Patent Application
20100247520
The present invention relates to a mutated netrin 4, and fragments thereof. It also relates to the use of said mutated netrin 4 and said fragments as drugs, in particular as anti-angiogenic agents.
Netrin 4 belongs to the netrins family, which are axons guiding molecules. To this day, 4 members of this family are known (netrins 1, G, 3, and 4). Netrin 4 is a protein consisting of a basic C-terminal domain interacting with heparin, 3 EGF-domains, and a laminin-domain (Yucchenco P D, Wadsworth W G (2004) Assembly and tissue functions of early embryonic laminins and netrins. Curr Opin Cell Biol. 16(5):572-9).
Patent application US 2003/0207347A1, published on Nov. 6, 2003, describes the native netrin 4 and uses thereof. More particularly, this application describes a netrin 4-derived polypeptide presenting properties for modulating angiogenesis, as well as the use of netrin 4 in a process of modulation of the vascular development, in particular of angiogenesis, and more particularly of inhibition of angiogenesis, in particular in tumors.
International patent application WO 2006/054000 describes the use of a mutated netrin 4 for the preparation of a drug for the prevention or the treatment of tumoral or non-tumoral pathologies, said mutated netrin 4 having an anti-angiogenic activity.
However, among sequences disclosed in this document, some improper mutated netrin 4 sequences comprise errors of sequencing.
An aim of the present invention is to provide new anti-angiogenic agents.
Another aim of the present invention is to provide a combination treatment allowing the increase of the treatments' efficiency involving angiogenesis, and in particular of usual anti-tumoral treatments, or of anti-angiogenic treatments used in pathologies other than tumors.
Until today, there is no known therapeutic agent able to interact with usual drugs as used for the treatment of age-related macular degeneration, or of other ocular diseases involving a neovascularization.
The present invention relates to a protein comprising or consisting of one of the following sequences:
The above-mentioned protein SEQ ID NO: 2 is a new protein corresponding to the mutated netrin 4 and the above-mentioned protein SEQ ID NO: 4 relates to the protein SEQ ID NO: 2 without a signal peptide.
The sequence SEQ ID NO: 2 comprises 628 amino acids and the sequence SEQ ID NO: 4 comprises 609 amino acids and is a fragment of SEQ ID NO: 2 from residue 20 to residue 628.
The mutated netrin 4, represented by the sequence SEQ ID NO: 2, corresponds to the netrin 4 protein represented by SEQ ID NO: 178 with the following 15 mutations:
The above-mentioned fragments, corresponding to protein sequences SEQ ID NO: 6 to SEQ ID NO: 72, are new fragments of the above-mentioned mutated netrin 4.
The above-mentioned sequences SEQ ID NO: 2q correspond to protein sequences SEQ ID NO: 6 to 72, and thus are the following protein sequences: SEQ ID NO: 6, SEQ ID NO: 8, SEQ ID NO: 10, SEQ ID NO: 12, SEQ ID NO: 14, SEQ ID NO: 16, SEQ ID NO: 18, SEQ ID NO: 20, SEQ ID NO: 22, SEQ ID NO: 24, SEQ ID NO: 26, SEQ ID NO: 28, SEQ ID NO: 30, SEQ ID NO: 32, SEQ ID NO: 34, SEQ ID NO: 36, SEQ ID NO: 38, SEQ ID NO: 40, SEQ ID NO: 42, SEQ ID NO: 44, SEQ ID NO: 46, SEQ ID NO: 48, SEQ ID NO: 50, SEQ ID NO: 52, SEQ ID NO: 54, SEQ ID NO: 56, SEQ ID NO: 58, SEQ ID NO: 60, SEQ ID NO: 62, SEQ ID NO: 64, SEQ ID NO: 66, SEQ ID NO: 68, SEQ ID NO: 70, and SEQ ID NO: 72.
The sequence SEQ ID NO: 6 is an EGF-fragment of the human mutated netrin 4, said fragment being coded by the nucleotide sequence SEQ ID NO: 5. This fragment comprises 255 amino acids and corresponds to a fragment of the mutated netrin 4 from residue 261 to residue 515 of sequence SEQ ID NO: 2.
The sequence SEQ ID NO: 8 is a fragment of the mutated netrin 4, being coded by the nucleotide sequence SEQ ID NO: 7. This fragment comprises 515 amino acids and corresponds to a fragment of the mutated netrin 4 from residue 1 to residue 515 of sequence SEQ ID NO: 2.
The fragments of the mutated netrin 4 corresponding to protein sequences SEQ ID NO: 10 to SEQ ID NO: 72, as well as the corresponding nucleotide sequences SEQ ID NO: 9 to SEQ ID NO: 71 are represented in the following table:
The above-mentioned proteins SEQ ID NO:185 to SEQ ID NO:209 are new proteins corresponding to the mutated netrin 4.
The mutated netrin 4, represented by the sequence SEQ ID NO: 185, corresponds to the netrin 4 protein represented by SEQ ID NO: 178 with the following 14 mutations:
The mutated netrin 4, represented by the sequence SEQ ID NO: 186, corresponds to the netrin 4 protein represented by SEQ ID NO: 178 with the following 13 mutations:
The mutated netrin 4, represented by the sequence SEQ ID NO: 187, corresponds to the netrin 4 protein represented by SEQ ID NO: 178 with the following 13 mutations:
The mutated netrin 4, represented by the sequence SEQ ID NO: 188, corresponds to the netrin 4 protein represented by SEQ ID NO: 178 with the following 12 mutations:
The mutated netrin 4, represented by the sequence SEQ ID NO: 189, corresponds to the netrin 4 protein represented by SEQ ID NO: 178 with the following 14 mutations:
The mutated netrin 4, represented by the sequence SEQ ID NO: 190, corresponds to the netrin 4 protein represented by SEQ ID NO: 178 with the following 14 mutations:
The mutated netrin 4, represented by the sequence SEQ ID NO: 191, corresponds to the netrin 4 protein represented by SEQ ID NO: 178 with the following 14 mutations:
The mutated netrin 4 corresponding to protein sequences SEQ ID NO: 2 and SEQ ID NO: 185 to SEQ ID NO: 191, and the native netrin 4 corresponding to protein sequence SEQ ID NO: 178, as well as the corresponding mutations are represented in the following table:
R
T
P
Y
Y
T
R
S
Y
K
A
E
S
A
S
T
P
Y
Y
T
R
S
Y
K
A
E
S
A
S
T
P
Y
Y
R
S
Y
K
A
E
S
A
S
T
P
Y
Y
T
S
Y
K
A
E
S
A
S
T
P
Y
Y
S
Y
K
A
E
S
A
S
T
P
Y
Y
T
R
S
K
A
E
S
A
S
T
P
Y
Y
T
R
S
Y
K
E
S
A
S
T
P
Y
Y
T
R
S
Y
K
The above-mentioned proteins corresponding to protein sequences SEQ ID NO: 185 to SEQ ID NO: 191 correspond to the substitution mutants of netrin 4 (sNET-4m) 1 to 7 (see example section).
The mutated netrin 4, represented by the sequence SEQ ID NO: 192, corresponds to the netrin 4 protein represented by SEQ ID NO: 178 with the following mutation:
The mutated netrin 4, represented by the sequence SEQ ID NO: 193, corresponds to mutated netrin 4 represented by SEQ ID NO: 192 wherein amino acids 516 to 618 have been deleted, and the mutated netrin 4, represented by the sequence SEQ ID NO: 204, corresponds to mutated netrin 4 represented by SEQ ID NO: 192 wherein amino acids 478 to 618 have been deleted.
The mutated netrin 4, represented by the sequence SEQ ID NO: 194, corresponds to the netrin 4 protein represented by SEQ ID NO: 178 with the following mutation:
The mutated netrin 4, represented by the sequence SEQ ID NO: 195, corresponds to mutated netrin 4 represented by SEQ ID NO: 194 wherein amino acids 516 to 618 have been deleted and the mutated netrin 4, represented by the sequence SEQ ID NO: 205, corresponds to mutated netrin 4 represented by SEQ ID NO: 194 wherein amino acids 478 to 618 have been deleted.
The mutated netrin 4, represented by the sequence SEQ ID NO: 196, corresponds to the netrin 4 protein represented by SEQ ID NO: 178 with the two following mutations:
The mutated netrin 4, represented by the sequence SEQ ID NO: 197, corresponds to mutated netrin 4 represented by SEQ ID NO: 196 wherein amino acids 516 to 618 have been deleted and the mutated netrin 4, represented by the sequence SEQ ID NO: 206, corresponds to mutated netrin 4 represented by SEQ ID NO: 196 wherein amino acids 478 to 618 have been deleted.
The mutated netrin 4, represented by the sequence SEQ ID NO: 198, corresponds to the netrin 4 protein represented by SEQ ID NO: 178 with the two following mutations:
The mutated netrin 4, represented by the sequence SEQ ID NO: 199, corresponds to mutated netrin 4 represented by SEQ ID NO: 198 wherein amino acids 516 to 618 have been deleted and the mutated netrin 4, represented by the sequence SEQ ID NO: 207, corresponds to mutated netrin 4 represented by SEQ ID NO: 198 wherein amino acids 478 to 618 have been deleted.
The mutated netrin 4, represented by the sequence SEQ ID NO: 200, corresponds to the netrin 4 protein represented by SEQ ID NO: 178 with the three following mutations:
The mutated netrin 4, represented by the sequence SEQ ID NO: 201, corresponds to mutated netrin 4 represented by SEQ ID NO: 200 wherein amino acids 516 to 618 have been deleted and the mutated netrin 4, represented by the sequence SEQ ID NO: 208, corresponds to mutated netrin 4 represented by SEQ ID NO: 200 wherein amino acids 478 to 618 have been deleted.
The mutated netrin 4, represented by the sequence SEQ ID NO: 202, corresponds to the netrin 4 protein represented by SEQ ID NO: 178 with the two following mutations:
The mutated netrin 4, represented by the sequence SEQ ID NO: 203, corresponds to mutated netrin 4 represented by SEQ ID NO: 198 wherein amino acids 516 to 618 have been deleted and the mutated netrin 4, represented by the sequence SEQ ID NO: 209, corresponds to mutated netrin 4 represented by SEQ ID NO: 202 wherein amino acids 478 to 618 have been deleted.
The mutated netrin 4 corresponding to protein sequences SEQ ID NO: 192 to SEQ ID NO: 203, as well as the corresponding mutations are represented in the following table:
The mutated netrin 4 of the invention and the fragments of the invention have an activity of inhibition of the angiogenesis.
The activity of inhibition of the angiogenesis is also called anti-angiogenic activity. This activity can for example be detected in vitro by showing the inhibition of the multiplication, as well as the migration, and the differentiation, of endothelial cells by the above-mentioned mutated proteins or fragments of the invention. Measurement of the inhibition of the endothelial cells multiplication can also be carried out by culturing endothelial cells in the presence of the protein or the fragment, the activity of which is to be tested. Measurement of the inhibition of the endothelial cells migration can also be tested by carrying out a “wound” on a carpet of endothelial cells and by then incubating the cells in the presence of the fragment to be tested. The number of cells that migrated on the wound is then measured. Measurement of the inhibition of the sprouting (tubulogenesis) of the endothelial cells can be carried out by measuring the length of tubules formed by endothelial cells cultured on gel in the presence of the fragment to be tested.
Among the classical models for measuring the angiogenesis, the following one can be cited (models by local administration):
Alternatively, the fragments of the invention can be injected by systemic route (intravenous, intra-peritoneal, and subcutaneous route) to animals by which an experimental angiogenic disease was created. The fragments of the invention can also be directly injected into a tumor. Alternatively, the fragments or the anti-idiotypic antibodies of the invention (described hereafter) can be administered by a gene therapy method by local or systemic route by any method allowing the expression of the fragments or of the anti-idiotypic antibodies of the invention. Alternatively, the fragments or the anti-idiotypic antibodies of the invention can be inserted into a plasmid which is transfected into cancer cells. All these measuring methods are in particular described in the article of Jain R K, Schlenger K, Hockel M, Yuan F (1997) Quantitative angiogenesis assays: progress and problems. Nat. Med. 3(11):1203-8.
The anti-tumoral activity designates an activity allowing the inhibition of tumor growth and/or the induction of the regression, and even the disappearance of tumors. For example, this activity can be detected in vivo by measuring the tumors mass, the development of which was induced in the mouse by the injection of tumor cells, in the presence and in the absence of the administration of peptide sequences of the invention and/or of nucleic acids that express the peptide sequences of the invention.
The mutated protein of the invention and the fragments of the invention are also characterized in that they have a pericytes activation activity.
This activity of activating the pericytes is in particular checked by the proliferation and migration tests as mentioned hereafter and in particular in the experimental part.
The present invention is in particular based on the fact that the netrins bind to the UNC5H4 receptors of pericytes and smooth muscle cells (SMC).
The present invention also relates to a nucleotide sequence coding for the above-mentioned protein, that is to say a nucleotide sequence coding for the mutated netrin 4.
A preferred nucleotide sequence of the invention is a nucleotide sequence characterized in that it comprises or consists of:
The above-mentioned sequences SEQ ID NO: 2q-1 code for the above-mentioned fragments of the mutated netrin 4, represented by SEQ ID NO: 2q, and they correspond to the following nucleotide sequences: SEQ ID NO: 5 coding for SEQ ID NO: 6, SEQ ID NO: 7 coding for SEQ ID NO: 8, SEQ ID NO: 9 coding for SEQ ID NO: 10, SEQ ID NO: 11 coding for SEQ ID NO: 12, SEQ ID NO: 13 coding for SEQ ID NO: 14, SEQ ID NO: 15 coding for SEQ ID NO: 16, SEQ ID NO: 17 coding for SEQ ID NO: 18, SEQ ID NO: 19 coding for SEQ ID NO: 20, SEQ ID NO: 21 coding for SEQ ID NO: 22, SEQ ID NO: 23 coding for SEQ ID NO: 24, SEQ ID NO: 25 coding for SEQ ID NO: 26, SEQ ID NO: 27 coding for SEQ ID NO: 28, SEQ ID NO: 29 coding for SEQ ID NO: 30, SEQ ID NO: 31 coding for SEQ ID NO: 32, SEQ ID NO: 33 coding for SEQ ID NO: 34, SEQ ID NO: 35 coding for SEQ ID NO: 36, SEQ ID NO: 37 coding for SEQ ID NO: 38, SEQ ID NO: 39 coding for SEQ ID NO: 40, SEQ ID NO: 41 coding for SEQ ID NO: 42, SEQ ID NO: 43 coding for SEQ ID NO: 44, SEQ ID NO: 45 coding for SEQ ID NO: 46, SEQ ID NO: 47 coding for SEQ ID NO: 48, SEQ ID NO: 49 coding for SEQ ID NO: 50, SEQ ID NO: 51 coding for SEQ ID NO: 52, SEQ ID NO: 53 coding for SEQ ID NO: 54, SEQ ID NO: 55 coding for SEQ ID NO: 56, SEQ ID NO: 57 coding for SEQ ID NO: 58, SEQ ID NO: 59 coding for SEQ ID NO: 60, SEQ ID NO: 61 coding for SEQ ID NO: 62, SEQ ID NO: 63 coding for SEQ ID NO: 64, SEQ ID NO: 65 coding for SEQ ID NO: 66, SEQ ID NO: 67 coding for SEQ ID NO: 68, SEQ ID NO: 69 coding for SEQ ID NO: 70, and SEQ ID NO: 71 coding for SEQ ID NO: 72.
The present invention relates to a recombinant vector, such as a plasmid, a cosmid, a phage or virus DNA, containing a nucleotide sequence as defined above, said recombinant vector being in particular characterized in that it contains the elements necessary for the expression in a host cell of the polypeptides encoded by the above-mentioned nucleotide sequences, inserted into said vector.
The present invention relates to a host cell, chosen in particular from bacteria, virus, yeasts, fungi cells, plant cells or mammal cells, said host cell being transformed, in particular using a recombinant vector as defined previously.
The present invention also relates to an antibody, characterized in that it is specifically directed against the protein of the invention, as mentioned above.
The present invention also relates to an anti-idiotypic antibody of an antibody as mentioned above.
The present invention also relates to a Fab fragment of the above-mentioned anti-idiotypic antibodies.
The present invention also relates to a pharmaceutical composition comprising as active substance:
The present invention also relates to the use as defined above of the mutated netrin 4, represented by sequence SEQ ID NO: 2 or SEQ ID NO: 4, for the preparation of a drug to be delivered at an amount from about 0.1 to about 2,000 μg/kg, in particular by intravenous, subcutaneous, systemic or intravitreal route, or by local route with infiltrations or a collyrium, and possibly in association with a electropermeation.
The mutated netrin 4 can also be delivered with an injection of a plasmid coding for the mutated netrin-4.
Alternatively, any one of the proteins or fragments of the invention can be delivered by any intravascular device (stents) after the fixation of the protein or the fragment on said device.
The present invention also relates to the use of:
for the preparation of a drug for the prevention or treatment of pathologies involving the inhibition of endothelial cell proliferation and/or migration, in particular for the prevention or treatment of pathologies chosen from the group consisting of: cancers and leukaemia, myopia-complicating choroidal neovascularization, cornea neovascularization, in particular graft rejection, glaucoma, diabetic retinopathies or premature retinopathies, rheumatoid arthritis, psoriasis arthritis, angioma, angiosarcoma, Castleman's disease, and Kaposi's sarcoma, or for the treatment of obesity or retinal neovascularization.
The expression “inhibition of endothelial cell proliferation” designates any substance able to slow down the proliferation of endothelial cells according to the proliferation test as described hereafter.
In one preferred embodiment, the present invention relates to the use of:
for the preparation of a drug for the prevention or treatment of pathologies involving the inhibition of endothelial cell proliferation and/or migration, in particular for the prevention or treatment of pathologies chosen from the group consisting of: cancers and leukaemia, in particular angioma, angio sarcoma, Castleman's disease, Kaposi's sarcoma and rheumatoid arthritis.
In one other preferred embodiment, the present invention relates to the use of:
for the preparation of a drug for the prevention or treatment of pathologies involving the inhibition of endothelial cell proliferation and/or migration, in particular for the prevention or treatment of pathologies chosen from the group consisting of: myopia-complicating choroidal neovascularization, cornea neovascularization, in particular graft rejection, glaucoma, diabetic retinopathies or premature retinopathies, psoriasis arthritis, or for the treatment of obesity or retinal neovascularization.
In one other preferred embodiment, the present invention relates to the use of:
for the preparation of a drug for the prevention or treatment of pathologies involving the inhibition of endothelial cell proliferation and/or migration, in particular for the prevention or treatment of pathologies chosen from the group consisting of: myopia-complicating choroidal neovascularization, cornea neovascularization, in particular graft rejection, glaucoma, diabetic retinopathies or premature retinopathies, psoriasis arthritis, or for the treatment of obesity or retinal neovascularization.
The present invention also relates to the use of:
for the preparation of a drug for the prevention or treatment of pathologies involving the stimulation of endothelial cell proliferation and/or migration, in particular for the prevention or treatment of pathologies chosen from the group consisting of: ischemic pathologies such as arteritis of lower limbs, myocardium infarct, cerebral vascular accidents, scleroderma, and Raynaud's disease.
Measurement of the activation of the endothelial cells proliferation can be carried out by placing the endothelial cells in an appropriate culture medium and by then measuring the total number of cells.
Measurement of the activation of the endothelial cells migration can be carried out by making a “wound” on a carpet of endothelial cells and then incubating the cells in the presence of the protein, the nucleotide sequence or the anti-idiotypic antibody to be tested. The number of cells having migrated onto the wound is then measured.
The present invention also relates to the use of:
for the preparation of a drug for the prevention or treatment of non-tumoral pathologies linked to or caused by a pericyte or smooth muscular cell rarefaction, and requiring an activation of pericyte or smooth muscular cell proliferation or migration, said non-tumoral pathologies being in particular chosen from the group consisting of:
The present invention also relates to the use as defined above, characterized in that the activity of activation of pericytes or smooth muscular cell proliferation or migration is measured according to the proliferation or migration test, and in that this activity of activation corresponds to at least 120% of the cells obtained in the absence of the protein, the nucleotide sequence, the antibody, the anti-idiotypic antibody or the Fab fragment of anti-idiotypic antibodies as defined above.
Measurement of the activation of the migration of pericytes or smooth muscular cells can be carried out by making a “wound” on a carpet of cells and then incubating the cells in the presence of the protein, the nucleotide sequence, the antibody, the anti-idiotypic antibody or the Fab fragment to be tested. The number of cells having migrated onto the wound is then measured.
Measurement of the activation of the proliferation of pericyte or smooth muscular cells can be carried out by placing the pericytes or smooth muscular cells in an appropriate culture medium, in particular in DMEM medium that does not contain any serum, and by measuring the total number of cells.
The present invention also relates to the use of:
in association with a chemotherapy agent, for the preparation of a drug for the treatment of cancers.
The present invention also relates to a pharmaceutical composition comprising a chemotherapy agent, in association with the protein represented by SEQ ID NO: 2 or SEQ ID NO: 4, or in association with a fragment of said sequence SEQ ID NO: 2 or SEQ ID NO: 4, represented by one of the sequences SEQ ID NO: 2q, q varying from 3 to 36,
said chemotherapy agent being in particular chosen from the group consisting of: doxorubicin, methotrexate, vinblastine, vincristine, cladribine, fluorouracil, cytarabine, anthracyclines, cisplatin, cyclophosphamide, fludarabine, gemcitabine, aromatase inhibitors, irinotecan, navelbine, oxaliplatin, taxol, and docetaxel.
The combination of an anti-angiogenic agent with a chemotherapy agent allows the obtaining of a synergic effect and the induction of a reduced resistance to the usual anti-tumoral treatments.
The present invention also relates to the use of:
in association with an anti-angiogenic agent chosen in particular from the group consisting of: AVASTIN (bevacizumab) manufactured by Genentech and Roche, MACUGEN (pegaptanib) manufactured by Eyetech and Pfizer, and LUCENTIS (ranibizumab) manufactured by Genentech and Novartis, or any other anti-VEGF agent, such as Sutent (Pfizer) or Sorafenib,), humanized antibodies against neuropiline-1 or any other anti-VEGF agent or any other anti-VEGF agent, such as SUTENT (sunitinib) or NEXAVAR (Sorafenib) as well as humanized antibodies against DLL4 or agents interfering with the angiopoietins pathways such as AM 386,
for the preparation of a drug for the prevention or treatment of tumoral or non-tumoral pathologies as defined above.
In the present invention, the doses of the mutated netrin-4 vary from about 10 to about 10,000 ng/injection, in particular from about 100 to about 5,000 ng/injection, every 6 weeks.
In the present invention, the doses of the anti-angiogenic agent (AVASTIN, MACUGEN or LUCENTIS for example) vary from about 0.3 to about 1 mg every 6 weeks.
The present invention also relates to a combination product comprising:
with an anti-angiogenic agent chosen in particular from the group consisting of: AVASTIN (bevacizumab), MACUGEN (pegaptanib), and LUCENTIS (ranibizumab), or any other anti-VEGF agent,), humanized antibodies against neuropiline-1 or any other anti-VEGF agent or any other anti-VEGF agent, such as SUTENT (sunitinib) or NEXAVAR (Sorafenib) as well as humanized antibodies against DLL4 or agents interfering with the angiopoietins pathways such as AM 386,
for a simultaneous, separated or sequential use for the treatment or prevention of tumoral or non-tumoral pathologies as defined above.
The present invention also relates to a protein comprising or consisting of: SEQ ID NO: 74, SEQ ID NO: 76, SEQ ID NO: 78, SEQ ID NO: 80, SEQ ID NO: 82, SEQ ID NO: 84, SEQ ID NO: 86, SEQ ID NO: 88, SEQ ID NO: 90, SEQ ID NO: 92, SEQ ID NO: 94, SEQ ID NO: 96, SEQ ID NO: 98, SEQ ID NO: 100, SEQ ID NO: 102, SEQ ID NO: 104, SEQ ID NO: 106, SEQ ID NO: 108, SEQ ID NO: 110, SEQ ID NO: 112, SEQ ID NO: 114, SEQ ID NO: 116, SEQ ID NO: 118, SEQ ID NO: 120, SEQ ID NO: 122, SEQ ID NO: 124, SEQ ID NO: 126, SEQ ID NO: 128, SEQ ID NO: 130, SEQ ID NO: 132, SEQ ID NO: 134, SEQ ID NO: 136, SEQ ID NO: 138, SEQ ID NO: 140, SEQ ID NO: 142, SEQ ID NO: 144, SEQ ID NO: 146, SEQ ID NO: 148, SEQ ID NO: 150, SEQ ID NO: 152, SEQ ID NO: 154, SEQ ID NO: 156, SEQ ID NO: 158, SEQ ID NO: 160, SEQ ID NO: 162, SEQ ID NO: 164, SEQ ID NO: 166, SEQ ID NO: 168, SEQ ID NO: 170, SEQ ID NO: 172, SEQ ID NO: 174, and SEQ ID NO: 176.
SEQ ID NO: 74 corresponds to a peptide construction comprising the signal peptide of native netrin 4 SEQ ID NO: 178, said signal peptide corresponding to the 19 first amino acids of said native netrin 4, and the fragment SEQ ID NO: 56.
SEQ ID NO: 76 corresponds to a peptide construction comprising the signal peptide of native netrin 4 SEQ ID NO: 178, and the fragment SEQ ID NO: 58.
SEQ ID NO: 78 corresponds to a peptide construction comprising the signal peptide of native netrin 4 SEQ ID NO: 178, and the fragment SEQ ID NO: 60.
SEQ ID NO: 80 corresponds to a peptide construction comprising the signal peptide of native netrin 4 SEQ ID NO: 178, and the fragment SEQ ID NO: 62.
SEQ ID NO: 82 corresponds to a peptide construction comprising the signal peptide of native netrin 4 SEQ ID NO: 178, and the fragment SEQ ID NO: 64.
SEQ ID NO: 84 corresponds to a peptide construction comprising the signal peptide of native netrin 4 SEQ ID NO: 178, and the fragment SEQ ID NO: 66.
SEQ ID NO: 86 corresponds to a peptide construction comprising the signal peptide of native netrin 4 SEQ ID NO: 178, and the fragment SEQ ID NO: 68.
SEQ ID NO: 88 corresponds to a peptide construction comprising the signal peptide of native netrin 4 SEQ ID NO: 178, and the fragment SEQ ID NO: 70.
SEQ ID NO: 90 corresponds to a peptide construction comprising the signal peptide of native netrin 4 SEQ ID NO: 178, and the fragment SEQ ID NO: 72.
SEQ ID NO: 92 corresponds to a peptide construction comprising the signal peptide of native netrin 4 SEQ ID NO: 178, and the fragment of the protein SEQ ID NO: 182 from residue 20 to residue 515.
SEQ ID NO: 94 corresponds to a peptide construction comprising the signal peptide of SEQ ID NO: 178, and the fragments of the protein SEQ ID NO: 182 from residue 20 to residue 260, and from residue 516 to residue 628.
SEQ ID NO: 96 corresponds to a peptide construction comprising the signal peptide of SEQ ID NO: 178, and the fragments of the protein SEQ ID NO: 182 from residue 20 to residue 320, and from residue 516 to residue 628.
SEQ ID NO: 98 corresponds to a peptide construction comprising the signal peptide of native netrin 4 SEQ ID NO: 178, and the fragments of the protein SEQ ID NO: 182 from residue 20 to residue 260, from residue 332 to residue 387, and from residue 516 to residue 628.
SEQ ID NO: 100 corresponds to a peptide construction comprising the signal peptide of native netrin 4 SEQ ID NO: 178, and the fragments of the protein SEQ ID NO: 182 from residue 20 to residue 260, from residue 394 to residue 445, and from residue 516 to residue 628.
SEQ ID NO: 102 corresponds to a peptide construction comprising the signal peptide of native netrin 4 SEQ ID NO: 178, and the fragments of the protein SEQ ID NO: 182 from residue 20 to residue 320, from residue 332 to residue 387, and from residue 516 to residue 628.
SEQ ID NO: 104 corresponds to a peptide construction comprising the signal peptide of native netrin 4 SEQ ID NO: 178, and the fragments of the protein SEQ ID NO: 182 from residue 20 to residue 260, from residue 332 to residue 387, from residue 394 to residue 445, and from residue 516 to residue 628.
SEQ ID NO: 106 corresponds to a peptide construction comprising the signal peptide of native netrin 4 SEQ ID NO: 178, and the fragments of the protein SEQ ID NO: 182 from residue 20 to residue 320, from residue 394 to residue 445, and from residue 516 to residue 628.
SEQ ID NO: 108 corresponds to a peptide construction comprising the signal peptide of native netrin 4 SEQ ID NO: 178, and the fragments of the protein SEQ ID NO: 182 from residue 20 to residue 320, from residue 332 to residue 387, from residue 394 to residue 445, and from residue 516 to residue 628.
SEQ ID NO: 110 corresponds to a peptide construction comprising the signal peptide of SEQ ID NO: 178, and the protein sequence SEQ ID NO: 4.
SEQ ID NO: 112 corresponds to a peptide construction comprising the signal peptide of SEQ ID NO: 178, and the protein sequence SEQ ID NO: 184.
SEQ ID NO: 114 corresponds to a peptide construction comprising the signal peptide of SEQ ID NO: 178, and the fragment SEQ ID NO: 6.
SEQ ID NO: 116 corresponds to a peptide construction comprising the signal peptide of SEQ ID NO: 178, and the fragment SEQ ID NO: 10.
SEQ ID NO: 118 corresponds to a peptide construction comprising the signal peptide of SEQ ID NO: 178, and the fragment SEQ ID NO: 12.
SEQ ID NO: 120 corresponds to a peptide construction comprising the signal peptide of SEQ ID NO: 178, and the fragment SEQ ID NO: 16.
SEQ ID NO: 122 corresponds to a peptide construction comprising the signal peptide of SEQ ID NO: 178, and the fragment SEQ ID NO: 18.
SEQ ID NO: 124 corresponds to a peptide construction comprising the signal peptide of SEQ ID NO: 178, and the fragment SEQ ID NO: 20.
SEQ ID NO: 126 corresponds to a peptide construction comprising the signal peptide of SEQ ID NO: 178, and the fragment SEQ ID NO: 22.
SEQ ID NO: 128 corresponds to a peptide construction comprising the signal peptide of SEQ ID NO: 178, and the fragment SEQ ID NO: 24.
SEQ ID NO: 130 corresponds to a peptide construction comprising the signal peptide of SEQ ID NO: 178, and the fragment SEQ ID NO: 26.
SEQ ID NO: 132 corresponds to a peptide construction comprising the signal peptide of SEQ ID NO: 178, and the fragment SEQ ID NO: 34.
SEQ ID NO: 134 corresponds to a peptide construction comprising the signal peptide of SEQ ID NO: 178, and the fragment SEQ ID NO: 36.
SEQ ID NO: 136 corresponds to a peptide construction comprising the signal peptide of SEQ ID NO: 178, and the fragment SEQ ID NO: 38.
SEQ ID NO: 138 corresponds to a peptide construction comprising the signal peptide of SEQ ID NO: 178, and the fragment SEQ ID NO: 46.
SEQ ID NO: 140 corresponds to a peptide construction comprising the signal peptide of SEQ ID NO: 178, and the fragment SEQ ID NO: 48.
SEQ ID NO: 142 corresponds to a peptide construction comprising the signal peptide of SEQ ID NO: 178, and the fragment SEQ ID NO: 50.
SEQ ID NO: 144 corresponds to a peptide construction comprising the signal peptide of SEQ ID NO: 178, and the fragment SEQ ID NO: 54.
SEQ ID NO: 146 corresponds to a peptide construction comprising the signal peptide of SEQ ID NO: 178, and the fragment of the protein SEQ ID NO: 182 from residue 261 to residue 515.
SEQ ID NO: 148 corresponds to a peptide construction comprising the signal peptide of SEQ ID NO: 178, and the fragment of the protein SEQ ID NO: 182 from residue 32 to residue 515.
SEQ ID NO: 150 corresponds to a peptide construction comprising the signal peptide of SEQ ID NO: 178, and the fragment of the protein SEQ ID NO: 182 from residue 32 to residue 628.
SEQ ID NO: 152 corresponds to a peptide construction comprising the signal peptide of SEQ ID NO: 178, and the fragment of the protein SEQ ID NO: 182 from residue 261 to residue 628.
SEQ ID NO: 154 corresponds to a peptide construction comprising the signal peptide of SEQ ID NO: 178, and the fragments of the protein SEQ ID NO: 182 from residue 332 to residue 387, and from residue 516 to residue 628.
SEQ ID NO: 156 corresponds to a peptide construction comprising the signal peptide of SEQ ID NO: 178, and the fragments of the protein SEQ ID NO: 182 from residue 32 to residue 260, and from residue 516 to residue 628.
SEQ ID NO: 158 corresponds to a peptide construction comprising the signal peptide of SEQ ID NO: 178, and the fragments of the protein SEQ ID NO: 182 from residue 32 to residue 320, and from residue 516 to residue 628.
SEQ ID NO: 160 corresponds to a peptide construction comprising the signal peptide of SEQ ID NO: 178, and the fragments of the protein SEQ ID NO: 182 from residue 32 to residue 260, from residue 332 to residue 387, and from residue 516 to residue 628.
SEQ ID NO: 162 corresponds to a peptide construction comprising the signal peptide of SEQ ID NO: 178, and the fragments of the protein SEQ ID NO: 182 from residue 32 to residue 260, from residue 394 to residue 445, and from residue 516 to residue 628.
SEQ ID NO: 164 corresponds to a peptide construction comprising the signal peptide of SEQ ID NO: 178, and the fragments of the protein SEQ ID NO: 182 from residue 261 to residue 320, from residue 332 to residue 387, and from residue 516 to residue 628.
SEQ ID NO: 166 corresponds to a peptide construction comprising the signal peptide of SEQ ID NO: 178, and the fragments of the protein SEQ ID NO: 182 from residue 332 to residue 387, from residue 394 to residue 445, and from residue 516 to residue 628.
SEQ ID NO: 168 corresponds to a peptide construction comprising the signal peptide of SEQ ID NO: 178, and the fragments of the protein SEQ ID NO: 182 from residue 261 to residue 320, from residue 332 to residue 387, from residue 394 to residue 445, and from residue 516 to residue 628.
SEQ ID NO: 170 corresponds to a peptide construction comprising the signal peptide of SEQ ID NO: 178, and the fragments of the protein SEQ ID NO: 182 from residue 32 to residue 320, from residue 332 to residue 387, and from residue 516 to residue 628.
SEQ ID NO: 172 corresponds to a peptide construction comprising the signal peptide of SEQ ID NO: 178, and the fragments of the protein SEQ ID NO: 182 from residue 32 to residue 260, from residue 332 to residue 387, from residue 394 to residue 445, and from residue 516 to residue 628.
SEQ ID NO: 174 corresponds to a peptide construction comprising the signal peptide of SEQ ID NO: 178, and the fragments of the protein SEQ ID NO: 182 from residue 32 to residue 320, from residue 394 to residue 445, and from residue 516 to residue 628.
SEQ ID NO: 176 corresponds to a peptide construction comprising the signal peptide of SEQ ID NO: 178, and the fragments of the protein SEQ ID NO: 182 from residue 32 to residue 320, from residue 332 to residue 387, from residue 394 to residue 445, and from residue 516 to residue 628.
The present invention also relates to the use of:
The present invention also relates to the use of:
The present invention also relates to the use of:
The present invention also relates to a pharmaceutical composition comprising pericytes or vascular smooth muscle cells, in association with the protein represented by SEQ ID NO: 2 or SEQ ID NO: 4, or in association with a fragment of said sequence SEQ ID NO: 2 or SEQ ID NO: 4, represented by one of the sequences SEQ ID NO: 2q, q varying from 3 to 36, or in association with one of the sequences SEQ ID NO:185 to SEQ ID NO:209.
The present invention also relates to a combination product comprising:
with pericytes or vascular smooth muscle cells,
for a simultaneous, separated or sequential use for the treatment of cancers.
The present invention also relates to the use of:
for the preparation of a drug for the prevention or treatment of pathologies involving the inhibition of endothelial cell proliferation and/or migration, in particular for the prevention or treatment of pathologies chosen from the group consisting of: cancers and leukaemia, myopia-complicating choroidal neovascularization, cornea neovascularization, in particular graft rejection, glaucoma, diabetic retinopathies or premature retinopathies, rheumatoid arthritis, psoriasis arthritis, angioma, angiosarcoma, Castleman's disease, and Kaposi's sarcoma, or for the treatment of obesity or retinal neovascularization.
The above-mentioned sequences SEQ ID NO: 2q correspond to protein sequences SEQ ID NO: 74 to 88, and thus are the following protein sequences: SEQ ID NO: 74, SEQ ID NO: 76, SEQ ID NO: 78, SEQ ID NO: 80, SEQ ID NO: 82, SEQ ID NO: 84, SEQ ID NO: 86, SEQ ID NO: 88, SEQ ID NO: 90, SEQ ID NO: 92, SEQ ID NO: 94, SEQ ID NO: 96, SEQ ID NO: 98, SEQ ID NO: 100, SEQ ID NO: 102, SEQ ID NO: 104, SEQ ID NO: 106, SEQ ID NO: 108, SEQ ID NO: 110, SEQ ID NO: 112, SEQ ID NO: 114, SEQ ID NO: 116, SEQ ID NO: 118, SEQ ID NO: 120, SEQ ID NO: 122, SEQ ID NO: 124, SEQ ID NO: 126, SEQ ID NO: 128, SEQ ID NO: 130, SEQ ID NO: 132, SEQ ID NO: 134, SEQ ID NO: 136, SEQ ID NO: 138, SEQ ID NO: 140, SEQ ID NO: 142, SEQ ID NO: 144, SEQ ID NO: 146, SEQ ID NO: 148, SEQ ID NO: 150, SEQ ID NO: 152, SEQ ID NO: 154, SEQ ID NO: 156, SEQ ID NO: 158, SEQ ID NO: 160, SEQ ID NO: 162, SEQ ID NO: 164, SEQ ID NO: 166, SEQ ID NO: 168, SEQ ID NO: 170, SEQ ID NO: 172, SEQ ID NO: 174, and SEQ ID NO: 176.
The above-mentioned sequences SEQ ID NO: 2q-1 code for the above-mentioned mutated netrin 4, represented by SEQ ID NO: 2q, and they correspond to the following nucleotide sequences: SEQ ID NO: 73, SEQ ID NO: 75, SEQ ID NO: 77, SEQ ID NO: 79, SEQ ID NO: 81, SEQ ID NO: 83, SEQ ID NO: 85, SEQ ID NO: 87, SEQ ID NO: 89, SEQ ID NO: 91, SEQ ID NO: 93, SEQ ID NO: 95, SEQ ID NO: 97, SEQ ID NO: 99, SEQ ID NO: 101, SEQ ID NO: 103, SEQ ID NO: 105, SEQ ID NO: 107, SEQ ID NO: 109, SEQ ID NO: 111, SEQ ID NO: 113, SEQ ID NO: 115, SEQ ID NO: 117, SEQ ID NO: 119, SEQ ID NO: 121, SEQ ID NO: 123, SEQ ID NO: 125, SEQ ID NO: 127, SEQ ID NO: 129, SEQ ID NO: 131, SEQ ID NO: 133, SEQ ID NO: 135, SEQ ID NO: 137, SEQ ID NO: 139, SEQ ID NO: 141, SEQ ID NO: 143, SEQ ID NO: 145, SEQ ID NO: 147, SEQ ID NO: 149, SEQ ID NO: 151, SEQ ID NO: 153, SEQ ID NO: 155, SEQ ID NO: 157, SEQ ID NO: 159, SEQ ID NO: 161, SEQ ID NO: 163, SEQ ID NO: 165, SEQ ID NO: 167, SEQ ID NO: 169, SEQ ID NO: 171, SEQ ID NO: 173, and SEQ ID NO: 175.
In one preferred embodiment, the invention relates to the use of:
for the preparation of a drug for the prevention or treatment of pathologies involving the inhibition of endothelial cell proliferation and/or migration, in particular for the prevention or treatment of pathologies chosen from the group consisting of: cancers and leukaemia, in particular angioma, angiosarcoma, Castleman's disease, Kaposi's sarcoma and rheumatoid arthritis.
In another preferred embodiment, the invention relates to the use of:
for the preparation of a drug for the prevention or treatment of pathologies involving the inhibition of endothelial cell proliferation and/or migration, in particular for the prevention or treatment of pathologies chosen from the group consisting of: myopia-complicating choroidal neovascularization, cornea neovascularization, in particular graft rejection, glaucoma, diabetic retinopathies or premature retinopathies, psoriasis arthritis, or for the treatment of obesity or retinal neovascularization.
In another preferred embodiment, the invention relates to the use of:
for the preparation of a drug for the prevention or treatment of pathologies involving the inhibition of endothelial cell proliferation and/or migration, in particular for the prevention or treatment of pathologies chosen from the group consisting of: myopia-complicating choroidal neovascularization, cornea neovascularization, in particular graft rejection, glaucoma, diabetic retinopathies or premature retinopathies, psoriasis arthritis, or for the treatment of obesity or retinal neovascularization.
The white column corresponds to Buffer; the black column corresponds to DeltaC NET-4m purified from transfected CHO cells; the gray column corresponds to NET-4m purified from CHO transfected cells.
The y-axis represents the optical density (OD) at 590 nm. The x-axis represents the volume (in micro-litters; μL) of conditioned medium added by well containing pgs A-745 CHO cells.
Curve with diamond corresponds to sNET-4m no: 2, curve with square corresponds to sNET-4m no: 3, curve with triangle corresponds to sNET-4m no: 4, curve with cross corresponds to sNET-4m no: 5 and curve with star corresponds to sNET-4m no: 6.
Experimental Part
Materials
The molecules netrin 4 (SEQ ID NO: 178), and the mutated netrin 4 (SEQ ID NO: 2) are recombinant proteins. The molecule netrin 4 is available by R&D.
The isoform of 165 amino acids of VEGF is produced by the infection of insect cells SF9 by a recombinant baculovirus containing the corresponding cDNA (Plouët J, Moro F, Coldeboeuf N, Bertagnolli S, Clamens S, Bayard F (1997) Extracellular cleavage of the vascular endothelial growth factor 189 aa form by urokinae is required for its mitogenic activity. J. Biol. Chem., 272, 13390-13396).
Human umbilical arterial endothelial cells (HUAEC) were isolated from umbilical arteries which were perfused with collagenase (Sigma) to digest the basal membrane. HUAEC cells were maintained in EBM medium (Clonetics), to which 15% of heat-inactivated foetal calf serum (FCS), 100 μg/ml of penicillin, and 100 μg/ml of streptomycin at 37° C. in 10% CO2 were added. The stem cultures received 2 ng/ml of VEGF at each even day.
Smooth muscular cells from aorta were maintained in DMEM medium to which 15% of heat-inactivated foetal calf serum (FCS), 100 μg/ml of penicillin, and 100 μg/ml of streptomycin at 37° C. in 10% CO2 were added. The stem cultures received 2 ng/ml of FGF-2 every other day.
Identification of a Mutated Netrin 4
Cloning of the Mutated Netrin 4
Total RNA of cells of the artery of human umbilical cord (HUAEC) were extracted with TriPure (Roche). Then the RNAs were transcribed by using the RT-PCR kit (AMV) of Roche according the manufacturer's indications.
Primers (5′)-TT CTA GAC ATG GGG AGC TGC GCG CGG-(3′) (sense) and (5′)—C ATT AAC GTC GAA CTG ACA GGT ATC-(3′) (antisense) were used for the amplification of the sequence 1-1039 of the netrin 4, while the primers (5′)-AG CAC TGT GCC CCG TTA TAC AAT GA-(3′) (sense) and (5′)—CGG GAT CCA CTT GCA CTC TCT TTT TAA AAT ATC C-(3′) (antisense) were used for the amplification of the sequence 914-1884 of the netrin 4.
The conditions for the amplification were: 35 cycles with denaturation at 94° C. for 1 minute; hybridization at 55° C. for 1 minute; and extension at 72° C. for 1 minute.
The products as obtained were mixed and used for a new PCR with the primers (5′)-TT CTA GAC ATG GGG AGC TGC GCG CGG-(3′) (sense), and (5′)—CGG GAT CCA CTT GCA CTC TCT TTT TAA AAT ATC C-(3′) (antisense) in the same conditions as described previously, with 25 cycles instead of 35. This PCR product containing the whole sequence of the netrin 4 was cloned in the intermediary vector pCR2.1 (Invitrogen). After the digestion by Xba I and Bam HI, the sequence of the netrin 4 was extracted from this vector and inserted into the pcDNA3.1 (−)/His myc C vector, said vector being digested by the same restriction enzymes. This last vector that contains the whole sequence of the mutated netrin 4 was used to transfect cells. An identical manipulation has led to the obtaining of an expression vector of the wild netrin 4 by using the sequence of the wild netrin 4.
The mutated netrin 4 was produced by the transfection of pgsA 745 CHO cells with the vector containing the sequence of the wild netrin 4 according to the protocol as described in: Plouët J, Moro F, Coldeboeuf N, Bertagnolli S, Clamens S, Bayard F (1997) Extracellular cleavage of the vascular endothelial growth factor 189-amino acid form by urokinase is required for its mitogenic activity. J. Biol. Chem., 272, 13390-13396. The protein was purified by heparin-sepharose affinity chromatography and eluted with a discontinuous gradient of NaCl (0.3, 1.0, and 2.0 M NaCl). The mutated netrin 4 of the invention (NET 4m) is eluted with NaCl2M, and it has a purity degree higher than 90%.
The biological activity of the mutated netrin 4 (NET 4m) was compared to the activity of the wild netrin 4 (NET 4) according to the proliferation test of the smooth muscular cells.
Construction of Deletion Mutants for the Mutated Netrin 4
The vector pcDNA3.1 (−)/His myc C containing the whole sequence of the mutated netrin 4 (628 amino acids) was digested by the restriction enzyme BamH1, treated with the fragment klenow of the polymerase 1, then digested with the restriction enzyme PshA1. The linearized fragment that corresponds to the vector pcDNA3.1 (−)/His myc C containing the sequence of the mutated netrin 4 from which the Cter domain (478-628) was deleted was then isolated after migration on agarose gel and is purified on a Qiagen column. After ligation, an expression vector pcDNA3.1 (−)/His myc C containing the sequence of the mutated netrin 4 from which the Cter domain was deleted (1-477) was obtained. The vector pcDNA3.1 (−)/His myc C containing the whole sequence of the mutated netrin 4 (628 aa) was digested by the restriction enzyme Xcm1. This enzyme that cuts the internal sequence of the netrin 4 in two sites (aa288/aa488) enables the deletion of the central domain of the protein (domain V with EGF motifs). After the purification and the ligation of the fragment, an expression vector pcDNA3.1 (−)/His myc C containing the sequence of the mutated netrin 4 from which the central domain was deleted (288/488) was obtained. However as the ligation of both sites Xcm1 leads to the onset of a stop codon (aa 313), this vector codes for a mutated netrin 4 that is truncated of 312 amino acids and that contains the sequence of the laminin domain (1-288) and a protein sequence of 24 amino acids.
Production of Anti-Idiotypic Antibodies
Firstly, a neutralizing antibody of the mutated netrin 4 of the invention or of a fragment of said mutated netrin 4 (SEQ ID NO: 2 to 72) was prepared by injecting to an animal, in particular a mouse, said mutated netrin or said fragment in admixture with Freund's complete adjuvant (1 volume for a volume of netrin or netrin fragment). A quantity of mutated netrin or netrin fragment varying from 10 to 500 μg/kg of body weight was used to immunize the animal. The same procedure was carried out after 15 and 30 days, except that the complete adjuvant was replaced by incomplete adjuvant. At day 40 the animals were bled, the serum was separated, and the immunoglobulins were purified by any usual fractionation method, in particular ammonium sulphate precipitation, protein A- or protein G-affinity chromatography. The neutralizing activity of the immunoglobulins was measured by any described test (for an example, for the mutated netrin 4 of the invention or one of its fragments: bonding of labelled netrin 4 to the extracellular domain of any one of its receptors, proliferation, migration, cell adhesion). Thus a group of immunoglobulins was considered as neutralizing when it was able to inhibit the interaction of the mutated netrin 4 either with the extracellular domain of dcc, neogenin, UNC5A, UNC5B, UNC5C or UNC5D.
Then anti-idiotypic antibodies of the mutated netrin 4 or of one of its fragments were prepared by injecting to mice by subcutaneous route 1 to 100 μg of the preparation of the immunoglobulins that neutralize the activity of said mutated netrin or said fragment as described previously, in association with 100 μl of adjuvant, in particular of Freund's complete adjuvant (Sigma). The injection was repeated 15, 30, and 45 days later. Fifty five days after the first injection, 10 μg of the same antibody were injected to mice by intraperitoneal route. Fifty eight days after the first injection, the mice were bled and their spleens were sampled and dilacerated in ISCOVE medium in order to release the splenocytes. The splenocytes were fused with mice's myeloma cells, in particular with AG8X 63 cells (Kearney J F, Radbruch A, Liesegang B, Rajewsky K (1979) A new mouse myeloma cell line that has lost immunoglobulin expression but permits the construction of antibody-secreting hybrid cell lines. J. Immunol. 123, 1548-50), and were incubated at a ratio of 100,000 cells/well. The fusion was carried out by adding 20 times a volume of 50 μl of polyethylene glycol (PEG) within an interval of 30 seconds. Four ml of 37° C.-preheated ISCOVE medium were then added dropwise to the cell suspension, and then, after an incubation time of 4 minutes at 37° C., 4 ml were added. The centrifugation was centrifuged then the cell centrifugation pellet was resuspended in 100 ml of ISCOVE medium complemented with 20% of foetal calf serum and HAT 1× (50×: Hypoxanthine 5 mM, Aminopterin 20 μM and Thymidine 0.8 mM) and distributed at a volume of 100 μl per well on macrophages. After 5 days, 100 μl of HAT medium were added, and after 8 to 14 days, the conditioned medium of each hybridoma was sampled to measure by ELISA the antibodies that were directed against the antibodies used as immunogenic agents, that is to say the anti-netrin antibodies. The activity of the anti-idiotypic antibodies was then measured by an ELISA test.
The Fab fragments of the anti-mutated netrin 4, prepared by any usual technique, in particular by papain digestion, were immobilized on microtitration plates (0.1-20 μg/ml in carbonate buffer 50 mM pH 9.6). After saturation of the non-specific sites by a solution of albumin serum diluted at 5 mg/ml in the same buffer, the supernatants of hybridomas cultures were added as half-diluted in PBS buffer (phosphate buffer) containing 0.05% Tween 20. After rinses, the anti-idiotypic antibodies were revealed by the addition of an appropriate concentration of mice peroxydase-coupled anti-Fc antibodies. The amount of fixed anti-idiotypic antibodies was then measured by revelation of the peroxydase and was proportional to the intensity of the colorimetric reaction.
The hybridomas were selected according to their ability to secrete antibodies against anti-mutated netrin 4 antibodies, and these selected hybridomas were then cloned: more precisely, the cells were grown in limit dilution condition (5 cells/ml) in a volume of 0.1 ml per well. The medium was changed after 10 days. After 15 days, some of the wells contained cell groups that grew from the starting cell, and these cells thus were identical and originated from the same clone. When the surface that was covered by the cells was at least half of the total surface of the well, the medium was sampled and analysed.
A second ELISA test was then carried out: goat immunoglobulins directed against the Fc domains of the human IgG were incubated on microtitration plates (0.1-20 μg/ml in carbonate buffer 50 mM pH 9.6). After saturation of the non-specific sites by a solution of albumin serum diluted at 5 mg/ml in the same buffer, the proteins containing the extracellular domains of the netrins receptors fused to a Fc sequence of human IgG were immobilized on the microtitration plates (incubation at a concentration of 1 to 100 μg/ml). The supernatants of the hybridomas cultures were added as half-diluted in PBS buffer containing 0.05% Tween 20. After rinses, the anti-idiotypic antibodies were revealed by the addition of an appropriate concentration of mice peroxydase-coupled anti-Fc antibodies. The amount of fixed anti-idiotypic antibodies was then measured by revelation of the peroxydase and was proportional to the intensity of the colorimetric reaction.
As soon as the clones were identified, their monoclonal state was confirmed by the usual procedure that consisted in seeding a 96 wells-plate with cells originating from the same clone and diluted in limit conditions as described previously. The secreting clones should thus secrete an antibody with the same specificity in order to be considered as monoclonal. A third cloning was then carried out exactly in the same conditions to ensure that the clones were monoclonal.
The monoclonal anti-idiotypic antibodies were screened by a series of tests, in particular by an ELISA test on extracellular domains of the known netrins receptors (dcc, neogenin, UNC5-A, UNC5-B, UNC5-C, UNC5-D), or measurement of the inhibition of the proliferation or migration of HUAEC cells or in vivo measurement tests of an anti-angiogenic activity.
Thus the anti-idiotypic antibodies were mimes of the netrins domains. The aim was to design an “internal image” of a netrin domain and to obtain an antibody that binds to a netrin receptor but that does not bind to all the receptors. As soon as the specificity was confirmed, the agonist function of this antibody was ascertained by measuring its activity on cells, that is to say by inhibiting the HUAEC functions without inhibiting or stimulating the netrins functions on SMC and/or by stimulating SMC without affecting HUAEC.
The interest of these antibodies was to be able to mimic a function of the netrins on a cell target without affecting other targets. Thus it would be interesting to stimulate the pericytes functions without inducing the apoptosis of the endothelial cells or to inhibit their migration, their proliferation, their differentiation in some pathologies:
Inversely it would be interesting to inhibit the proliferation of endothelial cells without affecting the pericytes for the above-mentioned pathologies.
Angiogenenesis' s Inhibition
In order to increase the specific activity of netrin-4 point mutations were inserted in its sequence and produced various mutants in CHO cells. One of them referred as NET-4m (mutated netrin-4 of the invention) exhibited a 2000-fold gain of function in the matrigel assay (
In Vitro angiogenenesis
In vitro angiogenesis assays were performed using HUAEC (105 cells/cm2) seeded on growth factor-reduced Matrigel (BD Biosciences) and incubated at 37° C. for 1 hour. Then 50 ng/ml of VEGF was added, the samples incubated for 2 hours and 400 ng/ml of netrin-1 or netrin-4 was added and incubation continued for 5 more hours. The plates were then rinsed with PBS and fixed at room temperature with 1% glutaraldehyde. The mean microcapillary network was measured using an automated computer-assisted image analysis system, and the total length of the capillaries in each well was determined (μm) for each experimental condition. Experiments were performed in triplicate and repeated at least three times.
As shown in the
Comparison of the Activity of NET-4 and NET-4m on Proliferation (
Plates with 96 wells were seeded with 2,000 SMC (smooth muscular cells) per well in DMEM medium complemented with 10% FCS. After 6 hours the cells were transferred in DMEM medium containing 2% FCS and were then stimulated (or not) with various concentrations of netrin 4 (NET-4) or of mutated netrin 4 (NET-4m). After 5 days, the wells were rinsed with DMEM and the cells were fixed with 1% glutaldehyde, coloured with violet crystal and solubilized with acetic acid. The optical density was measured at 595 nm. Similar results were obtained in three independent experiments. The indicated values are mean optical densities of 6 wells±standard deviation (SD).
Proliferation Test of SMC (
The mutated netrin 4 was produced by transfecting pgsA 745 CHO cells with the vector containing the sequence of the wild netrin 4 according to a known procedure (Plouët J, Moro F, Coldeboeuf N, Bertagnolli S, Clamens S, Bayard F (1997) Extracellular cleavage of the vascular endothelial growth factor 189 aa form by urokinae is required for its mitogenic activity. J. Biol. Chem., 272, 13390-13396). The protein was purified by heparin-sepharose affinity chromatography and eluted with a discontinuous gradient of NaCl (0.3, 1.0 and 2.0 M NaCl). NET-4m was eluted with NaCl2M and has a purity degree greater than 90%.
The biological activity of NET-4m was compared with the activity of wild NET 4 according to the smooth muscular cells proliferation test.
Migration Test of the SMC (
The confluent monolayer of SMC is incubated during one night in the presence of DMEM. A wound is made in the monolayer with a rubber policeman and the wells were washed three times with DMEM and then incubated in the presence of varying concentrations of netrin 4 (NET-4) or mutated netrin 4 (NET-4m). After 24 hours, the wells were washed three times and coloured with May-Grunwald-Giemsa and photographed and the cells are counted in 8 hpf per condition. The results are given in number of cells per hpf.
It appears that half of the maximal stimulation is obtained with a concentration of 12 ng/ml of non-mutated netrin 4 (NET-4) and 0.004 ng/ml of mutated netrin 4 (NET-4m). This means that the chimiotactic activity of the mutated netrin 4 is 3,000 times as active as the non mutated netrin 4.
Transfection of PC3 Cancer Cells
Prostate cancer cells (PC3) and Colon carcinoma are grown in DMEM medium complemented with antibiotics and foetal calf serum 10%. The transfection protocol was established as follows:
After the checking that the clones transfected with netrin 4 (NET-4) or mutated netrin 4 (NET-4m) have an equivalent duplication time (26-30 hours), the content of netrin 4 of each medium was measured as described previously in the paragraph relating to the proliferation test. 4 μl of conditioned medium were added to 100 μl of culture medium. The results are given in proliferation percentage in comparison with the control (well containing 4 μl of DMEM medium).
According to the
On the other hand, the conditioned medium of the clones 1 and 5 of NET-4m as well as the clone 8 of NET-4 stimulate the proliferation of HUAEC cells of only 200%, which corresponds to about 50% of the proliferation as induced by the conditioned medium of PC3 cells.
Thus netrin 4 (NET-4) or mutated netrin 4 (NET-4m) induces the proliferation of HUAEC without modifying the proliferation of PC3 cancer cells.
Transfection of LS174.
Colon carcinoma LS 174 cells are grown in DMEM medium complemented with antibiotics and foetal calf serum 10%.
The transfection protocol was established as follows:
After the checking that the clones transfected with netrin 4 (NET-4) or mutated netrin 4 (NET-4m) have an equivalent duplication time (26-30 hours), the content of netrin 4 of each medium was measured as described previously in the paragraph relating to the proliferation test. 4 μl of conditioned medium were added to 100 μl of culture medium. The results are given in proliferation percentage in comparison with the control (well containing 4 μl of DMEM medium).
Several clones were selected for each transfection. FS2 and FS3 are two representative clones of LS174 cells transfected with NET-4m. DeltaC1 and DeltaC2 are two representative clones of LS174 cells transfected with NET-4m DeltaC.
Analysis of the Tumorigenicity of the Clones of Transfected PC3 Cells
Non-transfected PC3 cells and PC3 cells transfected with NET4m (clones 1 and 5) were injected to nude mice's flank (1 million of cells pro injection). The length (L) and width (1) of each tumor were measured with a caliper and the volume is expressed by the formula 0.52×L×12. According to
Thus the mutated netrin 4 of the invention (NET-4m) exerts an anti-tumoral activity through its anti-angiogenic activity. It appears that mutated netrin 4 decreases the ratio of proliferating cells by 30% (clone 1) and 60% (clone 5), respectively. It also appears that mutated netrin-4 expression in PC3 cells increases the pericyte coverage of endothelial cells by 1.3 (clone 1) and 2-fold (clone 5), respectively. In fact the decrease of the ratio CD31/desmin (
Analysis of the Tumorigenicity of the Clones of Transfected LS 174 Cells
Non-transfected LS 174 cells, LS 174 cells transfected with NET-4m (FS3 and FS4) and LS 174 cells transfected with DeltaC NET-4m (DeltaC1 and DeltaC2) were injected to nude mice's flank (1 million of cells pro injection). The length (L) and width (1) of each tumor were measured with a caliper and the volume is expressed by the formula 0.52×L×12. Tumor volumes were recorded at J 25 and expressed as percentage of the volume of non transfected LS174 tumors.
According to
Thus the mutated netrin 4 of the invention (FS2 and FS3) exerts an antitumoral activity through its anti-angiogenic activity. It appears that DeltaC deleted netrin4 tumors behave as the parental cells thus demonstrating that the C-terminus sequence of netrin4 is required for its anti tumor angiogenesis activity whereas it is not for its activity to inhibit choroidal angiogenesis.
Synergic Effect of NET-4m on the Inhibition of VEGF
It is now well known that VEGF is a major actor of the pathologic angiogenesis and that its inhibition is a major therapeutic pathway. An anti-VEGF antibody is commercialized under the name AVASTIN®. Knowing that the netrin 4 (NET-4) acts through a mechanism of action differing from the one of the VEGF, the synergic effect of the netrin 4 (NET-4) with an anti-VEGF antibody commercialized under the name of AVASTIN® was measured.
Mice received a graft of non-transfected colon carcinoma LS 174 cells or of LS 174 cells transfected with NET-4 (clones FS3 8, 10 or 15). As soon as the tumors had a volume greater than 400 mm3, the mice received a peritoneal injection of AVASTIN® (50 μg every 3 days), said dose corresponding to the therapeutic recommendations in human pathology (10 mg/kg/every other week) and the tumor volume was measured as described previously.
It appears on
Effect of NET-4m (MC4) on the Migration of SMC
pgsA-745 CHO cells were transfected with the PCDNA-3 expression vectors containing the whole sequence of mutated netrin 4 (MC4). After 16 hours, the cells were incubated with DMEM medium and the conditioned media were collected after 48 hours. The migration activity on SMC cells was measured as described previously (see
Choroid Neovascularization (
Eight-week old Brown Norway rats (Janvier, Le Genest-St Isle, France) were anesthetized by intraperitoneal injection of 0.14 ml sodium pentobarbital (Sanofi Santé Animale). The pupils were dilated with 1% tropicamide (Théa, Clermont-Ferrand, France). Photocoagulation lesions were created around the optic nerve 1 to 2 disc diameters away from the papillae with an argon laser photocoagulator (Quantel Medical Clermont-Ferrand, France) set at 532 nm, mounted on a slit lamp and with a cover glass fulfilling the role of contact lens (parameters fixed to 150 mW, 100 ms and 100 μm). In all treated eyes included in the study, a reactive bubble at the retinal surface was observed after laser delivery as evidence for appropriate focusing and an indication of the rupture of Bruch's membrane. Rats were injected with netrin4 (1 ng of NET-4m) in a volume of 5 μl under the subretinal space on days 7 and day 10 after laser photocoagulation. 14 days after laser treatment, all animals were perfused with 1 ml of PBS containing 50 mg/ml fluorescein-labelled dextran (FITC-dextran; average molecular mass, 2×106; Sigma-Aldrich) and sacrificed. The eyes were harvested and fixed in PBS 4% paraformaldehyde (PAF) solution (LADD, Inland Europe, Conflans-sur-Lanterne, France). Retinas and choroids were dissected, and fixed for an additional 15 minutes at room temperature in methanol. The enucleated eyes were sectioned at the equator and the anterior half, including the lens and vitreous, was discarded. The retinas were carefully peeled from the eyecup and optic nerve by using specialized scissors and forceps under a biomicroscope (Wild M3Z, Heerbrugg). The posterior eye segment containing the complex sclera-choroid and the retina was dissected into quarters by four radial cuts. After washing in PBS, the flat mounts were mounted with Gelmount® (Biomeda, Foster City, Calif., USA), air-dried and examined under a fluorescence microscope (BX51; Olympus, Melville, N.Y.) at 488 nm or 594 nm excitation wavelength as appropriate. The incidence and size of the CNV complex were scored by morphometric analysis of the images with Image J Software (v1.36, NIH, USA). Subretinal injections of NET-4m (1 ng), after the onset of angiogenesis, reduced choroid neovascularization, as visualized by dextran perfusion, by more than 70%.
C57BL/6 mice (Janvier, Le Genest-St Isle, France) were anesthetized by intraperitoneal injection of 0.14 ml sodium pentobarbital (Sanofi Santé Animale). The pupils were dilated with 1% tropicamide (Théa, Clermont-Ferrand, France). Photocoagulation lesions were created around the optic nerve 1 to 2 disc diameters away from the papillae with an argon laser photocoagulator (Quantel Medical Clermont-Ferrand, France) set at 532 nm, mounted on a slit lamp and with a cover glass fulfilling the role of contact lens (parameters fixed to 150 mW, 100 ms and 100 μm). In all treated eyes included in the study, a reactive bubble at the retinal surface was observed after laser delivery as evidence for appropriate focusing and an indication of the rupture of Bruch's membrane. Mice were injected with netrin4 20 pg of NET-4m) in a volume of 1 μl under the intravitreal space on days 7 and day 10 after laser photocoagulation. 14 days after laser treatment, all animals were perfused with 1 ml of PBS containing 50 mg/ml fluorescein-labelled dextran (FITC-dextran; average molecular mass, 2×106; Sigma-Aldrich) and sacrificed. The eyes were harvested and fixed in PBS 4% paraformaldehyde (PAF) solution (LADD, Inland Europe, Conflans-sur-Lanterne, France). Retinas and choroids were dissected, and fixed for an additional 15 minutes at room temperature in methanol. The enucleated eyes were sectioned at the equator and the anterior half, including the lens and vitreous, was discarded. The retinas were carefully peeled from the eyecup and optic nerve by using specialized scissors and forceps under a biomicroscope (Wild M3Z, Heerbrugg). The posterior eye segment containing the complex sclera-choroid and the retina was dissected into quarters by four radial cuts. After washing in PBS, the flat mounts were mounted with Gelmount® (Biomeda, Foster City, Calif., USA), air-dried and examined under a fluorescence microscope (BX51; Olympus, Melville, N.Y.) at 488 nm or 594 nm excitation wavelength as appropriate. The incidence and size of the CNV complex were scored by morphometric analysis of the images with Image J Software (v1.36, NIH, USA). Intravitreal injection of NET-4m or DeltaC NET-4m, after the onset of angiogenesis, reduced choroidal neovascularization, as visualized by dextran perfusion, by 74% and 62% respectively.
Data of
Peritoneous Carcimomatosis
106 LS 174 or mutated transfected LS 174 (FS3 as described above) were injected in the peritoneal cavity of Nod-Scid mice. After 21 days the carcinomatosis was recorded by the Sugerbaker's score (Observations concerning cancer spread within the peritoneal cavity and concepts supporting an ordered pathophysiology. Cancer Treat Res. 1996; 82:79-100). The ascite was collected and its volume was measured and photomicrographs of the peritoneal cavity were taken.
As shown on
Combination of Pericytes and Mutated Netrin-4 as an Anti-Cancer Agent
As shown in
Production of NET-4m Proteins
pgsA-745 CHO cells are grown in DMEM medium complemented with antibiotics and foetal calf serum 10%. The transfection protocol was established as follows:
Purification of NET-4m proteins.
Conditioned media were adjusted to pH 7.4 with 10 mM Hepes buffer and the 10 ml mixture were mixed with 200 μl of cation exchange matrix (Sp sepharose, Pharmacia) for 4 hours at 4° C. Then the mixture was centrifugated at 800 g for 5 minutes. The pellet was washed with 10 mM Hepes buffer containing 0.1 M NaCl. Then the NET-4m proteins were eluted from Sp Sepharose by stepwise NaCl gradient (0.2 M, 0.4 M, 0.8 M NaCl) under a final volume of 0.5 ml per fraction. The active NET-4m was eluted between 0.4 and 0.8 M NaCl. The active material (determined by an ELISA assay as aboved described) were diluted with 2 volumes of distilled H2O containing 10 mM Hepes and applied to an Heparin sepharose (Pharmacia) column chromatography (200 μl). The column was then washed with 1 ml of 10 mM Hepes containing 0.3 M NaCl. The NET-4m proteins were then eluted by 0.5 ml og 10 mM Hepes containing 1.2 M NaCl and assayed for their mitogenic activity on SMC cells.
| Number | Date | Country | Kind |
|---|---|---|---|
| 07290075.6 | Jan 2007 | EP | regional |
| Filing Document | Filing Date | Country | Kind | 371c Date |
|---|---|---|---|---|
| PCT/EP08/50662 | 1/21/2008 | WO | 00 | 7/14/2009 |
