WO2006015497A1 - Procedes d'utilisation d'une proteine dkk1, de polypeptides immunogenes de ladite proteine, d'acides nucleiques codant la proteine dkk1 ou des polypeptides, ou de ligands de ladite proteine, pour detecter des tumeurs et pour eliciter une reponse immunitaire contre des tumeurs - Google Patents

Procedes d'utilisation d'une proteine dkk1, de polypeptides immunogenes de ladite proteine, d'acides nucleiques codant la proteine dkk1 ou des polypeptides, ou de ligands de ladite proteine, pour detecter des tumeurs et pour eliciter une reponse immunitaire contre des tumeurs Download PDF

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WO2006015497A1
WO2006015497A1 PCT/CA2005/001251 CA2005001251W WO2006015497A1 WO 2006015497 A1 WO2006015497 A1 WO 2006015497A1 CA 2005001251 W CA2005001251 W CA 2005001251W WO 2006015497 A1 WO2006015497 A1 WO 2006015497A1
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dkk1
cells
protein
immunogenic polypeptide
dkk1 protein
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Réjean LAPOINTE
Diane Beauseigle
Marie-Andrée FORGET
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Val Chum SC
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Val Chum SC
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    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/575—Immunoassay; Biospecific binding assay; Materials therefor for cancer
    • G01N33/5758—Immunoassay; Biospecific binding assay; Materials therefor for cancer involving compounds serving as markers for tumours, cancers or neoplasias, e.g. cellular determinants, receptors, heat shock/stress proteins, A-protein, oligosaccharides or metabolites
    • A—HUMAN NECESSITIES
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    • A61K40/00—Cellular immunotherapy
    • A61K40/20—Cellular immunotherapy characterised by the effect or the function of the cells
    • A61K40/24—Antigen-presenting cells [APC]
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K40/00—Cellular immunotherapy
    • A61K40/40—Cellular immunotherapy characterised by antigens that are targeted or presented by cells of the immune system
    • A61K40/41—Vertebrate antigens
    • A61K40/42—Cancer antigens
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    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
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    • C07—ORGANIC CHEMISTRY
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    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
    • C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
    • C07K14/4701—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals not used
    • C07K14/4748—Tumour specific antigens; Tumour rejection antigen precursors [TRAP], e.g. MAGE
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/5005—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells
    • G01N33/5008—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics
    • G01N33/5044—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics involving specific cell types
    • G01N33/5047—Cells of the immune system
    • G01N33/505—Cells of the immune system involving T-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
    • A61K2039/525—Virus
    • A61K2039/5256—Virus expressing foreign proteins
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    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
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    • A61K2239/00—Indexing codes associated with cellular immunotherapy of group A61K40/00
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K2239/00—Indexing codes associated with cellular immunotherapy of group A61K40/00
    • A61K2239/46—Indexing codes associated with cellular immunotherapy of group A61K40/00 characterised by the cancer treated
    • A61K2239/50—Colon
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/20—Fusion polypeptide containing a tag with affinity for a non-protein ligand
    • C07K2319/21—Fusion polypeptide containing a tag with affinity for a non-protein ligand containing a His-tag
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • C12N2799/00—Uses of viruses
    • C12N2799/02—Uses of viruses as vector
    • C12N2799/021—Uses of viruses as vector for the expression of a heterologous nucleic acid
    • C12N2799/023—Uses of viruses as vector for the expression of a heterologous nucleic acid where the vector is derived from a poxvirus
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    • C12N2799/00—Uses of viruses
    • C12N2799/02—Uses of viruses as vector
    • C12N2799/021—Uses of viruses as vector for the expression of a heterologous nucleic acid
    • C12N2799/027—Uses of viruses as vector for the expression of a heterologous nucleic acid where the vector is derived from a retrovirus

Definitions

  • the present invention relates to methods of use of DKK1 protein, immunogenic polypeptides thereof, nucleic acid encoding the DKK1 protein or polypeptides, or ligands thereof for detecting tumors; and for eliciting immune response against tumors.
  • the present invention also relates to DKK1 immunogenic polypeptides and nucleic acids encoding same.
  • a Canadian has a lifetime risk of about 1 :80 of developing kidney cancer (www.ncic.cancer.ca).
  • An incomplete understanding of the molecular mechanisms underlying this disease limited up to date the development of successful non-surgical therapies. Surgery therefore remains the mainstay of treatment for kidney cancer. There is currently therefore a mortality rate of 20% within 1 year of kidney cancer diagnosis.
  • cancer is still a major killer. Early detection of cancer helps to improve treatment efficacy. Still alternative strategies need to be elaborated to improve these statistics.
  • biological cancer therapy defined as the use of biological materials or processes to fight tumors.
  • cancer immunotherapy is based on prompting the immune system to specifically recognize and kill tumors.
  • proteins specifically expressed by tumor cells have been identified. These proteins can serve as targets for the immune system, and immunotherapy has been found to be successful in a limited number of patients.
  • Tumor antigens can be defined as proteins expressed in cancer cells/tumors but absent or minimally expressed in normal tissues, such as the heart, lungs and brain. Shared gene expression between tumors and normal cells from important tissues, such as the heart and lung, should be avoided for TA, since immunization against such antigens could result in autoimmunity. However, co-expression in a tissue that is not crucial for survival can be tolerated. For example, immunization against tyrosinase-related protein-1 , which is expressed in melanoma and melanocytes, results in tumor rejection and skin depigmentation 2 . The capacity of the immune system to react against TA, referred to as immunogenicity, is an important prerequisite. Although not essential, TAs are also desirably involved in tumor progression.
  • MHC major histocompatibility complex
  • MHC class I molecules are ubiquitously expressed, such peptide presentation can potentially occur in every tissue.
  • MHC class Il presentation is mainly achieved by specialized cells of the immune system: the antigen presenting cells (APC). In this case, exogenous antigens are taken up by the APC and reach the endosomal/lysosomal compartment where they are degraded to peptides (see Figure 3 right panel). Again, only peptides with high affinity will bind to MHC class II, and the complex will migrate to the cell surface.
  • APC antigen presenting cells
  • the overall function of the MHC system is to present antigens to T lymphocytes, MHC class I presentation leading to the cellular response mediated by CD8 + T cells, and MHC class Il presentation resulting in the humoral response involving CD4 + T cells and B lymphocytes.
  • CD4 + T cells also strengthen the CD8 + T cell-mediated cytotoxic response.
  • Peptides can also be indirectly delivered by vaccination with viral vectors coding for the minimal MHC determinant from a TA. Vaccination of metastatic melanoma patients were performed with recombinant ALVAC virus bearing short MAGE-1 and MAGE-3 sequences coding for antigenic peptides presented by HLA- [0017] Peptides from multiple TA pulsed on DCs can be used as a mean of immunizing patients.
  • peptide immunization directly or presented by efficient dendritic cells (DCs)
  • DCs dendritic cells
  • the expression of the whole TA in APC enables a stable and continuous production of potentially all relevant epitopes.
  • the gene, the cDNA, or the mRNA coding for a TA expressed in APC can be used to induce a potent immune reaction and sometimes tumor regressions.
  • tumor responses have been observed in stage III or IV melanoma patients immunized with full length recombinant MAGE-3 protein.
  • the MAGE-3 recombinant protein was provided by GlaxoSmithKIine Biologicals (Rixensart, Belgium).
  • Expression of TA can be mediated by transfer in DCs by different means, such as viral vectors 31 , and TA-specific T cells can be expanded using these TA- expressing DCs 31 .
  • DCs are conveniently categorized as “immature” and “mature” cells and allow for an easy discrimination of two well characterized phenotypes.
  • Immature DCs are CDHc + , MHC class H + , CD86 + , CD80
  • CD40L and lipopolisacharrides or CD40L and poly I:C for example, CD80 and CD83 increase and they secrete high level of IL-12 33 .
  • DCs may need to be properly activated or matured 32 .
  • Matured-TA-expressing DCs could be a means of expanding TA- specific T lymphocytes dedicated for adoptive transfer 33 . It was shown that only DCs matured with a combination of CD40L and LPS were effective in generating IFN- ⁇ secreting MART- 1 -specific T lymphocytes, demonstrating the importance of delivering strong maturational signals to DCs to get efficient T cell activations.
  • TA-expressing DCs have been shown to be effective in vivo for immunization in preventive and treatment of a tumor expressing ⁇ -galactosidase as a model TA in mouse 34 .
  • Results demonstrated that only ⁇ -galactosidase- expressing DCs or DCs pulsed with a define ⁇ -galactosidase peptide were effective in treating pulmonary metastases. This was correlated by the increase in circulating lytic ⁇ -galactosidase-specific T lymphocytes.
  • Renal cell carcinoma patients were immunized with autologous DCs transfected with RNA coding for TA from RNA prepared from autologous tumors. Both immunological responses and clinical benefits were reported 35 . Interestingly, an increase of tumor-specific T cells after vaccination with renal tumor RNA- transfected DCs was noted, with specificity to telomerase and G250, two renal cell cancer TA. [0024] Patients with advanced metastatic colon cancer were treated with DCs transfected with mRNA coding for the carcinoembryonic antigen (CEA). Again, immunizations were well tolerated. Both evidences of immunological responses and clinical benefits were reported 36 .
  • CEA carcinoembryonic antigen
  • a phase l/ll clinical trial was performed using human autologous DC transfected with cDNA of the human tumor antigen mucin (MUC1) as a vaccine in 10 patients with advanced breast, pancreatic or papillary cancer. Evidences of immunizations were demonstrated and the formulation was well tolerated by patients 37 .
  • MUC1 human tumor antigen mucin
  • Adoptive T cell therapy involving the ex vivo selection and expansion of antigen-specific T cell clones derived from peripheral blood or from T cells infiltrating tumors, provides a mean of augmenting antigen-specific immunity without the in vivo constraints that can accompany vaccine-based strategies. It includes the transfer of T lymphocytes expanded in vitro, and genetically-modified T cells with a T cell receptor (TCR) specific to a TA.
  • TCR T cell receptor
  • T lymphocytes were expanded from tumor infiltrating lymphocytes (TIL) and adoptively transferred to melanoma patients following the administration of a nonmyeloablative lymphocyte depleting regimen 39 ' 40 .
  • TIL tumor infiltrating lymphocytes
  • 51% demonstrated objective clinical responses using this approach 41 .
  • All patients had assessable disease (measurable disease on computed tomography scan or by physical exam) refractory to standard treatments including high-dose IL-2 therapy (except one patient, who did not receive IL-2 before entry into this protocol).
  • Five (14.7%) of the 34 patients who received high-dose IL-2 initially responded to IL-2 therapy alone, but then exhibited progressive disease and were enrolled on this protocol.
  • T cells recognize peptides derived from TA presented by MHC complexes by a molecule called T cell receptor (TCR) (see Figure 3).
  • TCR is highly polymorphic to respond to a gigantic diversity of pathogens for example.
  • T lymphocytes emerge some having TCR with a capacity to react with TA-derived peptides.
  • T lymphocytes from an HLA-A*0201 donor were engineered to express a TCR ( ⁇ and ⁇ chains) specific to an HLA-A*0201 epitope from MART-1 42 or gp100 43 , both melanoma antigens.
  • Modified T cells were expanded to a very high number and they became reactive against TA-expressing tumors. Clinical assays exploiting these TCR-modified anti-tumor T cells are expected to start in a near future at the Surgery branch of the American National Cancer Institutes.
  • RNA isolated from the MART-1 -reactive T-cell clone was subjected to RACE (rapid amplification of cDNA ends) polymerase chain reaction (PCR) and DNA sequence analysis in order to determine TCR ⁇ and ⁇ chain usage.
  • RACE rapid amplification of cDNA ends
  • PCR primers for cloning of the individual chain full-length cDNAs were designed.
  • polyA + RNA was isolated from 1 X 10 7 M1 F2 T cells using the Poly (A) Pure mRNA purification kit (Ambion, Austin, TX).
  • RT-PCR Reverse transcription-polymerase chain reaction
  • pMSGVI The retroviral vector backbone used in this study, is a derivative of the vector pMSGV (MSCV-based splice-gag vector) that utilizes a murine stem cell virus (MSCV) long terminal repeat (LTR; Hawley et al., 1994), and contains the extended gag region and env splice site from vector SFGtcLuc + ITE4 " .
  • MSCV murine stem cell virus
  • LTR long terminal repeat
  • a modification incorporates a naturally occurring Kozak sequence to enhance translational efficiency.
  • Lymphocytes were purified by centrifugation on a Ficoll/Hypaque cushion, washed in Hanks' balanced saline solution (HBSS) and resuspended in AIM-V medium supplemented with 50 ng/ml of OKT3, 300 IU/ml IL-2 and 5% human AB serum at a concentration of 1 X 10 6 cells per milliliter.
  • the lymphocytes were cultured in 24-well plates (Costar, Cambridge, MA) for 48 hr prior to transduction.
  • Nontissue culture-treated six-well plates (Becton Dickinson Labware, Franklin Lakes, NJ) were treated with 25 ⁇ g/ml of recombinant fibronectin fragment as directed by the manufacturer (RetroNectin, Takara, Otsu, Japan). Retroviral vector supernatant (4-6 ml) was added and the plates were incubated at 32°C for 2-4 hr after storage at 4 0 C overnight. Plates were warmed to room temperature, supernatant was removed and 10 6 stimulated PBL per milliliter were added to each well with 3-5 ml per well. These plates were incubated overnight and the transduction process was repeated the following day.
  • TCR-transduced PBMC was determined by co-culture with T2 cells pulsed with either HLA-A2-restricted influenza peptide (GILGFVFTL), MART- 1 27 . 35 peptide or gp1 OO 2O9 -217(2I O M) in AIM-V and 5% human serum.
  • the responder cells and the stimulators were co-cultured in a ratio of 1 :1 with 100,000 cells each in 96-well U-bottom plate (Costar, Corning, NY) with a total volume of 0,2 ml for 24 hr except TNF- ⁇ , which was harvested at 6 hr.
  • Cytokine secretion IFN- ⁇ or GM-CSF was measured via enzyme-linked immunosorbent assay (ELISA; Endogen, Cambridge, MA).
  • breast cancer TA such as HER-2/neu, carcinoembryonic antigen (CEA) 3 , MUC-1 4 , mammaglobin 5 , NY-ESO-1 6 and MAGE-1 7 .
  • CEA carcinoembryonic antigen
  • MUC-1 4 mammaglobin 5
  • mammaglobin 5 mammaglobin 5
  • NY-ESO-1 6 mammaglobin 5
  • MAGE-1 7 MAGE-1 7
  • Wirths et al. have described human DKK1 , an antagonist of wingless/WNT signaling (involved in fetal development), as a marker for Hepatoblastomas and Wilms' tumors.
  • Other cell types normal and tumor cells
  • DKK1 -negative Breast carcinoma samples, particularly, tested negatively, leaving DKK1 as a putative marker for a very limited number of cancer types.
  • DKK1 is a marker for largely spread cancers like breast, prostate, lung, colon, kidney and skin cancers.
  • DKK1 is a TA usable in an antitumoral vaccine.
  • DKK1 binds LRP5/6 and a protein called Kremen (the product of Krm gene). This association inhibits the interaction of LRP5/6 to Fz (Frizzled), which is a key interaction for beta-cathenin pathway engagement.
  • the present invention seeks to meet these needs and other needs.
  • the . present invention concerns the use of DKK1 as a marker of tumors. It further concerns the use of DKK1 and of immunogenic polypeptides derived there from as a useful TA.
  • the tumors shown to be expressing DKK1 are melanomas, prostate carcinomas, breast carcinomas, lung carcinomas renal carcinomas, ovarian and colon cancer.
  • the present invention encompasses immunization of cancer patients with various means including administering to patients DKK1 protein or immunogenic polypeptides or cells expressing same, polynucleotides encoding DKK1 protein or immunogenic polypeptides thereof.
  • a polynucleotide encoding a DKK1 protein or an immunogenic polypeptide thereof can be included in a plasmid and linked to a promoter sequence/expression control sequence, such as that from the cytomegalovirus (CMV).
  • CMV cytomegalovirus
  • DKK1 under the control of a promoter can then be transferred in DCs by chemical (lipofectamine or other) means, or using non-viral or viral vectors (adenoviral or other).
  • DKK1 expressing DCs can then administered so that reaches the lymphatic circulation, under the skin or directly in the lymphatic circulation.
  • Vectors encoding DKK1 or an immunogenic polypeptide thereof can also be administered directly, alone or with other genes having immunogenicity enhancing capacity.
  • adoptive transfer in the patients of T cells activated with DKK1 protein, immunogenic polypeptides thereof, polynucleotides encoding DKK1 protein or immunogenic polypeptides thereof or APCs expressing DKK1 protein or immunogenic polypeptides thereof.
  • MHC class I or class Il restricted immunogenic polypeptides or a combination of such polypeptides can be administered s.c, or intradermal (i.d.) from 100 ⁇ g to 1 mg several times at different intervals.
  • Peptides can be administered as is, or in combination with adjuvants.
  • Polypeptides can be administered after loading on matured antigen presenting cells such as DCs.
  • adoptively transferred T cells can be T lymphocytes from an donor engineered to express a TCR ( ⁇ and ⁇ chains) specific to an epitope from DKK1.
  • DKK1 immunogenic polypeptide refers to peptides from 8 to 25 residues derived from DKK1 protein sequence that can be presented by MHC molecules to stimulate either CD4 + or CD8 + T lymphocytes or both. This includes modified peptides in which a substitution of one or more amino acids were made to allow a better binding to MHC molecules, or to improve interaction with TCR. Without being so limited, the residues at position 2, 6 and 9 ( Figure 9) which being the putative anchor residues of the HLA-A*0201 restricted DKK1 immunogenic polypeptides can be substituted by a leucine or methionine residue, a valine residue, or a valine or a leucine residues, respectively.
  • sequences of polypeptides more likely to promote these interactions can be routinely predicted exploiting tools such as the SYFPEITHI 48 (http://www.syfpeithi.de/) tool, or Dr. Kenneth Parker's algorithms 49 (http://bimas.dcrt.nih.gov/molbio/hla_bind/).
  • Valid epitopes after proteasome cleavage can also be predicted with the PAProCTM tool (http://www.paproc.de/) 46 ' 47 .
  • DKK1 immunogenic polypeptides are exemplified herein with HLA-A*0201 restricted DKK1 polypeptides
  • DKK1 -derived peptides from other HLA alleles can be predicted.
  • HLA-A1 or HLA-A3 restricted DKK1 immunogenic polypeptides can be routinely identified with bioinformatics tools such as those described above. '
  • immune response refers to any reaction of the immune system against a foreign biological material (i.e. antigen).
  • immune system refers to the collection of organs and tissues and cells involved in the adaptive defense of a body against foreign biological material. It may be broken down into the adaptive immune system, composed of four lymphoid organs (thymus, lymph nodes, spleen and submucosal lymphoid nodules) and the group motile cells that are involved in the body's defense against foreign bodies.
  • immune response include in vivo or ex vivo "T lymphocytes activation" in an antigen-specific manner by triggering of the TCR, as illustrated by T cell proliferation, or secretion of an array of cytokines such as but not limited to GM-CSF, TNF- ⁇ , IFN- ⁇ , IL-2, IL-4 and IL-10, or evidence of cytolytic activity such as but not limited to secretion of perforin, granzyme family members, or migration of CD107a (LAMP-1) to the cell surface or any functional assay demonstrating lysis of a relevant target.
  • Upregulation of some surface or intracellular molecules can also serve as T cell activation markers, such as but not limited to CTLA-4, CD25 (high affinity IL-2 receptor) KI-67, or MHC class Il molecules.
  • T lymphocytes Upon stimulation through the T cell receptor, T lymphocytes frequently proliferate and secrete an array of cytokines, which can be diverse, depending on cell polarization.
  • Helper CD4 + T cells (Th) and cytotoxic CD8 + T cells (Tc) progress from naive to effector, and memory T cells to Th1, Th2, Td or Tc2 profiles.
  • Th1/Tc1-type cells and associated cytokines such as interferon (IFN)- ⁇ and IL-2
  • IFN interferon
  • Th2/Tc2-type cells and cytokines such as IL-4, IL-5 and IL-10, are secreted by lymphocytes committed to an allergic reaction and to humoral responses.
  • cytokine secreted by a wide array of T cells such as Td and Tc2
  • Td and Tc2 granulocyte-macrophage colony- stimulating factor
  • Qualitative evaluation of cytokine secretion by T cells can be done by ELISA. In such assays, T cells are co- cultured with their target from which specificity is assumed.
  • T cells When T cells are cultured with their specific target, IFN- ⁇ is secreted and evaluated by ELISA. This approach is useful to assess T cell specificity in a cell population with a relatively high proportion of cytokine secreting antigen-specific T cells. Quantitative evaluation of cytokine secretion can also be performed by ELISPOT assays. In this assay, T cells are co-cultured with relevant targets such as DKK1 exposing cells such as tumor cells and DKK1 expressing DCs in a 96-well plate pre-coated with an antibody specific to a cytokine of interest. These cells are then removed after cytokine secretion, and cytokines captured by coated antibodies are revealed in a sandwich assay similar to an ELISA reaction.
  • relevant targets such as DKK1 exposing cells such as tumor cells and DKK1 expressing DCs in a 96-well plate pre-coated with an antibody specific to a cytokine of interest.
  • the subsequent point in time after having detecting for the first time DKK1 in the patient may be any convenient time after a previous detection of DKK1 in the patient. It may depend on the type of cancer and could be adapted to coincide with other recommended periodical tests for that cancer. Without being so limited, it could be any time after the previous detection of DKK1 in the patient from about 1 week to about 7 years. Without being so limited, when the cancer is a breast cancer, a convenient subsequent point in time could be one year or 15 months after the previous test. For lung cancer, a convenient subsequent point in time after could be about a month or two months after the previous test.
  • the present invention encompasses the use of a DKK1 polynucleotide sequences as disclosed in Figure 1 along with DKK1 polynucleotide sequences substantially identical to those. Namely any DKK1 polynucleotide sequence that has at least 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, or 79%, preferably at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, or 89%, more preferably at least 90%, 91%, 92%, 93%, or 94%, and most preferably at least 95%, 96%, 97%, 98%, or 99% sequence identity, compared to a reference sequence such as those disclosed in Figure 1 using one of the alignment programs described using standard parameters.
  • amino acid sequences for these purposes normally means sequence identity of at least 70%, more preferably at least 80%, 90%, and most preferably at least 95%.
  • polypeptides that are substantially identical are they hybridize to each other under stringent conditions (see below).
  • stringent conditions are selected to be about 5°C lower than the thermal melting point (T m ) for the specific sequence at a defined ionic strength and pH.
  • T m thermal melting point
  • Nucleic acids that do not hybridize to each other under stringent conditions are still substantially identical if the polypeptides they encode are substantially identical. This may occur, e.g., when a copy of a nucleic acid is created using the maximum codon degeneracy permitted by the genetic code.
  • One indication that two nucleic acid sequences are substantially identical is when the polypeptide encoded by the first nucleic acid is immunologically cross reactive with the polypeptide encoded by the second nucleic acid.
  • hybridizing specifically to refers to the binding, duplexing, or hybridizing of a molecule only to a particular nucleotide sequence under stringent conditions when that sequence is present in a complex mixture (e.g., total cellular) DNA or RNA.
  • Bod(s) substantially refers to complementary hybridization between a probe nucleic acid and a target nucleic acid and embraces minor mismatches that can be accommodated by reducing the stringency of the hybridization media to achieve the desired detection of the target nucleic acid sequence.
  • T m is the temperature (under defined ionic strength and pH) at which 50% of the target sequence hybridizes to a perfectly matched probe. Specificity is typically the function of post-hybridization washes, the critical factors being the ionic strength and temperature of the final wash solution.
  • T m can be approximated from the equation of Meinkoth and Wahl, 1984; T m 81.5°C + 16.6 (log M) +0.41 (%GC) - 0.61 (% form) - 500/L; where M is the molarity of monovalent cations, %GC is the percentage of guanosine and cytosine nucleotides in the DNA, % form is the percentage of formamide in the hybridization solution, and L is the length of the hybrid in base pairs.
  • T m is reduced by about 1°C for each 1% of mismatching; thus, T m , hybridization, and/or wash conditions can be adjusted to hybridize to sequences of the desired identity.
  • the T m can be decreased 1O 0 C.
  • stringent conditions are selected to be about 5 0 C lower than the thermal melting point I for the specific sequence and its complement at a defined ionic strength and pH.
  • severely stringent conditions can utilize a hybridization and/or wash at 1 , 2, 3, or 4°C lower than the thermal melting point I;
  • moderately stringent conditions can utilize a hybridization and/or wash at 6, 7, 8, 9, or 10°C lower than the thermal melting point I;
  • low stringency conditions can utilize a hybridization and/or wash at 11, 12, 13, 14, 15, or 2O 0 C lower than the thermal melting point I.
  • hybridization and wash compositions those of ordinary skill will understand that variations in the stringency of hybridization and/or wash solutions are inherently described. If the desired degree of mismatching results in a T of less than 45°C (aqueous solution) or 32°C (formamide solution), it is preferred to increase the SSC concentration so that a higher temperature can be used.
  • An extensive guide to the hybridization of nucleic acids is found in Tijssen, 1993.
  • highly stringent hybridization and wash conditions are selected to be about 5 0 C lower than the thermal melting point T m for the specific sequence at a defined ionic strength and pH.
  • An example of stringent wash conditions is a 0.2X SSC wash at 65 0 C for 15 minutes. Often, a high stringency wash is preceded by a low stringency wash to remove background probe signal.
  • An example medium stringency wash for a duplex of, e.g., more than 100 nucleotides, is 1X SSC at 45 0 C for 15 minutes.
  • An example low stringency wash for a duplex of, e.g., more than 100 nucleotides, is 4-6X SSC at 4O 0 C for 15 minutes.
  • stringent conditions typically involve salt concentrations of less than about 1.5 M, more preferably about 0.01 to 1.0 M, Na ion concentration (or other salts) at pH 7.0 to 8.3, and the temperature is typically at least about 3O 0 C and at least about 60°C for long robes (e.g., >50 nucleotides).
  • Stringent conditions may also be achieved with the addition of destabilizing agents such as formamide.
  • destabilizing agents such as formamide.
  • a signal to noise ratio of 2X (or higher) than that observed for an unrelated probe in the particular hybridization assay indicates detection of a specific hybridization.
  • Nucleic acids that do not hybridize to each other under stringent conditions are still substantially identical if the proteins that they encode are substantially identical. This occurs, e.g., when a copy of a nucleic acid is created using the maximum codon degeneracy permitted by the genetic code.
  • substantially identical in the context of a DKK1 polypeptide indicates that a polypeptide comprises a sequence with at least 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, or 79%, preferably 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, or 89%, more preferably at least 90%, 91%, 92%, 93%, or 94%, or even more preferably, 95%, 96%, 97%, 98% or 99%, sequence identity to the reference sequence over a specified comparison window.
  • optimal alignment is conducted using the homology alignment algorithm of Needleman and Wunsch (1970).
  • An indication that two peptide sequences are substantially identical is that one peptide is immunologically reactive with antibodies raised against the second peptide.
  • a polypeptide is substantially identical to a second peptide, for example, where the two polypeptides differ only by a conservative substitution e.g. not substantially affecting the folding, the charge, the molecular weight, the lipophilicity or the final composition (glycosylation for example) of a polypeptide.
  • sequence comparison typically one sequence acts as a reference sequence to which test sequences are compared.
  • test and reference sequences are input into a computer, subsequence coordinates are designated if necessary, and sequence algorithm program parameters are designated.
  • sequence comparison algorithm then calculates the percent sequence identity for the test sequence(s) relative to the reference sequence, based on the designated program parameters.
  • the invention encompasses isolated or substantially purified nucleic acid or protein compositions.
  • an "isolated” or “substantially purified” DNA molecule or an “isolated” or “substantially purified” polypeptide is a DNA molecule or polypeptide that, by the hand of man, exists apart from its native environment and is therefore not a product of nature.
  • An isolated DNA molecule or polypeptide may exist in a purified form or may exist in a non-native environment such as, for example, a transgenic host cell.
  • An isolated or purified DNA or polypeptide may be synthesized chemically, may be produced using recombinant DNA techniques and then isolated or purified or may be isolated or purified from its natural host.
  • nucleic acid molecule or protein, or biologically active portion thereof is substantially free of other cellular material, or culture medium when produced by recombinant techniques and, in some circumstances, further purified, or substantially free of chemical precursors or other chemicals when chemically synthesized.
  • an "isolated" nucleic acid is free of sequences (preferably protein encoding sequences) that naturally flank the nucleic acid (i.e., sequences located at the 5' and 3' ends of the nucleic acid) in the genomic DNA of the organism from which the nucleic acid is derived.
  • the isolated nucleic acid molecule can contain less than about 5kb, 4 kb, 3 kb, 2 kb, 1 kb, 0.5 kb, or 0.1 kb of nucleotide sequences that naturally flank the nucleic acid molecule in genomic DNA of the cell from which the nucleic acid is derived.
  • a DKK1 protein, or an immunogenic polypeptide thereof, that is purified or substantially free of cellular material includes preparations of protein or polypeptide having less than about 30%, 20%, 10%, 5%, (by dry weight) of contaminating protein.
  • culture medium represents less than about 30%, 20%, 10%, or 5% (by dry weight) of chemical precursors or non-protein of interest chemicals.
  • Recombinant DKK1 proteins or immunogenic polypeptides thereof encompassed by the present invention also include fusion protein comprising not only DKK1 proteins or. immunogenic polypeptides thereof but also heterologous domains for enhancing immunogenicity, including adjuvants and amino acid sequences enabling epitope cross-presentation.
  • a helper epitope from an other protein such a protein D from H. influenzae as described in Example 7 is useful for enhancing immunogenicity.
  • Recombinant DKK1 proteins or polypeptides encompassed by the present invention may further include domains for facilitating purification such as a histidine tag.
  • the DKK1 proteins useful in the present invention encompass mature a DKK1 protein i.e. without its signal peptide (see Figures 1).
  • the mature protein of the DKK1 presented in figure 2 consists of residues 32 to 266 of this protein.
  • Useful vectors for transfecting cells to be used as APCs include plasmids where DKK1 polynucleotide expression would be controlled by a promoter sequence, such as that from the cytomegalovirus (CMV). Viral vectors can also be exploited to transduce APCs, such as but not limited to adenoviruses, lentiviruses or classical retroviruses.
  • Useful vectors for direct injections (i.e. without APCs) of DKK1 or immunogenic polypeptides thereof include the ALVAC virus, and non-viral plasmids including those from VICAL technologies (See ⁇ http://www.vical.com/company/dnatech.htm>).
  • ligand when used herein in reference to a DKK1 protein or immunogenic polypeptide refers to antibodies, or a natural DKK1 -binding domain from natural receptors including LPR5/6 and the Kremen protein 51 , agonists, antagonists and to any molecule found to bind DKK1 protein or immunogenic polypeptide from an assay on a combinatorial library namely, from a phage display library.
  • ligand when used herein in reference to a DKK1 polynucleotide encoding a DKK1 protein or immunogenic polypeptide refers to a complementary strand to one of the strands of a DNA, a cDNA, or an RNA, the complementary strand being used as a probe or as an amplification primer.
  • Useful APCs for the present invention include any cells with the ability to engulf DKK1 protein or immunogenic polypeptide thereof and present them to the cells of the immune system in a form that can be recognized by those immunocompetent cells. Without being so limited it includes DCs expressing DKK1 protein or immunogenic polypeptide thereof and B lymphocytes. In both instances (ligand to polypeptide or to polynucleotide), the ligands may be labeled by any direct or indirect means for proper detection.
  • expression control sequence refers to a sequence that promotes expression of a DKK1 protein or an immunogenic polypeptide thereof in a host cell including a tumor cell and a natural or recombinant APCs. Without being so limited, such sequence includes the CMV expression control sequence.
  • the present invention also provides a pharmaceutical composition
  • a pharmaceutical composition comprising a immunogenically effective amount of DKK1 or immunogenic polypeptide thereof, and one or more pharmaceutically or physiologically acceptable carriers, adjuvants and diluents.
  • adjuvants include incomplete Freund's adjuvant (IFA, also called Montanide, commercialized as ISA- 51 , Seppic Company, Paris, France), CpG sequences such as but not limited to CpG 7909 (TCGTCGTTTTGTCGTTTTGTCGTT (SEQ ID NO: 5); Coley Pharmaceutical Group), different cytokines with T cells stimulatory capacity such as IL-2, IL-7, IL-12, IL-15, or others, cytokines promoting activation of antigen presenting cells such as GM-CSF, CD40L, FLT3L, or others.
  • IFA incomplete Freund's adjuvant
  • CpG sequences such as but not limited to CpG 7909 (TCGTCGTTTTGTCGTTTTGTCGTT (
  • Adjuvants can also include products derived from pathogens such as detoxified lipopolysaccharides (Monophosphoryl lipid A or MPL, Corixa Corporation) or mycobacterial walls (Bioniche). Dosage regime may be adjusted to provide the optimum therapeutic response. For example, several divided doses may be administered daily or weekly, or the dose may be proportionally reduced as indicated by the exigencies of the therapeutic situation.
  • the active compound may be administered in a convenient manner such as by the oral, intramuscular, subcutaneous, intranasal, intradermal or suppository routes or implanting (e.g., using slow release molecules or devices).
  • the DKK1 or immunogenic polypeptide thereof may also be administered parenterally or intraperitoneally.
  • Dispersions can also be prepared in glycerol, liquid polyethylene glycols, and mixtures thereof and in oils. Under ordinary conditions of storage and use, these preparations contain a preservative to prevent the growth of microorganisms.
  • the pharmaceutical forms suitable for injectable use include sterile aqueous solutions (where water soluble) or dispersions and sterile powders of the extemporaneous dispersion.
  • the form must be sterile and must be fluid to the extent that easy syringability exists. It must be stable under the conditions of manufacture and storage and must be preserved against the contaminating action of microorganisms such as bacteria and fungi.
  • the carrier can be a solvent or dispersion medium containing, for example, buffered saline such as PBS, water, ethanol, polyol (for example, glycerol, propylene glycol, and liquid polyethylene glycol, and the like), suitable mixtures thereof, and vegetable oils.
  • the proper fluidity can be maintained, for example, by the use of a coating such as lecithin, by the maintenance of the required particle size in the case of dispersion and by the use of superfactants.
  • the preventions of the action of microorganisms can be brought about by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, sorbic acid, thimerosal, and the like. In many cases, it will be preferable to include isotonic agents, for example, sugars or sodium chloride. Prolonged absorption of the injectable compositions can be brought about by the use in the compositions of agents delaying absorption, for example, aluminum monostearate and gelatin.
  • Sterile injectable solutions are prepared by incorporating the active compounds in the required amount in the appropriate solvent with various of the other ingredients enumerated above, as required, followed by filtered sterilization.
  • dispersions are prepared by incorporating the various sterilized active ingredients into a sterile vehicle which contains the basic dispersion medium and the required other ingredients from those enumerated above.
  • the preferred methods of preparation are vacuum drying and the freeze-drying technique which yield a powder of the active ingredient plus any additional desired ingredient from previously sterile-filtered solution thereof.
  • the present invention thus relates to a use of a purified or recombinant DKK1 protein or of an immunogenic polypeptide comprising at least 8 contiguous amino acids of said DKK1 protein, for activating T cells.
  • the DKK1 protein comprises an amino acid sequence as set forth in any one of SEQ ID NO: 8 and residues 32 to 266 of SEQ ID NO: 8.
  • the DKK1 immunogenic polypeptide consists of a maximum of 25 amino acid residues and comprises an amino acid sequence as set forth in any one of SEQ ID NOs: 9-15, 34 and 38-41.
  • the DKK1 immunogenic polypeptide consists of a maximum of 25 amino acid residues and comprises an amino acid sequence as set forth in any one of SEQ ID NOs: 9-15.
  • the purified or recombinant DKK1 protein or the immunogenic polypeptide is expressed on an antigen presenting cell (APC).
  • the APC is a dendritic cell transformed to express the purified or recombinant DKK1 protein or immunogenic polypeptide comprising at least 8 contiguous amino acids of the DKK1 protein.
  • an isolated polynucleotide comprising the coding sequence of a DKK1 protein, or the coding sequence of an immunogenic polypeptide comprising at least 8 contiguous amino acids of the DKK1 protein, for activating T cells.
  • said isolated polynucleotide is comprised in an expression vector.
  • the vector further comprises a polynucleotide encoding an immunogenicity enhancing polypeptide.
  • the coding sequence is as set forth in any one of SEQ ID NO: 7 and nucleotides 248 to 952 of SEQ ID NO: 6.
  • the coding sequence encodes an amino acid sequence as set forth in any one of SEQ ID NOs: 9-15, 34 and 38-41. In an other specific embodiment, the coding sequence encodes an amino acid sequence as set forth in any one of SEQ ID NOs: 9-15.
  • a use of a purified or recombinant DKK1 protein or of an immunogenic polypeptide comprising at least 8 contiguous amino acids of said DKK1 protein, in the making of a medicament for activating T cells there is also provided a use of an isolated polynucleotide comprising the coding sequence of a DKK1 protein, or the coding sequence of an immunogenic polypeptide comprising at least 8 contiguous amino acids of the DKK1 protein, in the making of a medicament for activating T cells.
  • a method for monitoring the progression of breast, lung, kidney, colon, melanoma, prostate, ovarian or colon cancer in a patient, with the proviso that the kidney cancer is not Wilms' tumor comprising the steps of: (a) obtaining from the patient a biological sample susceptible of containing tumor cells; (b) contacting the biological sample with a ligand to a DKK1 protein or an immunogenic polypeptide thereof, or to a polynucleotide encoding said protein or said polypeptide to form a complex; (c) measuring the amount of the complex if any, d) repeating steps (a) and (b) using a biological sample obtained from the patient at a subsequent point in time, (e) comparing the amount of the complex measured in step (c) with that in step (d) thereby monitoring the progression of breast, lung, kidney, colon, melanoma , , prostate, ovarian or colon cancer in the patient.
  • a method for detecting the presence of breast, lung, kidney, colon, melanoma, prostate, ovarian or colon cancer in a patient, with the proviso that the kidney cancer is not Wilms' tumor comprising the steps of: (a) obtaining from the patient a biological sample susceptible of containing tumor cells; (b) contacting the biological sample with a ligand to a DKK1 protein or an immunogenic polypeptide thereof, or to a polynucleotide encoding said protein or said polypeptide to form a complex; (c) determining the presence or absence of the complex, wherein the presence of the complex is an indication of the presence of a breast, lung, kidney, colon, melanoma, prostate, ovarian or colon cancer in the patient.
  • a method for monitoring the progression of breast, lung, kidney, colon, melanoma, prostate, ovarian or colon cancer in a patient, with the proviso that the kidney cancer is not Wilms' tumor comprising the steps of: (a) contacting a biological sample from the patient, susceptible of containing tumor cells, with at least two oligonucleotide primers under conditions wherein said oligonucleotide primers are effective for specifically amplifying a polynucleotide sequence of DKK1 in a reverse transcription polymerase chain reaction; (b) detecting in the sample an amount of polynucleotide amplified in step (a); (c) repeating steps (a) and (b) using a biological sample obtained from the patient at a subsequent point in time; and (d) comparing the amount of polynucleotide detected in step (c) to the amount detected in step (b) thereby monitoring the progression of breast, lung, kidney,
  • a method for activating T cells specific for a DKK1 protein, or an immunogenic polypeptide thereof comprising incubating the T cells with at least one component selected from the group consisting of: (i) a purified or recombinant DKK1 protein or an immunogenic polypeptide thereof; (ii) a polynucleotide comprising the coding sequence of a DKK1 protein or an immunogenic polypeptide thereof; and (iii) antigen presenting cells transformed to express a recombinant DKK1 protein or an immunogenic polypeptide thereof, whereby T cells are activated.
  • a method for activating T cells specific for a DKK1 protein, or an immunogenic polypeptide thereof in a patient comprising administering to the patient at least one component selected from the group consisting of: (i) a purified or recombinant DKK1 protein or an immunogenic polypeptide thereof; (ii) a polynucleotide comprising the coding sequence of a DKK1 protein or an immunogenic polypeptide thereof; and (iii) antigen presenting cells transformed to express a recombinant DKK1 protein or an immunogenic polypeptide thereof, whereby T cells are activated in the patient.
  • an expression vector comprising a polynucleotide encoding a DKK1 protein operably associated with an expression control sequence, and a polynucleotide encoding an immunogenicity enhancing polypeptide.
  • the expression vector comprises an immunogenic polypeptide derived from a DKK1 protein operably associated with an expression control sequence.
  • the expression vector further comprises a polynucleotide encoding an immunogenicity enhancing polypeptide.
  • a host cell expressing the vector of the present invention.
  • the cell is an antigen-presenting cell (APC).
  • a fusion protein comprising a DKK1 protein or an immunogenic polypeptide thereof and an immunogenicity enhancing polypeptide.
  • a composition comprising a physiologically acceptable carrier, and a second component selected from the group consisting of: (a) a purified or recombinant DKK1 protein or an immunogenic polypeptide of DKK1; (b) a polynucleotide comprising the coding sequence of a DKK1 protein or an immunogenic polypeptide of DKK1 ; (c) an antibody specific to a DKK1 protein or to an immunogenic polypeptide thereof; (d) a fusion protein of the present invention; (e) a T cell population activated by cells expressing a DKK1 protein or expressing an immunogenic polypeptide thereof; and (f) APC cells of the present invention.
  • the method comprises contacting the T cells with a composition of the present invention.
  • an immunogenic polypeptide derived from a DKK1 protein in a specific embodiment, the polypeptide consists of up to 25 amino acid residues and comprises an amino acid sequence as set forth in any one of SEQ ID NOs: 9-15, 34 and 38-41.
  • an isolated polynucleotide encoding a polypeptide of the present invention in a further aspect of the invention.
  • Figure 1 presents the DKK1 mRNA sequence (SEQ ID NO: 6), wherein residues 155 to 952 (SEQ ID NO: 7) represent the coding sequence, and the DKK1 polypeptide sequence (SEQ ID NO: 8) obtained from GenbankTM accession number NM_012242 and SwissProtTM accession number: 094907, respectively;
  • Figure 2 presents the polypeptide sequence of DKK1 alone (SEQ ID NO: 8);
  • FIG 3 illustrates the presentation of endogenous antigens by MHC class I to CD8 + T cells (left panel) and of exogenous antigens by MHC class Il to CD4 + T cells (right panel) (From S. A. Rosenberg, The Cancer Journal from Scientific American, July/August 1995);
  • FIG. 4 shows the expression profile of DKK1 in tumor cell lines and PBMC.
  • A, B and C - mRNA was prepared from the indicated cell lines, and RT-PCR analyses were performed with the indicated specific primers.
  • Normal primary cell lines were prepared by stimulation of PBMC with anti-CD3 and IL-2 (T cells), or with soluble CD40L and IL-4, which stimulate B lymphocytes to proliferate (B cells).
  • Reverse transcriptase was omitted in the MDA231-RT group and HCC2218 EBVB cells (EBV-B -RT).
  • HCC2218 EBV-B and HCC1428 EBV-B are EBV-immortalized B lymphocytes prepared from breast cancer patients HCC2218 and HCC1428 respectively.
  • Amplification was detected by ethidium bromide staining after electrophoresis migration in agarose gel.
  • MeI melanoma
  • Figure 5 shows the expression profile of genes selected by the bio- informatic approach in tumor cell lines and PBMC.
  • mRNA was prepared from the indicated cell lines, and RT-PCR analyses were performed with the indicated specific primers.
  • Normal primary cell lines were prepared by stimulation of PBMC with anti-CD3 and IL-2 (T cells #1 and #2), or with soluble CD40L and IL-4, which stimulate B lymphocytes to proliferate (CD40B #1 and #2), and fresh PBMC (PBMC #1 and #2).
  • Reverse transcriptase was omitted in the MCF-7 -RT group.
  • HCC2218EBV and HCC1428EBV are EBV-immortalized B lymphocytes prepared from breast cancer patients HCC2218 and HCC1428 respectively. Amplification was detected by ethidium bromide staining after electrophoresis migration in agarose gel;
  • Figure 6 shows the expression profile of DKK1 in normal tissues.
  • a and B - mRNA was prepared from the indicated normal tissues (acquired from Origene Technologies in A, and from BD-Clontech in B), and controls.
  • RT-PCR analyses were performed with DKK1 and ⁇ -actin specific primers.
  • B - Expression was determined by quantitative real time RT-PCR (LightCyclerTM, Roche);
  • Figure 7 graphically shows the results of quantitative real-time, two- step RT-PCR analysis of DKK1 and ⁇ -actin from mRNA-prepared clinical samples.
  • cDNA from mRNA primed with oligo-dt were prepared from the indicated controls and clinical samples from breast (A), lung (B) or renal cell cancers (C).
  • Amplification was undertaken by real-time quantitative PCR and revealed by SYBr green staining. Standard curves for each gene were established to quantify the number of copies for each sample, and expression was considered only for the Ct of samples within the limit of each standard. Amplification of the relevant amplicon was further confirmed by separation on agarose gel revealed by ethidium bromide staining.
  • Samples from breast cancer patients (A) are clustered by ER and PR status, as evaluated by the clinical pathology service (score: -: negative, +: positive);
  • Figure 8 graphically shows a cluster analysis for breast cancer specimens. Samples were clustered according to A the expression of hormonal receptors; B - the reported family history or C - the histological grade. Legend: ER: estrogen receptor; PR: progesterone receptor; +: positive; -: negative; and
  • Figure 9 shows the determination of putative anchor residues for
  • DKK1 immunogenic polypeptides i.e. polypeptide 20 (SEQ ID NO: 9); polypeptide 40 (SEQ ID NO: 10); polypeptide 32 (SEQ ID NO: 11); polypeptide 37 (SEQ ID NO: 12); polypeptide 61 (SEQ ID NO: 13); polypeptide 68 (SEQ ID NO: 14); polypeptide 182 (SEQ ID NO: 15)) from an alignment of these polypeptides.
  • Residues two, six and nine (shaded) within a nine or 10-mer polypeptide sequence have been defined as anchor residues for HLA-A*0201 allele 44 .
  • the bio-informatic tool allows the analysis of expression profiles from the EST and SAGE databases by the clustering of libraries by origin, such as from normal or tumoral tissues.
  • Candidate genes were selected on the basis of high expression levels in available libraries prepared from human breast cancer, and absent or low expression levels in normal human tissues derived from important organs. A list of the most relevant candidates appears in Table I.
  • ⁇ -actin and ubiquitin C genes were included as positive controls, and, as expected, the reported expression levels are relatively equivalent in the normal and tumor tissues selected.
  • PDEF and CLSP were overexpressed in breast carcinomas according to both the EST and SAGE libraries.
  • some genes, such as DCD and DKK1 were detected in a higher proportion of breast cancer tissue samples based on the SAGE only, which was not correlated by expression assessment in the EST database.
  • some genes were found to be potentially overexpressed in breast cancer according to the EST database, which is in contrast to the SAGE approach.
  • Table I List of overexpressed candidate genes in breast cancer compared with normal tissues, identified by cDNA and the SAGE Digital Gene Expression Displayer (DGED) from the CGAP server, ⁇ -actin and ubiquitin C are referenced as ubiquitously-expressed genes.
  • DGED SAGE Digital Gene Expression Displayer
  • Hs.112408 S100 calcium-binding protein A7 17 1 088 0 0
  • a - Unigene ID and names when available - Number of positive sequences from libraries prepared from breast tumors or selected normal tissues, among all available tags or sequences. Values are reported as number of positives/1 000 000 sequences; NA: not available; c - Numbers are bolded and underlined when p ⁇ 0,05 AND five times the value from normal cells; d - No assigned Unigene ID (EST prepared from osteocarcinoma). The expression level is subjectively reported from 0 to +++++.
  • the melanoma cell lines 397mel, 537mel, 586mel, 888mel, 1087mel, 1088mel, 1278mel, 1300mel, 1337mel and MeIs-FB, the kidney cancer line RCC-W were established at the Surgery Branch (NCI/NIH), and the melanoma cell line SK23 was acquired from the ATCC.
  • the ovarian cancer line SKOV3 was also kindly provided by the Surgery Branch.
  • Negative controls included cells prepared from normal peripheral blood mononuclear cells (PBMC) as well as cultured activated lymphocytes to eliminate genes expressed in normal proliferating cells.
  • PBMC peripheral blood mononuclear cells
  • MUHC McGiII University Health Center
  • PBMC peripheral blood mononuclear cells
  • B cells from bulk PBMC were cultured with 500 ng/ml of a soluble trimeric CD40L (CD4OLs, Immunex Corporation, Seattle, WA) in complete media complemented with 200 U/ml of recombinant human IL-4 (Peprotech, Rocky Hill, NJ). Fresh complete media was added again on day three with IL-4 and 500 ng/ml CD4OLs. After the first round of proliferation (d5-8), cells were either frozen for future use or re- stimulated every two-three days when the culture reached a density of 1 ,5-2 X 10 6 cells/ml.
  • CD40L soluble trimeric CD40L
  • PBMC peripheral blood mononuclear cells
  • AIM-V medium Invitrogen
  • human AB serum heat-inactivated; Gemini Bio-Products; Calabasas, CA
  • 2 mM L-glutamine 100 U/ml penicillin/streptomycin and 10 ⁇ g/ml gentamicin (all from Invitrogen)
  • 300 IU/ml recombinant human IL-2 Choiron; Emeryville, CA
  • 30 ng/ml of an agonistic anti-CD3 OKT3, eBiosciences, San Diego, CA
  • PHA phytohemaglutinin
  • Intron-spanning PCR primers were designed from eight of the candidate genes presented in Table I above.
  • cDNA was first synthesized from mRNA (0,2 to 1 ⁇ g) with oligo-dt (Invitrogen) using the Omniscript Reverse Transcriptase Kit (QIAGEN) and then amplified using the HotStartTaq DNA Polymerase (QIAGEN).
  • the cycling conditions were 15 min at 95 0 C, 24 ( ⁇ -actin) or 32 (DKK1 and other candidate TA) cycles of 45 sec at 94 0 C, 45 sec at 55 0 C, 1 min at 72 0 C, with a final extension of 10 min at 72 0 C.
  • Primer sequences for ⁇ -actin 5 1 : GGAAGGCTGGAAGAGTGCC (SEQ ID NO: 16); 3': GTGATGGTGGGCATGGGTC (SEQ ID NO: 17), 700 bp amplicon. Amplification was detected by ethidium bromide staining after electrophoresis migration in agarose (2%) gel (all apparatus from Bio-Rad, Hercules, CA).
  • CLSP 5 1 primer: GTGAGCTGACTCCTGAGGAG (SEQ ID NO: 18); 3' primer: CTCGCGAACTCCTCGTAGTTC (SEQ ID NO: 19), amplicon 410 bp), MYL5 (5' primer: ACCAAGCAGGAGCTTAAGATG (SEQ ID NO: 20); 3' primer: AGAATGGTCTCCTCGGCGTC (SEQ ID NO: 21), amplicon 390 bp) ( Figure 5), Hs.199713 and Hs.1582 (data not shown) were expressed in several breast cancer lines but also in PBMC and activated lymphocytes (i.e.
  • the expression of PDEF (5' primer: TGACATG CTGTAC C CTGAG GA (SEQ ID NO: 22); 3' primer: GCTCTGGAAGGTCAGAGCAGCA (SEQ ID NO: 23), ampiicon 940 bp) and S100A7 (5' primer: AAGATGAG C AACACTC AAG CTG (SEQ ID NO: 24); 3' primer: GTCTCCCAGCAAGGACAGAAAC (SEQ ID NO: 25), ampiicon 247 bp) was confirmed in breast cancer lines but not in normal PBMC or activated lymphocytes (Figure 5).
  • S100A7 also known as psoriasin, has been described previously as being expressed preferentially in invasive breast cancer 20 .
  • PDEF has also been reported to be expressed with high frequency in breast cancer with no expression in important tissues 21 .
  • DKK1 (5' primer: ATTCCAACGCTATCAAGAACC (SEQ ID NO: 26); 3' primer: CCAAGGTGCTATGATCATTACC (SEQ ID NO: 27), ampiicon 383 bp) and DCD (5' primer: AGCATGAGGTTCATGACTCTC (SEQ ID NO: 28); 3' primer: CACGCTTTCTAGATCTTCGAC (SEQ ID NO: 29), ampiicon 284 bp) 22 were also expressed in breast cancer lines, but absent from normal cells tested ( Figure 4-A and Figure 5).
  • DKK1 is known to be expressed in fetal development, and its expression in mature humans has been detected in normal placenta and prostate, which are not targeted tissues in breast cancer immunotherapy 23 . DKKI's relative expression in normal cells was further determined here.
  • the cycling conditions were 15 min at 95 0 C, 40 cycles of 15 sec at 94 0 C, 30 sec at 55 0 C, 30 sec at 72 0 C and 5 sec at 82 0 C ( ⁇ -actin) or 84 0 C (DKK1).
  • the primer sequences used for the real-time PCR were the following: ⁇ - actin forward: AAGGCCAACCGCGAG (SEQ ID NO: 30) ; reverse: TAATGTCACGCACGATTCCCG (SEQ ID NO: 31); DKK1 5 1 : CTCGGTTCTCAATTCCAACG (SEQ ID NO: 32); 3': GCACTCCTCGTCCTCTG (SEQ ID NO: 33).
  • DKKI + and DKK1 " specimens were clustered according to different clinical parameters and to evaluate the discriminating potential for relevant clinical information. Interestingly, on the 70 specimens analyzed, only three were positive for HER-2/neu, however, the 18 DKK + tumors turned out to be negative for HER- 2/neu. This is of critical relevance considering the efficiency of HerceptinTM, a monoclonal antibody specific to HER-2/neu, which will be ineffective to DKKI + patients. This is relevant in so far that patients that will respond to TA vaccine from DKK1 would not have been responsive to HerceptinTM. Hence, at least a fraction of patients that were untreatable by HerceptinTM, namely that which expresses DKK1 , could now be treated.
  • stage III Five of the DKKI + tumors were scored stage III (28% of all DKKI + tumors) which is in contrast with only three stage III for 52 DKK1 " tumors (6%). The 5-year survival for stage III is about 50%; this illustrate again the fact that DKK1 is linked with the most aggressive breast tumors.
  • VHL ' ' " The occurrence of light renal cells carcinomas (VHL ' ' " ) is much higher (by 70 fold) than the occurrence of Wilms' tumors (about 500 cases per year in the USA). It is noted that although Wirths et al. (50) reported the presence of DKK1 in Wilms' tumors, Wirths did not disclose the absence or low expression of DKK1 in a sufficient number of normal cells to suggest that it would constitute a good TA. It further did not disclose its presence in other cancers..
  • DKK1 is expressed in melanomas (7/11), ovarian (Figure 4-C; SKOV3), and colon cancers (Figure 4-C; HCT116).
  • DKK1 expression was revealed in 2 prostate cancer cell lines derived from hormone-independent tumors (2/3; Figure 4-C).
  • DKK1 immunogenicity was evaluated by identifying T lymphocytes specific to putative human leukocyte antigens (HLA)-A2-restricted epitopes from DKK1 ( Figure 2).
  • HLA human leukocyte antigens
  • Figure 2 Putative HLA-A*0201 nine- and ten-mer epitopes were predicted by exploiting both the SYFPEITHI 48 (http://www.syfpeithi.de/) tool and Dr. Kenneth Parker's algorithms 49 (http://bimas.dcrt.nih.gov/molbio/hla_bind/).
  • Table III List of selected HLA-A*0201 epitopes predicted for DKK1 , using HLA- binding algorithms and a proteasome prediction tool (PAProCTM).
  • a Starting position of the peptide in the Dkk1 protein sequence.
  • b Score ranking according to the predicted half-time of dissociation to HLA-A2 molecules from the Biolnformatics & Molecular Analysis Section (BIMAS) server (http://thr.cit.nih.gov/molbio/hlaj-ind/), based on Dr. Kenneth Parker's algorithm.
  • BIMAS Biolnformatics & Molecular Analysis Section
  • ° Score ranking according to the peptide binding capacity to HLA-A2 molecules, from the SYFPEITHI server (http://www.syfpeithi.de/), based on Dr. Hans-Georg Rammensee's algorithm.
  • Polypeptides in Table III (polypeptides 20 (SEQ ID NO: 9), 36 (SEQ ID NO: 34), 11 (SEQ ID NO: 35), 18 (SEQ ID NO: 36), 40 (SEQ ID NO: 10), 182 (SEQ ID NO: 15), 25 (SEQ ID NO: 37), 52 (SEQ ID NO: 38), 246 (SEQ ID NO: 39), 191 (SEQ ID NO: 40), 37 (SEQ ID NO: 12), 68 (SEQ ID NO: 14), 32 (SEQ ID NO: 11), 61 (SEQ ID NO: 13), 58 (SEQ ID NO: 41)) were synthesized by "Le Service de Synthese de Peptides de I'Est du Quebec” (CHUL, Quebec, Canada) and were >80% pure.
  • the PBMC were prepared as mentioned earlier and frozen in fetal bovine serum (FBS, Invitrogen and Wisent) or Calf serum (Wisent) with 10% DMSO (Sigma; St- Louis, MO) at 5 to 10X10 6 cells/ml in liquid nitrogen.
  • FBS fetal bovine serum
  • DMSO 10% DMSO
  • PMBC were thawed and resuspended at 1x10 6 cells/ml in complete medium supplemented with 500 ng/ml of CD40L, and aliquoted in 16 tubes for each DKK1 peptides (15) (Table III), and one peptide derived from the influenza M-1 protein (M1-FLU: GILGFVFTL) as a positive control.
  • M1-FLU peptide derived from the influenza M-1 protein
  • Peptides were added at a concentration of 1 ⁇ M and plated in flat bottom 96-well culture cluster (Corning; Corning, NY) at 5 wells per peptides (100 or 150 ⁇ l/well). Individual cultures were restimulated with autologous peptide-pulsed CD40-B lymphocytes (CD40-B; method described above) seven to ten days later. CD40-B were pulsed with 1 ⁇ M peptides in ISCOVE (Invitrogen or Wisent) for three hours at room temperature. Peptide-pulsed CD40-B were washed in PBS, and added to corresponding T cell cultures. Two days later, 150 U/ml of IL-2 was added to the cultures and repeated every three days after.
  • CD40-B lymphocytes CD40-B lymphocytes
  • ISCOVE Invitrogen or Wisent
  • T cell cultures were evaluated cells by IFN- ⁇ ELISPOTTM assays. Co-cultures were performed in 96-well filtration Plates (MultiScreenTM-HTS; Millipore; Bedford, MA) according to manifacturer's instructions. ELISPOT plates were coated with anti- IFN- ⁇ imAb (5 ⁇ g/ml, Mabtech; Stockolm, Sweden) overnight at 4 0 C. After washing with 1X sterile PBS, plates were blocked with the complete medium (described earlier) and incubated for two hours at 37 0 C. Cultured T lymphocytes were washed and transferred (0,5x10 5 ce I Is/we 11) into coated ELISPOT plates.
  • HLA-A*0201 + TAP-deficient T2 cell lines ATCC, Manassas, VA
  • NCI/NIH Surgery Branch
  • Peptide-puised T2 or EBV-B were washed and added to the stimulated T lymphocytes and incubated overnight at 37 0 C.
  • Supernatants from ELISPOT recognition plates were harvested and frozen at -2O 0 C to evaluate the GM-CSF secretion by ELISA.
  • ELISPOT plates were then washed with PBS/0.01% Tween 20 (Sigma) and a biotinylated anti-IFN- ⁇ mAb (2 ⁇ g/ml, Mabtech) was added to each well. After a two-hour incubation at 37 0 C, plates were washed with the PBS-Tween and Streptavidine-HRP (Mabtech) was added. After a 45 min room temperature incubation, plates were washed with PBS/0.01% Tween 20 and once with PBS. Spots were revealed with the AEC substrate (0,1 N acetic acid, 0,1 M sodium acetate and H 2 Odd) for five minutes and then washed with water.
  • GM-CSF secretion was evaluated by ELISA from supernatant from the ELISPOT co-culture. ELISA were performed in Flat bottom 96-well (Nunc, Apogent Technologies; Portsmouth, NH) with paired antibodies for GM-CSF (Endogen; Wobum, MA), according to manufacturer's procedure.
  • GM-SCF secretion To be considered as a positive culture, GM-SCF secretion must be >50 pg/ml and twice the amount secreted when co-cultured with the control peptide.
  • the specificity of individual cultures was determined by IFN- ⁇ ELISPOTTM assays and GM-CSF ELISA, by co-culturing with T2 cells pulsed with the relevant peptide, or with an irrelevant control peptide.
  • a - only lines with > 10 spots and with a ratio > 2 were considered positive for ELISPOT assays, and GM-SCF secretion must be >50 pg/ml and twice the amount secreted when co-cultured with the control peptide, b - only wells demonstrating proliferating cells were tested for this patient.
  • Peptides recognized by 2 and more patient lymphocytes are Peptides recognized by 2 and more patient lymphocytes.
  • Tumors expressing both HLA-A2 and DKK1 serve as targets for peptide-specific T lymphocyte line or clones to determine naturally-processed epitopes.
  • peptide sequences of identified epitopes are optimized to enhance their binding capacity on anchor residues to HLA and T cell receptor (TCR) complexes, a procedure that frequently results in an increased immuno- stimulatory capacity in patient immunization.
  • Immunogenic peptides were aligned to define the most probable anchor residues (Figure 9).
  • immunogenic peptides from DKK1 can be defined as follows for the putative anchor residues: Position 2: Aliphatic residues (I or L), or a small residue (N, A, P or G); Position 6: Aliphatic residues (I or L), a polar residue (K or S) or a P; Position 9: Small hydrophobic residue (G or T), a small residue (N or P) or a L.
  • peptides will not necessarily be immunogenic in a given patient while it could be in another one because different HLA sub-types will bind different peptides with different affinities.
  • a putative immunogenic peptide will therefore be considered effective if it combines with the MHC complex of a given patient and if the lymphocytes are activated and capable of eliciting antibody production or killing activity towards the patient's cancer cells.
  • a recombinant DKK1 DNA is expressed in E. coli as a fusion protein with a protein D derived from H. influenzae at the N terminus, and a sequence of several histidine residues at the C terminus of the protein.
  • the inclusion of the first 109 residues of the protein D as a fusion partner is expected to improve the immunogenicity and to provide the vaccine protein with additional bystander help properties, whereas the inclusion of a His affinity tail facilitates the purification of the fusion protein.
  • the vaccination schedule comprises six vaccinations at three- week intervals.
  • An HLA-A*0101 -restricted DKK1 immunogenic polypeptide is administered in sterile, endotoxin-free PBS, at a concentration of 100 or 300 ⁇ g/ml, in three vaccination at one-month intervals.
  • the vaccine is divided between two sub cutaneous (s.c.) sites and two intradermal (i.d.) sites distant from the tumor. When possible, the injection sites is changed for each vaccination. For tumor- bearing patients who display tumor regression, additional immunizations are administered. This procedure is adapted from a successful method of the prior art.
  • Cancer patients receive DKK1 immunogenic polypeptides or modified version of these peptides (i.e. substitution in position two for I or M, six for V or nine for L to improve the binding to the HLA-A*0201 molecule) from the antigen DKK1.
  • Patients receive one mg of peptide emulsified in incomplete Freund's adjuvant in two to six immunizations at three weeks intervals.
  • Some of the patients also received high-dose IL-2 at 720000 IU/kg as an intravenous bolus over 15 minutes starting either one to five days after peptide injection. After two or four injections, all known sites of the disease are assessed.
  • a response is considered complete if all measurable lesions disappear.
  • a partial response is defined as 50% or greater decrease of the sum of the products of the longest perpendicular diameters of all legions, lasting at least one month without increase of any tumor or the appearance of new lesions. This procedure is adapted from a successful method of the prior art.
  • ALVAC virus bearing DKK1 sequences coding for HLA-A2-restricted DKK1 immunogenic polypeptides is produced by cloning cDNA encoding a DKK1 immunogenic polypeptide, is then ligated into a donor plasmid downstream of a vaccinia H6 early/late promoter element. The recombinant plasmid, harboring this expression cassette, is transfected into primary chick embryo fibroblasts, which are then infected with wild-type ALVAC virus. After successive rounds of plaque purification and selection, a recombinant ALVAC virus containing the appropriate expression cassette inserted into the C6 nonessential site, is isolated and amplified.
  • the viral vaccine is formulated as a lyophilized powder corresponding to a viral dose of 1 ,23x10 7 CCID50 (50% of the cell culture infectious dose).
  • the vaccine vials are kept stored at 4 0 C, and are reconstituted before administration with one ml of water for injection.
  • the vaccination schedule starts with four priming vaccinations with ALVAC miniDKKI at three-week intervals.
  • the fixed virus dose is determined by the titer of the available clinical batch.
  • the viral suspension is injected in two i.d. sites (0,1 ml each) and two subcutaneous sites (0,4 ml_ each), in the arms and the anterior aspect of the thighs.
  • the ALVAC vaccinations are followed after three weeks by three booster vaccinations with the DKK1 immunogenic polypeptides at three-week intervals.
  • Each peptide is injected once i.d. (60 ⁇ g) and once subcutaneously (240 ⁇ g), also in the arms and thighs. This procedure is adapted from a successful method of the prior art. 45
  • PBMC Peripheral blood mononuclear cells
  • FCS fetal calf serum
  • Non-adherent cells are removed and adherent cells are cultured for seven days in complete medium supplemented with GM-CSF (800 U/ml) and IL-4 (500 U/ml).
  • DCs are then harvested and pulsed with HLA-A2, HLA-A1 or HLA-A3 binding DKK1 immunogenic polypeptides, depending on the HLA type of the patient.
  • Peptides are pulsed at 50 ⁇ g/ml for two hours. Before injection, pulsed DCs are washed three times in sterile phosphate-buffered saline (PBS) and resuspended in a total volume of 0,5 ml of PBS (1X10 6 pulsed DCs). Patients receive four vaccinations at weekly intervals. The fifth vaccination is administered at week six, and subsequent vaccinations are performed monthly for up to 10 months. Vaccine preparation is administered intra-lymphatically into an inguinal lymph node under ultrasound control or is injected in close proximity to the regional lymph node. This procedure is adapted from a successful method of the prior art.
  • PBS sterile phosphate-buffered saline
  • HLA-A*0201 patients are immunized with DKK1 immunogenic polypeptides-pulsed autologous CD34-derived DCs.
  • the patients receive recombinant granulocyte-CSF 10 ⁇ g/kg/day s.c. for five days, for peripheral blood stem cell mobilization, and then undergo leukapheresis for two consecutive days to collect mobilized CD34 + HPCs.
  • the cells are processed using the CEPRATETM SC stem cell concentration system (CellPro Inc., Seattle, WA) to obtain an enriched population of CD34 + HPCs which are then cryopreserved.
  • CD34-derived DCs are generated from CD34 + HPC by culture at a concentration of 0,5 x 10 6 /ml culture medium (X-VIVO-15TM; BioWhittaker) supplemented with autologous serum, 10 "5 M 2- ⁇ -mercaptoethanol and 1% L-glutamine.
  • the following human recombinant cytokines, approved for clinical use, are used: GM-CSF (50 ng/ml; Immunex Corp.), FLT3-L (100 ng/ml; Immunex Corp.), and TNF (10 ng/ml; CellPro, Inc.).
  • the cells are centrifuged and resuspended in 9 ml of sterile saline for injection.
  • Vaccination is administered s.c. in three injection sites (both thighs and the upper arm). Limbs from which draining lymph nodes has been surgically removed and/or irradiated are not injected.
  • DCs are injected using a long spinal-cord needle and are spread over a 6- to 8-cm distance. This procedure is adapted from a successful method of the prior art.
  • Retroviral vectors can be exploited to transduce the nucleic acid coding for a DKK1 protein in DCs, derived from CD34 cells obtained as described in Example 13.
  • retroviral supernatant is added to cultured CD34 + cells on days two and three at a ratio of 1 :1 with culture medium.
  • GM-CSF, SCF, TNF- ⁇ and polybrene are added and cells are spun in the plate at 1 ,000 X g for one hour.
  • transduced DCs are resuspended in fresh complete DC medium in a six well plate and the differentiation is completed with cytokines. Cells are utilized on day 14 and DC phenotype is confirmed by morphological and FACS analyses.
  • the DKK1 -VSV- pseudotyped retroviral system is prepared by first inserting the complete DKK1 sequence in the pCLNC retroviral plasmid.
  • the pCLNC-DKK1 and pMDG-VSV plasmids are co-transfected in 293-gag-pol packaging cells using LipofectamineTM Plus (Life Technologies).
  • the 293-gag-pol packaging cells are cultured in DMEM supplemented with 10% heat inactivated FBS and antibiotics.
  • DKK1 -transduced CD34-derived DCs have the capacity to present both MHC class I and class Il epitopes, and are efficient in generating tumor-specific anti-DKK1 CD4 + and CD8 + T lymphocytes when co-cultured with autologous PBMC. This procedure is adapted from a successful method of the prior art.
  • Monocyte-derived DCs are cultured as described in Example 12 and are pulsed at 1 ⁇ g/ml for 90 minutes with DKK1 immunogenic polypeptides.
  • DKK1 polypeptides pulsed DCs are then washed and co-cultured in the same plate with 2 X 10 6 purified autologous T lymphocytes in the presence of a combination of CD40L.S and LPS.
  • the autologous T lymphocytes are isolated from cryopreserved PBMC using a Human T Cell Enrichment Column (R&D Systems) according to the manufacturer's protocol. After five days of incubation, 50% of the medium is replaced with fresh medium and the cells are transferred to a new 24 well plate.
  • IL-2 is then added to the culture at 10 CU/ml (Chiron, Emeryville, CA). This procedure is adapted from a successful method of the prior art.
  • DKK1 RNA transfection is carried out on the day of administration using cryopreserved and reconstituted monocyte-derived DCs as described in Example 12. Cryopreserved DCs are washed twice in PBS, counted, and spun at 300 x g for 10 min. Subsequently, DCs are resuspended at a concentration of 1 x 10 7 cells/ml in AIM-V medium and are coincubated for 60 min with 50 ⁇ g/ml RNA in a humidified incubator at 37°C/5% CO 2 .
  • DKK1 RNA-transfected DCs are administered at three proposed, escalating dose levels with the highest dose to be tested corresponding to the largest number of DCs that could be generated from the PBMCs of healthy volunteers isolated by a routine leukapheresis. Dose escalation is performed through an i.v. route using three vaccination cycles with 1 x 10 7 (low dose), 3 x 10 7 (medium dose), or 5 x 10 7 (high dose) cells applied at study weeks zero, two, and four.
  • EXAMPLE 18 Immunization of Cancer Patients with DKK1 cDNA Transfected- DCs [00121]
  • the plasmid pCMV DKK1 containing the cDNA of DKK1 under the control of the human CMV promoter, is produced under good manufacturing practice (GMP) conditions at Q-OneTM Biotech Ltd (Glasgow, U.K.).
  • DCs from the patients are prepared as described in Example 12 and transfected with pCMV DKK1 on day 5 of culture with cationic liposomes (Lipofectin; Gibco).
  • Lipofectin cationic liposomes
  • One million of these DCs per vaccine are cryopreserved 24 h later using GelifundolTM (Biotest, Dreieich, Germany) and DMSO at a final concentration of 10%.
  • DCs are evaluated by morphology and flow cytometry using the monoclonal antibodies (mAb) against CD1a, CD86 (Pharmingen), CD80, CD83 and HLA-DR (Immunotech, Coulter).
  • mAb monoclonal antibodies
  • CD80 CD86
  • CD83 CD83
  • HLA-DR Immunotech, Coulter
  • DKK1 epitopes is tested by flow cytometry analysis using a mAb against DKK1.
  • One million gene-transfected DCs are injected s.c. into the upper limb close to the inguinal lymph nodes on days one, 21 and 42, respectively. Delayed-type hypersensitivity (DTH) reactions comparing the vaccine, untransfected DC, and physiological saline are performed before and after vaccination (5X10 5 cells are injected i.d. in the patients' backs). This procedure is adapted from a successful method of the prior art.
  • DTH Delayed-type hypersensitivity
  • PBMCs are obtained and antigen-specific cytotoxic T lymphocytes (CTLs) are generated from autologous blood monocytes-derived DCs and pulsed with the HLA-A2-restricted DKK1 immunogenic polypeptides as described in Example 12. After three cycles of stimulation at weekly intervals, T cells are cloned by and expanded for in vitro testing. CTL clones demonstrating specific lysis of antigen-positive tumor targets in a chromium release assay are selected.
  • CTLs cytotoxic T lymphocytes
  • Clones are expanded in 14-day cycles by using anti-CD3 antibody (OKT3, OrthocloneTM; Ortho Biotech, Raritan, NJ) at 30 ng/ml, irradiated allogeneic PBMCs, at 10 6 cells/ml, irradiated allogeneic lymphoblastoid cell lines (2 x 10 5 cells/ml), and serial IL-2 (aldesleukin; Chiron) at 25-50 units/ml every two-three days. All patients selected express HLA-A2. A total of four T cell infusions is planned, the first without low-dose IL-2 and subsequent infusions (second, third, and fourth) co-administered with increasing doses of s.c.
  • anti-CD3 antibody OKT3, OrthocloneTM; Ortho Biotech, Raritan, NJ
  • irradiated allogeneic PBMCs at 10 6 cells/ml
  • irradiated allogeneic lymphoblastoid cell lines (2 x 10 5 cells/
  • IL-2 (0,25, 0,5, and 1 ,0 x 10 6 units/m 2 twice daily for 14 days. Patients are monitored closely by physical examination and serum chemistries for evidence of toxicity. Stopping rules included the appearance of serious (grade III toxicity by National Cancer Institute common toxicity criteria). This procedure is adapted from a successful method of the prior art.
  • G-CSF Granulocyte colony-stimulating factor
  • the cultures are maintained at cell concentrations between 5 x 10 5 and 2 x 10 6 cells per ml until several million TIL cells are available. Multiple independent cultures are screened by cytokine secretion assay for recognition of autologous tumor cells (if available) and HLA-A2 + tumor cell lines. Two to six independent TIL cultures exhibiting the highest cytokine secretion are further expanded in complete medium with 6000 IU per ml IL-2 until the cell number is over 5 x 10 7 cells (this cell number was typically reached three-six weeks after tumor excision).
  • TIL cultures that maintain specific tumor cell recognition are expanded for treatment using one cycle of a rapid expansion protocol with irradiated allogenic feeder cells, OKT3 (anti-CD3) antibody, and 6000 IU per ml IL-2.
  • This rapid expansion protocol typically results in 1000-fold expansions of cells by the time of administration 14-15 days after initiation of the expansions.
  • Patients who have DKK1 -specific TIL receive vaccination with 1 mg DKK1 immunogenic polypeptide in incomplete Freund's adjuvant (IFA) injected subcutaneously. This procedure is adapted from a successful method of the prior art.
  • IFA incomplete Freund's adjuvant
  • the present invention therefore relates the use of DKK1 or an immunogenic polypeptide thereof as a TA for tumors expressing DKK1.
  • DKK1 is directed to the use of a DKK1 protein or an immunogenic polypeptide thereof as a TA for tumors originating from the breast, the lung, the kidney, the skin, the ovary, the colon and the prostate.
  • DKK1 is considered to be a useful tool to: 1. detect aggressive tumors; 2. serve as a TA to be targeted in immunization strategies including preventive immunization of patients at risk for developing cancer, preventive immunization to prevent recurrences after surgical resection, or for immunotherapy of established tumors.
  • Prostate epithelium-derived Ets transcription factor mRNA is overexpressed in human breast tumors and is a candidate breast tumor marker and a breast tumor antigen.
  • Dermcidin a novel human antibiotic peptide secreted by sweat glands. Nat.lmmunol. 2, 1133-1137.
  • Retrovirally-transduced human dendritic cells can generate T cells recognizing multiple MHC class I and class Il epitopes from the melanoma antigen gp100. J.Immunol. 167, 4758-4764.

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Abstract

La présente invention se rapporte à l'utilisation d'une protéine DKK1 purifiée ou recombinée ou d'un polypeptide immunogène contenant au moins 8 acides aminés contigus de ladite protéine DKK1, ainsi qu'à l'utilisation d'un polynucléotide isolé contenant la séquence de codage d'une protéine DKK1 ou la séquence de codage d'un polypeptide immunogène contenant au moins 8 acides aminés contigus de la protéine DKK1, pour activer les lymphocytes T.
PCT/CA2005/001251 2004-08-13 2005-08-15 Procedes d'utilisation d'une proteine dkk1, de polypeptides immunogenes de ladite proteine, d'acides nucleiques codant la proteine dkk1 ou des polypeptides, ou de ligands de ladite proteine, pour detecter des tumeurs et pour eliciter une reponse immunitaire contre des tumeurs Ceased WO2006015497A1 (fr)

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US8476236B2 (en) 2006-12-07 2013-07-02 The United States Of America As Represented By The Secretary Of The Department Of Health And Human Services Treatment of skin conditions by Dickkopf1 (DKK1)
US20100129390A1 (en) * 2007-05-18 2010-05-27 Qing Yi Dkk1 as a universal tumor vaccine for immunotherapy of cancers
CN106220722A (zh) * 2016-08-16 2016-12-14 首都医科大学附属北京胸科医院 Dkk1自身抗体识别的抗原多肽及其用途
JP2021502414A (ja) * 2017-11-08 2021-01-28 ビオンテック ユーエス インコーポレイテッド Ｔ細胞を製造する組成物及び方法
KR20200109301A (ko) * 2017-11-08 2020-09-22 네온 테라퓨틱스, 인크. T 세포 제조 조성물 및 방법
WO2019094642A1 (fr) * 2017-11-08 2019-05-16 Neon Therapeutics, Inc. Compositions et procédés de production de lymphocytes t
US11162072B2 (en) 2017-11-08 2021-11-02 Biontech Us Inc. T cell manufacturing compositions and methods
JP7054418B2 (ja) 2017-11-08 2022-04-13 ビオンテック ユーエス インコーポレイテッド Ｔ細胞を製造する組成物及び方法
JP2022106722A (ja) * 2017-11-08 2022-07-20 ビオンテック ユーエス インコーポレイテッド Ｔ細胞を製造する組成物及び方法
KR102484433B1 (ko) 2017-11-08 2023-01-03 바이오엔테크 유에스 인크. T 세포 제조 조성물 및 방법
JP7420856B2 (ja) 2017-11-08 2024-01-23 ビオンテック ユーエス インコーポレイテッド Ｔ細胞を製造する組成物及び方法
US12258581B2 (en) 2017-11-08 2025-03-25 Biontech Us Inc. T cell manufacturing compositions and methods

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