WO2002089727A2 - Methodes d'utilisation de la cytokine c17 - Google Patents

Methodes d'utilisation de la cytokine c17 Download PDF

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Publication number
WO2002089727A2
WO2002089727A2 PCT/US2002/011869 US0211869W WO02089727A2 WO 2002089727 A2 WO2002089727 A2 WO 2002089727A2 US 0211869 W US0211869 W US 0211869W WO 02089727 A2 WO02089727 A2 WO 02089727A2
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WO
WIPO (PCT)
Prior art keywords
cells
cytokine
seq
patient
angiogenesis
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PCT/US2002/011869
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English (en)
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WO2002089727A9 (fr
WO2002089727A3 (fr
Inventor
Linzhao Cheng
Young Sung
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The John Hopkins University
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Priority to AU2002340544A priority Critical patent/AU2002340544A1/en
Publication of WO2002089727A2 publication Critical patent/WO2002089727A2/fr
Publication of WO2002089727A9 publication Critical patent/WO2002089727A9/fr
Publication of WO2002089727A3 publication Critical patent/WO2002089727A3/fr

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/52—Cytokines; Lymphokines; Interferons
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K48/00—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide

Definitions

  • a human hematopoietic secreted polypeptide called Cl 7 polypeptide was disclosed in Liu and Cheng., WO 00/63382 and Liu, et al., Genomics 65: 283-292 (2000). It is expressed in CD34 + cells but not in CD34 " cells. Thus it is expressed by those bone marrow and blood mononuclear cells which function as hematopoietic stem/progenitor cells.
  • the C17 polypeptide consists of 136 amino acid residues with a signal sequence. It is predicted to contain four ⁇ -helices, a characteristic of hematopoietic cytokines and interleukins. The gene for C17 polypeptide was mapped to human chromosome 4pl5-pl6.
  • Liu (WO 00/63382) disclosed that the polypeptide was a growth factor for mesenchymal stem cells in serum-free culture.
  • Mesenchymal stem cells are CD34-negative and C17-negative and do not differentiate into hematopoietic cells.
  • Liu (Genomics) speculated that C17 could act upon C17 expressing cells as well as C17 non-expressing cells.
  • a method for promoting angiogenesis is provided.
  • An expression vector comprising a polynucleotide sequence according to SEQ ID NO: 1 is administered to a patient in need thereof. Angiogenesis is thereby stimulated.
  • Another embodiment of the invention provides a method of inhibiting angiogenesis.
  • An antisense construct comprising all or a portion of at least 15
  • RNA is transcribed from the construct which is complementary to C17 mRNA.
  • Angiogenesis is inhibited by the complementary RNA.
  • another method is provided for promoting angiogenesis.
  • a C17 cytokine is administered to a patient in need thereof. Angiogenesis is thereby stimulated.
  • a method for inhibiting angiogenesis.
  • An antibody which specifically binds to a C17 cytokine is administered to a patient in need thereof. Angiogenesis is thereby inhibited.
  • a method is provided of expanding a population of cells in culture.
  • the cells are not mesenchymal stem cells.
  • the cells in culture are contacted with a C17 cytokine.
  • the cells are thereby stimulated to proliferate.
  • Other aspects of the invention provide an isolated and purified polynucleotide which encodes mouse C17 cytokine and has a sequence according to SEQ ID NO: 5 or which encodes mouse C17 cytokine according to SEQ ID NO: 6.
  • the invention provides an isolated and purified polynucleotide which encodes bovine C17 cytokine and has a sequence according to SEQ ID NO:4 or which encodes bovine Cl 7 cytokine according to SEQ ID NO: 3.
  • isolated and purified mouse C17 cytokine according to SEQ ID NO: 6 and isolated and purified bovine C17 cytokine according to SEQ ID NO:4.
  • fusion proteins comprising a detectable marker protein and a C17 cytokine selected from bovine, mouse, and human C17 cytokine.
  • the present invention provides an isolated and purified promoter which is transcriptionally active in CD34 + /C17 + cells but transcriptionally inactive in CD34 " /C17 " cells.
  • the promoter has a sequence according to SEQ ID NO: 7.
  • Fig. 1 shows the deduced peptide sequences of human, bovine and mouse C17 genes, as well as a consensus sequence (SEQ ID NOS: 2, 4, 6, 8).
  • Fig. 2 shows growth of C17- transduced TF1 human cells in serum free medium and in the optimal medium containing both serum and GM-CSF.
  • Fig. 3 shows that the Cl 7 promoter is sufficient to drive transgene expression specifically in C17 + /CD34 + cells.
  • Fig. 4 shows the 523 bp upstream genomic sequence which functions as a specific promoter.
  • Cl 7 polypeptide is a growth factor (cytokine) which stimulates endothelial cell growth.
  • cytokine a growth factor which stimulates endothelial cell growth.
  • C17 is a target for agents which block angiogenesis and neovascularization required for the progression of both solid tumors and leukemias.
  • C17 can be used to expand hematopoietic and endothelial cells and their precursors before transplantation for treating diseases.
  • Hematopoietic stem cells, endothelial progenitor cells, and hemangioblasts are currently difficult to impossible to expand in culture.
  • Cl 7 can be isolated from any mammalian source, including but not limited to humans, cows, mice, rats, guinea pigs, dogs, cats, monkeys, chimpanzees. It can also be made recombinantly using the natural, consensus, or synthetic gene sequences.
  • One particularly preferred method for producing C17 is using retrovirus expression vectors.
  • C17 nucleotide and amino acid sequences are provided in the appended sequence listing. These include:
  • Angiogenesis can be desirable or undesirable depending on the context. In the case of wound healing, for example, angiogenesis is desirable. In the case of tumors or other cancers, angiogenesis is undesirable. Thus, manipulation of C17 levels in a
  • 462904-1 mammal such as a human can provide a means to treat such conditions.
  • Increasing supplies of C17 can be obtained by means of supplying a polynucleotide sequence encoding C17. Typically this will be a cDNA sequence which is inserted in an expression vector.
  • Any expression vector can be used.
  • retroviruses including oncoretroviruses and lentiviruses, are particularly useful for this purpose.
  • retroviruses including oncoretroviruses and lentiviruses, are particularly useful for this purpose.
  • retrovirus murine stem cell virus (MSCV) retroviral vector which is superior to conventional MoMLV in LTR-mediated gene expression. Any vector known in the art for this purpose may, however, be used.
  • Fusion proteins of C17 have also been made and found to have biological activity. Thus, if fusion proteins are desirable, for facilitation of purification or monitoring, for example, then they can be used for treating disease states in which more angiogenesis is desired. Native, non-fusion Cl 7 protein can also be administered, as is desired.
  • Inhibiting, including reduction and total cessation, of angiogenesis can be accomplished by means of antisense or antibody administration.
  • Antisense oligonucleotides or antisense constructs can be administered.
  • the antisense molecules need not contain the entire coding (anti-coding) sequence of C17, but at least 18 contiguous nucleotides of C17.
  • Antisense oligonucleotides are complementary to the C17 mRNA.
  • Antisense constructs are typically double stranded coding sequences which are in the reverse orientation with respect to a promoter. Thus an mRNA is produced which is complementary to the native C17 mRNA.
  • the promoter used can be the native Cl 7 promoter or another promoter which has desirable properties for the therapeutic or experimental application.
  • Polypeptide C17 can be used to expand populations of responsive cells in culture.
  • Responsive cells include cells which are hematopoietic and cells which are not hematopoietic.
  • the responsive cells are C34 + , C17 + or both C34 + and C17 + .
  • Endothelial cells, including endothelial progenitor cells are C34 + .
  • the polypeptide used for expansion of the cell population can be administered in any form, including in a purified form, as a conditioned medium (CM), or by co-culture with C17-transfected cells.
  • CM conditioned medium
  • Cells to be expanded can also be transfected so that they express their own C17.
  • Polynucleotides which encode mouse and bovine C17 are provided. These polynucleotides are isolated and purified, i.e., they are not in chromosome 4p, but have been manipulated so that they are either separate or adjacent to other sequences than the native chromosome 4p sequences.
  • the polynucleotides can be in vectors, viral or non- viral, and/or in transfected host cells. Isolated and purified C 17 proteins encoded by these genes are also provided. They can be made from natural producing bovine or mouse cells or they can be made recombinantly or synthetically. The sequences of the proteins and polynucleotides are shown in SEQ ID NO: 3, 4, 5, and 6. Fusion proteins can also be formed recombinantly which couple C17 to desired epitopes or activities. Any such desirable moieties can be used, although myc, his, and GFP are preferred. Typically these are used to enhance purification, recovery, or detectability.
  • the promoter for human C17 cytokine has been isolated and analyzed. Its sequence is shown in SEQ ID NO: 7. It has been found to be transcriptionally active in CD34 + /C17 + cells but inactive in CD347C17 " cells. It can be used to specifically drive transcription in CD34 + /C17 + cells. Any gene which is desirably expressed in such CD34 + /C17 + cells can be coupled to the promoter and delivered to such cells.
  • C17 can enhance the growth of immature human hematopoietic cells.
  • TF1 human hematopoietic progenitor cell line
  • TF1 cells which were derived from bone marrow blastic cells of an erythroleukemia patient are multi-potent CD34 + cells.
  • FBS a hematopoietic cytokine
  • TF1 cells can proliferate indefinitely in RPMI-1640 medium.
  • TF1 cells can also differentiate into either erythroid-like cells or megakaryocytic-like cells upon different induction.
  • C17 mRNA is readily found in undifferentiated TF1 cells as in primary BM (bone marrow) or CB (cord blood) CD34 + cells. Upon induction of differentiation, the level of C17 mRNA was reduced by 10 fold 3 days after treatment.
  • TFI cells were transduced either by MGIN/C17 or the control vector MGIN (expressing only GFP). Greater than or equal to 80% TFI cells were stably 5transduced by MGIN/C17 of MGIN. Subsequently we selected stably-transduced TFI cells and examined whether MGIN/C17-transduced TFI cells gained any growth advantages.
  • MGIN/C17-transduced TFI cells grew similarly as MGIN-transduced TFI cells in the optimal medium containing 10% FBS and 1-2 ng/ml GM-CSF (data not shown).
  • serum-free we did not see significant difference of cell growth in the first 2-3 days, but we observed that MGIN/C17-transduced cells gained growth advantage after 5 days. Both transduced cells required a hematopoietic cytokine like GM-CSF for survival as the parental TFI cells.
  • C17 or precisely C17- 15GFP in MGIN/C17-transduced cells
  • the TFI growth data provided the first evidence that the Cl 7 can act as an autocrine cytokine for CD34 + immature hematopoietic cells as well for CD34 + endothelial cells.
  • the gene transduction approach which we have used for HUVEC and TFI cells provides a powerful tool to allow autonomous synthesis of Cl 7 proteins in candidate target cells in an 0autocrine fashion to affect subsequently the growth of target cells. This approach is particularly useful when the purified and active C17 cytokine proteins are limited in quantity.
  • bovine ESTs highly related to the human C17 cDNA.
  • the assembled bovine EST contains an open-reading frame, encoding a putatively peptide which is highly homology to the human C17. It appears that the 5 'end of bovine ESTs available is incomplete and at least two amino acids (including Methionine from the start 0codon) are missing.
  • the mouse (C57/B6 strain) homologue of the human C17 More recently we found the mouse (C57/B6 strain) homologue of the human C17.
  • the assembled mouse C17 cDNA (from 3 independent ESTs) has a structure (including the 5' un-translated region [UTR], the Kozak start codon, an open reading frame and 3' UTR) very similar to the human C17 cDNA.
  • the alignment of deduced peptide sequences of human, bovine, and mouse C17 is shown at Fig. 1.
  • C17 proteins 462904-1 (without signal peptides) of the mouse and human C17 proteins are 91% homologous if conservative amino acid substitutions are included. Moreover, C17 proteins from all the 3 species have a very similar 2 nd structure (predicted): 4 alpha-helix bundled structure which is characteristic of hematopoietic cytokines and interleukins. We did not find mouse C17 or a highly-related homology in Princeton's SCDb or GenBank's mouse EST database (April 27, 2001).
  • C17 expression is highly restricted to human CD34 + cells, and that the proximal 1.5kb flanking DNA fragment (from the cloned 129 kb genomic DNA) functions as a promoter to direct the expression of GFP or luciferase reporter gene in transiently transfected cells.
  • proximal 1.5kb flanking DNA fragment from the cloned 129 kb genomic DNA
  • GFP or luciferase reporter gene in transiently transfected cells.
  • lentiviral vectors with the self-inactivating (SIN) modification allow cell-specific transgene expression in stably transduced cells from a non-LTR promoter.
  • SIN self-inactivating
  • vector PI GFP which contains the 1.5 kb C17 promoter
  • vector P2.GFP which contains proximal the 852 bp genomic sequence
  • vector P3.GFP which contains the 512 bp genomic (plus 11 bp cDNA) C17 sequence
  • vector PO.GFP which does not have any known promoter sequence.
  • PGK.GFP which contains a strong constitutive promoter from a human housekeeping gene PGK.
  • EF.NGFR which contains the constitutive human EF la promoter and the NGFR (p75) cell-surface marker.
  • Each GFP-containing test vector and the internal control vector (1:1) were used to co- transfect 293T cells and the integrity of GFP reporter gene in each test vector was first confirmed in transfected 293T cells. Lentiviral virus supernatants were then collected and used to transduce CD34 + /C17 + and CD347C17 " cells.
  • GFP transgene expression (as well as the NGFR expression from the internal control, using an anti-NGFR
  • the vectors PGK.GFP and PO.GFP acted similarly either in TFI or HL60 cells. Contrary to their expression in TFI cells, the three Cl 7 promoter-containing vectors (PI .GFP, P2.GFP and P3.GFP) produced only background levels of GFP expression in HL60 cells. As in HL60 cell, similarly results were obtained in K562 cells which are CD34/C17X As in TFI cells, similar results were obtained in HUVEC which are CD34 + /C17 + . Combined together, the 523 bp C17 promoter is necessary and sufficient to direct transgene expression specifically in CD34 + /C 17 + cells after lentiviral-mediated gene transduction.
  • the C17 gene is also expressed in proliferating CD34+ endothelial cells.
  • the secreted C17 protein functions as a growth factor for CD34+ endothelial cells.
  • HUVEC from multiple healthy donors were cultured with an optimized medium (EGM-2, Clonetics) which contains 2/5 FBS, bFGF, VEGF, IGF-1 and many other bioactive molecules.
  • C17 conditioned medium had no effect on the growth of HUVEC cultured with this optimal EGM-2 medium.
  • C17 conditioned medium significantly enhanced HUVEC growth.
  • the growth enhancement was mainly due to the C17 protein, since CM from control 193 cells (control CM) was insufficient.
  • Cl 7/myc/his was used little effect was observed on HUVEC growth in ESFM, with or without VEGF.
  • C17 significantly enhanced growth. Purified protein also enhanced the growth in the optimal EGM-2 medium.
  • Equal amounts of transduced HUVEC by the MGIN or MGIN/C 17 vector were plated in 6-well plates either in EGM-2 complete medium or a suboptimal medium: ESFM+ bFGF. After 3 days, transduced cells cultured with EGM-2 first reacted confluence. These cells in duplicates were harvested, counted, and replated in flasks. Transduced HUVEC cultured with ESFM + bFGF divided slower and were harvested at day 6, together with replated HUVEC cultured with the EGM-2 medium. Mock- transduced HUVEC expanded 4 and 20 fold, respectively at day 3 and day 6 in the EGM-2 medium.
  • MGIN/C 17-transduced HUVEC expanded about 6 and 27 fold, while the MGIN-transduced HUVEC only expanded about 5 and 19 fold at day 3 and day 6, respectively.
  • the enhancement of C17/GFP to HUVEC growth is more evident when the transduced HUVEC were cultured in suboptimal medium for 6 days: MGIN/C 17-transduced HUVEC expanded about 4 fold, while mock- or MGIN-transduced HUVEC only expanded less than 2 fold.
  • Endothelial cells including HUVEC will migrate on the surface of Matrigel, connect to each other and form a capillary structure.
  • Matrigel Becton Dickinson

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Abstract

Nous avons trouvé que la protéine C17 stimule la croissance de cellules endothéliales et hématopoïétiques exprimant le marqueur CD34. Dans la moelle osseuse et le sang, des populations rares de cellules exprimant CD34 sont des précurseurs immatures à la fois des cellules hématopoïétiques et des cellules endothéliales, dont des cellules souches hématopoïétiques, des cellules souches endothéliales et des hémangioblastes. Cette découverte rend la C17 utile pour favoriser l'angiogenèse et fait de ladite C17 une cible pour des médicaments contre l'angiogenèse. Des molécules spécifiques de C17 seront donc utiles pour traiter des tumeurs et la leucémie, ainsi que pour traiter les blessures en favorisant la cicatrisation.
PCT/US2002/011869 2001-05-03 2002-05-01 Methodes d'utilisation de la cytokine c17 WO2002089727A2 (fr)

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US60/288,083 2001-05-03
US13269202A 2002-04-26 2002-04-26
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Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2006111198A1 (fr) * 2005-04-18 2006-10-26 Associazione Foresta Per La Ricerca Nella Riproduzione Umana Utilisation d'inhibiteurs pde-5 pour reparer des tissus endommages par un trauma ou par une maladie
WO2009055613A2 (fr) * 2007-10-26 2009-04-30 Genentech, Inc. Inhibition de l'activité de l'activateur du plasminogène de type urokinase (upa)
EP3012269A1 (fr) 2014-10-20 2016-04-27 Erhard Hofer Protéine de type 1 (CYTL1) et ses utilisations

Families Citing this family (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US9522217B2 (en) 2000-03-15 2016-12-20 Orbusneich Medical, Inc. Medical device with coating for capturing genetically-altered cells and methods for using same
WO2005107817A2 (fr) 2004-04-30 2005-11-17 Orbus Medical Technologies, Inc. Dispositif medical comprenant un revetement permettant de capturer des cellules genetiquement modifiees et ses procedes d'utilisation

Non-Patent Citations (2)

* Cited by examiner, † Cited by third party
Title
LIU X. ET AL.: 'Molecular cloning and chromosomal mapping of a candidate cytokine gene selectively expressed in human CD34+ cells' GENOMICS vol. 65, no. 3, 01 May 2000, pages 283 - 292, XP004439369 *
ZHAOHUI Y. ET AL.: 'Specific expression of a novel cytokine-like gene in human CD34+ cells' BLOOD vol. 96, no. 11, 16 November 2000, page 142B, XP002967535 Abstract No. 4311 *

Cited By (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2006111198A1 (fr) * 2005-04-18 2006-10-26 Associazione Foresta Per La Ricerca Nella Riproduzione Umana Utilisation d'inhibiteurs pde-5 pour reparer des tissus endommages par un trauma ou par une maladie
WO2009055613A2 (fr) * 2007-10-26 2009-04-30 Genentech, Inc. Inhibition de l'activité de l'activateur du plasminogène de type urokinase (upa)
WO2009055613A3 (fr) * 2007-10-26 2009-11-19 Genentech, Inc. Inhibition de l'activité de l'activateur du plasminogène de type urokinase (upa)
EP3012269A1 (fr) 2014-10-20 2016-04-27 Erhard Hofer Protéine de type 1 (CYTL1) et ses utilisations
WO2016062692A2 (fr) 2014-10-20 2016-04-28 Erhard Hofer Protéine cytokine-like 1 (cytl1) et ses utilisations

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AU2002340544A8 (en) 2009-07-30
WO2002089727A9 (fr) 2003-01-09
AU2002340544A1 (en) 2002-11-18
WO2002089727A3 (fr) 2009-06-11

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