EP2387415A1 - Impfstoffzusammensetzungen - Google Patents

Impfstoffzusammensetzungen

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Publication number
EP2387415A1
EP2387415A1 EP10708697A EP10708697A EP2387415A1 EP 2387415 A1 EP2387415 A1 EP 2387415A1 EP 10708697 A EP10708697 A EP 10708697A EP 10708697 A EP10708697 A EP 10708697A EP 2387415 A1 EP2387415 A1 EP 2387415A1
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EP
European Patent Office
Prior art keywords
cells
composition
cytokine
cancer
renca
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EP10708697A
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English (en)
French (fr)
Inventor
Andrzej Mackiewicz
Piotr Wysocki
Wiktoria Suchorska
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Agirx Ltd
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Agirx Ltd
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Priority to EP10708697A priority Critical patent/EP2387415A1/de
Publication of EP2387415A1 publication Critical patent/EP2387415A1/de
Withdrawn legal-status Critical Current

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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
    • A61K39/001116—Receptors for cytokines
    • 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
    • A61K39/001136—Cytokines
    • 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
    • A61K2039/51—Medicinal preparations containing antigens or antibodies comprising whole cells, viruses or DNA/RNA
    • A61K2039/515—Animal cells
    • A61K2039/5152—Tumor cells
    • 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
    • A61K2039/51—Medicinal preparations containing antigens or antibodies comprising whole cells, viruses or DNA/RNA
    • A61K2039/515—Animal cells
    • A61K2039/5156—Animal cells expressing foreign proteins
    • 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
    • A61K2039/555—Medicinal preparations containing antigens or antibodies characterised by a specific combination antigen/adjuvant
    • A61K2039/55511—Organic adjuvants
    • A61K2039/55522—Cytokines; Lymphokines; Interferons

Definitions

  • the invention relates to tumour therapy.
  • the present invention relates to vaccine compositions comprising allogenic cells modified with hypercytokines for the prophylaxis and treatment of cancer in general and in particular for the treatment of melanoma and renal cell cancer.
  • Cytokines and cytokine receptors Interleukin-11 (IL-I l) together with interleukin-6 (IL-6), Leukaemia Inhibitor Factor
  • Cardiotrophin 1 belongs to the family of hemopoietic cytokines (named IL-6-type or gpl30 cytokines), which share structural similarity and a common receptor subunit (gpl30) (Bazan et al., 1990). Although, each of the IL-6-type cytokines requires a specific (unique) receptor complex, at least one molecule of gpl30 is always engaged. Initially a ligand (IL-6, IL-I l, CNTF) binds specifically to its non- signaling receptor ⁇ subunit and next recruits the signaling receptor chain.
  • IL-6, IL-I l, CNTF binds specifically to its non- signaling receptor ⁇ subunit and next recruits the signaling receptor chain.
  • IL-6 and IL-1 1 use a gpl30 homodimer for transducing the signal, while LIF, CNTF, CT-I utilize a heterodimer gpl30/LIFR.
  • OSM either recruits gpl30/OSMR or gpl30/LIFR heterodimers (reviewed in Heinrich et al., 2003, Bravo et al., 2000).
  • the tertiary structure of IL-6-type cytokines has been intensely investigated during recent years.
  • IL-6-type cytokines have identified three receptor binding sites (termed I, II, III), which seem to be conserved among the gpl30 family (reviewed in Bravo et al, 2000).
  • Site I which enables ligand to bind to its non-signaling receptor, is formed by amino acids from the C-terminal part of the A-B loop and the C-terminal residues of the D helix.
  • Site II seems to be a universal g ⁇ l30 binding site for all members of IL-6-type cytokines and consists of exposed residues on helices A and C.
  • Site III is composed of an N-terminal half of helix D, the N- terminal part of the A-B loop and amino acid residues of the end of the C-D loop. This site is always occupied by the second signaling receptor: gpl30, LIFR or OSMR, depending upon the identity of the ligand.
  • the receptors involved in IL-6-type cytokine signaling belong to the type I membrane proteins. They possess an extracellular N-terminus and one transmembrane domain (with the exception of CNTFR, which is linked to the membrane by a lipid anchor (Davis et al, 1991). Because of a common structural motif in their extracellular region, they are classified as cytokine receptor class I family (Bazan et al, 1990). This family is characterized by the presence of at least one cytokine binding homology domain (CHD) consisting of two fibronectin-type-III-like domains (FNIII) termed D2 and D3.
  • CHD cytokine binding homology domain
  • FNIII fibronectin-type-III-like domains
  • the CHD is composed of approximately 200 amino acids, with four positionally conserved cysteine residues at the N-terminal domain and a characteristic conserved Trp-Ser-X-T ⁇ -Ser (WSXWS) motif at the C-terminal domain. Additionally each receptor subunit contains an Ig-like domain, which is located at the N-terminus of the membrane-proximal CHD.
  • the IL-6-type receptors are divided into two groups: ⁇ and ⁇ subunits. Receptors ⁇ (for IL-6, IL-I l and CNTF) are not involved in signal transduction.
  • Subunits ⁇ the signal transducing receptor chains, contain a considerably larger cytoplasmic part than ⁇ subunits and have three membrane-proximal FNIII domains that may play some role such as in stabilization and/or in orientation of the transmembrane receptor dimers (reviewed in Bravo et al, 2000, Heinrich et al, 2003).
  • membrane bound IL-6-type receptor subunits their soluble forms were found in biological fluids (reviewed in Marz et al, 1999). They are formed either by limited proteolysis (shedding) of membrane-bound receptors or by translation from differentially spliced mRNA.
  • Hyper- IL-6 is another example of a new designer agent, which consists of D2 and D3 domain of IL-6 receptor (IL-6 R) ⁇ chain connected to IL-6 via a polypeptide linker (Fischer et al, 1997 and WO 97/32891).
  • IL-6 soluble IL-6 receptor
  • sIL-6 R soluble IL-6 receptor
  • the average half-life of the IL-6/sIL-6 R complex might be shorter than the time needed to assemble the IL-6/sIL-6 R/gpl30 complex (Wells et al., 1996).
  • the stability of the IL-6/sIL-6 R complex was enhanced by linking both components in order to create a fusion protein (Hyper-IL-6) (WO 97/32891). Hyper-IL-6 can directly bind to its signal transducing receptor subunit and enhance IL-6 activity.
  • Hyper-IL-6 is a fully active fusion protein, which mediates response at 100 to 1000-fold lower dose compared to the combination of soluble IL-6 and sIL-6 R molecules (Fischer et al., 1997).
  • another superagonist has been designed for IL-6-type family named IL-11/R-FP (Plan et al, 1999).
  • IL-11/R-FP was created by covalently linking D2 and D3 domains of IL-I l R (position L/109 - G/318) with IL- 11 (position P/29 - L/199) using a 21 amino acid linker and demonstrated 50-fold higher activity in vitro than the combination of IL-I l and sIL-11 R.
  • this construct was composed of truncated segments of the human IL-I l R and IL-I l and, thus, lacks naturally existing parts of the respective receptor and cytokine.
  • the artificial linker used is no naturally occurring sequence, which contributes to the immunogenicity of IL-11/R-FP when used for treatment of human patients.
  • WO 99/02552 A2 (Yeda Research and Development Co. Ltd. (Revel M. et al.) "Chimeric interleukin-6 soluble receptor/ligand protein, analogs thereof and uses thereof, published 21 Jan 1999) relates to chimeric proteins comprising a fusion protein product of sIL-6R and IL-6 and biologically active analogs of such proteins. In these chimeric proteins sIL-6R may be directly fused to IL-6 or via specific linker peptides. WO 99/02552 A2 further discusses a potential use of said chimeric proteins or analogs as inhibitors of cancer cells.
  • chimeric proteins for the preparation of a medicament for treating mammalian cancers, for enhancement of bone marrow transplantation, for increasing hematopoeisis, or for treating liver or neurological disorders.
  • the specific fusion proteins sIL-6R/IL-6 and sIL-6R ⁇ Val/IL-6 produced and examined in the Example section of WO 99/02552 have also been studied in an article by Chebath et al. (1997).
  • a review article by Kallen KJ. et al. discusses the potential therapeutic applications of interleukin-6 hyperagonists and antagonists.
  • Said therapeutic applications comprise haematologic disorders, solid malignancies, cardiac ischaemia and transplantation, bone disease, glomerulonephritis and amyloidosis, acquired immunodeficiency syndrome, rheumatic disorders, autoimmunity, burns and major trauma, anaemia, expansion of immature haematopoietic stem cells in bone marrow transplantation and tumour therapy, inducing thrombopoiesis and liver regeneration.
  • Fusion proteins as those described above which comprise a cytokine and its physiological receptor are sometimes also called "hypercytokines" due to their high activity at lower doses as compared to the individual cytokine and/or a mixture of the cytokine with its soluble receptor.
  • RCC Renal Cell Cancer
  • tumour cell-based vaccines consisting of tumour cells admixed with a particular adjuvant (e.g., bacillus Calmette-Guerin, Corynebacterium parvum or IFNs); genetically modified tumour vaccines based on tumour cells expressing genes encoding immunostimulatory factors; and DCs modified with tumour-derived RNA, loaded with peptides/tumour lysates or fused with tumour cells.
  • adjuvant e.g., bacillus Calmette-Guerin, Corynebacterium parvum or IFNs
  • tumour vaccines e.g., bacillus Calmette-Guerin, Corynebacterium parvum or IFNs
  • tumour vaccines e.g., bacillus Calmette-Guerin, Corynebacterium parvum or IFNs
  • tumour vaccines e.g., bacillus Calmette-Guerin, Corynebacterium parvum or IFNs
  • tumour vaccines
  • Cancer vaccines based on irradiated tumor cells deliver a wide spectrum of tumor antigens and may efficiently activate anti-tumor immune responses (Mach, N. and Dranoff, G, 2000; Ward, S. et al., 2002).
  • Immunostimulatory potential of irradiated whole-cell vaccines depend on the uptake of tumor antigens by dendritic cells (DCs) and their presentation in regional lymph nodes to activate antigen-specific (as well as bystander) helper and cytotoxic T cells (Scheffer, S. R. et al., 2003).
  • DCs dendritic cells
  • Transduction of vaccine cells with genes encoding immunostimulatory molecules can substantially increase their immunogenicity.
  • IL-6 interleukin-6
  • IL-6R IL-6 receptor
  • gp80 membrane- bound subunits - ⁇
  • gpl30 membrane- bound subunits - ⁇ (gp80)
  • gpl30 membrane- bound subunits - ⁇ (gp80)
  • gpl30 unlike the gp80 subunit is expressed on all cells in the body so far studied (Taga, T. and Kishimoto, T., 1997).
  • sIL-6R A soluble variant of gp80 (sIL-6R) exists in human plasma. With IL-6 it may form a dynamic complex which directly stimulates gpl30-expressing cells (Mackiewicz, A. et al., 1992).
  • tumour vaccines designed for mass scale production.
  • Such tumour vaccines consist of established allogenic tumour cells which are irradiated and injected into tumour-bearing patients, hi studies with tumour cells genetically modified to express hypercytokines the inventors surprisingly found that a composition comprising two different genetically modified tumour cell lines has a synergistic effect at least on the IL-2 and INF - ⁇ production of peripheral blood lymphocytes. This increased production of IL-2 and INF - ⁇ causes a beneficial shift of the immune response towards a ThI immune response which is connected with cytotoxic activity.
  • the compositions of the present invention comprising a first and a second allogenic cell line genetically modified to express the same or different hypercytokines will therefore be better suited as medicaments for the treatment of tumours as those known from the prior art.
  • compositions comprising just one tumour cell line genetically modified to express a hypercytokine is already effective in the prophylaxis of cancer, such as renal cell cancer.
  • This composition is also a suitable drug for the treatment of cancer and for preventing recurrence of cancer.
  • the present invention relates to a composition
  • a composition comprising (1) one or more first cells modified to express a first hyper-cytokine and (2) one or more second cells modified to express a second hyper-cytokine, wherein the one or more second cells are different from the one or more first cells.
  • the present invention relates to a composition
  • a composition comprising (1) one or more first cells modified to express a first hyper-cytokine.
  • the present invention relates to a composition according to the first or second aspect for use in medicine.
  • the present invention relates to a pharmaceutical composition
  • a pharmaceutical composition comprising a composition according to the first, the second, or the third aspect additionally comprising pharmaceutically acceptable diluents, carriers, excipients, fillers, binders, lubricants, glidants, disintegrants, adsorbents, and/or preservatives.
  • the present invention relates to the composition according to the first, the second, or the third aspect or the pharmaceutical composition according to the fourth aspect for the treatment of cancer, the prevention of cancer, and/or the prevention of recurrence of cancer.
  • the present invention relates to the use of a composition according to the first, the second, or the third aspect for the preparation of a pharmaceutical composition for the treatment of cancer, the prevention of cancer and/or the prevention of recurrence of cancer.
  • Fig. 1 shows the T-cell proliferative response in an allogenic mixed tumour-lymphocyte reaction (AMTLR).
  • AMTLR allogenic mixed tumour-lymphocyte reaction
  • Fig. 2 shows the effect of anti-IL-2 antibody on the T-cell proliferation in an allogenic mixed tumour-lymphocyte reaction (AMTLR).
  • AMTLR allogenic mixed tumour-lymphocyte reaction
  • Fig. 3 shows the cytokine secretion by Michl-H6 and Mich2-H6 cells expressed as MFI and pg/ml.
  • Fig. 4 shows the cytokine secretion by PBLC isolated from healthy individuals expressed as MFI and pg/ml.
  • Fig. 5 shows results from the stimulation of PBLC cytokine production by Michl-H6 cells, by Mich2-H6 cells, and by a combination of Michl-H6 and Mich2-H6 cells. The results are expressed as MFI and in pg/ml.
  • Fig. 6 shows the influence of vaccination on the survival of mice bearing kidney RENCA tumors.
  • Fig. 6A Animals immunized prophylactically with two various placebo vaccines showed similar survival rates. RENCA w/t ⁇ ⁇ • ; RENCA-Ad ⁇ 7001 - ⁇ -; control (non-immunized) animals-*".
  • Fig. 6C Therapeutic immunization with RENCA-H6 vaccine initiated 7 days after subcapsular implantation of RENCA cells did not influence survival of mice.
  • An arrow i indicates start of immunotherapy RENCA-H6 "* * ; RENCA w/t • ⁇ • ; control (non-immunized)-*-.
  • Fig. 6D Immunotherapy with RENCA-H6 vaccine initiated 24 hours following subcapsular implantation of RENCA cells significantly prolonged survival of kidney tumor-bearing animals.
  • RENC A-H6 vs. RENCA-w/t (p 0.004)
  • Fig. 7 shows the influence of the post-nephrectomy administration of vaccine on the survival rate of mice bearing RENCA kidney tumors.
  • Fig. 7A Adjuvant treatment with RENCA-H6 vaccine significantly prolonged survival of nephrectomized animals. Arrows: 4 indicates nephrectomy, and i initiation of immunotherapy.
  • RENCA-H6 "A * ; RENCA w/t • ⁇ • ; control (non-immunized)-*-.
  • RENCA-H6 vs. RENCA-w/t (p 0.03).
  • nephrectomized mice cured with RENC A-H6 vaccine completely rejected subcutaneously implanted RENCA but not Meth-A tumors.
  • Fig. 8 shows an evaluation of the RENCA-H6 vaccine stimulated antigen-specific immune response at the site of injection.
  • Fig. 8A MatrigelTM 'tumors' containing RENCA-H6 vaccine were infiltrated by a significantly smaller population of Treg cells (CD4+CD25+Foxp3+) when compared with RENCA-w/t vaccine (placebo).
  • RENCA-H6 group 32.7% of CD4+CD25+ T cells expressed Foxp3 molecule while 89.2% in the placebo group.
  • Fig. 8B Compared to placebo, RENC A-H6 vaccine attracted a higher number of activated, mature DCs expressing CD40, CD80 and CD86 costimulatory molecules.
  • Fig 9 shows a flow cytometry analysis of T lymphocytes infiltrating s.c. RENCA tumors. Repeated immunization with RENCA-H6 vaccine increased the number of memory T lymphocytes CD4+CD62L low and CD8+ CD62L low in rejected subcutaneous tumors implanted distally from the vaccination site.
  • Fig. 10 shows a flow cytometry analysis of T lymphocytes infiltrating s.c.
  • RENCA tumors in control (non-immunized) and in mice repeatedly immunized (5 times) with RENCA- w/t (placebo) and RENCA-H6 vaccine.
  • RENCA tumors contained higher percentage of CD4+CD43+, CD8+CD43+ and CD8+CD69 T lymphocytes than non-immunized and placebo-immunized animals.
  • Fig. 11 shows that immunization with RENC A-H6 vaccine activates non-specific and antigen-specific cellular immune responses.
  • Fig. HB Splenocytes from non- immunized, RENCA-GFP and RENCA-H6-GFP immunized mice were stimulated with GFP peptide followed by anti-CD8 plus pentamer staining and then analyzed by flow cytometry.
  • Fig. 12 shows tumor growth after s.c. injection of RenCa wild-type and RenCa-Hl 1 cells (Fig. 12A). The survival time of animals after s.c. injection RenCa wild-type and RenCa-Hl l cells is shown in Fig. 12B.
  • Fig. 13 shows tumor growth after s.c. injection of RenCa wild-type and RenCa-Hl 1 cells in animals immunized with RenCa- WT and RenCa-Hl 1.
  • Fig. 14 shows survival time of animals after treatment with RenCa wild-type and RenCa- HI l cells (s.c, IxIO 6 cells).
  • Fig. 15 shows survival time of animals immunized with RenCaWT and RenCa-Hl 1 cells after subcapsular injection of RenCa wild-type cells (into left kidney 5xlO 4 cells).
  • Fig. 16 shows survival time of animals after subcapsular injection of RenCa wild-type cells (into left kidney 5x10 4 cells) after nephrectomy and adjuvant treatment with RenCa- WT and RenCa-Hl 1 cells (1x10 6 cells).
  • Fig. 17 shows survival time of animals after subcapsular injection of RenCa wild-type cells (into left kidney 5xlO 4 cells) after immunotherapy with RenCa-WT and RenCa-Hl l cells (IxIO 6 cells). Treatment: 9 vaccinations every 2 days.
  • Fig. 18 shows survival time of animals after subcapsular injection of RenCa wild-type cells (into left kidney 5x10 4 cells) after immunotherapy with RenCa- WT and RenCa-Hl l cells (1x10 6 cells). Treatment was continued after 9 th vaccination.
  • Fig. 19 shows detection of antigen-specific CD8+ T cells after immunization RenCa/GFP Or RenCa/GFP-Hl 1.
  • RenCa/GFP-Hl As a positive control splenocytes from RenCa/GFP-H6 animals were used.
  • Fig. 20 shows detection of Treg population.
  • Fig. 21 shows the flow cytometry analysis of dendritic cells infiltrated GMTV cells in matrigel: A) no difference in CDl lc+CD80+ population, B) activation of CDl lc+CD86+ cells.
  • Fig. 22 shows the expression CD54 adhesion molecule on CDl Ic+ cells infiltrating RenCa vaccine cells.
  • Fig. 23 shows an analysis of CD4+CD28+ and CD8+CD28+ expression in cells infiltrating tumors.
  • Fig. 24 shows an analysis of CD43 expression and CD69 expression on CD4+ cells (A) and CD8+ cells (B) infiltrating tumor cells.
  • Fig. 25 shows an analysis of CD62L expression on CD4+ cells and CD8+ cells infiltrating tumor cells.
  • Fig. 26 shows a flow cytometry analysis of population of NK cells infiltrating tumor in group of animals immunized RenCa- WT and RenCa-Hl 1.
  • Fig. 27 shows bioimaging scans of mice that had been challenged with cells capable of emitting luminescence signals.
  • Fig. 27A Day 0; dose: IxIO 7 irradiated cells; exposition time: 10 seconds.
  • Fig. 27B Day 0; dose: O.5xlO 7 irradiated cells; exposition time: 10 seconds.
  • Fig. 27C Day 0; dose: 0.1x10 7 irradiated cells; exposition time: 10 seconds.
  • Fig. 27D Day 7; dose (from left to right): IxIO 7 , 0.5xl0 7 , O.lxlO 7 irradiated cells; exposition time: 60 seconds.
  • Fig. 27E Day 14; dose (from left to right): IxIO 7 , 0.5xl0 7 , O.lxlO 7 irradiated cells, negative control (no cells); exposition time: 60 seconds.
  • Fig. 27F Day 0; dose (from left to right): 2xlO 7 , IxIO 7 , O.5xlO 7 , O.lxlO 7 non-irradiated cells; exposition time: 10 seconds.
  • Fig. 27G Day 7; dose (from left to right): 2xlO 7 , IxIO 7 , O.5xlO 7 , O.lxlO 7 non-irradiated cells; exposition time: 10 seconds.
  • Fig. 27H Day 7; dose (from left to right): 0.5xl0 7 , O.lxlO 7 non-irradiated cells; exposition time:
  • Fig. 271 Day 13; dose (from left to right): 2xlO 7 , IxIO 7 , O.5xlO 7 , O.lxlO 7 irradiated cells; exposition time: 10 seconds.
  • the terms used herein are defined as described in "A multilingual glossary of biotechnological terms: (IUPAC Recommendations)", Leuenberger, H. G. W, Nagel, B. and K ⁇ lbl, H. eds. (1995), Helvetica Chimica Acta, CH-4010 Basel, Switzerland).
  • hypo-cytokine refers to a fusion protein comprising, essentially consisting or consisting of (a) a soluble part of a cytokine receptor, and (b) a cytokine which can bind under physiological conditions to said soluble part of a cytokine receptor and an optional peptide linker positioned between the soluble cytokine receptor and the cytokine.
  • said cytokine is GM-CSF, IL-6, IL-I l, IL-15, anti-TGF, EPO, interferon, LIF, OSM, CNTF, CT- 1.
  • soluble cytokine receptor refers to a soluble fragment of the cytokine receptor, e.g.
  • the receptor fragment is soluble, if it is not or essentially not inserted into the membrane of a mammalian cell, preferably a human cell, expressing the receptor fragment. If the cytokine receptor is located N-terminally with respect to the cytokine it is preferred that the cytokine receptor still comprises its secretion signal, which will be cleaved during maturation of the protein, i.e. the mature hypercytokine protein will not comprise the secretion signal.
  • the cytokine receptor is located C-terminally with respect to the cytokine it is preferred that the cytokine receptor does not comprise its secretion signal.
  • the hyper-cytokine optionally may comprise a peptide linker positioned between the cytokine receptor and the cytokine.
  • said peptide linker has a low immunogenicity or is non-immunogenic. More preferably, said peptide linker is non-immunogenic to human beings.
  • the soluble cytokine receptor is located at the amino-terminal part of the hyper-cytokine and the cytokine is located at the carboxy-terminal part of the hypercytokine.
  • hyper-cytokine activity refers to the activity of the fusion protein. While particularly preferred hypercytokines have based on the same molar amount a 100- to 1000-fold higher activity in the same assays as the cytokine on which they are based or as a mixture of the cytokine and the cytokine receptor, i.e. the unfused parts forming the hypercytokine, not every hypercytokine will show such a dramatic improvement, which will depend among others on the length of the parts of cytokine and soluble cytokine receptor included and the length of the protein linker, if any, present.
  • it has at the same molar amount at least 20-fold, 30-fold, 40-fold, 50-fold, 60-fold, 70-fold, 80-fold, 90-fold, 100-fold, 150-fold, 200-fold, 250-fold, 300-fold, 350-fold, 400-fold, 450-fold, 500-folde, 550-fold, 600-fold, 650- fold, 700-fold, 750-fold, 800-fold, 850-fold, 900-fold, 950-fold or 1000-fold the activity of the cytokine on which it is based or of a combination of the cytokine and the soluble cytokine receptor.
  • Suitable assay systems include, e.g. for IL-6 hypercytokine the induction of proliferation of BAF-3/cells as described in Fischer M. et al. (1997).
  • sequence identity used throughout the specification preferably refers to a sequence identity of at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% to the respective reference polypeptide.
  • sequence identity is to be calculated with reference to the longer of the two sequences to be compared. If the reference sequence is indicated, the sequence identity is determined on the basis of the full length of the reference sequence indicated by SEQ ID.
  • a peptide sequence consisting of 21 amino acids compared to the amino acids of full length IL-6 according to SEQ ID NO: 2 may exhibit a maximum sequence identity percentage of 9.9% (21 : 212) while a sequence with a length of 106 amino acids may exhibit a maximum sequence identity percentage of 50% (106:212).
  • HMMER package http://hmmer.wustl.edu/
  • CLUSTAL algorithm Thimpson J.D. et al., 1994
  • sequence matching may be calculated using e.g. BLAST, BLAT or BlastZ (or BlastX).
  • sequence matching analysis may be supplemented by established homology mapping techniques like Shuffle-LAGAN (Brudno M., 2003) or Markov random fields.
  • a “peptide linker” in the context of the present invention refers to an amino acid sequence of between 1 and 100 amino acids.
  • a peptide linker according to the present invention has a minimum length of at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 amino acids.
  • a peptide linker according to the present invention has a maximum length of 100, 95, 90, 85, 80, 75, 70, 65, 60, 55, 50, 45, 40, 35, 34, 33, 32, 31, 30, 29, 28, 27, 26, 25, 24, 23, 22, 21, 20, 19, 18, 17, 16, or 15 amino acids or less.
  • the above-indicated preferred minimum and maximum lengths of the peptide linker according to the present invention may be combined, if such a combination makes mathematically sense.
  • the peptide linker of the present invention is non-immunogenic; in particularly preferred embodiments, the peptide linker is non- immunogenic to humans.
  • RENCA refers to a murine renal carcinoma cell line.
  • the spellings “RENCA”, “Renca” and “RenCa” are used interchangeably herein.
  • the present invention provides a composition comprising, essentially consisting, or consisting of:
  • one or more second cells modified to express a second hyper-cytokine, wherein the one or more second cells are different from the one or more first cells.
  • the present invention provides a composition comprising, essentially consisting, or consisting of: (1) one or more first cells modified to express a first hyper-cytokine.
  • the second cells are different from the first cells, if the first and the second cells are derived from different cell lines and/or if the second cells carry a different genetic modification than the first cells, e.g. first and second cells have been modified to express different hypercytokines.
  • the first and second cells are derived from tissue of two different individuals, preferably from two different humans. It is preferred that the two tissues, preferably tumour tissues are of the same type.
  • tissue refers to both solid tissue like, e.g. skin, liver, brain, kidney, lung, stomach, colon, bladder, or testes, as well as mobile cell populations like, e.g. lymphocytes, or stem cells.
  • the cells are autologous or allogenic, it is particularly preferred that the first and/or the second cells are allogenic.
  • allogenic or "allogeneic” in an alternative spelling characterizes the relation between the cells and a patient receiving the cells. Cells from a particular individual will be allogenic to any other patient, while they will be autologous to that particular individual. Allogenicity is a prerequisite for industrial large scale production of any cell based vaccine, since otherwise each cellular vaccine would have to be produced individually from cells isolated and cultured from the respective patient to be treated. Allogenic cells provide additional advantages, which include that allogenic cells tend to induce a stronger immune response in a patient than autologous cells.
  • first cells and second cells refer to an individual cell, to a clonal population of that cell and to an assortment of similar cells.
  • the cells are derived from primary tissue, preferably a primary tumour
  • the cells will not all be clonal, but will be composed of one, two, three or more clonal cell populations belonging to a particular cell and/or tumour type.
  • tumour cells show a high genetic variability upon propagation and, thus, it is common that cells within one established cell line are not entirely identical genetically. These cells are an example of an assortment of similar cells.
  • Another example are primary tumour cells originating from one tumour, which have undergone 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more cycles of subcultivation in vitro, which will lead to selection of proliferating cell subtypes and, thus, to reduction of heterogeneity of the cell population, i.e. render the assortment of cells more similar.
  • the first and/or second cells are derived from primary tissue, preferably the same type of primary tissue, in particular tumour tissue, the first and second cells are considered different, if they are derived from two different individuals, preferably from two different humans.
  • modified to express indicates that a gene encoding the respective hyper- cytokine has been stably introduced into the cell in a form which allows stable expression of the gene encoding the hypercytokine and, subsequently, production of the respective hypercytokine.
  • the gene encoding the hypercytokine is introduced into an expression vector for use in mammalian cells, which ordinarily include an origin of replication (as necessary, see below), a promoter located in front of the gene to be expressed, optionally an enhancer in trans, along with any necessary ribosome binding sites, RNA splice sites, polyadenylation site, and transcriptional terminator sequences.
  • an expression vector may then be used to modify the cell to express the respective hypercytokine.
  • the expression vector of the present invention comprises, essentially consists or consists of plasmids; phagemids; phages; cosmids; artificial chromosomes, in particular artificial mammalian chromosomes or artificial yeast chromosomes; knock-out or knock-in constructs; viruses, in particular adenovirus, vaccinia virus, attenuated vaccinia virus, canary pox virus, lentivirus (Chang and Gay, 2001), herpes virus, in particular Herpes simplex virus (HSV-I, Carlezon, et al, 2000), baculovirus, retrovirus, adeno-associated-virus (AAV, Carter and Samulski.
  • viruses in particular adenovirus, vaccinia virus, attenuated vaccinia virus, canary pox virus, lentivirus (Chang and Gay, 2001), herpes virus, in particular Herpes simplex virus (HSV-I, Carlezon, et al, 2000),
  • viral vectors like adenoviral vectors, lentiviral vectors, baculovirus vectors or retroviral vectors (Lindemann et al, 1997, and Springer et al., 1998).
  • plasmids which allow the generation of such recombinant viral vectors
  • examples of plasmids which allow the generation of such recombinant viral vectors include pFastBacl (Invitrogen Corp., Carlsbad CA), pDCCMV (Wiznerowicz et al, 1997) and pShuttle-CMV (Q-biogene, Carlsbad, California).
  • the coding sequences may be ligated to an adenovirus transcription/translation control complex, e.g., the late promoter and tripartite leader sequence.
  • the hypercytokine gene may be inserted in the genome of an adenovirus by in vitro or in vivo recombination.
  • Insertion in a non-essential region of the viral genome (e. g., region El, E3, or E4) will result in a recombinant virus that is viable and capable of expressing the respective hypercytokine in infected cells. It is preferred that the viral vector used is modified to be replication incompetent in order to prevent that first and/or second cells modified to express the hypercytokine produce viral particles.
  • the expression vector either has to be provided with an origin of replication, which allows replication independent from the genome of the cell or has to be integrated into the genome of the first and/or second cells.
  • an origin of replication which allows replication independent from the genome of the cell or has to be integrated into the genome of the first and/or second cells.
  • the expression vector is maintained episomally.
  • Suitable origins of replication may be derived from SV40 or other viral (e. g., Polyoma, Adeno, CMV, VSV, BPV) source. In the latter case, if the expression vector is integrated into the genome, e.g. a chromosome, it is not required to provide an origin of replication.
  • Suitable promoters may be derived from the genome of mammalian cells (e. g., metallothionein promoter) or from mammalian viruses (e.g., the adenovirus late promoter; the vaccinia virus 7.5K promoter or the cytomegalovirus promoter).
  • mammalian viruses e.g., the adenovirus late promoter; the vaccinia virus 7.5K promoter or the cytomegalovirus promoter.
  • the early and late promoters of S V40 virus are particularly useful because both are obtained easily from the virus as a fragment which also contains the SV40 viral origin of replication.
  • Smaller or larger SV40 fragments may also be used, provided there is included the approximately 250 bp sequence extending from the Hindll ⁇ site toward the BgHl site located in the viral origin of replication. Further, it is also possible, and may be desirable, to utilize promoter or control sequences normally associated with the cytokine or cytokine receptor encoding polynucleotide on which the hypercytokine is based.
  • operatively linked means incorporated into a genetic construct so that expression control sequences effectively control expression of a coding sequence of interest.
  • Specific initiation signals may also be required for efficient translation of hypercytokine coding sequences. These signals include the ATG initiation codon and adjacent sequences. Exogenous translational control signals, including the ATG initiation codon, may additionally be needed. One of ordinary skill in the art would readily be capable of determining this and providing the necessary signals. It is well known that the initiation codon must be in-frame (or in-phase) with the reading frame of the desired coding sequence to ensure translation of the entire insert. These exogenous translational control signals and initiation codons can be of a variety of origins, both natural and synthetic. The efficiency of expression may be enhanced by the inclusion of appropriate transcription enhancer elements and transcription terminators.
  • polyadenylation site e.g., 5'-AATAAA-3'
  • the poly A addition site is placed about 30 to 2000 nucleotides "downstream" of the termination site of the protein at a position prior to transcription termination.
  • cells can be transformed with vectors controlled by appropriate expression control elements (e.g., promoter, enhancer, sequences, transcription terminators, polyadenylation sites, etc.), and a selectable marker.
  • appropriate expression control elements e.g., promoter, enhancer, sequences, transcription terminators, polyadenylation sites, etc.
  • engineered cells may be allowed to grow for 1-2 days in an enriched medium, and are then switched to a selective medium.
  • the selectable marker in the recombinant plasmid confers resistance to the selection and allows cells to stably integrate the plasmid into their chromosomes and grow to form foci which in turn can be cloned and expanded into cell lines.
  • a number of selection systems may be used including, but not limited to, the herpes simplex virus thymidine kinase (tk), hypoxanthine-guanine phosphoribosyltransferase (hgprt) and adenine phosphoribosyltransferase (aprt) genes, in tk-, hgprt-, or aprt-cells, respectively.
  • tk herpes simplex virus thymidine kinase
  • hgprt hypoxanthine-guanine phosphoribosyltransferase
  • aprt adenine phosphoribosyltransferase
  • antimetabolite resistance can be used as the basis of selection for dihydrofolate reductase (dhfr), that confers resistance to methotrexate; gpt, that confers resistance to mycophenolic acid; neomycin (neo), that confers resistance to the aminoglycoside G-418; and hygromycin (hygro), that confers resistance to hygromycin.
  • the expression vector used to transform, transfect or infect the cell to be modified comprises the gene encoding the selectable marker as one transcript with the gene encoding the hypercytokine. To ascertain the individual expression of the selectable marker and the hypercytokine an internal ribosome entry site (IRES) is placed between the two coding sequences.
  • IRS internal ribosome entry site
  • the cells to be included in the composition of the present invention are preferably propagated separately. Preferably they are propagated in vitro in one of two modes: as non- anchorage dependent cells growing in suspension throughout the bulk of the culture or as anchorage-dependent cells requiring attachment to a solid substrate for their propagation (i.e., a monolayer type of cell growth).
  • the appropriate growth conditions are determined by the cell type and can be determined by the skilled person using routine experimentation.
  • the first and/or second hyper-cytokine is a fusion protein comprising, consisting essentially of or consisting of a soluble cytokine receptor and a cytokine.
  • the first hyper-cytokine is a fusion protein comprising, consisting essentially of or consisting of a soluble cytokine receptor and a cytokine.
  • the soluble cytokine receptor is independently selected from (a) the group consisting of sIL-6R, sIL-l lR, sOSM-R, sCNTF-R, and sCT-l-R; or (b) a polypeptide exhibiting at least 90% sequence identity to a polypeptide according to (a); and the cytokine is independently selected from (c) the group consisting of IL-6, IL-I l, OSM, CNTF, and CT-I; or (d) a polypeptide exhibiting at least 90% sequence identity to a polypeptide according to (c), and optionally a peptide linker between the soluble cytokine receptor and the cytokine, wherein the resulting fusion protein has hyper-cytokine activity.
  • the arrangement is from the N- terminal end to the C-terminal end of the fusion protein as follows: soluble cytokine receptor - optional peptide linker - cytokine.
  • the hypercytokine comprises at least one natural or artificial secretion signal. Since all cytokines are secreted they naturally comprise such a secretion signal. Similar signalling peptides are also found in cytokine receptors. Preferably this secretion signal is located at the N-terminal end of the fusion protein. It will be cleaved during processing and/or secretion of the hypercytokine.
  • sIL-6R When referring to sIL-6R, sIL-l lR, sOSM-R, sCNTF-R, and sCT-l-R the respective soluble parts of IL-6R, IL-I lR, OSM-R, CNTF-R, and CT-I-R, preferably of human origin are meant, the sequence of which are indicated herein as SEQ ID NO: 1 for IL-6R and SEQ ID NO: 3 for IL-I IR.
  • the sequences of all other cytokine receptors can be accessed on NIH GenBank or EMBL databanks, e.g.
  • OSM-R GenBank Acces.: NP_003990
  • CNTF-R GenBank Acces.: NP_001833
  • SEQ ID NO: 2 for IL-6
  • SEQ ID NO: 4 for IL-11.
  • the sequences of all other cytokines can be accessed on NIH or EMBL databanks, e.g. for OSM (GenBank Acces. NO: P 13725), CNTR (GenBank Acces.: NP_000605), and CT-I (Swiss-Prot Acces. No.: Q16619).
  • the hyper-cytokine is a fusion protein comprising, consisting essentially of or consisting of:
  • IL-6R part exhibiting at least 90% sequence identity to human soluble IL-6 receptor (sIL-6R), wherein said sequence identity is calculated over the entire length of the polypeptide sequence from Pl 13 to A323 of SEQ ID NO: 1,
  • IL-6 part exhibiting at least 90% sequence identity to human interleukin-6 (IL-6), wherein said sequence identity is calculated over the entire length of the polypeptide sequence from P29 to M212 of SEQ ID NO: 2, and
  • the fusion protein has hyper-cytokine activity, preferably at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 100% or more of the activity of the Hyper-IL-6 fusion protein according to SEQ ID NO: 9, when tested in a relevant assay of IL-6 activity, e.g. the induction of proliferation of BAF-3/cells as described in Fischer M. et al. (1997).
  • the hyper- cytokine is a fusion protein comprising, consisting essentially of or consisting of:
  • an IL-I IR part exhibiting at least 90% sequence identity to human soluble IL-11 receptor (sIL-l lR), wherein said sequence identity is calculated over the entire length of the polypeptide sequence from Ml to G365 of SEQ ID NO: 3,
  • IL-I l part exhibiting at least 90% sequence identity to human interleukin-11 (IL-I l), wherein said sequence identity is calculated over the entire length of the polypeptide sequence from Al 9 to L 199 of SEQ ID NO: 4, and
  • the fusion protein has hyper-cytokine activity, preferably at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 100% or more of the activity of the sIL-11 R-IL-11 fusion protein according to SEQ ID NO: 11, when tested in a relevant assay of IL-11 activity.
  • the hyper-cytokine comprises, consists essentially of or consists of: (a) a polypeptide having the amino acid sequence according to SEQ ID NO: 5 to 11; or
  • the polypeptide having the amino acid sequence according to SEQ ID NO: 5 consists of the part from Pl 13 to A323 of IL-6R according to SEQ ID NO: 1, a glycine-rich linker sequence of 13 amino acids as shown in SEQ ID NO: 13, and the part from P29 to M212 of IL-6 according to SEQ ID NO: 2 (Fischer et al., 1997). Further sIL-6R and IL-6 fusion proteins having the amino acids sequences according SEQ ID NO: 6, 7 and 8 have been described by Chebath et al.
  • the polypeptide according to SEQ ID NO: 6 comprises a 3 amino acid linker sequence (EFM)
  • the polypeptide according to SEQ ID NO: 7 comprises a 13 amino acid linker sequence (EFGAGLVLGGQFM; SEQ ID NO: 12)
  • the polypeptide according to SEQ ID NO: 8 contains no linker sequence.
  • Additional fusion proteins as shown in SEQ ID NO: 9 and in SEQ ID NO: 10 have been described in WO 97/32891 and comprise amino acids Ml to A323 of sIL-6R according to SEQ ID NO: 1 and amino acids P29 to M212 of IL-6 according to SEQ ID NO: 2 linked by different linker sequences, namely either by a 13 amino acid linker sequence (RGGGGSGGGGSVE, SEQ ID NO: 13) or by a 18 amino acid linker sequence (RGGGGSGGGGSGGGGSVE; SEQ ID NO: 14), respectively.
  • the fusion protein having the amino acid sequence according to SEQ ID NO: 11 comprises the region from Ml to Q365 of sIL-l lR and the region from Al 9 to L 199 of IL-I l.
  • the sIL-6R IL-6 fusion according to SEQ ID NO: 5 is a particularly preferred embodiment and is referred herein as "Hyper-IL-6" or "H6".
  • a preferred expression cassette of the invention comprises Hyper-IL-6 and the Neo selectable marker both under the control of the CMV immediate early promoter. This cassette may be comprised in a variety of vectors, preferably viral vectors like retroviral vectors.
  • the first hypercytokine and the second hypercytokine are the same hypercytokine.
  • the first and/or the second cells are allogenic cells. In a preferred embodiment of the second aspect, the first cells are allogenic cells.
  • the second cell preferably the allogenic second cell
  • HLA human leukocyte antigen
  • MHC human major histocompatibility complex
  • the group of genes encoding this complex resides on chromosome 6, and encodes cell-surface antigen-presenting proteins and many other genes.
  • the major HLA antigens are essential elements in immune function.
  • class I antigens A, B & C
  • present peptides from inside the cell including viral peptides if present
  • class II antigens (DR, DP, & DQ) present phagocytosed antigens from outside of the cell to T-lymphocytes
  • HLAs proteins on the outer part of body cells that are (effectively) unique to that person.
  • the immune system uses the HLAs to differentiate self cells and non-self cells. Any cell displaying that individuals' HLA type belongs to that individual (and therefore is not an invader). Long before PCR based gene sequencing and gene identification were available, the HLA antigens were recognized as factors interfering with or, occasionally, permitting successful transplantion.
  • Donor organs transplanted into recipients elicit antibodies against the donor's tissues and turning the donor's HLA receptors into antigens of the recipients immune system, hence the name 'human leukocyte antigens'.
  • the types of receptors could be classified based on the antibodies that they induced. These antibodies, particularly to donors who were homozygotes of a particular class II haplotype can be used to identify different receptor types and isoforms.
  • HLA HLA
  • MHC class I and MHC class II MHC class II (or rarely, D locus).
  • D locus D locus
  • This classification is based on sequence information from the respective HLA loci. Accordingly, the skilled person is well aware how to determine, whether two groups of cells have the same or a different HLA type.
  • the first and the second cell line have a different HLA type based on the antibody type classification system.
  • the one or more first cells and/or the one and more second cells is a tumour cell.
  • the one or more first cells is a tumour cell.
  • the tumour cell is selected independently for each cell from the group consisting of a melanoma cell, a renal carcinoma cell, a prostate cancer cell, a colon cancer cell, a lung cancer cell, a pancreas cancer cell, a liver cancer cell, a brain cancer cell, a head and neck cancer cell, and a sarcoma cell.
  • the first and the second cells are selected from the same tumour cell type but either from different tumours within an individual or from two different individuals.
  • the first cells, which are modified to express a hypercytokine are the human (Homo sapiens) melanoma derived cells Michl, deposited on April 24, 2007 under accession number DSM ACC2837 with the "Deutsche Sammlung von Mikroorganismen und Zellkulturen” (DSMZ), Inhoffenstr. 7 B, 38124 Braunschweig, Germany and/or the second cells, which are modified to express a hypercytokine are the human (Homo sapiens) melanoma derived cells Mich2, deposited on April 24, 2007 under accession number DSM ACC2838 with the DSMZ. Michl and Mich2 originate from different patients.
  • DSMZ Deutsche Sammlung von Mikroorganismen und Zellkulturen
  • the first cells are Michl or Mich2.
  • the first cells are Michl -H6, deposited on April 24, 2007 under accession number DSM ACC2839 with the DSMZ.
  • the second cells are Mich2-H6, deposited on April 24, 2007 under accession number DSM ACC2840 with the DSMZ.
  • Michl -H6 and Mich2-H6 have respectively been derived from infection of Michl and Mich2 with a retrovirus comprising the expression cassette according to SEQ ID NO: 15 and expressing Hyper-IL-6 according to SEQ ID NO: 9 under the control of the CMV promoter.
  • the first cells are Michl -H6 or Mich2- H6.
  • the first cells are Michl -H6 or Mich2- H6.
  • compositions comprising first and/or second cells, in particular tumour cells, modified to express a hypercytokine can be further enhanced, if the first and/or second cells are engineered to comprise at least one further polynucleotide encoding an antigen, preferably a tumour antigen, a cytokine, in particular GM-CSF, IL-2, IL-6, IL-7, IL-I l, IL-15, IL-21, anti-TGF, EPO, interferon, in particular INF- ⁇ , LIF, OSM, CNTF, CT-I or a hypercytokine different from the first hypercytokine comprised in the respective cell.
  • an antigen preferably a tumour antigen, a cytokine, in particular GM-CSF, IL-2, IL-6, IL-7, IL-I l, IL-15, IL-21, anti-TGF, EPO, interferon, in particular INF- ⁇ , LIF, OSM, CNTF, CT-I or
  • the engineering is preferentially achieved by using a vector, in particular one of the expression vectors indicated above with respect to hypercytokines and the subsequent or simultaneous introduction of this/these vector(s) into the first and/or second cells to be modified.
  • the one or more additional polynucleotide can be comprised in a separate vector or can be comprised within the same vector as the hypercytokine encoding polynucleotide. It is preferred that the host cells simultaneously express both the hypercytokine and the at least one further protein encoded by the at least one further polynucleotide.
  • tumour antigen comprises all substances, which elicit an immune response against a tumour.
  • Particular suitable substances are proteins or protein fragments which are enriched in a tumour cell in comparison to a healthy cell. These substances are preferably present within and/or are accessible on the outside of the tumour cell. If the tumour antigen is only present within a tumour cell, it will still be accessible for the immune system, since the antigen or fragments thereof will be presented by the MHC system at the surface of the cell.
  • the tumour antigen is almost exclusively or exclusively present on and/or in the tumour cell and not in a healthy cell of the same cell type. It is particularly preferred that the tumour antigen is exclusively present on and/or in the tumour cell and is not present in any healthy cell of any cell type.
  • Suitable tumour antigens can be identified, for example, by analyzing the differential expression of proteins between tumour and healthy cells of the same cell type using a microarray-based approach (Russo et al., Oncogene. 2003, 22:6497-507), by PCR- or microarray-based screening for tumour specific mutated cellular genes (Heller, Annu. Rev. Biomed. Eng. 2002, 4:129-53) or by serological identification of antigens by recombinant expression cloning (SEREX; Tureci et al., MoI Med Today. 1997, 3:342-349 ).
  • tumour cell which include for example, oncogenes like, for example truncated epidermal growth factor, folate binding protein, melanoferrin, carcinoembryonic antigen, prostate-specific membrane antigen, HER2-neu.
  • oncogenes like, for example truncated epidermal growth factor, folate binding protein, melanoferrin, carcinoembryonic antigen, prostate-specific membrane antigen, HER2-neu.
  • tumour antigens are selected to be expressed in the first and/or second cells of the compositions of the invention, which elicit a strong immune response.
  • Antigens eliciting a strong immune response will induce at least 1%, preferably at least 5%, more preferably at least 10% and most preferably at least 15% IFN ⁇ -producing CD8+ T or CD4+ T cells isolated from mice previously immunized with the antigen, upon challenge with the antigen and/or will induce preferably at least 5%, and most preferably at least 15% of B-cells cells isolated from mice previously immunized with the antigen, upon challenge with the antigen to proliferate.
  • Antigens fulfilling these criterions are candidates to be expressed in the cancer vaccine compositions of the present invention.
  • tumour antigen is selected from the group consisting of T-cell-defined cancer-associated antigens belonging to unique gene products of mutated or recombined cellular genes, in particular cyclin-dependent kinases (e.g.
  • composition according to the first aspect or the second aspect of the present invention When the composition according to the first aspect or the second aspect of the present invention is administered to a patient it is administered to elicit an immune response both against the first and/or second cells and any tumour cells, which share epitopes and/or tumour antigens with the first and/or second cells. It is, thus, expected that the cells of the compositions will only survive for a limited time within the recipient of the compositions of the present invention and are then cleared from the organism of the recipient by the immune system of the recipient. Nevertheless, it is preferred for safety reasons that the proliferation of the first, preferably allogenic and/or the second, preferably allogenic cells has been inhibited prior to the administration of these cells to a patient.
  • the term “inhibition" comprises both the slowing down of the proliferation rate and the complete cessation of proliferation.
  • the skilled person is aware of a larger number of chemical and physical methods, which affect the growth rate of cells, these include without limitation radiation, e.g. ⁇ -irradiation or cross-linking, e.g. by psoralen or aldehyde.
  • the level of inhibition should preferably be such, that transcription and translation of the transgenes introduced into the first and second cells is not completely shut down, i.e. the transgenes should be expressed at a level of at least 5%, preferably at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 100% or more of the expression level in the first and/or second cells prior to inhibition.
  • the cells are capable to continue to go through 1 to 5, i.e. 1, 2, 3, 4, or 5, replication cycles after the chemical or physical method for inhibition of proliferation is administered.
  • compositions according to the first and second aspect of the present invention can be further enhanced, if one or more additional cells, which may be autologous or allogenic, which are different from the first and/or the second cells are also included in the composition.
  • these cells originate from a further individual, preferably having a HLA type different from the HLA types of the first and/or second cell types.
  • these cells are tumour cells, preferably from the same tumour type as the first and/or second cells.
  • the proliferation of the one or more additional, preferable allogenic cells has been inhibited, preferably as outlined above.
  • the one or more cells of the one or more additional allogenic cells have been modified to express a cytokine, a cytokine receptor, a hypercytokine and/or a tumour antigene.
  • the cytokine is selected from the group consisting of GM- CSF, IL-2, IL-6, IL-7, IL-I l, IL-15, IL-21, anti-TGF, EPO, interferon, in particular INF- ⁇ , LIF, OSM, CNTF, CT-I and the cytokine receptors or soluble parts thereof are those receptors corresponding to the indicated cytokines.
  • the hypercytokine is selected from the group consisting of hyper- IL-6, e.g. according to SEQ ID NO: 5, 6, 7, 8, 9 or 10, IL-2, hyper- IL- 11, e.g. according to SEQ ID NO: 11, hyper CNTF, and hyper-OSM.
  • compositions of the present invention elicit an immune response
  • the composition may further comprise adjuvants, which are commonly used in vaccines to enhance the immunizing effect.
  • Preferred adjuvants are selected from the group consisting of un- methylated DNA, in particular unmethylated DNA comprising CpG dinucleotides (CpG motif), in particular CpG ODN with phosphorothioate (PTO) backbone (CpG PTO ODN) or phosphodiester (PO) backbone (CpG PO ODN); gel-like precipitates of aluminum hydroxide (alum); bacterial products from the outer membrane of Gram-negative bacteria, in particular monophosphoryl lipid A (MPLA), lipopolysaccharides (LPS), muramyl dipeptides and derivatives thereof; synthetic lipopeptide derivatives, in particular Pam 3 Cys; lipoarabinomannan; peptidoglycan; zymosan; heat shock proteins (HSP), in particular HSP 70; dsRNA and synthetic derivatives thereof
  • Particular preferred adjuvants which can be comprised in the compositions of the present invention are selected from the group consisting of unmethylated DNA, in particular unmethylated DNA comprising CpG dinucleotides (CpG motif), in particular CpG ODN with phosphorothioate (PTO) backbone (CpG PTO ODN) or phosphodiester (PO) backbone (CpG PO ODN) and synthetic lipopeptide derivatives, in particular Pam 3 Cys.
  • CpG motif CpG dinucleotides
  • PTO phosphorothioate
  • PO phosphodiester
  • the present invention concerns a composition according to the first or second aspect of the present invention for use in medicine.
  • the present invention concerns a pharmaceutical composition
  • a pharmaceutical composition comprising a composition according to the first or second aspect of the invention additionally comprising pharmaceutically acceptable diluents, carriers, excipients, fillers, binders, lubricants, glidants, disintegrants, adsorbents, and/or preservatives.
  • the pharmaceutical composition is formulated for parenteral use, preferably in the form of a sterile aqueous solution which may contain other substances, for example, enough salts or glucose to make the solution isotonic with blood.
  • the aqueous solutions should be suitably buffered (preferably to a pH of from 3 to 9), if necessary.
  • a particularly preferred aqueous solution is phosphate buffered saline (PBS).
  • a unit dose of a composition or pharmaceutical composition of the present invention comprises between at least 1 x 10 5 and 1 x 10 9 cells of first cells, preferably at least 2 x 10 5 , 3 x 10 5 , 4 x 10 5 , 5 x 10 5 , 6 x 10 5 , 7 x 10 5 , 8 x 10 5 , 9 x 10 5 , 1 x 10 6 , 2 x 10 6 , 3 x 10 6 , 4 x 10 6 , 5 x 10 6 , 6 x 10 6 , 7 x 10 6 , 8 x 10 6 , 9 x 10 6 , 1 x 10 7 , 2 x 10 7 , 3 x 10 7 , 4 x 10 7 , 5 x 10 7 , 6 x 10 7 , 7 x 10 7 , 8 x 10 7 , 9 x 10 7 , and 1 x 10 8 .
  • composition of the present invention comprises between 1 x 10 to 1 x 10 first cells, preferably 2.5 x 10 7 .
  • the unit dose of a composition or pharmaceutical composition of the present invention comprises between at least 1 x 10 5 and 1 x 10 9 cells of second cells, preferably at least 2 x 10 5 , 3 x 10 5 , 4 x 10 5 , 5 x 10 5 , 6 x 10 5 , 7 x 10 5 , 8 x 10 5 , 9 x 10 5 , 1 x 10 6 , 2 x 10 6 , 3 x 10 6 , 4 x 10 6 , 5 x 10 6 , 6 x 10 6 , 7 x 10 6 , 8 x 10 6 , 9 x 10 6 , 1 x 10 7 , 2 x 10 7 , 3 x 10 7 , 4 x 10 7 , 5 x 10 7 , 6 x 10 7 , 7 x 10 7 , 8 x 10 7 , 9 x 10 7 , and
  • a particular preferred unit dose of a composition or pharmaceutical composition of the present invention comprises between 1 x 10 7 to 1 x 10 8 second cells, preferably 2.5 x 10 7 .
  • the composition or pharmaceutical composition of the present invention comprises about the same number of first cells and second cells for a total of 2 x 10 5 to 2 x 10 8 cells per unit dose.
  • a particular preferred unit dose of a composition or pharmaceutical composition of the present invention comprises between 2 x 10 7 to 2 x 10 8 first and second cells, preferably 5 x 10 7 .
  • the total volume of the unit dose is preferably between 0.5 to 20 ml, preferably, 1 to 5 ml, e.g. 1, 2, 3, 4, or 5 ml.
  • the present invention relates to the compositions of the present invention or the pharmaceutical composition of the present invention for the treatment of cancer, the prevention of cancer, and/or the prevention of recurrence of cancer.
  • Preferred cancers treatable or preventable with a composition according to the present invention are selected from the group consisting of cancer of the gastrointestinal or colorectal tract, liver, pancreas, kidney, bladder, prostate, endometrium, head and neck cancer, ovary, testes, prostate, skin, eye, melanoma, dysplastic oral mucosa, invasive oral cancer, small cell and non-small cell lung cancer, hormone-dependent breast cancer, hormone independent breast cancer, transitional and squamous cell cancer, neurological malignancy, including neuroblastoma, glioma, astrocytoma, osteosarcoma, soft tissue sarcoma, hemangioma, endocrinological tumour, hematologic neoplasia including leukemia, lymphoma, and other mye
  • melanoma particularly preferred is the treatment or prevention of melanoma, renal cell carcinoma, prostate cancer, colon cancer, lung cancer, pancreas cancer, liver cancer, brain cancer, head and neck cancer, or sarcoma. Even more preferred is the treatment or prevention of melanoma, pancreas cancer and renal cell cancer.
  • the present invention relates to the use of the compositions of the present invention for the preparation of a pharmaceutical composition for the treatment of cancer, the prevention of cancer, and/or the prevention of recurrence of cancer.
  • compositions of the invention can be used in the treatment and/or prevention of a wide variety of different cancers, however, preferred cancers treatable or preventable according to the present invention are selected from the group consisting of cancer of the gastrointestinal or colorectal tract, liver, pancreas, kidney, bladder, prostate, endometrium, head and neck cancer, ovary, testes, prostate, skin, eye, melanoma, dysplastic oral mucosa, invasive oral cancer, small cell and non-small cell lung cancer, hormone-dependent breast cancer, hormone independent breast cancer, transitional and squamous cell cancer, neurological malignancy, including neuroblastoma, glioma, astrocytoma, osteosarcoma, soft tissue sarcoma, hemangioma, endocrinological tumour, hematologic neoplasia including leukemia, lymphoma, and other myeloproliferative and lymphoproliferative diseases, carcinoma in situ, hyperplastic lesion, adenoma
  • melanoma particularly preferred is the treatment or prevention of melanoma, renal cell carcinoma, prostate cancer, colon cancer, lung cancer, pancreas cancer, liver cancer, brain cancer, head and neck cancer, or sarcoma. Even more preferred is the treatment or prevention of melanoma, pancreas cancer and renal cell cancer.
  • cancer vaccine In particular in the context of the treatment and/or prevention of cancer it is envisionable that patients are immunized with a "cancer vaccine" prior to the development of any symptoms of a disease, i.e. receive a protective immunization, or after they have developed symptoms of the disease, i.e. receive a therapeutic vaccination. It is further envisioned that the cancer vaccine can be administered to patients after symptoms of the disease have disappeared in said patients in order to prevent recurrence of the disease. In particular, the cancer vaccine can be administered to the patients after surgical excision of the tumour to prevent cancer recurrence.
  • the compositions and pharmaceutical compositions of the present invention can be used as such cancer vaccines.
  • the expression of at least one further cytokine, in particular GM-CSF, by the first and/or second cells expressing a hypercytokine, preferably Hyper-IL-6 can provide in the context of certain tumours, in particular melanoma and renal cancer an even stronger in vivo anti-tumour response than cells expressing only the hypercytokine. Therefore, in a preferred use the first and/or the second cells expressing hypercytokine are modified to express at least one further cytokine are used for the production of a medicament to prevent or treat a proliferative disease.
  • first and the second cells are from the same type of tissue, preferably tumour tissue but have a partially or completely different HLA type than the first cell and/or the second cell.
  • Example 1 Hyper-IL-6 (H6) augments T-cell proliferative response in allogenic mixed tumour-lymphocyte reaction (AMTLR)
  • Irradiated tumour cells were mixed with unprimed, allogenic lymphocytes in the presence or absence of IL-6 (1 ng/ml) or purified H6 (1 ng/ml). After three days, T-cells were assayed for proliferation by 3 H-thymidine incorporation, determined as counts per minute (cpm). The results of this experiment are shown in Fig. 1. Columns 1 to 3 show the results of spontaneous T-cell proliferation, i.e. in the absence of tumour cells. Apparently, spontaneous T-cell proliferation does not occur to a significant extent (columns 1 to 3), irrespective whether IL-6 (column 2) or H6 (column 3) are added to the mixture.
  • Example 2 T-cell proliferation in allogenic mixed tumour-lymphocyte reaction is dependent on IL-2.
  • Irradiated tumour cells were mixed with unprimed, allogenic lymphocytes in the presence or absence of IL-6 (1 ng/ml), purified H6 (1 ng/ml) and anti-IL-2 antibody (1 ⁇ g/ml). After three days T-cells were assayed for proliferation by 3 H-thymidine incorporation, determined as cpm. The results of this experiment are shown in Fig. 2.
  • FIG. 1 of Fig. 2 show the results of T-cell proliferation in response to allogenic tumour cells in the absence of anti-IL-2 antibody.
  • the results from this experiment are almost identical to the results presented in Fig.l, columns 4 to 6.
  • T-cells show a very strong proliferation in the presence of allogenic tumour cells (column 1 of Fig. 2).
  • the addition of IL-6 has no apparent effect on the proliferation of T-cells (column 2 of Fig. 2).
  • the addition of H6 leads to an almost two-fold increase in T-cell proliferation (column 3 of Fig. 2).
  • Example 3 Hyper-IL-6 augments IL2 and IFN- ⁇ production by T-cells in allogenic mixed tumour-lymphocytic reaction
  • Hyper-IL-6 H6
  • results obtained indicate that hyper-IL-6 increases immunostimulatory potential of allogenic melanoma cells.
  • Hyper-IL-6 is not only more potent than IL-6 but also displays qualitatively different biological activity.
  • native IL-6 which is a known Th2 inducer
  • hyper- IL-6 appears to reduce IL-IO expression while increasing IFN- ⁇ and IL-2 production by peripheral blood lymphocytes (PBLC) which is characteristic of a ThI response.
  • PBLC peripheral blood lymphocytes
  • Test Articles A375 melanoma cells and their derivative A375-H6 cells; PBLC isolated from a healthy volunteer.
  • FBS FBS
  • PBS GIBCO/Invitrogen
  • DMEM GIBCO/Invitrogen
  • Trypsin EDTA Tissue culture flask 25cm 2 (Sarstedt), 24 well plate (Nunc), Lymphocyte separation medium (ICN), BD Cytometric Bead Array (CBA) Human ThI /Th2 Cytokine Kit-II (Becton Dickinson), IL-6 (Pharmingen), Flow cytometer (Becton Dickinson), FACSAriaTM (Becton Dickinson).
  • PBLC drawn from a healthy volunteer were separated from whole blood by centrifugation over lymphocyte separation medium. Cells were washed twice in PBS and counted by standard procedures in a haemocytometer. Lymphocytes were re-suspended at 2 x 10 6 cells per ml in DMEM culture medium supplemented with 2% FBS. Tumour cells were trypsinized, washed twice in PBS and re-suspended at 2 x 10 6 cells per ml in DMEM medium supplemented with 2% FBS. 0.5 ml of lymphocyte suspension was mixed with 0.5 ml of tumour cells and seeded on a 24 well plate to give 1 ml of mixed cell culture.
  • 0.5 ml of lymphocyte suspension + 0.5 ml of culture medium (control) 0.5 ml of lymphocyte suspension + 0.5 ml of culture medium + 10 ng of IL-6 0.5 ml of lymphocyte suspension + 0.5 ml of A375 tumour cells suspension 0.5 ml of lymphocyte suspension + 0.5 ml of A375 cells suspension + 10 ng of IL-6 0.5 ml of lymphocyte suspension + 0.5 ml of A375-H6 cells suspension 0.5 ml of A375 tumour cells suspension + 0.5 ml of culture medium 0.5 ml of A375-H6 tumour cells suspension + 0.5 ml of culture medium
  • the mixed cells were cultured for three days in a humidified cell incubator at 37 0 C, 5% CO 2 / 95% air. After 3 days cell-free supernatant was collected and analyzed for cytokine content. Collected supernatants were properly marked and immediately frozen at -20 C until analysis. Cytokine content was determined by CBA within one month according to the instruction provided in the CBA manual. The results of this example are summarized in Table 1 below. They show that the A375 tumour cells stimulate allogenic T-cells to produce IL-2, IL-6, IL-IO and IFN- ⁇ . The presence of hyper-IL6 but not IL-6 significantly augmented IL-2 and IFN- ⁇ production in T-cells and at the same time reduced IL-10 secretion.
  • hyper-IL-6 is not only more potent then IL-6 but also displays a qualitatively different biological activity.
  • hyper-IL-6 appears to reduce IL-10 expression while increasing IFN- ⁇ and IL-2 production characteristic for ThI response.
  • This type of T-helper response i.e. ThI is of primary importance during cytotoxic T-cell induction and development and is therefore a desirable response in anti -tumour vaccines.
  • Example 4 Synthesis of cytokines, growth factors and vascular factors by melanoma cells and H6-modified melanoma cells
  • the aim of this example was to assess the synthesis of selected cytokines, growth and vascular factors by melanoma cells which can be used as components in a vaccine.
  • a further aim was to evaluate the effect of the H6 gene modification on the synthesis of above factors by said melanoma cells.
  • Test Articles Michl cells, Mich2 cells, Michl -H6 cells and Mich2-H6 cells were thawed and cultured for two days and passaged for the next 3 days. Then cells were trypsinized and frozen. These cells (passage 1 - Pl) were used in the experiment.
  • Methodology The cell lines studied were thawed and seeded in culture flasks. Cells were cultured until confluency and then maintained in serum (FBS) free medium (DMEM) for 48 hr in 5% CO 2 humidified atmosphere. Then the media were collected and analyzed for the above- listed factors using multiplex particle-based immunoassay with FC readout.
  • FBS serum
  • DMEM free medium
  • Table 3 Secretion of different cytokines and growth factors by melanoma cells and H6- modifed melanoma cells
  • IL-2, IL-4 and IL-10 were either not secreted or secreted at extremely low levels which were below the detection limit of the assay.
  • IL-2, IL-4 and IL-IO were not detectable in the assay.
  • Michl cells Significant qualitative (IL-2, IL-10) and quantitative differences between Michl and Michl -H6 cells are observed. Except for GM-CSF which was decreased, expression of all other factors studied was significantly increased in H6 modified cells.
  • Mich2 cells Significant qualitative (IL-2, IL-10, INF- ⁇ ) and quantitative differences between Mich2 and Mich2-H6 cells are observed. Except for IL- 12 and VEGF which were at the same level, expression of all factors studied was significantly increased. In contrast, Mich2-H6 cells did not express INF- ⁇ . Summary of results from example 4
  • IL-2 and IL-IO additional proteins
  • IL-8 a very strong chemoattractant increasing recruitment of immune cells into the vaccine injection site.
  • GM-CSF is a major stimulator of dendritic cell maturation, hence inducing antigen presentation.
  • VEGF is a signaling protein involved in vasculogenesis and angiogenesis. It is also capable of stimulating monocyte/macrophage migration.
  • IL-2, IL- 12 and INF - ⁇ display immunomodulatory functions on T cells. Modified cells also secreted IL-10 which is considered to be an immunoinhibitory factor. However, experimental studies demonstrated that murine melanoma cells modified with IL-10 cDNA elicited specific anti-melanoma immune responses indicating that in such setting IL-10 provides a stimulatory signal for T lymphocytes. Moreover, modified cells secreted significant quantities of IL-6. However, additional identification studies are necessary since anti-IL-6 antibodies may react with H6 protein. Accordingly, high IL-6 levels detected by the employed method in the culture medium may reflect secretion of the transgenic H6 protein but not necessarily the native IL-6 protein by vaccine cells.
  • Example 5 Cytokine production of a mixture of two H6-modified melanoma cell lines in an AMTLR
  • the aim of this example included the analysis of the effect of a mixture of two melanoma cell lines (Michl -H6 and Mich2-H6) on cytokine production by PBLC isolated from various healthy individuals with different HLA haplotypes. Moreover, the effect of a mixture of two cell lines was compared with each line used alone. Cytokine production was assessed by measurement of cytokine content in culture medium by CBA (Cytometry Bead Assay).
  • Michl -H6 cells and Mich2-H6 cells were obtained from the same source as described in Example 4 and were irradiated; PBLC were isolated from 4 healthy volunteers (3 males and 1 female).
  • DMEM fetal calf serum
  • FBS GIBCO/ Invitrogen
  • PBS GIBCO/Invitrogen
  • Lymphocytes Separation Medium ICN
  • 3 H-thymidine Amersham Biosciences
  • Trypsin EDTA GEBCO/Invitrogen
  • 96 well plate Sarstedt
  • 75 ml tissue culture flasks Corning
  • BD Cytometric Bead Array CBA
  • Human Thl/Th2 Cytokine Kit-II BD Biosciences
  • FACS AriaTM BD Biosciences
  • Scintillation Counter Phase-contrast microscope (Olympus); CO 2 Incubator (Sanyo); Centrifuge (Sorvall).
  • Cell preparation Cells were cultured per standard procedures in the lab under GMP-Like conditions by a qualified research worker.
  • Michl-H6 and Mich2-H6 cells were plated separately into culture flasks in DMEM culture medium supplemented with 10% FBS at a seeding density of approximately 1.67x10 cells per cm 2 .
  • the cells were grown in culture until confluency (3-5 days) and were then trypsinized, washed twice with PBS, re-suspended at 2xlO 6 cells per ml in DMEM medium supplemented with 2% FBS for cytokine production record, then irradiated at 80Gy ( 60 Co).
  • PBLCs peripheral blood cells were separated from the whole blood collected from 4 healthy individuals (coded: A, D, M and N) by centrifugation over lymphocyte separation medium. Cells were washed twice with PBS and counted by standard procedures in a haemocytometer. PBLCs were then re- suspended at 5x10 5 cells per ml in DMEM culture medium supplemented with 2% FBS for cytokine production record.
  • the test was performed on one 96 well plate for the IL-2, IL-4, IL-6, IL-IO, TNF- ⁇ and IFN- ⁇ cytokine production panel.
  • the cell suspension was transferred into the 96 well plate in a volume of lOO ⁇ l per each cell line in one row for each of four individuals. Into each row the cells were transferred in concentration of 2 x 10 5 cells per well. Each cell line was reduplicated in four variant columns (four samples for each cell line). There were no melanoma cells added into the last but one 16 wells (4 per each of four individuals), i.e. in Row 5, with these wells acting as a negative control (Control -) for spontaneous PBLC proliferation (the control wells contained DMEM + 2% FBS in quantity of lOO ⁇ l and PBLC in concentration of 5x10 4 cells per well). There were no PBLCs added into the last 12 wells, i.e.
  • PBLCs were added to each well including the negative control wells (but except positive control wells) in a volume of lOO ⁇ l and at concentration of 0.5 x 10 5 cells per well.
  • the total volume in each well was 200 ⁇ l (i.e. lOO ⁇ l of PBLC plus lOO ⁇ l of melanoma cells solutions).
  • Table 4 Arrangement of samples and controls
  • the mixed cells were co-cultured for 3 days for cytokine production analysis, as it is optimal period for incubation, in a humidified incubator at 37°C in 5% CO 2 /95% air atmosphere.
  • Cytokines accumulated in the medium were measured using BDTM Biosciences Cytometric Bead Array (CBA) Human Thl/Th2 Cytokine Kit- II Assay within one month according to the instruction provided in the CBA manual.
  • CBA Cytometric Bead Array
  • Michl-H6 cells did not produce IFN- ⁇ , TNF- ⁇ , IL-10 and IL-2.
  • these cytokines were also not seen in culture media of mixtures of both cell lines.
  • low quantities of TNF- ⁇ and IL-10 were secreted by Mich2-H6 cells.
  • Both cell lines produced IL-6 and IL-4 in very high and moderate quantities, respectively. These two cytokines were produced at comparable levels by each cell line.
  • Fig. 4 the results of cytokine production by PBLCs are shown.
  • the PBLC did not produce IFN- ⁇ , IL-10 or IL-2.
  • Fig. 5 shows results of the effect of Michl-H6 and Mich2-H6 cells alone and in combination on PBLC cytokine secretion.
  • Incubation of PBLC with melanoma cell lines led to the modulation of cytokine production by PBLC for most of the cytokines studied. Only IL-4 and likely IL-6 secretion were not affected.
  • PBLCs from all four donors a synergistic effect of a mixture of both cell lines as compared to each cell line used separately was observed with respect to IL-2 production. A similar synergistic effect was seen in 3 out of 4 donors on IFN- ⁇ production.
  • the combination of Michl-H6 and Mich2-H6 cells increases in vitro immunogeneicity of the vaccine as compared to each cell line used alone by shifting the immune response towards a ThI type as demonstrated by the synergistic effect on IL-2 and INF- ⁇ production by PBLC.
  • a designer cytokine e.g. a hypercytokine such as H6, and the combination of multiple tumour cell lines increases the therapeutic potential of an allogenic vaccine.
  • Example 6 Immunotherapy with irradiated RENCA cells modified with Hyper-IL-6 gene
  • Example 6 female Balb/c Fl mice, 8-12 weeks of age were used. The animals were purchased from the Polish Academy of Sciences (Warsaw/Poland). Animals were kept under constant pathogen-free conditions in rooms with 12-h day/night cycle with unlimited access to food and water. All experiments were performed according to the guidelines approved by the Local Ethical Committee for Animal Research at the University of Medical Sciences, Poznan, Tru.
  • RENCA renal cell carcinoma
  • Method 6 murine sarcoma cells
  • DMEM medium Invitrogen Corporation, USA
  • culture medium 10% heat- inactivated fetal bovine serum, antibiotics and 2 mM of L-glutamine (all from Invitrogen), hereafter referred to as culture medium.
  • Cells were cultured in 78 cm 2 culture plates at 37°C in a fully humidified atmosphere of 5% CO 2 /95% air and passaged every 3>-A days.
  • An El -deleted adenoviral recombinant of the human strain 5 was obtained from Dr. Frank Graham (IRBM-Merck, Italy). The vector was modified to encode H6 (AdH6). An empty (without transgene) adenoviral vector Ad ⁇ 7001 was kindly provided by Dr. H. Ertl (Wistar Institute, Philadelphia, PA). The viruses were propagated and titrated on El-transfected 293 cells as described previously (Kowalczyk, D. W. et al., 2001).
  • Lentiviral vector encoding GFP protein was obtained after cotransfection of 293FT packaging cells with 3 vectors - (i) pMD2.G encoding VSV-derived capside, (ii) p8.91 encoding HIV-derived gag and pol genes, and (iii) pWPXL encoding GFP. Following cotransfection 293FT cells were cultured in X-VIVO medium (Lonza, Walkersville, MD). Subsequently, 24 and 48 hours after cotransfection the culture medium was collected, concentrated using Amicon column (Milipore, Canada) and used for transduction of RENCA or RENCA-H6 cells. Transduction was carried out on 96-well plate and was repeated 3 times. Cells with high-level expression of GFP were selected. RENCA-GFP or RENCA-H6-GFP were then used for vaccination experiments.
  • RENCA-H6 cDNA encoding Hyper-IL-6
  • RENCA-H6 cDNA encoding Hyper-IL-6
  • RENCA and RENCA-H6 cells were transduced or co-transduced with Lenti-GFP, respectively. 24 hours following transduction cells were irradiated with a dose of 80Gy (Co 60 ). Following irradiation cells were stored in liquid nitrogen until use. in vivo studies
  • mice In order to establish an orthotopic tumor model Balb/c mice underwent a kidney subcapsular injection of RENCA cells. Prior to inoculation of tumor cells, mice were anesthetized with Avertin anesthesia according to a standard protocol (Weiss, J. and Zimmermann, F., 1999). Briefly, mice received i.p. injection (0.7 mg/g) of Avertin working solution (2,2,2- tribromoethanol diluted in tert-amyl alcohol). Skin of the anesthetized mice at left lumbar region was shaved with an electric shaver. Next, the skin and subcutaneous tissue were cut with a scalpel and the left kidney was exposed.
  • Avertin working solution 2,2,2- tribromoethanol diluted in tert-amyl alcohol
  • RENCA cells suspended in 10 ⁇ L of PBS were injected subcapsularly into the exposed kidney. Finally, the wound was closed with 2-3 surgical stitches.
  • 10 days after tumor implantation mice were again anesthetized.
  • the tumor-bearing kidney was exposed and following ligation of renal artery and vein a nephrectomy was performed according to uro- oncological guidelines.
  • an autopsy was performed in order to determine the cause of death.
  • mice were immunized according to two regimes.
  • mice were given into left hip a single subcutaneous (s.c.) injection of IxIO 6 of placebo (RENCA w/t) or vaccine (RENCA-H6) cells suspended in 100 ⁇ L of PBS.
  • mice were immunized 5 times in 3-day intervals into both hips with 1x10 6 of placebo or vaccine cells suspended in 100 ⁇ L of PBS.
  • survival of mice was monitored twice a week.
  • mice cured by adjuvant treatment with RENCA-H6 cells were used. 25 weeks after adjuvant therapy mice received s.c. injection of RENCA or Meth-A cells (5xlO 5 in 100 ⁇ L of PBS). In a control group naive Balb/c mice received a s.c. injection of RENCA cells (5xlO 5 ) [Fig. 7B].
  • mice were administered subcutaneously (s.c.) with placebo (RENCA-w/t) or RENCA-H6 cells (1x10 6 ) suspended in 100 ⁇ L of liquid MatrigelTM at 4°C according to a procedure published by Kowalczyk et al. Following implantation, Matrigel at body temperature becomes solid and forms palpable tumors that can be easily excised for isolation of vaccine-infiltrating cells. 10 days later Matrigel+placebo or Matrigel+vaccine mice were sacrified, and Matrigel 'tumors' were excised, minced, pooled (8 mice per group) and 'tumor' infiltrating mononuclear cells were isolated by gradient centrifugation.
  • the single-cell suspension was then stained with anti-CDl lc, anti-CD25 (PE); anti-CD4, anti-CD40, anti-CD54, anti-CD80, anti-CD86, anti- MHCI, anti-MHCII (FITC); oraz anti-Foxp3 (APC) monoclonal antibodies (Pharmingen/Becton Dickinson, USA).
  • the cells were subsequently analyzed in a flow cytometry (FACSCanto, BD Biosciences, USA)
  • mice were immunized 5 times in 3-day intervals with placebo or RENC A-H6 cells. After 14 days mice in control (non-immunized), placebo and vaccine groups received s.c. injection of 100 ⁇ L Matrigel containing IxIO 6 of RENCA cells. 7 days later, mice were sacrif ⁇ ed, and Matrigel 'tumors' were excised, minced, pooled (8 mice per group) and tumor infiltrating lymphocytes were isolated by gradient centrifugation.
  • the single-cell suspension was then stained with anti-CD4, anti-CD8, anti-NKl.l (FITC); anti-CD40, anti-CD43, anti-CD62L, anti-CD69 (PE) monoclonal antibodies (Pharmingen/Becton Dickinson, USA), and analyzed by flow cytometry.
  • FITC anti-CD4, anti-CD8, anti-NKl.l
  • PE anti-CD40, anti-CD43, anti-CD62L, anti-CD69
  • PE monoclonal antibodies
  • a stable RENCA-GFP cell line was used for preparation of placebo and vaccine cells. Mice were immunized 5 times in 3-day intervals. On day 20, splenocytes from non- immunized, RENCA-GFP-w/t- and RENCA-GFP -H6-immunized mice were isolated and restimulated in vitro with ⁇ -galactosidase 96-103 (DAPIYTVN) CTL epitopic peptide (0.2 ⁇ g/ml) for 7 days.
  • DAPIYTVN ⁇ -galactosidase 96-103
  • RENCA-w/t and RENCA- Ad ⁇ 7001 cells were compared in order to select a proper placebo for further experiments.
  • Median survival of mice in control (non- immunized) group was 10 weeks (all animals died within 12 weeks).
  • the median survival of RENCA-w/t immunized animals was 12 weeks (22% survived until the end of experiment), and in RENCA-Ad ⁇ 7001 group 13 weeks (11% of mice survived) [Fig. 6A].
  • OS overall survival
  • Prophylactic immunization with RENCA-H6 vaccine significantly prolongs survival of kidney tumor-bearing animals.
  • Prophylactic immunization with RENCA-H6 vaccine significantly prolonged survival of mice with kidney tumors [Fig. 6B].
  • Median survival of control (non-immunized) mice was 7.5 weeks (all animals died within 10 weeks).
  • In all animals that died during the experiment autopsy revealed numerous lung metastases and ascites associated with neoplastic infiltration of peritoneum.
  • mice which were cured by adjuvant administration of the vaccine: one group was challenged with RENCA cells, the other group with MethA cells. Animals immunized 5 months earlier with RENCA-H6 vaccine completely rejected s. c. implanted RENCA tumors. However, all previously immunized mice developed MethA tumors. In the control group, 100% of na ⁇ ve mice developed subcutaneous RENCA tumors [Fig 7B].
  • RENC A-H6 vaccine inhibits induction of Foxp3+ in CD4+CD25+ T lymphocytes
  • CD4+CD25+Foxp3+ Treg may inhibit induction of specific immune response.
  • An increase in the number and activity of Treg cells may significantly impede the therapeutic efficacy of cellular vaccines.
  • In order to evaluate the participation of Treg lymphocytes in the development of anti-tumor immune response we have analyzed the expression of Foxp3 molecule on CD4+CD25+ T lymphocytes infiltrating the vaccine cells at the site of administration.
  • Matrigel containing RENCA-H6 cells we observed significantly smaller population of CD4+CD25+Foxp3+ cells as compared with placebo (RENCA w/t). Expression of Foxp3 molecule was detected on 32.7% and 89.2% of CD4+CD25+ at the site of RENCA-H6 or RENCA administration, respectively [Fig. 8A]
  • RENCA-H6 vaccine provides a strong stimulatory signal for DCs
  • NK cells, activated T lymphocytes and memory T lymphocytes are engaged in the process of tumor rejection in RENC A-H6 immunized animals
  • RENCA-H6 vaccine augments generation of antigen-specific anti-tumor immune response
  • the orthotopic animal model of RCC employed in these experiments mimics the clinical course of renal cancer in human, in terms of growth characteristics, metastatic potential and responsiveness to systemic treatment (Ahn, K. S. et al., 2001).
  • Other orthotopic tumor models such as prostate cancer have also indicated the importance and specificity of a microenvironment on tumor development and its biology (Vieweg, J. et al., 1994).
  • the inventors have analyzed the efficacy of RENCA-H6 vaccine in different settings which resemble particular clinical conditions observed in renal cell cancer patients.
  • the first set of experiments carried out in a prophylactic setting clearly demonstrated that the vaccine was able to induce tumor rejection mechanisms. It also confirmed reports of other groups that RENCA w/t cells are immunogenic per se and may prevent development of implanted tumors in a small subset of immunized mice (AIi, S. A. et al., 2000).
  • RENCA-H6 vaccine Evaluation of the therapeutic potential of RENCA-H6 vaccine was carried out in palliative and adjuvant settings.
  • efficacy of immunotherapy in mice with established renal cell cancer Such a situation may be observed in patients with locally-advanced, inoperable kidney tumor.
  • Immunotherapy with RENC A-H6 vaccine proved effective in inducing rejection of renal cell cancer only if the treatment was initiated 24 hours following subcapsular inoculation of tumor cells. Since generation of antitumor, specific immune response requires approximately 14 days (Su, Z. et al., 2003), vaccination on day 7 following tumor cell inoculation induced immune response on day 21.
  • RENCA-H6 vaccine was more pronounced when compared with other vaccines or molecular therapies in preclinical studies carried out in analogous tumor models (AIi, S. A. et al., 2000; Hara, I. et al., 2000; Kausch, I. et al., 2004).
  • nephrectomy was carried out up to 2 weeks following inoculation.
  • nephrectomy was carried out on day 10 after subcapsular inoculation.
  • the 25%-survival rate of non- immunized animals after resection of primary kidney tumor resembles clinical conditions, where 10-year survival rate of localized renal cancer patients who underwent nephrectomy varies between 20-50% (Interferon-alpha and survival in metastatic renal carcinoma: early results of a randomised controlled trial.
  • DCs dendritic cells
  • CD4+ and CD8+ T lymphocytes are responsible for processing and presentation of phagocytosed antigens to T lymphocytes in the context of respective patients own MHC molecules.
  • Single mature DC expressing surface costimulatory molecules is capable of activating from 100 to 3000 T lymphocytes (Banchereau, J. and Steinman, R. M., 1998). Intense infiltration by mature DCs of the site of RENCA-H6 administration indicated that H6 stimulated maturation of DCs that translated into increased efficacy of the vaccine.
  • H6-mediated anti-melanoma activity was partially related to GM-CSF, which is known to be a major factor responsible for DCs maturation (32) (Ozbek, S. et al., 2001).
  • DCs exposed to IL-6 prime T cells against cryptic determinants and broaden the spectrum of target antigens which may be recognized by effectory T cells (33) (Drakesmith, H. et al., 1998). Accordingly - without wishing to be bound by any theory - the inventors believe that H6 secreted by the vaccine stimulated presentation of cryptic antigens by DCs.
  • CD4+CD25+Foxp3+ T regulatory lymphocytes may directly suppress activation and maturation of DCs (34) (Shevach, E. M. et al., 2002). Transformation of naive CD4+CD25- T lymphocytes into Treg is facilitated by TGF- ⁇ (35, 36) (Chen, W. et al., 2003; Pyzik, M. and Piccirillo, C. A., 2007). RENCA-w/t vaccine cells were intensively infiltrated by CD4+CD25+Foxp3+ cells.
  • RENCA tumors An efficient rejection of RENCA tumors following vaccination requires both T helper and antigen specific cytotoxic lymphocytes (19, 23, 38) (AIi, S. A. et al., 2000; Hara, I. et al., 2000; Mendiratta, S. K. et al., 2000). Indeed, in mice immunized with RENCA-H6 vaccine, rejected tumors were densely infiltrated with activated CD4+ and CD8+ T lymphocytes. In addition RENCA-H6 immunization led to a generation of antigen specific CD8+ T lymphocytes. Moreover RENCA cells upregulate MHC class II complex in response to IFN- ⁇ (39) (Hillman, G. G.
  • CD4+ T lymphocytes may directly recognize antigens presented on tumor cells.
  • immunization with RENCA-H6 cells generated larger populations of memory T cells (CD4+CD62L
  • Example 7 Immunotherapy with irradiated RENCA cells modified with Hyper-IL-11 gene
  • RenCa Renal cell carcinoma
  • DMEM Invitrogen Corp., Carlsbad, CA
  • FBS heat-inactivated fetal bovine serum
  • BVP heat-inactivated fetal bovine serum
  • HepG2 HepG2 and 293 cell lines
  • Cells were maintained as described above and culture medium for BaD and Baf3/gpl30 cell lines were supplemented with 10% medium conditioned IL-3. Conditioned medium was collected from BVP cell culture.
  • RenCaLuc cell line was obtained by transduction of RenCa cells with lentiviral vector encoding firefly luciferase. Expression of luciferase was assesed in vitro using Luciferase Assay Kit (Sigma-Aldrich).
  • RenCa cells were cultured in culture plates until 85% confluence. Following medium change, cells were incubated with AdHl 1 or AdH6. 48 h after transduction cells were harvested and irradiated with 80 Gy to prevent clonogenic survival in vivo after vaccination (GammaCell 1000). After irradiation, cells were counted and a working cell bank was created. All GMTVs (genetically modified tumour vaccines) used in the study were from the same batch.
  • H-I l and H-6 were identified by Western Blot using rabbit anti-human antibodies against IL-11 or IL- 6, and sIL-6R (Santa Cruz Biotechnology). Concentrations of both transgenic proteins were assessed using ELISA for IL-I l and sIL-6R (Anogen). Biological activity of H-I l and H-6 was evaluated in two bioassays.
  • One bioassay utilized Baf3 and Baf3/gpl30 cells proliferation assay and the other HepG2 human hepatoma cells which produce ⁇ -1-antichymotrypsin upon induction with H-6 and H-I l (Mackiewicz et al., 1992).
  • H-2K d -restricted enhanced green fluorescent protein (EGFP) 200-208 (HYLSTQSAL; SEQ ID NO: 15) CTL epitopic peptide was purchased from Prolmmune Ltd. (Oxford, United Kingdom).
  • mice Female BALB/c Fl mice, aged 8 to 12 weeks, were used. Animals were purchased from the Polish Academy of Sciences, Animal Facility (Warsaw, Tru). Animals were kept under constant pathogen-free conditions in rooms with 12-hour day/night cycle with unlimited access to food and water. All experiments were carried out according to the guidelines approved by the Regional Ethical Committee at the University of Life Sciences (Poznan, Tru).
  • mice were inoculated s.c. with RenCa cells into left hip on day 0 (established amount of cells suspended in 100 ⁇ L PBS).
  • Orthotopic model mice anesthetized with Avertin anesthesia received renal subcapsular injection of RenCa cells. Briefly, mice received i.p. injection (0.7 mg/g) of Avertin working solution (2,2,2-tribromoethanol diluted in tert-amyl alcohol). Skin of the anesthetized mice at left lumbar region was shaved with an electric shaver. Next, the skin and s.c.
  • tissue were cut with a scalpel and the left kidney was exposed. Using a tuberculin syringe, 1 ⁇ l ⁇ 4 RenCa cells suspended in 10 ⁇ L PBS were injected subcapsularly into the exposed kidney. Finally, the wound was closed with two to three surgical stitches.
  • mice were inoculated s.c. into left hip on day 0 with IxIO 6 of RenCa and RenCa-Hl l cells suspended in 100 ⁇ L PBS. Tumor growth and animal survival were monitored.
  • mice were immunized with GMTV 3 times in 4 days intervals. At day 14 after immunization, tumor cells were inoculated s.c. into left hip of mice (IxIO 6 of RenCa cells). Time to tumor formation and average tumor volume were analyzed.
  • the therapeutic potency of GMTV was studied. Mice were inoculated s.c. into left hip with 1x10 of RenCa cells on day 0. After 24h GMTV was injected into right hip of the mice in 48h intervals. Tumor growth and animal survival were monitored.
  • mice were immunized with GMTV 3 times in 4 days intervals. 14 day after immunization wild tumor cells (10 4 RenCa cells) were injected subcapsular into the exposed kidney.
  • wild tumor cells (10 4 RenCa cells) were injected subcapsular into the exposed kidney.
  • mice were again anesthetized, the tumor-bearing kidney was exposed, and following ligation of renal artery and vein a nephrectomy was performed according to uro- oncological guidelines. The following day the treatment with GMTV started and comprised of 9 vaccinations every 48h.
  • mice were administered with 10 4 RenCa cells into the kidney as described above. After 24h mice received GMTV 9 times every 2 days.
  • mice were immunized s.c. with GMTV (1x10 6 in lOO ⁇ l) 3 times every 72h. Then wild- type RenCa cells (IxIO 6 ) suspended in lOO ⁇ l of Matrigel were injected intracutaneously into mice. After 14 days, cells were isolated and stained with anti-CD4 (FITC), anti-CD8 (APC-Cy7), anti-NKl.l, anti-CD28, anti-CD40, anti-CD43, anti-CD62L, anti-CD69 (PE) (Becton Dickinson, San Diego, CA) and analyzed using FACSCan flow cytometer.
  • FITC anti-CD4
  • APC-Cy7 anti-CD8
  • PE Anti-CD69
  • RenCa cells were transduced by Lentivirus encoding Luciferase (LVLuc). Luciferase positive cells were selected using serial dilutions of cells. RenCa cells were inoculated subcapsularly into kidney as described above. Tumors and metastases were monitored weekly using Bioimager (Caliper Life Sciences). Before analyses mice were injected with D-Luciferine according to protocol (Caliper Life Sciences).
  • H-I l Concentration of H-I l was estimated by IL-I l ELISA.
  • Interleukin-11 is one of the components of H-I l fusion protein. Concentration of IL-I l accumulated in RenCa-Hl l medium was 170ng IL-I l (per 10 6 cells/24h). IL-11 concentration in RenCa- WT medium was 65pg per 10 6 cells/24h.
  • H-I l and H-6 Activity of H-I l and H-6 was assessed using HepG2 cells protein expression assay (rocket electrophoresis) and Baf3/gpl30 cells proliferation assay.
  • H-I l induced expression of ⁇ — 1— antichymotrypsin in HepG2 cells. There was no ⁇ -1-antichymotripsin stimulation by medium from RenCa-WT cell culture; normal human serum and Hyper-IL-6 were used as control.
  • Proliferation of Ba ⁇ /gpl30 in presence of medium from RenCa-Hl 1 was very high compared to proliferation of Baf3/gpl30 supplemented with medium from RenCa- WT. Medium containing H-6 was used as a positive control.
  • mice were immunized three times with RenCa-WT and RenCa-Hl l (1x10 6 cells per mouse). 14 days after immunization, non- irradiated RenCa-WT cells were inoculated (1x10 cells per mouse). Non-immunized mice served as a control. Only 12.5% of animals immunized with GMTV RenCa-Hl 1 developed tumors. Tumors in this group were small ( ⁇ 0,3cm ) and have grown slower than in animals immunized with RenCa-WT (Fig. 13). In this group 50% of mice developed tumors after 30 th day of experiment. All mice from non-immunized control group developed tumors before 21 st day of experiment.
  • Non-irradiated RenCa-WT cells were injected in three groups of animals. 24 hours after inoculation of cells, the treatment started. All non-treated mice died before week 7, mice treated with RenCa-WT lived no longer than 11 weeks, and 75% of mice treated with RenCa-Hl 1 were still alive in 16 th week after injection of cells (Fig.14).
  • H-11 inhibits tumor growth in prophylactic mouse orthotopic renal cancer model
  • Mice were immunized with RenCa-Hl l or RenCa-WT (IxIO 6 in lOO ⁇ l PBS). After 14 days RenCa cells were implanted subcapsularly into kidney (5x10 4 in lO ⁇ l PBS) in both groups of mice and in control group (naive mice). 50% of non-immunized animals developed a tumor after 4 weeks. Maximum survival time in this group was 8 weeks. 80% of mice immunized with RenCa- WT developed tumors after 14 weeks. Only in 12% of mice immunized with RenCa-Hl 1 tumors were observed. Tumor growth was inhibited in both groups of animals, i.e. in animals immunized with modified and non-modified RenCa cells. During 20 weeks of experiment survival rate of mice immunized with RenCa-Hl 1 was 100% (Fig. 15).
  • RenCa-Hl 1 moderately increases survival time in adjuvant orthotopic murine renal cancer model
  • mice were inoculated subcapsularly with RenCa cells (5xlO 4 ) into kidney. After 10 days, nephrectomy was performed. Immunization with RenCa-WT and RenCa- HI l resulted in an extension of survival time of mice after nephrectomy (Fig.16). All animals of the non-treated group (i.e. without nephrectomy) died within 5 weeks after inoculation, and all animals of the group undergoing nephrectomy without adjuvant treatment died within 6 weeks after inoculation.
  • RenCa-Hl 1 and RenCa-WT vaccine were treated with RenCa-Hl 1 and RenCa-WT vaccine.
  • some mice were injected with PBS only. It was observed that animals without treatment survived a maximum of 6 weeks compared to a group treated with RenCa-Hl 1 and RenCa-WT vaccine. Animals from the RenCa-Hl l and RenCa-WT groups showed tumor progression when the treatment was stopped after the ninth vaccination, i.e. during the third week of the experiment. However, survival rate was better in the RenCa-Hl l group than in the RenCa-WT group.
  • mice were treated with RenCa-Hl l, RenCa-H6 (positive control), and RenCa-WT vaccine. Mice were administered with GMTV 9 times every 2 days and after 9 th vaccination the treatment was continued every 7 days. As a control group, some mice were injected with PBS only. Animals without treatment lived at most 11 weeks. Maximum time of survival of RenCa-WT mice was 12 weeks. It was observed that continuation of treatment in both groups of animals receiving immunotherapy with vaccine led to significant longer survival times. 50% of mice treated with RenCa-H6 and 70% of mice treated RenCa-Hl 1 survived 16 weeks (Fig. 18).
  • RenCa-Hl l As a model antigen was used.
  • Control, RenCa-Hl l treated, and RenCa-H6 treated mice were immunized with the recombinant adenoviral vector encoding GFP.
  • the population -f GFP-specific, CD8+ splenocytes RenCa vaccine-treated animals 14 days after immunization followed by 7 days of in vitro restimulation was 2-fold higher than that of control mice (Fig.19).
  • mice received the RenCa-Hl l suspended in Matrigel. After 14 days, Matrigel was removed and infiltrating cells were isolated and stained. It was observed that Treg population was about 2-fold smaller in vaccine treated group of mice than in the group of control mice (Fig. 20).
  • mice were immunized s.c. with GMTV. Wild-type RenCa cells were suspended in Matrigel and injected intracutaneously into mice. 14 days later cells isolated from Matrigel were stained and analyzed using FACSDiva flow cytometer. Increase of cells expression CD4+CD28+ antigen was observed, however there was no difference in CD8+CD28+ cells (Fig. 23).
  • MichlH ⁇ Luc and Mich2H6Luc were mixed 1 :1.
  • the cell mixture either irradiated (80 Gy) or non-irradiated was injected subcutaneously in doses of 2x10 7 (only non-irradiated), IxIO 7 , 0.5xl0 7 and O.lxlO 7 cells into Balb/C mice.
  • 2x10 7 only non-irradiated
  • IxIO 7 0.5xl0 7
  • O.lxlO 7 cells O.lxlO 7 cells
  • Fig. 27D shows mice 7 days after injection with IxIO 7 , 0.5xl0 7 or O.lxlO 7 irradiated cells.
  • the luminescence scale to the right of the pictures ranges from about 1,000 to about 20,500 counts. This picture was taken with an exposition time of 60 seconds.
  • Fig. 27H shows mice 7 days after injection with 0.5x10 7 or O.lxlO 7 non-irradiated cells.
  • the luminescence scale to the right of the pictures ranges from about 1,000 to about 18,500 counts. This picture was taken with an exposition time of only 10 seconds.
  • Fig. 27H showing mice challenged with non-irradiated cells has an almost identical luminescence scale as Fig. 27D but this scale is reached in Fig. 27H with only one sixth of the exposition time used in Fig. 27D (10 sec vs. 60 sec). Therefore, it can be estimated that mice challenged with non-irradiated cells contain six times as much cells as compared to mice challenged with irradiated cells. Hence it can be concluded that irradiated cells are eliminated much faster than non-irradiated cells.
  • the aim of the above experiments was to analyse the fate of vaccine cells injected in vivo.
  • the concept of therapeutic cellular vaccines is to deliver a "message" and specific stimulation to the immune system.
  • Costimulatory molecules such as hyper-cytokines need to be provided locally due to their systemic toxicity (on one hand) and on the other hand cells which provide cancer antigens and transgenic proteins need to be alive for some time. Accordingly, these cells are irradiated with sterilizing doses - doses which do not kill the cells but allow them to divide 2- 4 times. Due to safety reasons it has to be demonstrated that these cancer cells will be eliminated rather than form tumors at the site of injection or spread in the body. In vitro studies demonstrated that irradiated cells die within 7 days.
  • Tumor cells engineered to express interleukin-6 exhibit a reduced tumorigenicity depending on the tumor cell model.
  • Cell MoI Biol Noisy-le-grand), 42: 169-11%, 1996.
  • a fusion protein of interleukin- 11 and soluble interleukin- 11 receptor acts as a superagonist on cells expressing gpl30.
  • TGF -beta 1 modulates Foxp3 expression and regulatory activity in distinct CD4+ T cell subsets. J Leukoc Biol, 82: 335-346, 2007.
  • Human Homo sapiens melanoma cell line stably transformed with a retrovirus to express H-IL-6 and the
  • Neomycin resistance gene under the control of a CMV-promoter
  • Human Homo sapiens melanoma cell line stably transformed with a retrovirus to express H-IL-6 and the
  • Neomycin resistance gene under the control of a CMV-promoter

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