WO2003059949A2

WO2003059949A2 - A protein that mediates angiogenesis and tumour growth inhibition, its cdna, alone or in complex with an expression vector

Info

Publication number: WO2003059949A2
Application number: PCT/IB2003/000116
Authority: WO
Inventors: Paola Romagnani; Sergio Romagnani; Mario Serio
Original assignee: Universita degli Studi di Firenze
Current assignee: Universita degli Studi di Firenze
Priority date: 2002-01-18
Filing date: 2003-01-20
Publication date: 2003-07-24
Anticipated expiration: 2004-07-18
Also published as: AU2003235574A8; AU2003235574A1; WO2003059949A3; ITFI20020008A1

Abstract

It is described a novel type of protein exerting therapeutics and diagnostic role in human tumors, its cDNA, alone or in complex with an expression vector.

Description

A PROTEIN THAT MEDIATES ANGIOGENESIS AND TUMOUR GROWTH INHIBITION, ITS CDNA, ALONE OR IN COMPLEX WITH AN EXPRESSION VECTOR

Field of the invention The invention refers to a protein [SEQ. ID. N° 1], its use in diagnosis and therapy of tumours, and its cDNA [SEQ. ID. N° 2]. State of the art

As it is known CXCR3 mediates the angiostatic and anti tumour effects of chemokines IP-10, Mig and l-TAC and it is also known that, depending on the cell type expressing it, it can mediate completely different effects (chemotaxis, proliferation, adhesion, inhibition of proliferation).

In view of the role played by this kind of protein structure for the treatment of various pathologies, in particular tumours, it is evident the importance to identify and make available new proteins capable of controlling the vascular and tumour growth induced by the above said chemokines. Brief description of the drawings

Fig.1 shows the sequence of the protein according to the invention. Fig.2 shows the sequence of the DNA expressing the protein of Fig. 1. Fig.3 shows schematically an expressing vector containing the cDNA sequence coding for CXCR3-B.

Detailed description of the invention

It was now surprisingly found and it is an object of the present application, that a new protein (hereinafter named CXCR3-B) [SEQ: 1] mediates the inhibition of endothelial cell growth induced by angiostatic chemokines IP-10, Mig, l-TAC and PF-4 and probably controls tumour growth.

The present invention is related to the complexes containing the above said protein and the expression vectors and the plasmides containing such complexes. The analysis of the "reading frame" of the gene of the sole chemokine receptor known up to now CXCR3, identified the existence of a possible splicing variant corresponding to a new mRNA.

The existence of this new variant was confirmed by rapid amplification of cDNA ends performed on the total RNA of human thymus and endothelial tissue. The Northern blotting analysis using a probe for the sequence common to the two variants, permitted to reveal the existence of two bands of mRNA of the expected length; on the contrary using a probe selective for a portion of the sequence of the new variant a new RNA of 1 ,8 kb was identified. Operating as described the expression of the new mRNA of CXCR3 was identified in various human tissues as heart, skeletal muscle, kidney and liver. Thereafter a dosage in quantitative RT-PCR (Taq-Man) was realised, using oligonucleotides selective for the sequences of the two isoforms in various types of human cells. It could be noted that the CXCR3-B is selectively expressed by the human capillary endothelial tissue and by some types of tumour cell of epithelial origin cultivated in vitro. Therefore the CXCR3 expressed by the capillary endothelial tissue which is capable of mediating the endothelial growth inhibition in vitro is not the one known up to now but a distinct variant identified for the first time by the present invention. Using the RT-PCR the total sequence was obtained in the form of cDNA, starting from atg to the stop codon (tga) [SEQ: ID: 2] and it was cloned in an expressing vector of the type usually employed for the transcription of recombinant molecules or for creating transient or stabile transfectants. An expressing vector according to the invention is, for example, a plasmide containing a promoter and a polyadenilation sequence (normally of viral origin) and a resistance to antibiotics for prokaryotes and/or eucaryotes (see Fig. 3).

The correctness of the sequence and its orientation were confirmed by automatic sequencing. Obviously instead of cloning the whole sequence in the expressing vector it is possible to clone a part of it as, for example, it is normally done when probes are to be obtained.

An immortalised cell line of human capillary endothelial cells was transfected with the expressing vector coding for CXCR3-B, an other vector codifying for the known CXCR3 protein (CXCR3-A) and respectively the two vectors deprived of the portion coding for the two proteins. Stable transfectants were obtained by selecting the cells with antibiotics G-418 and hygromicine and in such transfectants the expression of the two types of CXCR3 was examined by using flux cytofluorimetry in the presence of two types of monoclonal antibodies anti-CXCR3. Numerous clones expressing the two receptor variants were selected, however it was evident that the endothelial cells expressing the new type (CXCR3-B) rapidly died while the clones expressing the known type of CXCR3 (CXCR3-A) all had an high proliferation rate, similar or even superior to the one of the respective "mock. Six of the obtained clones, all expressing low intensity CXCR3-B, could survive some steps, showing a replicative rhythm 4-8 folds lower of the one of the corresponding "mock" and an high trend to precocious cell senescence and death. Other studies, both using the immunofluorescence or the RT-PCR techniques, could confirm that what observed was due to the fact that the cell lines were capable of spontaneously producing under culture high levels of l-TAC, and lower levels of IP-10; which were responsible of the high proliferation of the cells transfected with the known type of CXCR3 (CXCR3-A) and of the growth inhibition of the cells transfected with the new type (CXCR3-B) respectively. In fact an antibody specifically anti-l-TAC was capable of making the transfectants of CXCR3-B to proliferate and to reduce the proliferation of the cells transfected with CXCR3-A. The newly identified receptor (CXCR3-B) is capable of binding all the three chemokines specific for CXCR3 (IP-10, Mig and l-TAC) which can induce inhibition of angiogenesis with various affinity while it can not bind other 17 different and not related chemokines. However, rather surprisingly, the transfectants of CXCR3-B are capable of binding the chemokine PF-4 with high affinity, similar to the one of IP-10, while the transfectants of CXCR3-A bind PF-4 with a very low affinity. This is particularly important to note since PF-4, together with IP-10, Mig and l-TAC, is the fourth chemokine having a strong angiostatic and anti tumour effect. However the receptor capable of mediating its effect was unknown. The data show that CXCR3- B is the receptor which can mediate the effect of the four angiostatic chemokines on the endothelial cells. The chemokines IP-10, Mig, l-TAC and PF-4 activate the same transduction pathways of the signal in the transfectants of CXCR3-B, that is the increase the production of cAMP and of the inhibitor of the cell cycle p21Cip1/waf1 in a way which is p53-independent.

In order to investigate the existence in vivo, of this protein monoclonal antibodies specific for the last 51 amino acids of its terminal NH2 extremity were prepared. Such antibodies, if used in immunohystochemistry, are capable of recognising the activated endothelial tissue in tumour and inflammations characterised by increased angiogenesis but not in normal tissues. The expression of CXCR3-B is inversely correlated to the tumour extension, to its rate of lymph nodes invasion and to the angiogenesis degree present in the tissue samples of the tumour analysed in 30 patients affected by "non small cell" lung cancer. Experimental part Northern blot analysis 2 μg of poly-A⁺ RNA were separated on a 2.0% agarose gel containing 0.7 M formaldehyde and ethidium bromide 0.5 mg/ml, the RNA was transferred on a nylon membrane, hybridised with a [α-³²P]dCTP labelled cDNA and revealed by autoradiography.

Rapid Amplification of cDNA ends (RACE) Was performed using oligonucleotides specific for the cDNA portion which is typical of the CXCR3-B alone followed by amplification towards the extremity 5^* and 3'.

Real-Time Quantitative RT-PCR according to Tag-Man

The total RNA was retro-transcripted using a oligo dT16. The cDNA was thereafter amplified. During the PCR cycles the probes hybridise specifically to the sample and are cut by the 5'-3'esonuclease activity of Taq polymerase, inducing a higher emission of fluorescence proportional to the quantity of specific PCR product. The fluorescence signal was monitored in real time using the 7700 Sequence detector (PE Applied BioSystem, Foster City, CA, USA). donation of the gene seguence of the new receptor in an expression vector and generation of stable transfectants.

The known sequence of CXCR3 and the one of its new variant were cloned in expression vectors and transfected by electroporation in a cell line of endothelial cells immortalised with SV40. Clones of stable transfectants for the two isoforms were thereafter generated using resistance induced to various antibiotics. Incorporation of r³H1-Thvmidine

The cells were plated in 96 wells and incubated with various stimuli. At the end of the incubation 1 μCi/ml of [³H]-Thymidine are added in the wells and the radioactivity due to the incorporation of the [³H]-Thymidine was analysed with a beta particles counter. Binding

The binding was performed using 2 x 10⁵ transfected endothelial cells. The cells were plated and incubated overnight in a standard medium, washed and incubated with a constant concentration (85 pM) of ¹²⁵l- labelled chemokines in the presence of increasing concentrations of unlabelled chemokines. After incubation at room temperature for 90 minutes the cells were washed and lysed in NaOH 1N. The radioactivity was determined using a gamma particles counter and the data were analysed with MacLigand software.

Preparation of monoclonal antibodies specific for CXCR3-B BALC/c mice were immunised with a peptide corresponding to the last 51 amino acids of the terminal NH2 portion of CXCR3-B which is selectively expressed by this receptor. After repeated immunisations the mice were sacrificed the spleen was collected and fusion with mielomatose cells was performed in order to obtain the ibridomas. The positivity of the various cloned ibridomas was analysed by ELISA tests on the peptide used for the immunisation. The two selected antibodies were highly specific in recognising the CXCR3-B as demonstrated by the immunohystochemistry test performed on transfectants of CXCR3-A and B and on the mock.

Claims

1. Protein having the amino acids sequence reported in SEQ. ID 1.

2. Portion of cDNA, expressing the protein according to Claim 1, having the nucleotide sequence reported in SEQ ID 2.

3. Complexes consisting of the protein according to claim 1 of CXCR3-B and an expression vector.

4. Complexes consisting of the cDNA sequence according to claim 2 and an expression vector.

5. Complexes according to claims 3 and 4 wherein the expression vectors are those used for the transcription of recombinant molecules or for creating transient or stabile transfectants.

6. Plasmides containing the SEQ ID 1 according to claim 1, or parts thereof, a promoter, a polyadenilation sequence and a resistance to antibiotics for prokaryotes and/or eucaryotes.

7. Plasmides according to claim 6 wherein such polyadenilation sequence is of viral origin.

8. Use of the protein according to claim 1 for the preparation of molecules capable to binding to it.

9. Use of complexes according to claims 3 and 4 as transfectants agents.

10. Use of the plasmides according to claims 6 and 7 as probes.

11. Prokaryotes and/or eucaryote cells transfected with complexes according to claims 3 and 4.

12. Use of the protein according to claim 1 for the preparation of formulations for the indirect treatment of epithelial and vascular tumours , for controlling: angiogenesis, the growth of leukaemia and lymphomas and various hematopoietic tumours, in the transplant rejection acute and chronic and in other cutaneous diseases showing an high angiogenensis, in the chronic inflammatory diseases of the liver (cirrhosis) and kidney (glomerulonephritis, hemolytic-uric syndrome and other vasculopathies) atherosclerosis and vascular alterations, secondary or associated to endothelial damage, miocardio ischaemia.

13. Use of the protein ad diagnostic medium of tumours or diseases expressing a protein according to claim 1.

14. Use of the monoclonal antibodies specific anti-CXCR3-B (clones PLIand PL2) for diagnostic, prognostic analysis and as search reactives.

15. Use of the monoclonal antibodies specific anti-CXCR3-B (clones PL1 and PL2) as pharmacological agents capable of blocking the functions mediated by such receptor CXCR3-B.

16. Complex consisting of a protein according to claim 1 and the chemokine PF-4.

17. Use of the complex according to claim 16 for the synthesis of molecules capable of interacting with the protein according to Claim 1.

18. Use according to claim 17 wherein such molecules have pharmacological action.

19. Use according to claim 17 wherein such molecules have diagnostic action.