Robo1-Fc fusion protein for use in
the treatment of hepatocarcinoma
The present invention relates to a recombinant protein Robo1-Fc and to the use
thereof for treating diseases in which a Slit protein is overexpressed. In particular,
the present invention relates to the use of a Robo-Fc protein for treating cancer,
especially hepatocarcinomas. It also relates to a composition comprising such a
recombinant protein. Another aspect of the invention consists of the use of a
Robo1-Fc molecule as a diagnostic tool for detecting the overexpression of a
molecule of the Slit family in a patient.
Slit ligands were first of all described for their role in repelling axonal growth in
neuronal development. Since then, the Robo/Slit regulatory pathway has also been
described in tumour angiogenesis. Specifically, the Robo4/Slit2 pathway has been
described for inhibiting the response of endothelial cells to VEGF (Jones C.A. et al.
Nat. Med 14, 448-453 (2008)). Regulation by the Robo/Slit pathway makes it
possible to channel the excessive proliferation of endothelial non-mature
neovessels or buds (non-productive angiogenesis) and to mature these vessels.
The expression of Slit2 at the transcriptional level has been demonstrated in
several human cancer lines, in particular HCT1 16 derived from colon carcinoma,
Skov-3 derived from ovarian carcinoma, HeLa derived from cervical cancer, MDAMB-
435 derived from melanoma, Hec-1A derived from uterine cancer and, finally,
769-P derived from renal carcinoma (Stella MC et al., Mol Biol Cell. 2009 Vol. 20,
Issue 2 , 642-657). Overexpression of the Slit2 protein has also been demonstrated
on human tissues derived from carcinomas: oral carcinoma (Wang et al. 2008,
Cancer Sci. 2008 Mar; 99(3): 510-7), prostate carcinoma (Latil et al. 2003 Int J
Cancer. 2003 Jan 20; 103(3): 306-15), colon carcinoma (Wang et al. 2003, Cancer
Cell, Volume 4 , Issue 1, July 2003, Pages 19-29), and liver carcinoma (Avci et al,
2008, BMC cancer, 8 : 392). More recently, overexpression of the Slit2 protein has
been shown on samples of endometriosis (Shen et al, 2009, AJP 175 (2): 479).
The Robol protein exists as two isoforms a and b. The extracelllar domain of the
Robol protein isoform b (NP_598334) comprises 5 immunoglobulin domains: Ig1 ,
Ig2, Ig3, Ig4 and Ig5. The Robo protein interacts with the Slit ligands at the level of
the Ig1 and Ig2 domains. Liu et al. (2004, Mol. Cell Neurosci, 26: 232-240) have
demonstrated the importance of the Ig2 domain in the interaction with Slit and in the
activity of Robo (chemorepulsion).
The use of antibodies specific for the Robol and the Slit2 proteins has been
described in application WO2003/075860. These antibodies make it possible to
inhibit tumour angiogenesis.
However, it would be advantageous to propose an alternative approach for treating
cancer, in particular a molecule capable of inhibiting the Slit2 signalling pathway.
The inventors have developed a strategy of soluble chimeric Robol receptors
capable of binding Slit ligands and consequently, of inhibiting the intracellular
signalling of the Robo/Slit pathway. Surprisingly, the Robo1-Fc molecules according
to the invention have an anti-angiogenesis effect by preventing the formation of
mature vessels and not by inhibiting the proliferation of endothelial cells.
The inventors have shown that the Robo1-Fc molecules also have an antitumour
effect in lung and liver cancer.
DETAILED DESCRIPTION OF THE INVENTION
A subject of the present invention is a Robo-Fc recombinant protein comprising the
extracellular domain of the Robol protein, isoform b or a part of this domain, a
junction region (linker) and an immunoglobulin Fc domain.
In one particular embodiment, the extracellular domain of the Robol protein,
isoform b consists of the Ig1 and Ig2 domains. These domains correspond to the
peptide of SEQ ID NO.2 encoded by the nucleotide sequence SEQ ID N0.1 , or a
sequence having at least 80%, 85%, 90%, 95% or 99% identity with the sequence
SEQ ID NO.2.
The fusion protein also comprises a junction region, also called "linker". In the
context of the present invention, the linker makes it possible to give the
recombinant protein stability, in particular by limiting in vivo cleavage. Linkers that
can be used in a Robo1-Fc molecule are, for example, GluArgProSerPheVal and
GlyGlyGlyGlySer. Those skilled in the art have sufficient knowledge to select a
linker suitable for this use.
The Fc domain of the Robo1-Fc recombinant molecules according to the invention
corresponds to a crystallizable fragment of an immunoglobulin. This Fc fragment
can come from various immunoglobulins lgG1 , lgG2, lgG3 or lgG4. It is responsible
for the effector function of the immune response (WO2008/065543).
In one embodiment according to the invention, the Fc domain comes from an lgG4
immunoglobulin. In one particular embodiment, at least one point mutation or
deletion has been introduced into the lgG4 Fc domain so as to increase the stability
of the molecule, in particular by stabilizing the hinge region made up of the two Fc
domains (Angla et al., 1993, Mol. Immunol., 30: 105-108), to reduce or eliminate the
residual activity of the lgG4-Fc, in particular the effector activity (WO 97/09351),
and to increase the homogeneity during the production of the recombinant protein.
In particular, at least two point mutations, preferably three point mutations, have
been introduced into the lgG4 Fc domain. In the Robo1-Fc L1, Robo1-Fc L2 and
Robo1-Fc L3 molecules according to the invention, the preferred mutations are the
following:
S241 P (Kabat numbering) in order to stabilize the disulphide-bridge bonding
in the Fc hinge region;
L248E (Kabat numbering) in order to eliminate the residual effector activity
of the lgG4-Fc domain;
- absence of the C-terminal lysine in order to reduce the heterogeneity of the
protein.
Robo1-Fc molecules according to the invention comprising the Ig1 and Ig2 domains
of Robol isoform b, a linker and a mutated lgG4 domain as described above are
Robo1-Fc L 1 , Robo1-Fc L2 and Robo1-Fc L3.
Robol -Fc L 1 corresponds to the protein of sequence SEQ ID NO.4 , encoded by the
nucleotide sequence SEQ ID NO.3 .
Robo1-Fc L2 corresponds to the protein of sequence SEQ ID NO.6 , encoded by the
nucleotide sequence SEQ ID NO.5 .
Robol -Fc L 1 and Robol -Fc L2 differ by virtue of the nature of the linker.
Robo1-Fc L3 corresponds to the protein of sequence SEQ ID NO.24, encoded by
the nucleotide sequence SEQ ID NO.23.
In one particular embodiment of the invention, one or more point mutations or
deletions have been introduced in order to increase the homogeneity during
production. In particular, one amino acid, preferably two amino acids, have been
truncated in the N-terminal position. Such a Robo-1-Fc molecule according to the
invention is the Robo1-Fc L3 molecule, in which the two amino acids (Ser20 and
Arg21) have been truncated and in which the amino acid Leu 22 has been fused to
the signal peptide of interleukin 2 .
The homogeneity of the Robo1-Fc molecules according to the invention is also
increased by deleting the C-terminal Lys as previously described.
A subject of the invention is also proteins of which the protein sequence
corresponds to the sequences SEQ ID NO.2 , SEQ ID NO.4, SEQ ID NO.6 or SEQ
ID N0.24 or has at least 80%, 85%, 90%, 95% or 99% identity with the sequences
SEQ ID NO.2, SEQ ID NO.4, SEQ ID NO.6 or SEQ ID N0.24. These protein
variants have the same biological activity as the proteins having the sequence SEQ
ID NO.2, SEQ ID NO.4, SEQ ID NO.6 or SEQ ID N0.24, in particular their ability to
interact with the proteins of the Slit family.
The Robo1-Fc proteins according to the invention can be produced by transfection
of expression plasmids encoding these proteins into any cell type suitable for the
expression of eukaryotic recombinant proteins: HEK293, CHO, etc., and then
recovered in the culture supernatant and purified according to conventional
methods.
Characterization of the Robo1-Fc L 1 , Robo1-Fc L2 and Robo1-Fc L3 proteins
according to the invention has made it possible to confirm that they have the
qualities required for their administration as a biotherapeutic agent.
A Robo1-Fc protein according to the invention has the ability to interact with a
protein of the Slit family.
The Robo1-Fc proteins according to the invention specifically recognize the human
Slit1, Slit2 and Slit3 proteins and the murine Slit2 protein, in particular by interaction
with their D2 domain. Their affinity is similar with respect to the 3 members of the
Slit family.
Another subject according to the invention corresponds to the nucleic acid
molecules encoding the proteins according to the invention.
Thus, the nucleic acid molecules corresponding to the sequences SEQ ID N0.1 ,
SEQ ID NO.3, SEQ ID NO.5 or SEQ ID N0.23 or having at least 80%, 85%, 90%,
95% or 99% identity with the molecules having the sequence SEQ ID N0.1 , SEQ
ID NO.3, SEQ ID NO.5 or SEQ ID N0.23 are part of the invention.
Another subject according to the invention consists of the use of a Robo1-Fc
protein according to the invention for treating diseases in which a protein of the Slit
family is overexpressed.
The proteins of the Slit family which can be targeted by the Robo1-Fcs according to
the invention are Slit1 , Slit2 or Slit3.
It has been shown that Robol can interact with the various members of the Slit
family. Consequently, it is advantageous to note that the Robo1-Fc proteins
according to the invention are capable of simultaneously inhibiting the signalling
pathways mediated by Slit1 , Slit2 and Slit3, which makes it possible to broaden the
therapeutic spectrum in comparison with the antibodies which are specific for only
one of these pathways.
In another embodiment, a Robo1-Fc protein is used for treating diseases in which a
protein of the Slit family is overexpressed, by inhibiting angiogenesis without
inhibiting endothelial cell proliferation. This anti-angiogenic activity linked to a
vessel maturation defect is called "non-productive angiogenesis".
Specifically, the experimental studies have shown that the Robol -Fc molecules
according to the invention are capable of inhibiting the formation of tubules, without
inhibiting endothelial cell proliferation. They make it possible to very significantly
reduce the tumour volume in a murine model of lung cancer.
In one preferred embodiment, the Robol -Fc proteins that can be used for treating
diseases in which a Slit protein is overexpressed are Robo1-Fc L 1 , Robo1-Fc L2
and Robo1-Fc L3, and the molecules which are derived therefrom, include, in
particular, point mutations or deletions aimed at increasing the homogeneity during
production without significantly modifying the biological properties of these
molecules.
In general, the diseases that can be treated with a Robo-1-Fc protein according to
the invention are all the diseases in which inhibition of the Slit pathway can have a
therapeutic effect, in particular the diseases in which a protein of the Slit family is
overexpressed, and in particular those in which Slit2 is overexpressed.
Since the Robo1-Fc molecules according to the invention specifically bind the Slit2
molecule, it is interesting to note that said molecules are capable of simultaneously
inhibiting the two pathways in which Slit2 is involved, namely the Robo1/Slit2 and
Robo4/Slit2 pathways.
In a specific embodiment of the invention, the Robo1-Fc proteins according to the
invention is used for treating cancer, in particular pancreatic cancer, colon cancer,
colorectal cancer with or without lymphatic metastasis, breast cancer, lung cancer
and lung metastases, ovarian cancer, cervical cancer, melanomas, renal cancer,
oral cancer, prostate cancer, liver cancer, etc. In a specific embodiment, said
cancer is a cancer in which Slit2 is overexpressed.
In one particular embodiment, the Robo-Fc proteins are used for treating lung
cancer.
In another particular embodiment, the Robo-Fc proteins are used for treating liver
cancer, such as e.g. hepatocarcinoma, hepatocellular carcinoma (HCC),
fibrolamellar carcinoma (a pathological variant of hepatocellular carcinoma),
cholangiocarcinoma, hepatoblastoma, or angiosarcoma of the liver.
The Robo1-Fc molecules according to the invention can also be used as an
anticancer medicament as an alternative to or as a supplement to the current
therapies.
In particular, these molecules can be administered in combination (optionally
simultaneously) with anticancer compounds.
Another subject of the invention relates to a composition comprising a Robo1-Fc
protein as defined above and one or more pharmaceutically acceptable excipients.
The Robo1-Fc proteins according to the invention can be formulated in
pharmaceutical compositions with a view to topical, oral, parenteral, intranasal,
intravenous, intramuscular, subcutaneous, intraocular, etc., administration.
Preferably, the pharmaceutical compositions contain pharmaceutically acceptable
vehicles for an injectable formulation. They may in particular be isotonic sterile
saline solutions (monosodium or disodium phosphate, sodium, potassium, calcium
or magnesium chloride, etc., or mixtures of such salts), or dry, in particular freezedried,
compositions which, by addition, as appropriate, of sterilized water or of
physiological saline, allow the formation of injectable solutes.
Another aspect of the invention consists of the use of a Robo1-Fc molecule as a
diagnostic tool for detecting the overexpression of a molecule of the Slit family in a
patient. This is because it has been shown that the Slit pathway is implicated in
many cancers. The provision of a test for evaluating a disturbance of the Slit
signalling pathway is very useful for the purpose of selecting patients capable of
responding to a treatment based on the administration of a Robo1-Fc molecule.
This diagnostic tool may be in the form of a ready-to-use kit, comprising a Robol-
Fc molecule in a form suitable for it to be brought into contact with a biological
sample from a patient (blood, urine, tumour biopsy) liable to exhibit an
overexpression of a Slit molecule. The Robo-Fc molecule can optionally be
prelabelled, and the combination of Robo-Fc and Slit is detected so as to evaluate
an increase in the expression of a Slit protein in the biological sample in
comparison with a control sample. This kit can, for example, be in the form of an
ELISA kit.
DESCRIPTION OF THE FIGURES
Figure 1 : Expression plasmids for the Robo1-Fc L 1 , Robo1-Fc L2 and Robo1-Fc
L3 proteins.
Figure 2 : Evaluation of the interaction of the Robo1-Fc fusion protein variants with
the Slit2 protein by ELISA.
Figure 3 : Affinity of Robo1-Fc for HEK293 cells not expressing Slit2, measured by
FACS.
Figure 4 : Affinity of Robo1-Fc for HEK293 cells expressing Slit2, measured by
FACS.
Figure 5 : Pharmacokinetic profile of the Robo1-Fc proteins after an iv injection in
mice. A. Administration of Robo1-Fc L 1 , B. Administration of Robo1-Fc L2.
Figure 6 : Effect of the Robo1-Fc L 1 molecule in a coculture test with endothelial
cells and mesenchymal cells. A. Robo1-Fc L 1 inhibits tubule formation in culture. B.
Robo1-Fc L 1 significantly inhibits VEGF-induced pseudotubule formation.
Figure 7 : Evaluation of the effect of the Robo1-Fc L 1 molecule on an ex vivo aortic
ring model in mice. A : Description of the protocol for preparing an aortic ring and for
measuring tubules. B : a . Control; b. Robo1-Fc Slit2-minus 500 mg/mL + VEGF
10 ng/mL; c . Robo1-Fc L 1 500 mg/mL + VEGF 10 ng/mL.
Figure 8 : Mean tumour weight (HepG2 cells in the left lobe of the liver of SCID
mice) 28 days after injection of the cells, in the control group and in the group
having received 4 injections of Robo1-Fc at 25 mg/kg.
Figure 9 : Mean tumour weight (Hep3B cells one lobe of the liver of SCID mice) 28
days after injection of the cells, in the control group and in the group having
received 5 mg/kg of Robo1-Fc twice a week.
Figure 10: Plasmatic AFP concentration in the control group and in the group
having received 5 mg/kg of Robo1-Fc twice a week.
EXAMPLES
Example 1: Preparation of the Robo1-Fc proteins
a . Constructs allowing the expression of the Robo1-Fc recombinant proteins
used as biotherapeutic agents.
The Robo1-Fc recombinant proteins consist of a fusion between the first two
immunoglobulin domains (Ig1-lg2) of the human Robol protein, isoform b
(NP_598334) and the Fc domain of human immunoglobulin G4 (hlgG4-Fc,
SwissProt IGHG4_HUMAN).
In order to obtain the Robol -Fc L 1 construct, a fragment of the cDNA (SEQ ID NO.1)
corresponding to the immunoglobulin (Ig) domains Ig1 and Ig2 of this protein (SEQ
ID NO.2) followed by a GluArgProSerPheVal linker was amplified by PCR using the
human foetal heart cDNA library (ref. HL5042T, Clontech). This amplified fragment
was then cloned into the eukaryotic expression vector pXL4904 (described in Figure
1) such that the two Ig domains of Robol are expressed as a fusion with the Fc
domain of human lgG4 in the C-terminal position. Three point mutations were
introduced into the lgG4-Fc domain in order to obtain the following characteristics:
S241P (Kabat numbering) in order to stabilize the disulphide-bridge bonding in the
Fc hinge region; L248E in order to eliminate the residual effector activity of the lgG4-
Fc domain; absence of the C-terminal lysine in order to reduce the heterogeneity of
the protein. The cDNA sequence used to express this recombinant protein
corresponds to the sequence SEQ ID NO.3 . The recombinant protein obtained is
called Robo1-Fc L 1 and corresponds to the protein sequence SEQ ID NO.4 .
In order to obtain the Robol -Fc L2 construct, the same cDNA corresponding to the
Ig1-lg2 domains but without the linker mentioned was cloned into the eukaryotic
expression vector pXL4909 (described in Figure 1), which allows the expression of
these Ig domains as a fusion with the same Fc domain of lgG4 containing the 3 point
mutations described in the construction of Robol -Fc L1, but this time introducing a
GlyGlyGlyGlySer linker upstream of the Fc domain. The cDNA sequence used to
express this recombinant protein corresponds to the sequence SEQ ID NO.5 . The
recombinant protein obtained is called Robol -Fc L2 and corresponds to the protein
sequence SEQ ID NO.6.
b. Construction of the Robo1-Fc proteins used as controls
In order to obtain the Robo1-Fc Slit2-minus construct, the cDNA previously cloned
into the pXL4904 plasmid was modified by PCR so as to introduce the point
mutations allowing the substitutions of the leucine at position 38 to glutamine and the
phenylalanine at position 89 to tyrosine. The cDNA sequence used to express this
recombinant protein corresponds to the sequence SEQ ID NO.7 . The recombinant
protein obtained is called Robo1-Fc Slit2-minus and corresponds to the protein
sequence SEQ ID NO.8 .
In order to obtain the Robo1-Fc heparin-minus construct, the cDNA cloned into the
pXL4904 plasmid was modified by PCR so as to introduce the point mutations
allowing the substitutions of the arginine at position 97 to aniline and the lysine at
position 98 to alanine. The cDNA sequence used to express this recombinant protein
corresponds to the sequence SEQ ID NO.9 . The recombinant protein obtained is
called Robo1-Fc heparin-minus and corresponds to the protein sequence
SEQ ID NO. 10.
c . Production of the various Robo1-Fc proteins
The two proteins Robo1-Fc L 1 and Robo1-Fc L2 were produced by transient
transfection in the HEK293 line (Freestyle 293-F cells ref 51-0029, Invitrogen,
according to the supplier's recommendations) using the pXL4904 and pXL4909
plasmids, respectively, and the helper plasmids pXL4544 and pXL4551 allowing the
expression of two N-glycan glycosylation enzymes, i.e. a-2,3-sialyltransferase and
-1 as has been described in application WO2008/065543.
These proteins were also produced by transfection in the CHO line (CHO-S cells, ref
11619-012, Invitrogen, according to the supplier's recommendations) using the
pXL4904 and pXL4909 plasmids, respectively.
The Robo1-Fc L 1 and Robo1-Fc L2 proteins expressed in the culture natant of the
HEK293 cells were purified by chromatography on a protein A affinity column
(MabSelect ref. 17-5199-02, Amersham Biosciences) and elution in 20 mM NaCI /
100 mM acetic acid buffer, and then formulated in PBS buffer (ref. 14190-094,
Invitrogen).
The Robo1-Fc Slit2-minus and Robo1-Fc heparin-minus recombinant proteins were
produced and purified in the same way.
d . Physicochemical characterization of the Robo1-Fc recombinant proteins
SDS-PAGE and gel permeation analysis made it possible to show that the proteins
were dimeric and more than 96% pure. Mass spectrometry analysis made it possible
to demonstrate the identity of these proteins, the measured weight of the
deglycosylated protein being in perfect agreement with the weight calculated in
silico. Analysis of the monosaccharide composition and quantification of the
N-glycan sialic acids as described by Saddic et al. 2002. (Methods Mol. Biol. 194:
23-36 and Anumula et al. 1998. Glycobiology 8 : 685-694) made it possible to
demonstrate that the proteins were sialylated to a very great extent. The results are
given in table 1. It will be noted that the N-terminal analysis of the Robo1-Fc L 1 and
Robo1-Fc L2 molecules showed that these purified proteins contained a variable
proportion (0 to 40%) of a form with the first 2 residues (Ser20 and Arg21) truncated.
Table 1: Robo1-Fc protein identity
Example 2 : Preparation of the Slit proteins used as ligand
The cDNA encoding the human Slit2 protein corresponds to the reference protein
NP_004778. Fragments of this cDNA were amplified by PCR using the human brain
cDNA library (ref. 639300, Clontech).
The cDNA corresponding to the D2 domain was cloned into the eurkaryotic
expression vector pXL491 1 in order to express this domain containing a His tag in
the C-terminal position. The cDNA sequence used to express this recombinant
protein corresponds to the sequence SEQ ID NO.1 1. The recombinant protein
obtained is called Slit2-D2 and corresponds to the protein sequence SEQ ID NO.12.
The cDNA corresponding to the D1-D2 domains was cloned into the eukaryotic
expression vector pXL4912 in order to express these two domains containing a His
tag in the C-terminal position. The cDNA sequence used to express this recombinant
protein corresponds to the sequence SEQ ID NO. 13. The recombinant protein
obtained is called Slit2-D1 D2 and corresponds to the protein sequence SEQ ID
NO.14.
The cDNA corresponding to the extracellular part (Nt) of the Slit2 protein was cloned
into the eukaryotic expression vector pXL5033 in order to express this protein with a
His tag in the C-terminal position. The cDNA sequence used to express this
recombinant protein corresponds to the sequence SEQ ID NO. 15. The recombinant
protein obtained is called Slit2-N and corresponds to the protein sequence SEQ ID
NO.16.
The cDNA encoding the D2 domain of the murine Slit2 protein, and corresponding to
the D2 domain of the described reference protein NP_848919, was obtained from
the cDNA cloned into the pXL491 1 plasmid. Four fragments making it possible to
generate the five point mutations were generated by PCR with pXL491 1 as template,
and then these fragments were used as template to amplify the cDNA encoding the
complete D2 domain by sequential PCR. The protein carries the Thr31 1Ser,
Lys313Arg, Ne329Leu, e4 1 1Val and Pro418Ala mutations allowing it to be identical
to the D2 domain of the murine Slit2 protein. This plasmid makes it possible to
express the D2 domain of the murine Slit2 protein with a His tag in the C-terminal
position. The cDNA sequence used to express this recombinant protein corresponds
to the sequence SEQ ID NO. 17. The recombinant protein obtained is called mSlit2-
D2 and corresponds to the protein sequence SEQ ID NO. 18.
The cDNA encoding the human Slit1 protein corresponds to the described reference
protein NP_003052. A fragment of this cDNA was amplified by PCR using the
human brain cDNA library (ref. 639300, Clontech). The cDNA corresponding to the
D2 domain was cloned into the eukaryotic expression vector pXL5020 in order to
express this domain containing a His tag in the C-terminal position. The cDNA
sequence used to express this recombinant protein corresponds to the sequence
SEQ ID NO. 19. The recombinant protein obtained is called Slit1-D2 and corresponds
to the protein sequence SEQ ID NO.20.
The cDNA encoding the human Slit3 protein corresponds to the described reference
protein NP_003053. A fragment of this cDNA was amplified by PCR using the
human brain cDNA library (ref. 639300, Clontech). The cDNA corresponding to the
D2 domain was cloned into the eukaryotic expression vector pXL5021 in order to
express this domain containing a His tag in the C-terminal position. The cDNA
sequence used to express this recombinant protein corresponds to the sequence
SEQ ID NO.21. The recombinant protein obtained is called Slit3-D2 and corresponds
to the protein sequence SEQ ID NO.22.
The three proteins called Slit2-D2, Slit2-D1 D2 and Slit2-N were produced by
transient transfection in the HEK293 line (Freestyle 293-F cells according to the
supplier's recommendations, Invitrogen) using the pXL491 1 plasmid (respectively,
pXL.4912 or pXL5033).
The Slit2-D2 and Slit2-D1D2 proteins expressed in the culture supernatant of the
HEK293 cells were purified by chromatography on an Ni-chelating sepharose
column (ref. 17-0409-03, Amersham Biosciences) and elution in imidazole buffer and
then formulated in PBS buffer (ref. 14190-094, Invitrogen) adjusted to 1M NaCI.
Mass spectrometry analysis (LC/MS) made it possible to demonstrate the identity of
these proteins.
The three proteins called mSlit2-D2, Slit1-D2 and Slit3-D2 were produced, purified
and characterized in a comparable manner.
Example 3 : Study of the affinity of the Robo1-Fc recombinant proteins for the
Slit proteins and for heparin by means of three methods: ELISA, SPR and
FACS
a . Affinity of the Robo1-Fc proteins for heparin
In order to determine the affinity of the Robo1-Fc constructs for heparin, 2 mg of
Robo1-Fc protein, purified and formulated in 10 mM phosphate, pH 7.0, were
chromatographed on a heparin column ( 1 ml_ HiTrap Heparin-Sepharose HP, GE
Heathcare) by elution with a linear gradient of NaCI of from 0 to 1.5 M.
Table 2 indicates the NaCI concentration of 448 mM necessary for eluting the
Robo1-Fc L 1 protein as described in the literature (Fukuhara, N. et al. 2008 J. Biol.
Chem. 283 p16226- 16234).
Table 2 : Affinity of Robo1-Fc for heparin
These results show that the Robo1-Fc heparin-minus protein is not retained on this
column, and therefore that it no longer has any affinity for heparin. This protein is
therefore a heparin-negative mutant.
b. Evaluation of the interaction of the Robo1-Fc protein variants with the D2
domain of the human Slit2 protein
This example describes the interaction of the two variants called Robo1-Fc L 1 and
Robo1-Fc L2 with their natural ligand (in these experiments, the D2 domain of
human Slit2) by ELISA assay.
The human Slit2-D2 protein was bound to lmmulon-4 enzyme-linked plates (VWR
Scientific Inc. Swedesboro, NJ). A concentration range (from 20 mg/mL to
0.02 mg/mL) of the Robo1-Fc L 1 and Robo1-Fc L2 variants was added and then
detected by means of the peroxidase-conjugated goat anti-human IgG antibody
(Sigma; ref. A0170, dilution to 1:50 000). Visualization was carried out with the TMBH
20 2 substrate (Interchim; ref UP664780) and the measurements were carried out
with a plate reader at 450 nm. The results are reported in Figure 2 . They show that
the two variants Robo1-Fc L 1 and L2 specifically interact with the human Slit2
protein (in particular with the D2 domain).
c . Evaluation of the interaction of the Robo1-Fc protein with the human
variants of the Slit family, namely Slit1 and Slit3
This example describes the interaction of the Robo1-Fc L 1 fusion protein with the
Slit1-D2, Slit2-D2 and Slit3-D2 variants by ELISA assay.
The D2 domain of the human Slit variants was bound to lmmulon-4 enzyme-linked
plates (VWR Scientific Inc. Swedesboro, NJ). A concentration range (from 1 mg/mL
to 0.001 mg/mL) of the Robo1-Fc L 1 fusion protein was added and then detected
using the peroxidase-conjugated goat anti-human IgG antibody (Sigma; ref. A0170,
dilution to 1:50 000). Visualization was carried out with the TMB-H20 2 substrate
(Interchim; ref UP664780) and the measurements were carried out with a plate
reader at 450 nm. Similarly, the Robo1-Fc Slit2-minus variant, which is mutated at
the level of the Slit2-binding site, was evaluated according to a concentration range
(from 20 mg/mL to 0.02 mg/mL) by ELISA assay under the same conditions described
above. The results are reported in table 3 below.
Table 3 : Affinity of Robo1-Fc for the human variants of the Slit protein
These results show that the Robo1-Fc protein interacts specifically with the three
proteins of the family, Slit1, Slit2 and Slit3 (in particular with their D2 domain).
In addition, the Robo1-Fc Slit2-minus protein no longer has affinity for heparin and it
is therefore a heparin-negative mutant.
d . Evaluation of the interaction of the Robo1-Fc protein with the murine
Slit2 protein
This example describes the interaction of the Robo1-Fc L 1 fusion protein with the
murine protein mSlit2-D2 by ELISA assay.
The murine protein mSlit2-D2 was bound to an lmmulon-4 enzyme-linked plate
(VWR Scientific Inc. Swedesboro, NJ). A concentration range (from 2 mg/mL to
0.002 m -.) of the Robo1-Fc L 1 fusion protein was added and then detected using
the peroxidase-conjugated goat anti-human IgG antibody (Sigma; ref. A0170, dilution
to 1:50 000). Visualization was carried out with the TMB-H20 2 substrate (Interchim;
ref UP664780) and the measurements were carried out with a plate reader at
450 nm. The results are reported in table 4 below.
Table 4 : Affinity of Robo1-Fc L 1 for the murine Slit2 protein
These results show that the Robo1-Fc protein interacts specifically with the murine
Slit2 protein
e. Affinity of Robo1-Fc for the Slit protein measured by SPR
This example describes the determination of the affinity constant of the Robo1-Fc L 1
fusion protein with the human Slit2 protein (in this experiment, Slit2-D2) by SPR
(Surface Plasmon Resonance; BIAcore 2000). The interaction between the Robol-
Fc protein and the human Slit2 protein was analysed after having bound the Robol-
Fc fusion protein to a CM5 chip. The kinetic measurements were carried out
according to the protocol of Canziani et al. (2004. Anal. Biochem. 325: 301-307).
Table 5 : Affinity constant of Robo-Fc L 1 with human Slit2-D2 by SPR (steady-state
analysis)
A second method, which consists in determining the affinity constant between the
Robo1-Fc L 1 fusion protein and the human Slit2 protein, was analysed after having
bound the Slit2-D2 protein to the CM5 chip. The kinetic measurements are carried
out according to the protocol of Canziani et al. (2004. Anal. Biochem. 325: 301-307)
using the Scatchard method according to a model with two non-equivalent binding
sites.
Table 6 : Affinity constant of Robo1-Fc with human Slit2-D2 by SPR according to the
two-phase Scatchard model
f . Affinity of Robo1-Fc for Slit measured by FACS
This example describes the affinity of the Robo1-Fc protein on HEK293 mammalian
cells expressing Slit2.
The HEK293 cells described and used as in example 1 were transfected either with
a ballast plasmid, having no cDNA that encodes in the mammalian cell, or the
pXL491 1 plasmid encoding the Slit2-D2 protein, or the pXL4912 plasmid encoding
the Slit2-D1D2 protein, or pXL5033 encoding the Slit2-N protein described in
example 2 . The cells were distributed into 96-well plates 48 hours post-transfection,
and the Robo1-Fc protein was added in a concentration range of from 0.01 to
3Dmg/L for 30 min at 4°C. The Robo1-Fc protein is either the Robo1-Fc L 1
biotherapeutic agent, or the Robo1-Fc Slit2-minus mutant, or the Robo1-Fc heparinminus
mutant. The cells were washed and the anti-human Fc antibody labelled with
Alexa 488 (ref: A-1 1013, Invitrogen) was incubated for 30 min at 4°C. The labelled
HEK293 cells are then quantified by FACS (Geomean).
Figure 3 describes the binding of the HEK293 cells to the Robo1-Fc protein, via the
fluorescence measured by the FACS in the absence of Slit2 expression. The Robol-
Fc protein and also the Robo1-Fc Slit2-minus mutant bind to the HEK293 cells,
whereas the Robo1-Fc heparin-minus mutant does not bind. Robo1-Fc therefore
binds partly to the HEK293 cells via heparin binding at the low Robo1-Fc
concentrations of 0.3 to 0.03 mg/L.
Figure 4 describes the binding of the HEK293 cells to the Robo1-Fc protein, via the
fluorescence measured by FACS when Slit2-N is expressed by transient
transfection. Only the Robo1-Fc protein binds to the HEK293 cells expressing Slit2-
N. The Robo1-Fc Slit2-minus and Robo1-Fc heparin-minus mutants do not bind (or
virtually not) in the Robo1-Fc concentration range of 3.0 to 0.3 mg/L, compared with
the biotherapeutic Robo1-Fc L 1 protein.
Table 7 describes the affinity constants measured by FACS for the Robo1-Fc protein
when the Slit2-N, Slit2-D1 D2 or Slit2-D2 proteins are expressed in the HEK293 line.
Table 7 : Affinity of Robo1-Fc for the Slit2 protein on cells by FACS
As in the previous examples, the Robo1-Fc Slit2-minus mutant proved to be Slit2-
negative and the Robo1-Fc heparin-minus mutant has a weaker affinity for Slit2 than
the biotherapeutic protein.
Robo1-Fc binds to Slit2-N and Slit2-D1 D2 expressed by the HEK293 cells with
comparable affinities which are better than the affinity with Slit2-D2.
Example 4 : Pharmacokinetic properties of the Robo1-Fc L 1 and Robo1-Fc L2
proteins
This example describes the pharmacokinetic profile and the plasma concentration
of the Robo1-Fc protein injected once in mice intravenously (iv).
Three Balb/C mice (for each time) were injected, via the caudal vein, with each of
the Robo1-Fc proteins at 2.5 mg/mL in a proportion of 100 m I_/10 g (~ 25 mg/kg). At
the predetermined times (0.5, 1, 6 , 24, 48 and 72 h after administration), the mice
were anaesthetized, and blood was sampled and collected in a tube containing
10 m I_ of citrate (CPD-A, C-4431 Sigma) and 2 m I_ of protease inhibitors (Complete
Protease Inhibitor Mix, Roche Molecular Biochemical). The tubes were centrifuged
and the plasma samples were collected and frozen at -20°C.
The wells of 96-well plates were coated with the Slit2 protein (Slit2-D2), and the
plasma samples, diluted to 1/5000 and 1/20 000, were brought into contact for one
hour at 37°C. The peroxidase-conjugated anti-human Fc antibody (ref. 31413,
Pierce) was subsequently incubated and then visualized with TMB-H20 2 (ref
UP664780, Interchim) and the absorbance was read at 450 nm. A calibration range
was prepared with each purified Robo1-Fc protein.
The plasma concentrations of the Robo1-Fc L 1 and Robo1-Fc L2 proteins are
represented in Figure 5 . The pharmacokinetic parameters are described in the
following table 8 and show that the protein is stable after injection in mice.
Table 8 : Pharmacokinetic parameters of the Robo1-Fc proteins after iv injection in
mice
Example 5 : Description of the Robo1-Fc biotherapeutic protein improved with
respect to its homogeneity in the N-terminal position
This example describes the expression plasmid, the production and the
physicochemical characterization of another Robo1-Fc protein, called Robo1-Fc L3,
which is different from the Robo1-Fc L 1 protein by virtue of the absence of the first
two residues Ser20 and Arg21.
The cDNA cloned into the pXL4904 plasmid was modified by PCR in order to
eliminate the Ser20 and Arg21 codons, and to fuse the next codon (Leu22) to the
coding sequence of the peptide signal of interleukin 2 . The pXL5004 expression
plasmid was then generated, see Figure 1. The cDNA sequence used to express this
recombinant protein corresponds to the sequence SEQ ID NO.23.
The Robo1-Fc L3 protein was produced, purified and characterized as described in
example 1. The N-terminal analysis showed that this purified protein was perfectly
homogeneous. The recombinant protein obtained is called Robo1-Fc L3 and
corresponds to the protein sequence SEQ ID NO.24.
Example 6 : Evaluation of the interaction of the Robo1-Fc L3 protein with the
human Slit2 protein
This example describes the interaction of the Robo1-Fc L3 fusion protein with the
human Slit2 protein (Slit2-D2) by ELISA assay.
The human Slit2-D2 protein was bound to lmmulon-4 enzyme-linked plates (VWR
Scientific Inc. Swedesboro, NJ). A concentration range (from 1 mg/mL to
0.001 mg/mL) of the Robo1-Fc L3 fusion protein was added and then detected by
means of the peroxidase-conjugated goat anti-human IgG antibody (Sigma; ref.
A0170, dilution to 1:50 000). The visualization was carried out with the TMB-H20 2
substrate (Interchim; ref UP664780) and the measurements were carried out with a
plate reader at 450 nm. The results obtained are reported in table 9 below.
Table 9 : Affinity of the Robo1-Fc L3 protein for the Slit2 protein - Comparison with
the Robo1-Fc L 1 protein
These results show that the affinities of the two variants Robo1-Fc L 1 and Robol-
Fc L3 for the Slit2-D2 protein are comparable.
Example 7 : Evaluation of the activity of the Robo-Fc protein on
neovascularization
a . In vitro endothelial cell and fibroblast coculture model: specific activity of
the Robo1-Fc L 1 molecule
The in vitro angiogenesis model corresponds to a rearrangement of human vein
endothelial cells on a monolayer of human dermal fibroblasts. Briefly, the fibroblasts
(Lonza) are seeded into 24-well plates (Becton Dickinson) at 40 000 cells/well in
1 ml of medium. After 3 days of proliferation (D3), human vein endothelial cells
(HUVEC-Lonza) are seeded onto the fibroblast cell monolayer at 10 000 cells/well
in 500 m I of EGM® medium (endothelial basal medium, Lonza) + 2% FCS (foetal calf
serum - Lonza) + 10 mg/ml hEGF (recombinant human epidermal growth factor -
Lonza). The cells are stimulated with 30 ng/mL of VEGF (R&D Systems), with the
Robo1-Fc L 1 molecule or with a Robo1-Fc Slit2-minus negative control at the
concentration of 500 mg/ml (D3 to D9). After 3 days, the medium is replaced and the
wells are stimulated according to the conditions of the experiment.
After 2 days, the cells are fixed with ethanol and labelled with an anti-CD31
antibody specific for HUVECs, followed by an anti-alkaline phosphatase antibody,
and then visualized with an alkaline phosphatase substrate (D1 1). A quantification
of the tubules labelled with the anti-CD31 antibody is carried out by means of image
acquisitions made under a microscope (x4 objective) and the length of the
pseudotubules is analysed using image analysis software (Biocom Visiolab 2000
software) (Figure 6).
In this in vitro angiogenesis assay, Robo1-Fc L 1 (500 mg/ml) shows an inhibitory
activity on the formation of the tubules formed by the HUVECs. This inhibition
amounts to 82% and it is statistically significant compared with the effect obtained
with the Robo1-Fc Slit2-minus molecule (negative control).
b. Ex vivo mouse aortic ring model: specific activity of the Robo1-Fc L 1
molecule
The Robo1-Fc L 1 molecule was evaluated in a mouse aortic ring model. Briefly,
mouse aortas are removed and cleaned, and cut into sections of 1 mm (DO). These
rings are embedded in rat collagen in the presence of VEGF at 10 ng/ml, of the
Robo1-Fc L 1 molecule at the concentration of 500 mg/ml or of a Robo1-Fc Slit2-
minus negative control at the concentration of 500 mg/ml. Tubules will form from the
ring, thus mimicking in vitro, the formation of neovessels. After 6 days, a
quantification of the labelled tubules is carried out by means of image acquisitions
made under a microscope (x3 objective) (Figure 7A) and the length of the
pseudotubules is analysed using image analysis software (Biocom Visiolab
software 2000) (Figure 7).
Under these experimental conditions, Robo1-Fc L 1 (500 mg/ml) shows a strong
inhibitory activity on the formation of the tubules formed, in comparison with the
Robo1-Fc Slit2-minus molecule used as a negative control.
These results suggest that Robo1-Fc L 1 is capable of inhibiting the formation of
neovessels without inhibiting endothelial cell proliferation. This anti-angiogenic
activity linked to a vessel maturation defect is called "non-productive angiogenesis".
Example 8 : Evaluation of the Robo1-Fc L1 protein in a lung tumour model in
mice
The Robo1-Fc L 1 molecule was evaluated in a model of a lung cancer tumour in
C57/BI6 mice.
a . Murine lung tumour model
In order to establish the murine lung tumour model, 8-week-old female C57/BI6
mice were anaesthetized. The area at the level of the left scapula of the mouse was
shaved and disinfected. A 1 cm incision was made above the scapula.
The cells to be injected are derived from a Lewis lung carcinoma tumour line
(ATCC, CRL-1642). The cells were mixed with Matrigel® in a ratio of 1 vol of
Matrigel to 4 vol of cells. The cell concentration was 62 500 cells/ml. The cells were
injected into the lung at a rate of 20 m I per mouse, and then the wound was sutured.
After 23 days, the mice were euthanized. The ribcage was opened up, and the left
lung and the mediastinal lymph nodes were removed. The tumour present on the
left lung was measured using an electronic calliper rule in order to determine the
tumour volume according to the formula: I2XLX0.52. The mediastinal lymph nodes
are weighed. The results are expressed as mean value ± standard deviation from
the mean. The statistical analysis was carried out by means of a parametric
Student's test.
b. Treatment of the mice bearing a lung tumour with the Robo1-Fc
recombinant protein
The treatment using the Robo1-Fc protein was carried out as follows: a preparation
containing the Robo1-Fc protein was injected at the dose of 25 mg/kg/day,
intravenously, on D10, D14, D17 and D21 post-injection of the tumour cells. The
control group was injected with PBS buffer (10 ml/kg).
c . Results
On D23, the mean volume of the tumours obtained in the group treated with the
Robo1-Fc recombinant protein was 2 1.45 ± 2.16 mm3; the mean volume of the
tumours obtained in the control group was 39.93 ± 8.41 mm3. The reduction in
tumour volume in the animals treated with the Robo1-Fc protein is 46%. This
difference is statistically significant (p<0.05). The mean weight of the mediastinal
lymph nodes (metastatic lymph nodes) obtained in the group treated with the
Robo1-Fc protein is 12.50 ± 1.26 mg. The mean weight of the mediastinal lymph
nodes obtained in the control group is 30.67 ± 7.69 mg. The reduction in weight of
the mediastinal lymph nodes for the group treated with the Robo1-Fc protein is 59%
at the limit of significance (p=0.07).
Example 9 : Evaluation of the Robo1-Fc L 1 protein in two models of human
hepatocarcinoma
The Robo1-Fc L 1 molecule was evaluated in two models of human
hepatocarcinoma in SCID mice.
A. Orthotopic model of human hepatocarcinoma in SCID mice using HepG2
cell line
a . Description of the model
Female SCID mice are anaesthetized (with a mixture of ketamine/xylazine) and the
area of the incision is shaved. The skin is cleaned and disinfected before making a
1 cm subcostal incision in the skin and in the muscle wall. A part of the lobe of the
liver is carefully extracted from the peritoneum in order to carry out the injection of
the cells. A 50 m I volume of the cell suspension (HepG2 originating from ATCC,
human hepatoblastoma cells mixed with Matrigel ® at 40 106 cells/ml) is injected
into the left lobe. The lobe is put back into the abdominal cavity. The peritoneum is
closed with surgical glue and then the skin is sutured with staples. The mice are
treated with Robo1-Fc solubilized in PBS at 25 mg/kg, i.p. injection twice a week.
The treatment begins two weeks after the implantation of the cells. Four weeks after
inoculation with the cells, the animals are sacrificed for autopsy.
b. Treatment of the mice bearing a human hepatocarcinoma with the
Robo1-Fc recombinant protein
Robo1-Fc was evaluated in this model with a treatment twice a week at the dose of
25 mg/kg. The weight of the mice is measured on the first and last day of the
protocol. The weight of the tumours was measured 28 days after the injection of the
cells, after sacrificing the animals
c . Results
The tumour growth leads to a considerable loss of weight by the control animals
during the protocol, whereas the animals treated with Robo1-Fc have an identical
weight at the end of the experiment (Table 10). At the end of the experiment, after
4 i.p. injections of Robo1-Fc, the mean tumour weight of the Robo-1-Fc-treated
group is significantly reduced by 30% (p < 0.05) in comparison with the control
group having received injections of solvent (Figure 8).
Table 10: Weight of animals at the beginning and end of the protocol
Mean weight of mice (g)
Mean
(+/- standard deviation of the
DO D28
mean)
Control (n=1 1) 19.24 ± 0.48 16.46 ± 0.54
Robo1-Fc (n=13) 19.17 ± 0.31 19.07 ± 0.63
B. Orthotopic model of human hepatocarcinoma in SCID mice using Hep3B
cell line
a . Description of the model
In this HCC model, Hep3B cell line was used. This tumor cell line is a commercial
cell line (from ATCC) coming from an hepatoblastoma of a young patient. The same
protocol of cell injection was used as previously described in example 9 A a). The
initial concentration of cells is 2.106 in an injected volume of 50m I.
b.Treatment of mice bearing Hep3B tumors with Robo1-Fc
Robo1-Fc was evaluated in this model by a treatment twice a week at 5 mg/kg. The
mice were weighted before treatment and each time before the next treatment in
order to adjust the dosage with the injected volume. The mice were also weighted
the last day of the protocol, before sacrifice. The lobe with the tumor was measured
after sacrifice on the last day of the protocol. Furthermore, before sacrifice, the
blood of mice were withdrawn and the serum was discarded for afetoprotein (AFP)
determination; AFP is secreted by the cancer liver cells and it is currently accepted
as a biomarker of HCC development in humans (Fimus et al., 2004, Mol
Diagn8(4):207-12. Review).
c . Results
The mice did not gain weight during the tumoral development for 35 days. The
treatment of Robo1-Fc at 5 mg/kg showed no effect on the mice weight (Table 11).
At the end of the experiment, the mean tumoral weight of the control group was 895
±156 mg. the mean tumoral weight of the Robo1-Fc treated group was 483±55 mg
(Figure 9). Treatment with Robo1-Fc induced a significant reduction of the tumor
burden of 46%.
Furthermore the plasmatic AFP concentration was 316 ± 73 mg/ml in control tumor
bearing mice and 139 ± 28 mg/ml in Robo1-Fc treated mice, showing a 56 %
inhibition of plasmatic AFP in treated animals (Figure 10). This inhibition of
plasmatic AFP correlated well with the anti tumoral activity of Robo1-Fc.
Table 11 : Mice weight at the beginning and at the end of the protocol
Mice weight
DO D35
Mean +/- sem
Control group (n=14) 19.08 ± 0.30 19.12 ± 0.76
Robo1-Fc group (n=14) 19.36 ± 0.36 20.37 ± 0.31
Claims
1. Recombinant protein comprising the extracellular domain derived from
Robol protein or a part of this domain, for use in the treatment of cancer.
2. Recombinant protein for use according to Claim 1, wherein said cancer is
hepatocarcinoma.
3. Recombinant protein for use according to either one of Claims 1 or 2 ,
which does not inhibit endothelial cell proliferation.
4. Recombinant protein for use according to any one of Claims 1 to 3 ,
wherein said extracellular domain is an extracellular domain derived from
isoform b of the Robol protein.
5. Recombinant protein for use according to Claim 4 , in which the
extracellular domain of Robol or part thereof consists in the Ig1 and Ig2
immunoglobulin domains of said extracellular domain.
6. Recombinant protein for use according to Claim 5 , in which the
extracellular domain of Robol has at least 80% identity with the sequence
SEQ ID NO.2.
7. Recombinant protein for use according to any one of the preceding
Claims, wherein said protein further comprises a linker and an
immunoglobulin Fc domain.
8. Recombinant protein for use according to Claim 7 , in which the Fc domain
comes from human immunoglobulin G4.
9. Recombinant protein for use according to Claim 8 , in which the Fc domain
contains the S241 P and L248E mutations and in which the lysine located
in the C-terminal position is absent.
10. Recombinant protein for use according any one of the preceding Claims,
the sequence of which has at least 80% identity with the sequence SEQ
ID NO. 4 , SEQ ID NO.6 or SEQ ID NO. 24.
11. Recombinant protein for use according any one of the preceding Claims,
the sequence of which is the sequence SEQ ID NO. 4 , SEQ ID NO.6 or
SEQ ID NO. 24.