Patent Application
20060104964
The present invention relates to a recombinant nucleic acid sequence encoding both a specific angiogenesis factor antagonist and a vascular endothelial structure regulator; its preparation; protein expression; and the use of the sequence or the protein in the inhibition of angiogenesis and/or the treatment of cancer.
Angiogenesis, the formation of new blood vessels, is a key in the development and progression of cancer. Angiogenesis is governed by a range of angiogenic factors and anti-angiogenic factors. Angiogenic factors are known to include a range of cytokines, such as vascular endothelial growth factor (VEGF), fibroblast growth factor (FGF) eg Basic FGF (bFGF), interleukins (ILs, eg IL-8) and hepatocyte growth factor/scatter factor (HGF/SF). Without new blood vessels, a tumour can not grow beyond 2 mm in diameter, due to limited blood supply and nutrient/oxygen diffusion.
Furthermore, tumour cells may disseminate in the body and produce micro- and macro-metastasis in organs and tissues, but remain invisible for from months to years. Once new blood vessels grow into these quiescent tumours, they will grow at a much faster speed, begin to manifest clinical symptoms and become lethal to patients. New blood vessels in the tumour provide not only nutrients and oxygen, but also a passage for tumour cells to enter the circulation and therefore aid the process of metastasis.
Therefore, anti-angiogenesis has become a focus in the development of new anti-cancer drugs. The fundamental importance of angiogenesis in cancer development and metastasis has prompted the discovery of a large number of angiogenesis inhibitors, including agents specifically designed as anti-angiogenesis agents (such as anti-VEGF antibody, anti-bFGF antibody, fumagillin and recombinant products based on a single gene, such as angiostatin), and those discovered unintentionally (such as beta-inteferon, tamoxifen and interleukins-4 and -12).
Some of the angiogenic factor antagonists are suitable for the purpose of anti-angiogenesis, but others are not. For example, each antagonist works specifically on only one particular angiogenic factor, whereas there are about 20-40 angiogenic factors in the body, in any given tumour. Another problem is that using a specific antagonist will result in a balance switch in which the targeted angiogenic factor is suppressed, but other factor(s) increase in compensation. Hence, the balance shifts from the targeted angiogenic factor to another or others, resulting in resistance to anti-angiogenesis therapy.
Accordingly, the present invention is directed to an agent to suppress angiogenesis, obtainable by genetically engineering two important regulators of angiogenesis, an angiogenic factor antagonist and an endothelial structure regulator (such as vascular endothelial cadherin).
We have therefore genetically engineered a recombinant molecule that comprises both a sequence capable of expressing a specific antagonist and a sequence capable of expressing a specific endothelial cell marker, being a vascular endothelial structure regulator, which is essential to the formation of new blood vessels. The recombinant products (referred to collectively herein as KV products, such as those referred to as KVEn, wherein n is an integer, K represents the angiogenic factor antagonist, V represents vascular endothelial cells and E represents the expression vector, and others referred to by J numbers) both retain the antagonistic properties of an anti-angiogenic factor; and also specifically recognise cells that produce new blood vessels, ie vascular endothelial cells.
Therefore, the recombinant products will work on the general mechanism for forming new blood vessels as well as on a specific mechanism operated by a specific angiogenic factor; and have the further advantage in preventing the balance switch and angiogenesis resistance that currently faces anti-angiogenesis therapy.
Accordingly, the present invention provides an isolated, purified or recombinant nucleic acid sequence (hereinafter, a KV sequence) comprising:
By ‘homologous’ herein is meant a sequence having at least 80% identity of nucleotides (or, in the case of an amino acid sequence, bases) in the same order within the sequence. Preferably, the sequence has at least 85% and more preferably at least 90%, such as over 95% homology.
The present invention further provides a polypeptide (protein) sequence (of amino acids) encoded by a nucleotide sequence of the invention.
Specific embodiments of the present invention will therefore now be described with reference to the accompanying Figures, in which:
Particular oligonucleotides (d) that are included in this invention are those encoding the vascular endothelial structure regulator. The endothelial structure regulator is suitably derived from VE-cadherin, E-selectin, occludin, claudin-5 and/or vascular cell adhesion molecule (VCAM), especially VE-cadherin, occluding and claudin-5.
Accordingly, the present invention further provides an isolated, purified or recombinant nucleic acid sequence (hereinafter, a KV sequence) comprising:
The antagonist fragment is suitably derived from VEGF, bFGF, hepatocyte growth factor/scatter factor (HGF/SF) and/or chemokines. Preferably, the antagonist fragment is derived from VEGF and/or HGF/SF. Particularly preferred antagonist fragments are KS2101 and KS2105, as defined below.
In general, such products may be prepared by using a conventional recombinant DNA technique. For example, first, a plurality of separate DNA fragments are prepared, at least one of which comprises a sequence encoding an antagonist and at least one of which comprises a sequence encoding the endothelial structure regulator. This may be carried out with specific primers that allow a further recombinant to be prepared, which generates a new recombinant gene. In particular, certain recombinant genes, referred to hereinbelow as KVE702, J35, J36 and J37, have been generated from DNA fragments cloned from human fibroblasts and vascular endothelial cells. The new KVE702 gene and its fragments have been used to transfect human epithelial cells and to generate products that may be suitable for angiogenesis intervention.
Accordingly, the present invention further provides an isolated, purified or recombinant nucleic acid sequence comprising:
The MRC-5 cell line is available from the European Collection of Animal Cell Cultures, and HUVEC is obtainable by extraction from fresh umbilical cord.
Preferably, the sequence (a) is selected from the following:
as shown in
as shown in
as shown in
as shown in
That part of the sequence according to the invention [KVE702 sequence] derived from the MRC-5 cell line is shown in
That part of the sequence according to the invention [J35 sequence] derived from the MRC-5 cell line is shown in
That part of the sequence according to the invention [J36 sequence] derived from the MRC-5 cell line is shown in
That part of the sequence according to the invention [J37 sequence] derived from the MRC-5 cell line is shown in
Using these cloned products, it is possible to transfect a suitable cell and establish stable transfectants to see whether the transfection affects the motile behaviour of the transfected cells. Determination of a suitable cell for transfection is carried out by usual trial-and-error methods known in the art in which, for example, human epithelial, fibroblast or leukaemic cells are transfected with a plasmid carrying both the gene and an antibiotic resistance gene, to which toxic antibiotics (such as G418, available from InVitrogen) are added. Cells that are able to incorporate the gene will therefore die as a result of the antibiotic, thereby allowing exclusion of cells unsuitable for transfection. An example of such suitable cells is the human breast cancer cell line, MCF-7. The transfectants can then be used to generate recombinant proteins for testing in an angiogenesis assay and subsequent selection for therapeutic and/or diagnostic use.
Accordingly, the present invention further provides an isolated, purified or recombinant construct incorporating a KV sequence according to the above description, in particular, one wherein the nucleic acid sequence is linked operably with nucleotides enabling expression and secretion in a cellular host of a protein (hereinafter, the KV protein) encoded by the KV sequence.
Furthermore, this invention provides DNA or RNA, especially cDNA or mRNA, according to any of the aforementioned sequences or constructs; and a method for preparing such DNA or RNA as described herein, together with such DNA or RNA preparable by such a method.
Accordingly, the present invention also provides a method for preparing a KV sequence, which method comprises:
(a) generating a fragment of cDNA encoding a specific angiogenesis factor antagonist;
(b) generating a fragment of cDNA encoding a specific vascular endothelial structure regulator, which fragments (a) and (b) are complementary at one end thereof; and
(c) combining the fragments to generate a recombinant gene capable of expressing the corresponding KV protein.
Especially, the present invention provides an isolated, purified or recombinant polypeptide comprising both an angiogenic factor antagonist and a vascular endothelial structure regulator, such as those mentioned herein; and, in particular, an isolated, purified or recombinant polypeptide comprising KV protein, or a mutant or variant thereof having substantially the same activity as KV protein.
For example, there is provided an isolated, purified or recombinant polypeptide comprising an amino acid sequence selected from
It will be apparent that the invention therefore further provides a cell, plasmid, virus or live organism that has been genetically-engineered to produce a KV protein, said cell, plasmid, virus or live organism having incorporated expressibly therein a KV nucleotide sequence according to this invention; a vector comprising such a sequence; and/or a host cell transformed or transfected with such a vector.
For gene therapy, preferably a viral vector would be chosen and genetically-engineered to produce the KV protein. Both retroviral and adenoviral vectors could be used, such as the Retro-X™ or Adeno-X™ systems from Clontech (USA).
Furthermore, this invention provides a process for obtaining a substantially homologous source of KV protein, which process comprises culturing cells having incorporated expressibly therein a KV nucleotide sequence according to this invention, and thereafter recovering the cultured cells.
The KV protein(s) according to this invention may therefore be used in connection with any condition associated with angiogenesis, such as cancer, for the regulation of the development of blood vessels and their formation, whether in the vascular endothelium and/or a tumour. A suitable dose may be determined according to conventional techniques known to those skilled in the art of pharmacy, but may conveniently be in the range of from 0.5 to 10 mg/kg bodyweight, administered in a suitable regime, such as from once to seven times per week.
Accordingly, the present invention still further provides such a KV protein, optionally in association with a pharmaceutically acceptable carrier therefor, for use in therapy, such as for use in any of the conditions mentioned herein. It is especially preferred that, for protein therapy, there is provided a pharmaceutical formulation comprising such a protein (KV protein) in association with a pharmaceutically acceptable carrier therefor. Preferred formulations include those for parenteral administration, such as injections and infusions for intravenous or intramuscular administration. Other suitable formulations are well-known to those skilled in the art of pharmaceuticals.
Also provided are:
a method for preparing such formulations, which method comprises bringing the protein (KV protein) into association with the carrier;
a method for the prophylaxis or treatment of a mammal, including man, comprising the administration to said mammal of a non-toxic, effective amount of such a protein (KV protein);
a KV protein for use in medicine, such as in the inhibition of angiogenesis and/or the treatment of cancer; and
the use of a KV protein in the preparation of a medicament, suitable for use in the inhibition of angiogenesis and/or the treatment of cancer.
The following examples are provided by way of illustration of the present invention.
Cells Used
Human fibroblasts (an established cell line, MRC-5) and human vascular endothelial cells (HUVEC) (extracted from human umbilical vein) (Cai J et al. Gamma linolenic acid inhibits expression of VE-cadherin and tube formation in human vascular endothelial cells. Biochemical and Biophysical Research Communications, 1999, 258, 113-118) were used.
Preparation of Human mRNA and cDNA Template
mRNA was isolated from fibroblasts or endothelial cells using an mRNA extraction kit (Sigma Chemicals, Poole, Dorset, UK). Complementary DNA (cDNA) was prepared from the mRNA using a reverse transcription kit (Promega).
Oligonucleotides (Primers) Used in PCR Reaction and Recombinant PCR
Sets of PCR primers were designed to amplify the areas of interest from the prepared cDNA, ie antagonist from the fibroblasts and endothelial marker/antagonist from the endothelial cells. The PCR primers were designed in such way that the products of each reaction would be used in the subsequent recombination. The primers were synthesised by Life Technologies and used exclusively for this work. Those primers designed for the cloning of the recombinant gene named herein KVE702 were as follows:
The amplification was carried out at three separate settings: first, to generate a fragment from fibroblast cDNA and, secondly, to generate a fragment from HUVEC cDNA, both products being complementary at one end. The final step was to generate a recombinant product by joining the two fragments. Each reaction was performed under special conditions in order to generate the desired products, as follows:
Setting 1 (to generate antagonist): 95° C. for 5 minutes, then 36 cycles of 95° C. for 1 minute, 61° C. for 1 minute and 72° C. for 2 minutes, followed by 72° C. for 7 minutes.
Setting 2 (to generate endothelial marker): 95° C. for 5 minutes, then 36 cycles of 95° C. for 40 seconds, 58° C. for 2 minutes and 72° C. for 2 minutes, followed by 72° C. for 10 minutes.
Setting 3 (to generate recombinant gene): without primer at 95° C. for 5 minutes, then
4 cycles of 95° C. for 40 seconds, 35° C. for 1 minute and 72° C. for 90 seconds, followed by 72° C. for 90 seconds, then
4 cycles of 95° C. for 40 seconds, 40° C. for 1 minute and 72° C. for 90 seconds, followed by 72° C. for 90 seconds, then
4 cycles of 95° C. for 40 seconds, 45° C. for 1 minute and 72° C. for 90 seconds, followed by 72° C. for 90 seconds, then
4 cycles of 95° C. for 40 seconds, 50° C. for 1 minute and 72° C. for 90 seconds, followed by 72° C. for 90 seconds, then
4 cycles of 95° C. for 40 seconds, 55° C. for 1 minute and 72° C. for 90 seconds, followed by 72° C. for 90 seconds, then
4 cycles of 95° C. for 40 seconds, 60° C. for 1 minute and 72° C. for 90 seconds, followed by 72° C. for 90 seconds, then
4 cycles of 95° C. for 40 seconds, 64° C. for 1 minute and 72° C. for 90 seconds, followed by 72° C. for 90 seconds, followed by 72° C. for 10 minutes;
then, with primers added, 35 cycles of 95° C. for 40 seconds, 59° C. for 1 minute and 72° C. for 90 seconds, followed by 72° C. for 10 minutes.
From these reactions, the following products were generated:
1. DNA fragments were isolated from fibroblasts using RT-PCR, referred to herein as: KS2101 (
2. DNA fragments from HUVEC cells, referred to herein as: VC503 (
3. The recombinant sequence: A further step was carried out to generate the recombinant sequence, by joining KS2101 and VC503, using the aforementioned recombinant technique. This generated a specific recombinant sequence, namely KVE702 (
Cloning of the Specific Products
Each fragment and the recombinant gene were cloned into a mammalian expression vector (pcDNA3.1/V5/His-TOPO, available from InVitrogen) and transfected into a competent E. coli. Colonies that carried the desired products were detected using PCR. The positive colonies were further expanded and grown in large volume. Plasmids resulting from cloning the genes into the vector (ie that carried the specific products) were then purified from these E. coli preparations.
KS2101, KS2105, VC1, VC503 and KVE702 gene-carrying plasmids were transfected into mammalian epithelial cells. A transfection agent, Transfast (Promega), was used. After series testing, MCF-7 cells (well-known as a human breast cancer cell line) were found to be the most suitable and acceptable cell for this purpose and chosen to be the cells for transfection in the current study. The optimal transfection condition was at Transfast:DNA=2:1.
After transfection, cells that retained these new genes were selected using a selection medium containing G418 (from InVitrogen or Calbiochem), which caused cells that had no new genes gradually to die out whilst those with new genes carried on dividing. Cells expressing these new genes of interest were obtained after over 4 weeks' selection (so-called stable transfectants). It was observed that wild type (non-transfected) cells were almost all dead after two weeks, whilst between 10-30% of the cells transfected with the genes of interest remained viable. In approximately 4 weeks, enough of these viable cells were available for biological testing.
In order to test whether the stably transfected cells (prepared according to Example 2) were different from the wild type, a technique known as the cell spreading/colony scattering assay was carried out (Jiang et al Monocyte conditioned media possess a novel factor which increases motility of cancer cells Int. J. Cancer 53 426-431 (1993)). Briefly, wild type or transfectants were plated in tissue cultureware at low density and then allowed to form colonies (clusters). These were then treated either with medium as control or with a scatter-inducing faction (HGF/SF). After 24 hours, cells were fixed and digitised images were obtained using a digital camera. The spreading and scattering were quantified as described by Jiang et al. (in Gamma linolenic acid selectively regulates the expression maspin and motility of cancer cells Biochemical and Biophysical Research Communications 237 639-644 (1997)) using an image analysis package (Optimas 6 from Optimas UK); the results for each culture were as follows:
Wild Type
Wild type MCF-7 cells formed tightly packed clusters in culture with apparent cell-cell joining. A scattering inducer (HGF/SF) can disperse the colonies, ie cells apparently move away from each other.
VC1 Transfectants
VC1 transfected cells appeared as much tighter clusters, compared with the wild type. VC1 transfectant substantially reduced their response to HGF/SF (5, 10, 2 and 50 ng/ml). Cells appeared as small, tightly-packed clusters, with cell-cell joining remaining visible.
KS2105 AND KS2101 Transfectants
KS2105 transfectant exhibited a similar cell morphology, when compared with controls. These cells, however, reduced their response significantly to HGF/SF. A similar response, although to a lesser degree, was seen with KS2101 transfectants.
KVE702 Transfectants
The established KVE702 revealed a similar morphology to control. Scattering inducer HGF/SF failed to induced a significant change. Hence, transfection did not alter the morphology of the cell, but reduced its response to HGF/SF.
The data obtained therefore clearly show that MCF-7 cells transfected with VC1, KS2101 and KS2105 did not significantly change their morphology. In fact, the cells appeared to reduce their response to stimulation. The data thus indicate that transfection did not alter the aggressiveness of MCF-7 cells.
The study used a technique known as in vitro tubule formation analysis, to test the effect of recombinant materials on the formation of blood vessel-like structures (Kanayasu et al Eicosapentaenoic acid inhibits tube formation of vascular endothelial cells in vitro. Lipids, 26 271-276 (1991); Bach et al VE-cadherin mediates endothelial cell capillary tube formation in fibrin and collagen gels Exp Cell Res 238 324-334 (1998)).
24 multi-well plates were first coated with Matrigel™ (available from Beckton Dickinson) (200 μg/well) and allowed to form a thin gel layer. 5×104 HUVEC cells in 0.5 ml of DMEM with 10% foetal calf serum (FCS) were then added over Matrigel™ for 24 hours. The medium was aspirated, and a further 0.5 ml of Matrigel™ was overlaid with a further 0.5 ml of medium, which contained either medium, HGF, NK4, or NK4 and HGF in combination. Cell cultures were observed under a phase-contrast microscope after 24 hours. Each well was photographed four times at random and tubule length was measured using an image analysis software (Optimas 6 from Optimas UK). A known angiogenesis inducer, HGF/SF, and conditioned medium from the stable transfectants were then tested on the cells. The results of the study were as follows:
VC1 and KS2105 Products Reduced the Tubule Formation
Conditioned medium from the VC1 transfectant reduced the tubule forming that was induced by HGF/SF. The conditioned medium on its own appeared to have some minor effect on tubule formation. Interestingly, KS2105 supernatant reduced tubule formation both with and without angiogenesis inducer.
KVE702 Reduced Tubule Formation
Conditioned medium from KVE702 increased tubule length, although to a small degree. However, when an angiogenic factor was included, which significantly increased tubule length, KVE702 supernatant exerted a significant inhibitory effect on tubule formation.
Conclusion: Therefore, it was observed that HGF/SF significantly increased tubule formation from vascular endothelial cells. Supernatants from the stable transfectants can reduce this increase in tubule formation. Hence, the present invention may present a new opportunity to produce anti-cancer agents.
Using the techniques described above in Example 2, MCF-7 (human breast cancer cells) were transfected with KVE702 gene and a stable transfectant selected using G418. The cells were then tested using the established Matrigel™ invasion assay described by Jiang et al in Cancer Research, 55 5043-8 (1995). It was found that the transfected cells had a reduced invasiveness compared with wild MCF-7 cells or with MCF-7 cells that had been transfected with a control plasmid carrying the Lac-z gene (available from InVitrogen). The response of the transfected cells to HGF/SF was also found to be significantly reduced, compared to wild and control cells.
Since invasiveness of cancer cells is directly related to the progression and metastasis of a cancer, its reduction indicates a potential future in cancer therapy for the recombinant product of the invention.
Following the method of Example 1, the title recombinant product was prepared, using the following primers in place of i-iv of Example 1:
From these reactions, the following products were generated:
1. DNA fragments were isolated from fibroblasts using RT-PCR: J9 (antagonist) (
2. DNA fragments were isolated from HUVEC cells: J12 (endothelial marker) (
3. A recombinant gene: A further step was carried to generate a recombinant gene, by joining J9 and J12, using the aforementioned recombinant technique. This generated a specific recombinant gene, namely: J35 (recombinant product) (
Following the method of Example 1, the title recombinant product was prepared, using the following primers in place of i-iv of Example 1:
From these reactions, the following products were generated:
1. DNA fragments were isolated from fibroblasts using RT-PCR: J10 (antagonist) (
2. DNA fragments were isolated from HUVEC cells: J11 (endothelial marker) (
3. A recombinant gene: A further step was carried to generate a recombinant gene, by joining KS2101 and J11, using the aforementioned recombinant technique. This generated a specific recombinant gene, namely: J36 (recombinant product) (
Following the method of Example 1, the title recombinant product was prepared, using the following primers in place of i-iv of Example 1:
From these reactions, the following products were generated:
1. DNA fragments were isolated from fibroblasts using RT-PCR: J6 (antagonist) (
2. DNA fragments were isolated from HUVEC cells: J8 (endothelial marker) (
3. A recombinant gene: A further step was carried to generate a recombinant gene, by joining KS2101 and J8, using the aforementioned recombinant technique. This generated a specific recombinant gene, namely: J37 (recombinant product) (
| Number | Date | Country | Kind |
|---|---|---|---|
| 0010630.2 | May 2000 | GB | national |
This application is a division of co-pending application Ser. No. 10/275,296, filed on Jun. 9, 2003. Application Ser. No. 10/275,296 is the national phase of PCT International Application No. PCT/GB01/01956 filed on May 4, 2001 under 35 U.S.C. § 371, which claims priority of British Application No. 0010630.2 filed May 4, 2000. The entire contents of each of the above-identified applications are hereby incorporated by reference.
| Number | Date | Country | |
|---|---|---|---|
| Parent | 10275296 | Jun 2003 | US |
| Child | 11328070 | Jan 2006 | US |
