WO2002089727A2

WO2002089727A2 - Methods of using cytokine c17

Info

Publication number: WO2002089727A2
Application number: PCT/US2002/011869
Authority: WO
Inventors: Linzhao Cheng; Young Sung
Original assignee: The John Hopkins University
Priority date: 2001-05-03
Filing date: 2002-05-01
Publication date: 2002-11-14
Also published as: WO2002089727A9; AU2002340544A8; WO2002089727A3; AU2002340544A1

Abstract

We have found that C17 protein stimulates the growth of endothelial and hematopoietic cells expressing the CD34 marker. In bone marrow and blood, rare populations of cells expressing CD34 are immature precursors for both hematopoietic and endothelial cells, including hematopoietic stem cells, endothelial progenitor cells and hemangioblasts. This finding makes C17 useful for promotion of angiogenesis and makes it a target for drugs for antiangiogenesis. Thus C17 specific molecules will be useful for treating tumors and leukemias, as well as for treating wounds to encourage healing.

Description

METHODS OF USING CYTOKTNE C17

This application claims benefit of U.S. provisional application serial number

60/288,083 filed May 3, 2001. This invention was made using funding from the National Institutes of Health, grant no. CA06973. Therefore the U.S. government retains certain rights in the invention.

BACKGROUND OF THE INVENTION

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 α-helices, a characteristic of hematopoietic cytokines and interleukins. The gene for C17 polypeptide was mapped to human chromosome 4pl5-pl6. However, the biological functions of the polypeptide were not known. 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.

There is a need in the art for elucidation of the biological function of C17 in the mammalian body.

SUMMARY OF THE INVENTION

According to one embodiment of the invention a method is provided for promoting angiogenesis. 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

462904-1 contiguous nucleotides of SEQ ID NO: 1 in reverse orientation with respect to a promoter is administered to a patient in need thereof. An RNA is transcribed from the construct which is complementary to C17 mRNA. Angiogenesis is inhibited by the complementary RNA. In yet another embodiment of the invention another method is provided for promoting angiogenesis. A C17 cytokine is administered to a patient in need thereof. Angiogenesis is thereby stimulated.

In still another embodiment, a method is provided for inhibiting angiogenesis. An antibody which specifically binds to a C17 cytokine is administered to a patient in need thereof. Angiogenesis is thereby inhibited.

According to another aspect of the invention 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.

Similarly, 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.

Further provided by the present invention are 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.

Also provided by the invention are fusion proteins comprising a detectable marker protein and a C17 cytokine selected from bovine, mouse, and human C17 cytokine.

Finally, 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.

BRIEF DESCRIPTION OF THE DRAWINGS

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).

462904-1 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.

DETAILED DESCRIPTION OF THE DRAWINGS

It is a discovery of the present invention that Cl 7 polypeptide is a growth factor (cytokine) which stimulates endothelial cell growth. Thus it provides a tool for stimulating angiogenesis and neovascularization in wound healing and other vascular deficient diseases. In addition, C17 is a target for agents which block angiogenesis and neovascularization required for the progression of both solid tumors and leukemias.

One of the exciting aspects of these findings, is that 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.

Examples of C17 nucleotide and amino acid sequences are provided in the appended sequence listing. These include:

SEQ ID NO: 1 human nucleotide SEQ ID NO: 2 human amino acid

SEQ ID NO: 3 bovine nucleotide

SEQ ID NO: 4 bovine amino acid

SEQ ID NO: 5 mouse nucleotide

SEQ ID NO: 6 mouse amino acid SEQ ID NO: 7 human promoter

SEQ ID NO: 8 consensus amino acid

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. The inventors have found that retroviruses, including oncoretroviruses and lentiviruses, are particularly useful for this purpose. One such preferred retrovirus is 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. Preferably the responsive cells are C34⁺, C17⁺ or both C34⁺ and C17⁺. Endothelial cells, including endothelial progenitor cells are C34⁺ . We found that they are responsive to C17 and can be expanded, i.e., stimulated to proliferate in culture. Such cells can be used for transplantation, whether autologous or heterologous. 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. Cells to be expanded can also be transfected so that they express their own C17.

462904-1 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.

The invention is further described with respect to experimental particulars in the following examples.

EXAMPLES

Example 1

C17 can enhance the growth of immature human hematopoietic cells.

We have used a human hematopoietic progenitor cell line, TF1 , as an initial model system for studying expression and functions of C17. TF1 cells which were derived from bone marrow blastic cells of an erythroleukemia patient are multi-potent CD34⁺ cells. In the presence of 10% FBS and a hematopoietic cytokine (such as GM-CSF or IL-3), 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.

462904-1 We have examined whether C17 functions as an autocrine cytokine for TFI cells. For this purpose, we used the retroviral vector MGIN/CI7 which constitutively expresses a C17-GFP fusion protein. 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. During the transduction, selection and subsequent culture, 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). We next tested their ability to survive and grow lOin the sub-optimal (without serum and/or GM-CSF) and optimal medium. Under the conditions used (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. In the presence of GM-CSF, however, C17 (or precisely C17- 15GFP in MGIN/C17-transduced cells) enhanced cell survival and/or growth.

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. Example 2

We have cloned bovine and mouse homologues of human Cl 7 cDNA. 5

Recently we found two 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. 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. The mature forms

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).

Example 3

We have made several lentiviral vectors containing the Cl 7 promoter to direct GFP reporter expression specifically in CD34^¥/C17* cells.

We found that 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. In order to efficiently transfect/transduce CD34⁺ human cells and achieve transgene expression from chromosomal templates, we decided to use retroviral/lentiviral-mediated gene transduction techniques.

We and others have found that lentiviral vectors with the self-inactivating (SIN) modification allow cell-specific transgene expression in stably transduced cells from a non-LTR promoter.Therefore we decided to construct SIN lentiviral vectors containing the C17 promoter and GFP reporter gene, and to test the cell-specific transgene expression in a variety of cultured and primary CD34⁺ cells. In addition to vector PI. GFP which contains the 1.5 kb C17 promoter, we also made 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, and vector PO.GFP which does not have any known promoter sequence. As a positive control in parallel, we used vector PGK.GFP which contains a strong constitutive promoter from a human housekeeping gene PGK. In addition, we used an internal control vector 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

462904-1 antibody conjugated with R-PE) was analyzed 5-10 days after transduction. An example is shown in Fig 3 with TFI (CD34⁺/C17⁺) and HL60 (CD347C17 ) hematopoietic cells. The percentages of NGFR⁺ cells were similar among different test samples in each cell type (data not shown), whereas the levels of GFP expression varied among cells transduced by different test (GFP) vectors. In TFI cells, 3 vectors containing different lengths of the C17 promoter acted similarly and directed a medium-level of GFP expression (as compared to PGK.GFP). The PO.GFP vector produced -5% GFP⁺ cells which expressed GFP at a very low level. 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.

Example 3

The C17 gene is also expressed in proliferating CD34+ endothelial cells.

We found that the C17 gene is also expressed in cultured proliferating HUVEC (from Clonetics). Example 4

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. We found that C17 conditioned medium had no effect on the growth of HUVEC cultured with this optimal EGM-2 medium. However, under suboptimal conditions (serum-free ESFM medium (Gibco/BRL)), 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. When purified Cl 7/myc/his was used little effect was observed on HUVEC growth in ESFM, with or without VEGF. However, in the presence of bFGF, C17 significantly enhanced growth. Purified protein also enhanced the growth in the optimal EGM-2 medium.

Example 5

462904-1 MGIN/C17-transduced HUVEC secrete C17/GFP proteins and display a growth advantage.

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. We observed that the 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.

Example 6

Cl 7 expression in HUVEC does not alter their differentiation potential.

Endothelial cells including HUVEC will migrate on the surface of Matrigel, connect to each other and form a capillary structure. Matrigel (Becton Dickinson

Labware) is soluble extracts of basement membrane matrix and is rich in extracellular matrix proteins and cytokines such as TGF/J. Single cell suspensions were added onto solidified Matrigel. MGIN/C 17-transduced HUVEC cultured either in the EGM-2 or ESFM + bFGF medium for 6 days formed the same capillary structure as untreated HUVEC. Therefore the Cl 7 stimulation did not alter the HUVEC potential to display a differentiation phenotype upon Matrigel induction.

462904-1

Claims

1. A method of promoting angiogenesis comprising: administering an expression vector comprising a polynucleotide sequence according to SEQ ID NO: 1 to a patient in need thereof, whereby angiogenesis is stimulated.

2. The method of claim 1 wherein the patient has a wound in need of healing.

3. The method of claim 2 wherein the expression vector is administered directly to the wound site.

4. A method of inhibiting angiogenesis comprising: administering to a patient in need thereof an antisense construct comprising all or a portion of at least 18 contiguous nucleotides of SEQ ID NO: 1 in reverse orientation with respect to a promoter, whereby an RNA is transcribed from the construct which is complementary to C 17 mRNA, whereby angiogenesis is inhibited. .

5. The method of claim 4 wherein the patient carries a tumor.

6. The method of claim 5 wherein the construct is administered to the tumor site.

7. The method of claim 6 wherein the construct is administered by intratumoral injection.

8. The method of claim 4 wherein the patient has leukemia.

9. A method of promoting angiogenesis comprising: administering a C17 cytokine to a patient in need thereof, whereby angiogenesis is stimulated.

10. The method of claim 9 wherein the patient has a wound in need of healing.

11. A method of inhibiting angiogenesis comprising: administering an antibody which specifically binds to a C17 cytokine to a patient in need thereof, whereby angiogenesis is inhibited.

12. The method of claim 11 wherein the patient has a tumor.

13. The method of claim 11 wherein the patient has leukemia.

14. A method of expanding a population of cells in culture, comprising: contacting cells in culture with a C17 cytokine, whereby the cells are stimulated to proliferate, wherein the cells are not mesenchymal stem cells.

15. The method of claim 14 wherein the cells are not hematopoietic cells.

462904-1

16. The method of claim 15 wherein the cells are hematopoietic stem cells.

17. The method of claim 15 wherein the cells are endothelial progenitor cells.

18. The method of claim 14 wherein the cytokine is purified.

19. The method of claim 14 wherein the cytokine is produced by recombinant host cells which comprise an expression vector comprising a polynucleotide sequence according to SEQ ID NO: 1.

20. The method of claim 19 wherein the recombinant host cells are co-cultured with said cells in culture.

21. The method of claim 14 further comprising transplanting the proliferated cells to a host.

22. The method of claim 21 wherein the transplanting is autologous.

23. An isolated and purified polynucleotide which encodes mouse C17 cytokine and has a sequence according to SEQ ID NO: 5.

24. An isolated and purified polynucleotide which encodes mouse C17 cytokine according to SEQ ID NO: 6.

25. An isolated and purified polynucleotide which encodes bovine C17 cytokine and has a sequence according to SEQ ID NO:4.

26. An isolated and purified polynucleotide which encodes bovine C17 cytokine according to SEQ ID NO: 3.

27. An isolated and purified mouse C17 cytokine according to SEQ ID NO: 6.

28. An isolated and purified bovine C17 cytokine according to SEQ ID NO:4.

29. A fusion protein comprising a detectable marker protein and a C17 cytokine selected from bovine, mouse, and human C17 cytokine.

30. The fusion protein of claim 29 wherein the marker protein is green fluorescent protein.

31. An isolated and purified promoter which is transcriptionally active in CD34⁺/C17⁺ cells but transcriptionally inactive in CD347C17^" cells, which has a sequence according to SEQ ID NO: 7.

32. The promoter of claim 31 which is operably linked to a protein coding sequence other than for human C 17 cytokine.

462904-1