997-04-03 Immunological rejection of transfected tumour cells

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
AUPN9123 1996-04-03
AUPN9123A AUPN912396A0 (en) 1996-04-03 1996-04-03 Complement mediated rejection of transfected tumour cells

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WO1997038109A1 true WO1997038109A1 (fr) 1997-10-16

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Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2002042468A3 (fr) * 2000-11-27 2002-11-21 Geron Corp Vecteurs de glycosyltransferase pour traitement du cancer
US9458231B2 (en) 2010-09-03 2016-10-04 Stemcentrx, Inc. Modulators and methods of use

Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1991003484A1 (fr) * 1989-08-31 1991-03-21 The Biomembrane Institute Determination du genotype abo

Patent Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1991003484A1 (fr) * 1989-08-31 1991-03-21 The Biomembrane Institute Determination du genotype abo

Non-Patent Citations (4)

* Cited by examiner, † Cited by third party
Title
BIOCHEMICAL & BIOPHYSICAL RESEARCH COMMUNICATIONS, 15 May 1991, Volume 176, Number 3, L. MANGLE-GAW et al., "Genomic Structure & Expression of Human beta-1,4-Galactosyltransferase". *
CANCER RESEARCH, 1 July 1996, Volume 56, D.C. LATEMPLE et al., "Synthesis of alpha-Galactosyl Epitopes by Recombinant alpha1,3-Galactosyltransferase for Opsonization of Human Tumor Cell Vaccines by Anti-galactose", pages 3069-3074. *
GLYCOBIOLOGY, 1995, Volume 5, Number 8, U. GALILI & F. ANARAKI, "alpha-Galactosyl (Gal alpha1-3Galbeta1 - 4G1cNAC-R) Epitopes on Human Cells: Synthesis of the Epitope on Human Red Cells by Recombinant Primate alpha1,3Galactosyltransferase Expressed in E-Coli", pages 775-782. *
INTERNATIONAL JOURNAL OF BIOCHEMISTRY, 1995, Volume 27, Number 3, S.K. CHATTERJEE et al., "Analysis of the Sequences of Human beta-1,4-Galactosyltransferase cDNA Clones", pages 329-336. *

Cited By (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2002042468A3 (fr) * 2000-11-27 2002-11-21 Geron Corp Vecteurs de glycosyltransferase pour traitement du cancer
US6713055B2 (en) * 2000-11-27 2004-03-30 Geron Corporation Glycosyltransferase vectors for treating cancer
US9458231B2 (en) 2010-09-03 2016-10-04 Stemcentrx, Inc. Modulators and methods of use
US10017565B2 (en) 2010-09-03 2018-07-10 Abbvie Stemcentrx Llc Modulators and methods of use

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