ORIGINAL
NOVEL ANTI-IGF-IR ANTIBODIES AND USES THEREOF
The present invention relates to novel antibodies capable of binding specifically
to the human insulin-like growth factor I receptor IGF-IR andlor capable of specifically
5 inhibiting the tyrosine kinase activity of said IGF-IR, especially monoclonal antibodies
of murine, chimeric and humanized origin, as well as the amino acid and nucleic acid
sequences coding for these antibodies. The invention likewise comprises the use of
these antibodies as a medicament for the prophylactic andfor therapeutic treatment of
cancers overexpressing IGF-IR or any pathology connected with the overexpression of
1 o said receptor as well as in processes or kits for diagnosis of illnesses connected with the
overexpression of the IGF-IR. The invention finally comprises products andlor
compositions comprising such antibodies in combination with anti-EGFR antibodies
andlor anti-VEGF antibodies andlor antibodies directed against other growth factors
involved in tumor progression or metastasis andor compounds and/or anti-cancer
15 agents or agents conjugated with toxins and their use for the prevention andlor the
treatment of certain cancers.
The insulin-like growth factor I receptor called IGF-IR is a well described
receptor with tyrosine kinase activity having 70% homology with the insulin receptor IR.
IGF-IR is a glycoprotein of molecular weight approximately 350,000.
20 It is a hetero-tetrarneric receptor of which each half-linked by disulfide bridges is
composed of an extracellular a-subunit and of a transmembrane &subunit. IGF-IR binds
IGFl and IGF2 with a very high affinity (Kd #I nM) but is equally capable of binding to
insulin with an affinity 100 to 1000 times less. Conversely, the IR binds insulin with a
very high affmity although the IGFs only bind to the insulin receptor with a 100 times
2 5 lower affinity. The tyrosine kinase domain of IGF-IR and of IR has a very high sequence
homology although the zones of weaker homology respectively concern the cysteinerich
region situated on the a-subunit and the C-terminal part of the @-subunit. The
sequence differences observed in the a-subunit are situated in the binding zone of the
ligands and are therefore at the origin of the relative -ties of IGF-IR and of IR for
3 0 the IGFs and insulin respectively. The differences in the C-terminal part of the &subunit
result in a divergence in the signalling pathways of the two receptors; IGF-IR mediating
mitogenic, differentiation and anti-apoptosis effects, while the activation of the IR
principally involves effects at the level of the metabolic pathways (Baserga et al.,
Biochim. Biophys. Acta, 1332:F105-126, 1997; Baserga R, Exp. Cell. Res., 253:l-6,
1999).
The cytoplasmic tyrosine kinase proteins are activated by the binding of the
5 ligand to the extracellular domain of the receptor. The activation of the kinases in its
turn involves the stimulation of different intra-cellular substrates, including IRS-1, IRS-
2, Shc and Grb 10 (Peruzzi F. et al., J. Cancer Res. Clin. Oncol., 125:166-173, 1999).
The two major substrates of IGF-IR are IRS and Shc which mediate, by the activation of
numerous effectors downstream, the majority of the growth and differentiation effects
10 connected with the attachment of the IGFs to this receptor. The availability of substrates
can consequently dictate the final biological effect connected with the activation of the
IGF-IR. When IRS-1 predominates, the cells tend to proliferate and to transform. When
Shc dominates, the cells tend to differentiate (Valentinis B. et al., J. Biol. Chem.
274: 12423-12430, 1999). It seems that the route principally involved for the effects of
15 protection against apoptosis is the phosphatidyl-inositol 3-kinases (PI 3-kinases) route
(Prisco M. et al., Horm. Metab. Res., 31:80-89, 1999; Peruzzi I?. et al., J. Cancer Res.
Clin. Oncol., 125: 166- 173, 1999).
The role of the IGF system in carcinogenesis has become the subject of intensive
research in the last ten years. This interest followed the discovery of the fact that in
20 addition to its mitogenic and antiapoptosis properties, IGF-IR seems to be required for
the establishment and the maintenance of a transformed phenotype. In fact, it has been
well established that an overexpression or a constitutive activation of IGF-IR leads, in a
great variety of cells, to a growth of the cells independent of the support in media
devoid of fetal calf serum, and to the formation of tumors in nude mice. This in itself is
25 not a unique property since a great variety of products of overexpressed gen& can
transform cells, including a good number of receptors of growth factors. However, the
crucial discovery which has clearly demonstrated the major role played by IGF-IR in
the transformation has been the demonstration that the R- cells, in which the gene
coding for IGF-IR has been inactivated, are totally refractory to transformation by
30 different agents which are usually capable of transforming the cells, such as the E5
protein of bovine papilloma virus, an overexpression of EGFR or of PDGFR, the T
antigen of SV 40, activated ras or the combination of these two last factors (Sell C. et
al., Proc. Natl. Acad. Sci., USA, 90: 11217-1 1221, 1993; Sell C. et al., Mol. Cell. Biol.,
14:3604-3612, 1994; Morrione A. J., Virol., 695300-5303, 1995; Coppola D. et al.,
Mol. Cell. Biol., 14:4588-4595, 1994; DeAngelis T. et al., J. Cell. Physiol., 164:214-
221,1995).
5 IGF-IR is expressed in a great variety of tumors and of tumor lines and the IGFs
amplify the tumor growth via their attachment to IGF-IR. Other arguments in favor of
the role of IGF-IR in carcinogenesis come from studies using murine monoclonal
antibodies directed against the receptor or using negative dominants of IGF-R. In
effect, murine monoclonal antibodies directed against IGF-IR inhibit the proliferation of
10 numerous cell lines in culture and the growth of tumor cells in vivo (Arteaga C. et al.,
Cancer Res., 49:6237-6241, 1989; Li et al., Biochem. Biophys. Res. Corn., 196:92-98,
1993; Zia I?. et al., J. Cell. Biol., 24:269-275, 1996; Scotlandi K. et al., Cancer Res.,
5834127-41 3 1,1998). It has likewise been shown in the works of Jiang et al. (Oncogene,
18:6071-6077, 1999) that a negative dominant of IGF-IR is capable of inhibiting tumor
15 proliferation.
Cancer pathologies are characterized by an uncontrolled cellular growth. In
several cancer, growth factors are specifically binding with their receptors and then
transmit growth, transformation andlor survival signals to the tumoral cell. The growth
factor receptors over-expression at the tumoral cell surface is largely described
2 0 (Salomon D.S. et al., Crit. Rev. Oncol. Hematol., 1995, 19:183; Burrow S. et al., J.
Surg. Oncol., 1998, 69:21; Hakam A. et al., Hum. Pathol., 1999, 30:1128; Railo M.J. et . .
al., Eur. J. Cancer, 1994, 30:307; Happerfield L.C. et al., J. Pathol., 1997, 183:412).
This over-expression, or abnormal activation, leading to a direct perturbation of cellular
growth regulation mechanisms, can also affect the cell sensibility to induced apoptose
2 5 by classical chemotherapies or radiotherapies.
During last few years, it has been shown that the targeting of growth factor
receptors, like EGFR or Her2lneu over-expressed on the tumoral cell surface, with
respectively humanized (hercepW) or chimeric (C225) antibodies results. in an
significant inhibition of the tumoral growth in patients and in a significant increase of
30 the efficacity of classical chemotherapy treatments (Carter P., Nature Rev. Cancer,
2001, 1(2):118; Hortobagyi G. N., Semin. Oncol., 2001, 28:43; Herbst R S. et al.,
Semin. Oncol., 2002, 29:27). Other receptors like IGF-IR or VEGF-R (for vascular
endothelial growth factor receptor) have been identified as potential target in several
preclinical studies.
More particularly, IGF-IR is part of the tyrosine kinase receptors. It shows a
high homology with the Insulin receptor (17) which exist under two isoforms A and B.
5 Sequences of isoforms A and B, are registered under Accession Numbers
X02160 and M10051, respectively, in the NCBI Genbank. Other data, without
limitations, relating to IR are incorporated herein by references (Vinten et al., 1991,
Proc. Natl. Acad. Sci. USA, 88:249-252; Belfiore et al., 2002, The Journal of Biological
Chemistry, 277:39684-39695; Dumesic et al., 2004, The Journal of Endocrinology &
10 Metabolism,89(7):3561'-3566).
The IGF-IR and IR are tetrameric glycoproteins composed of two extracellular
a- and two transmembrane P-subunits linked by disulfide bonds. Each a-subunit,
containing the ligand-binding site is approximately 130- to 135-kDa; whereas each gsubunit
containing the tyrosine kinase domain is approximately 90- to 95- kDa These
15 receptors share more than 50% overall amino acid sequence similarity and 84%
similarity in the tyrosine kinase domain. After ligand binding, phosphorylated receptors
recruit and phosphorylate docking proteins, including the insulin receptor substrate-1
protein family (IRSl), Gab1 and Shc (Avruch, 1998, Mol.Cell. Biochem., 182, 31-48;
Roth et al., 1988, Cold Spring Harbor Symp. Quant. Biol. 53, 537-543; White, 1998,
20 Mol. Cell. Biochem., 182, 3-11; Laviola et al., 1997, 1. Clin. Invest. 99, 830-837;
Cheatham et al., 1995, Endocr. Rev. 16, 117-142), leading to the activation of dierent
intracellular mediators. Although both the IR and IGF-IR similarly activate major
signalling pathways, differences exist in the recruitment of certain docking proteins and
intracellular mediators between both receptors (Sasaoka et al., 1996, Endocrinology
25 137, 4427-4434; Nakae et al., 2001, Endocr. Rev. 22, 818-835; ~ u ~ oanidt L e Roith
2001, Horm. Res. 55, Suppl. 2, 22-26; Koval et al., 1998, Biochem. J. 330, 923-932).
These differences are the basis for the predominant metabolic effects elicited by IR
activation and the predominant mitogenic, transforming and anti-apoptotic effects
elicited by IGF-IR activation @e Meyts et al., 1995, Ann. N.Y. Acad. Sci., 766, 388-
30 401; Singh et al., 2000, Prisco et al., 1999, Horm. Metab. Res. 31, 80-89; Kido et al.
2001, J. Clin. Endocrinol. Metab., 86, 972-979). Insulin binds with high amnity to the
IR (100-fold higher than to the IGF-IR), whereas insulin-like growth factors (IGF1 and
IGF2) bind to the IGF-IR with 100-fold higher affinity than to the IR.
The human IR exists in two isoforms, IR-A and IR-B, generated by alternative
splicing of the IR gene that either excludes or includes 12 amino acid residues encoded
by a small exon (exon 11) at the carboxy-terminus of the IR a-subunit. The relative
abundance of IR isoforms is regulated by tissue specific and unknown factors (Moller et
al., 1989, Mol. Endocrinol., 3, 1263-1269; Mosthaf et aL, 1990, EMBO J., 9, 2409-
2413). R-B is the predominant R isoform in normal adult tissues (adipose tissue, liver
and muscle) that are major target tissues for the metabolic effects of insulin (Moller et
al., 1989; Mosthaf et al., 1990). IR-A is the predominant isoform in fetal tissues and
mediates fetal growth in response to IGF2 (Frasca et al., 1999, Mol. Cell. Biol., 19,
3278-3288), as also suggested by genetic studies carried out in transgenic mice
(DeChiara et al., 1990, Nature 345, 78-80; Louvi et al., 1997, Dev. Biol. 189, 33-48).
Moreover, when cells transform and become malignant, dedifferentiation is often
associated with an increased IR-A relative abundance (Pandini et al., 2002, The Journal
of Biological Chemistry, Vol. 277, No 42, pp39684-39695).
Given the high degree of homology, the insulin and IGF-I half-receptors
(composed of one a- and one P-subunit) can heterodirnerize, leading to the formation of
insulin/IGF-I hybrid receptors (Hybrid-R) (Soos et al., 1990, Biochem J., 270,383-390;
Kasuya et al., 1993, Biochemistry 32, 1353 1-13536; Seely et al., 1995, Endocrinology
136,1635-1641; Bailyes et al., 1997, Biochem J. 327,209-215).
Both IR isoforms are equally able to form hybrids with IGF-IR. Hybrid-R,
however, have different finctional characteristics. Hybrid-RsB has reduced afEnity for
IGFl and especially for IGF2. In contrast, Hybrid-RsA has a high affinity for IGFl and
bind also IGF2 and insulin at a physiological concentration range. The expression of
Hybrid-RsA up-regdates the IGF system by two different mechanisms i) binding (with
high m t y ) and activation by both IGFl and IGF2 (which do not occur with the
Hybrid-RsB), ii) activation of the IGF-IR pathway after insulin binding. Insulin binding
to Hybrid-RsA phosphorylates the IGF-IR P-subunit and activates an IGF-IR-specific
substrate (CrMI) so that Hybrid-RsA shifts insulin to IGF-IR signaling (Pandini et al.,
2002).
In several tissues, like liver, spleen or placenta, Hybrid-R are more represented
than IGF-IR (Bailyes et al., 1997). As tumor tissues overexpress, or present an abnormal
activation, both IGF-IR and IR-A (Frasca et al., 1999; Sciacca et al., 1999, Oncogene
18,2471-2479; Vella et al., 2001, Mol. Pathol., 54, 121-124), Hybrid-RsA may also be
5 overexpressed in a variety of human malignancies, including thyroid and breast cancers
providing a selective growth advantage to malignant cells able to respond by a type
IGF-IR signalisation following a stimulation by IGFl andlor IGF2 but also by insulin at
physiological concentrations (Bailyes et al., 1997; Pandini et al., 1999, Clin. Cancer
Res., 5, 1935-1944; Belfiore et al., 1999, Biochimie (Paris) 81, 403-407; Frasca et al.
10 1999, Sciacca et al., 1999; Vella et al., 2001).
The realisation of such "therapeutic tools" able to block in the same time the two
receptors is of particular interest as they will allow to avoid the escape phenomena
mediated by the expression, or abnormal activation, in a same tumor of IGF-IR and
hybrid-R.
15 Regarding the increasing interest on IGF-IR and, more particularly, monoclonal
antibodies able to bind to, or inhibit the tyrosine kinase activity of, IGF-IR, the
applicants have already developed and characterized a humanized monoclonal antibody
called 7C10 or h7C10 (coded F50035). An international patent application
PCTER 03/00178 relating to this antibody and its uses have been filed and published on
2 0 24 July 2003 under the publication number WO 03/05995 1. The content of this patent
application is incorporated herein by reference.
The object of the present invention is to be able to have available other murine
monoclonal antibodies, preferably cherized or humanized antibodies, which will
recognize IGF-IR specifically and with great affinity. These antibodies will interact
2 5 little or not at all with the IR. Their attachment will be able to inhibit in vitro the growth
of tumors expressing IGF-IR by interacting principally with the signal transduction
pathways activated during IGFlIIGF-IR and IGM/IGF-IR interactions. These
antibodies will be able to be active in vivo on all the types of tumors expressing IGF-IR
including estrogen-dependent tumors of the breast and tumors of the prostate.
30 The present invention also allows to jointly block the hybrid-R and IGF-IR
activity by generating a compound, and more particularly antibodies, of high afhity
able to bind to said two receptors and also to block their activation by IGF1, IGF2 or
Insulin.
The present invention also deals with the use of isolated antibodies according to
the present invention, or a hgment thereof; said antibodies or fragment being able to
5 bind to i) human IGF-IR ,and/or to inhibit the binding of its native ligands, preferably
IGF 1 andlor IGF2, and/or also able to inhibit specifically the tyrosine kinase activity of
said IGF-IR and/or ii) hybrid-R, and/or to inbibit the binding of its native ligands,
preferably IGF1, IGF;! andlor Insulin, andlor also able to specifically inhibit the tyrosine
kinase activity of said hybrid-R.
10 According to another preferred embodiment, said antibodies are used for cancer
therapy, more particularly breast cancer therapy.
Actually, it is known that breast tumoral ce-l ls specifically present on their
surface IGF-IR but also a great number of Insulin receptor and, as a consequence, a
great number of Hybrid-R (Frasca et al., 1999; Sciacca et al., 1999; Vella et al., 2001).
15 More particularly, the present invention concerns four different anti-IGF-IR
monoclonal antibodies.
fn a first aspect, a subject of the present invention is an isolated antibody, or one
of its functional hgments, said antibody or one of its said hgments being capable of
binding specifically to the human insulin-like growth factor I receptor and, if necessary,
2 0 preferably moreover capable of inhibiting the natural attachment of the ligands IGFl
a d o r IGF2 of IGF-IR and/or capable of specifically inhibiting the tyrosine kinase
activity of said IGF-IR, characterized in that it comprises a light chain comprising at
least one complementarity determining region CDR chosen from the CDRs of amino
acid sequence SEQ ID Nos. 1,2 and 3, or at least one CDR whose sequence has at least
25 80%, preferably 85%, 90%, 95% and 98% identity, after optimum alignment, with the
sequence SEQ ID Nos.'l, 2 and 3, or in that it comprises a heavy chain comprising at
least one CDR chosen from the CDRs of amino acid sequence SEQ ID Nos. 4,s and 6,
or at least one CDR whose sequence has at least 80%, preferably 85%, 90%, 95% and
98% identity, after optimum alignment, with the sequence SEQ ID No. 4,5 and 6.
30 In the present specification and corresponding exemples, this antibody will be
referred as 13F5. .
In the present description, the terms "to bind" and "to attach" have the same
meaning and are inter-changeable,
In the present description, the terms polypeptides, polypeptide sequences,
peptides and proteins attached to antibody compounds or to their sequence are
5 interchangeable.
It must be understood here that the invention does not relate to the antibodies in
natural form, that is to say they are not in their natural environment but that they have
been able to be isolated or obtained by purification hm natural sources, or else
obtained by genetic recombination, or by chemical synthesis, and that they can then
10 contain unnatural amino acids as will be described further on.
By CDR regions or CDR(s), it is intended to indicate the hypervariable regions
of the heavy and light chains of the immunoglobulins as defined by Kabat et al. (Kabat
et al., Sequences of proteins of immunological interest, 5th Ed., U.S. Department of
Health and Human Services, NM, 1991, and later editions). Three heavy chain CDRs
15 and 3 light chain CDRs exist. The term CDR or CDRs is used here in order to indicate,
according to the case, one of these regions or several, or even the whole, of these
regions which contain the majority of the amino acid residues responsible for the
binding by affinity of the antibody for the antigen or the epitope which it recognizes.
By "percentage of identity" between two nucleic acid or amino acid sequences
20 in the sense of the present invention, it is intended to indicate a percentage of
nucleotides or of identical amino acid residues between the two sequences to be
compared, obtained after the best alignment (optimum alignment), this percentage being
purely statistical and the differences between the two sequences being distributed
randomly and over their entire length. The comparisons of sequences between two
2 5 nucleic acid or amino acid sequences are traditionally carried out by comparing these
sequences after having aligned them in an optimum manner, said comparison being able
to be carried out by segment or by "comparison window". The optimum alignment of
the sequences for the comparison can be carried out, in addition to manually, by means
of the local homology algorithm of Smith and Waterman (1981) [Ad. App. Math.
3 0 2:482], by means of the local homology algorithm of Neddleman and Wunsch (1970) [J.
. Mol. Biol. 48:443], by means of the similarity search method of Pearson and Lipman
(1988) [Proc. Natl. Acad. Sci. USA 85:2444), by means of computer software using
these algorithms (GAP, BESTFIT, FASTA and TFASTA in the Wisconsin Genetics
Software.Package, Genetics Computer Group, 575 Science Dr., Madison, WI, or else by
BLAST N or BLAST P comparison sohare).
The percentage of identity between two nucleic acid or amino acid sequences is
5 determined by comparing these two sequences aligned in an optimum manner and in
which the nucleic acid or arnino acid sequence to be compared can comprise additions
or deletions with respect to the reference sequence for an optimum alignment between
these two sequences. The percentage of identity is calculated by determining the
number of identical positions for which the nucleotide or the arnino acid residue is
10 identical between the two sequences, by dividing this number of identical positions by
the total number of positions in the comparison window and by multiplying the result
obtained by 100 in order to obtain the percentage of identity between these two
sequences.
For example, it is possible to use the BLAST program, "BLAST 2 sequences"
15 (Tatusova et al., "Blast 2 sequences - a new tool for comparing protein and nucleotide
sequences", FEMS Microbiol Lett. 174:247-250) available on the site
http://www.ncbi.nlm.nih.gov/ gor~l2.html;th e parameters used being those given by
default (in particular for the parameters "open gap penalty": 5, and "extension gap
penalty": 2; the matrix chosen being, for example, the matrix "BLOSUM 62" proposed
2 0 by the program), the percentage of identity between the two sequences to be compared
being calculated directly by the program.
By amino acid 'sequence having at least 80%, preferably 85%, 90%, 95% and
98% identity with a reference amino acid sequence, those having, with respect to the
reference sequence, certain modifications, in particular a deletion, addition or
2 5 substitution of at least one amino acid, a truncation or an elongation are preferred. In the
case of a substitution of one or more consecutive or nonconsecutive amino acid(s), the
substitutions are preferred in which the substituted amino acids are replaced by
"equivalent" amino acids. The expression "equivalent amino acids" is aimed here at
indicating any amino acid capable of being substituted with one of the amino acids of
3 0 the base structure without, however, essentially modifying the biological activities of
the corresponding antibodies and such as will be defined later, especially in the
examples. These equivalent amino acids can be determined either by relying on their
structural homology with the amino acids which they replace, or on results of
comparative trials of biological activity between the different antibodies capable of
being carried out.
By way of example, mention is made of the possibilities of substitution capable
5 of being carried out without resulting in a profound modification of the biological
activity of the corresponding modified antibody. It is thus possible to replace leucine by
valine or isoleucine, aspartic acid by glutamic acid, glutatnine by asparagine, arginine
by lysine, etc., the reverse substitutions being naturally envisageable under the same
conditions.
10 In a second aspect, a subject of the present invention is an isolated antibody, or
one of its functional fiagrnents, said antibody or one of its said fragments being capable
of binding specifically to the human insulin-like growth factor I receptor and, if
necessary, preferably moreover capable of inhibiting the natural attachment of the
ligands IGFl and/or IGF2 of IGF-IR and/or capable of specifically inhibiting the
15 tyrosine kinase activity of said IGF-IR, characterized in that it comprises a light chain
comprising at least one complementarity determining region CDR chosen from the
CDRs of amino acid sequence SEQ ID Nos. 7, 8 and 9, or at least one CDR whose
sequence has at least 80%, preferably 85%, 90%, 95% and 98% identity, after optimum
alignment, with the sequence SEQ ID Nos. 7, 8 and 9, or in that it comprises a heavy
2 0 chain comprising at least one CDR chosen from the CDRs of amino acid sequence SEQ
ID Nos. 10, 11 and 12, or at least one CDR whose sequence has at least 80%, preferably
85%, 90%, 95% and 98% identity, after optimum alignment, with the sequence SEQ ID
Nos. 10,ll and 12.
In the following specification, this antibody will be referred as 12D5.
25 In a third aspect, a subject of the present invention is an isolated antibody, or one
of its hctional fragments, said antibody or one of its said hgments being capable of
binding specifically to the human insulin-like growth factor I receptor and, if necessary,
preferably moreover capable of inhibiting the natural attachment of the ligands IGFl
andlor IGF2 of IGF-IR andlor capable of specifically inhibiting the tyrosine kinase
30 activity of said IGF-IR, characterized in that it comprises a light chain comprising at
least one complementarity determining region CDR chosen fiom the CDRs of amino
acid sequence SEQ ID Nos. 13, 14 and 15, or at least one CDR whose sequence has at
least 80%, preferably 85%, 90%, 95% and 98% identity, after optimum alignment, with
the sequence SEQ ID Nos. 13, 14 and 15, or in that it comprises a heavy chain
comprising at least one CDR chosen from the CDRs of amino acid sequence SEQ ID
Nos. 16, 17 and 18, or at least one CDR whose sequence has at least 80%, preferably
5 85%, 90%, 95% and 98% identity, after optimum alignment, with the sequence SEQ ID
No. 16,17 and 18.
In the following specification, this antibody will be referred as 2D10.
Last, in yet another aspect, a subject of the present invention is an isolated
antibody, or one of its functional fragments, capable of binding specifically to the
10 human insulin-like growth factor I receptor and, if necessary, preferably moreover
capable of inhibiting the natural attachment of the ligands IGFl andlor IGF2 of IGF-IR
and/or capable of specifically inhibiting the tyrosine kinase activity of said IGF-IR,
characterized in that it consists in the antibody called 21E3 and registered at the CNCM
as thereafter mentioned.
15 The antibodies according to the present invention, i.e. 13F5, 12D5,2D10 and
21E3 are preferably specific monoclonal antibodies, especially of murine, chimeric or
humanized origin, which can be obtained according to the standard methods well known
to the person skilled in the art.
In general, for the preparation of monoclonal antibodies or their functional
20 fkagments, especially of murine origin, it is possible to refer to techniques which are
described in particular in the manual "Antibodies" (Harlow and Lane, Antibodies: A
- Laboratory Manual, Cold Spring Harbor Laboratory, Cold Spring Harbor NY, pp. 726,
1988) or to the technique of preparation fi-om hybridomas described by Kohler and
Milstein (Nature, 256:495-497, 1975).
25 The monoclonal antibodies according to the invention can be obtained, for
example, from an animal cell immunized against the IGF-IR, or one of its fragments
containing the epitope specifically recognized by said monoclonal antibodies according
to the invention. Said IGF-IR, or one of its said fiagrnents, can especially be produced
according to the usual working methods, by genetic recombination starting with a
3 0 nucleic acid sequence contained in the cDNA sequence coding for the IGF-IR or by
peptide synthesis starting f?om a sequence of amino acids comprised in. the peptide
sequence of the IGF-IR.
The monoclonal antibodies according to the invention can, for example, be
purified on an aflinity column on which the IGF-R or one of its fragments containing
the epitope specifically recognized by said monoclonal antibodies according to the
invention has previously been immobilized More particularly, said monoclonal
5 antibodies can be purified by chromatography on protein A andlor G, followed or not
followed by ion-exchange chromatography aimed at eliminating the residual protein
contaminants as well as the DNA and the LPS, in itself followed or not followed by
exclusion chromatography on Sepharose gel in order to eliminate the potential
aggregates due to the presence of dimers or of other multimers. In an even more
10 preferred manner, the whole of: these techniques can be used simu1taneously or
successively.
Chimeric or humanized antibodies are likewise included in antibodies according
to the present invention.
By chimeric antibody, it is intended to indicate an antibody which contains a
15 natural variable (light chain and heavy chain) region derived fiom an antibody of a
given species in combination with the light chain and heavy chain constant regions of an
antibody of a species heterologous to said given species.
The antibodies or their fiagrnents of chimeric type according to the invention
can be prepared by using the techniques of genetic recombination. For example, the
20 chimeric antibody can be produced by cloning a recombinant DNA containing a
promoter and a sequence coding for the variable region of a non-human, especially . -
murine, monoclonal antibody according to the invention and a sequence coding for the
constant region of human antibody. A chimeric antibody of the invention encoded by
such a recombinant gene will be, for example, a mouse-man chimera, the specificity of
25 this antibody being determined by the variable region derived fiom the murine DNA
and its isotype determined by the constant region derived from the human DNA. For the
methods of preparation of chimeric antibodies, it is possible, for example, to refer to the
document Verhoeyn et al. (BioEssays, 8:74,1988).
By humanized antibody, it is intended to indicate an antibody which contains
30 CDR regions derived fiom an antibody of nonhuman origin, the other parts of the
antibody molecule being derived fiom one (or &om several) human antibodies.
Moreover, some of the residues of the segments of the skeleton (called FR) can be
modified in order to conserve the affinity of the binding (Jones et al., Nature, 321:522-
525, 1986; Verhoeyen et al., Science, 239:1534-1536, 1988; Riechmann et al., Nature,
332:323-327,1988).
The humanized antibodies according to the invention or their fragments can be
5 prepared by techniques known to the person skilled in the art (such as, for example,
those described in the documents Singer et al., J. Immun. 150:2844-2857, 1992;
Mountain et al., Biotechnol. Genet. Eng. Rev., 10: 1-142, 1992; or Bebbington et al.,
Biorrechnology, 10: 169- 175, 1992). Such humanized antibodies according to the
invention are preferred for their use in in vitro diagnostic methods, or iir vivo
1 0 prophylactic andlor therapeutic treatment.
By functional fragment of an antibody according to the invention, it is intended
to indicate in particular an antibody fragment, such as Fv, scFv (sc for single chain),
Fab, F(abY)2F, ab', scFv-Fc fragments or diabodies, or any fragment of which the halflife
time would have been increased by chemical modification, such as the addition of
15 poly(allcy1ene) glycol such as poly(ethy1ene) glycol ('PEGylationyy) (pegylated
fragments called Fv-PEG, scFv-PEG, Fab-PEG, F(abY)2-PEGo r Faby-PEG)( "PEG" for
Poly(Ethy1ene) Glycol), or by incorporation in a liposome, said fragments having at
least one of the characteristic CDRs of sequence SEQ ID Nos. 1 to 6,7 to 12 or 13 to 18
according to the invention, and, especially, in that it is capable of exerting in a general
2 0 manner an even partial activity of the antibody firom which it is descended, such as in
particular the capacity to recognize and to bind to the IGF-IR, and, if necessary, to
.. inhibit the activity of the IGF-IR.
Preferably, said f?unctional fiagments will be constituted or will comprise a
partial sequence of the heavy or light variable chain of the antibody fiom which they are
25 derived, said partial sequence being sufficient to retain the same specificity of binding
as the antibody from which it is descended and a sufficient sanity, preferably at least
equal to 11100, in a more preferred manner to at least 1/10, of that of the antibody from
which it is descended, with respect to the IGFW2. Such a functional fragment will
contain at the minimum 5 amino acids, preferably 10, 15, 25, 50 and 100 consecutive
3 0 amino acids of the sequence of the antibody hm which it is descended.
Preferably, these functional fiagments will be hgments of Fv, scFv, Fab,
F(ab')l, F(abY), scFv-Fc type or diabodies, which generally have the same specificity of
binding as the antibody from which they are descended. According to the present
invention, antibody fragments of the invention can be obtained starting from antibodies
such as described above by methods such as digestion by enzymes, such as pepsin or
papain and/or by cleavage of the disulfide bridges by chemical reduction. In another
5 manner, the antibody fragments comprised in the present invention can be obtained by
techniques of genetic recombination likewise well known to the person skilled in the art
or else by peptide synthesis by means of, for example, automatic peptide synthesizers
such as those supplied by the company Applied Biosystems, etc..
More particularly, the invention comprises the antibodies, or their hctional
10 fiagrnents, according to the present invention, especially chimeric or humanized
antibodies, obtained by genetic recombination or by chemical synthesis.
According a first approach, the antibody will be define by its heavy chain
sequence.
In a first preferred manner, the present invention relates to an antibody or one of
15 its fUnctiona1 fi-agrnents, according to the invention, characterized in that it comprises a
heavy chain comprising at least two of the three CDRs or the three CDRs of sequence
SEQ ID Nos. 4 to 6, or at least two of three CDRs or three CDRs of sequence
respectively having at least 80% identity after optimum alignment with the sequenceh
SEQ ID Nos. 4 to 6.
20 In a second preferred manner, the present invention relates to an antibody or one
of its functional fragments, according to the invention, characterized in that it comprises
a heavy chain comprising at least two of the three CDRs or the three CDRs of sequence
SEQ ID Nos. 10 to 12, or at least two of three CDRs or three CDRs of sequence
respectively having at least 80% identity after optimum alignment with the sequence
In a third preferred manner, the present invention relates to an antibody or one of
its functional fragments, according to the invention, characterized in that it comprises a
heavy chain comprising at least two of the three CDRs or the three CDRs of sequence
SEQ ID Nos. 16 to 18, or at least two of three CDRs or three CDRs of sequence
3 0 respectively having at least 80% identity after optimum alignment with the sequence
SEQ ID Nos. 16 to 18.
According a second approach, the antibody will be now define by its light chain
sequence.
In a likewise preferred first embodiment, the antibody or one of its hctional
fragments, according to the invention, is characterized in that it comprises a light chain
5 comprising at least one CDR chosen from the CDRs of sequence SEQ ID Nos. 1 to 3, or
a CDR whose sequence has at least 80% identity after optimum alignment with the
sequence SEQ ID Nos. 1 to 3.
In a second embodiment, the antibody or one of its fimctional fi-agments,
according to the invention, is characterized in that it comprises a light chain comprising
10 at least one CDR chosen. from the CDRs of sequence SEQ ID Nos. 7 to 9, or a CDR
whose sequence has at least 80% identity after optimum alignment with the sequence
SEQ ID Nos. 7 to 9.
In a third preferred embodiment, the antibody or one of its functional fi-agments,
according to the invention, is characterized in that it comprises a light chain comprising
15 at least one CDR chosen fi-om the CDRs of sequence SEQ ID Nos. 13 to 15, or a CDR
whose sequence has at least 80% identity after optimum alignment with the sequence
SEQ ID Nos. 13 to 15.
According a third approach, the antibody will be now define both by its light
chain sequence and its heavy chain sequence.
20 In a first preferred manner, the antibody or one of its functional fi-agnents
according to the invention is characterized in that it. comprises a heavy chain comprising
the three CDRs of sequence SEQ ID Nos. 4 to 6, or three CDRs of sequence having at
least 80% of identity after optimum alignment with the sequence SEQ ID Nos. 4 to 6,
and in that it moreover comprises a light chain comprising the three CDRs of sequence
25 SEQ ID Nos. 1 to 3, or three CDRs of sequence having at least 80% of identity after
optimum alignment with the sequence SEQ ID Nos. 1 to 3.
In a second preferred manner, the antibody or one of its functional fragments
according to the invention is characterized in that it comprises a heavy chain comprising
the three CDRs of sequence SEQ ID Nos. 10 to 12, or three CDRs of sequence having
3 0 at least 80% of identity after optimum alignment with the sequence SEQ ID No. 10 to
12 and in that it moreover comprises a light chain comprising the three CDRs of
sequence SEQ ID Nos. 7 to 9, or three CDRs of sequence having at least 80% of
identity after optimum alignment with the sequence SEQ ID Nos. 7 to 9.
In a third preferred manner, the antibody or one of its functional fiagments
according to the invention is characterized in that it comprises a heavy chain comprising
5 the three CDRs of sequence SEQ ID Nos. 16 to 18, or three CDRs of sequence having
at least 80% of identity after optimum alignment with the sequence SEQ ID No. 16 to
18 and in that it moreover comprises a light chain comprising the three CDRs of
sequence SEQ ID Nos. 13 to 15, or three CDRs of sequence having at least 80% of
identity after optimum alignment with the sequence SEQ ID Nos. 13 to 15.
10 In yet another preferred embodiment, the antibody or one of its functional
fiagments according to the invention and called 13F5 is characterized in that it
comprises a heavy chain of sequence comprising the amino acid sequence SEQ ID
No. 20, and in that it moreover comprises a light chain of sequence comprising the
amino acid sequence SEQ ID No. 19.
15 In yet another preferred embodiment, the antibody or one of its functional
fiagments according to the invention and called 12D5 is characterized in that it
comprises a heavy chain of sequence comprising the amino acid sequence SEQ ID
No. 22 or 23, and in that it moreover comprises a light chain of sequence comprising the
amino acid sequence SEQ ID No. 21.
20 In yet another preferred embodiment, the antibody or one of its functional
fiagments according to the invention and called 2D10 is characterized in that it
comprises a heavy chain of sequence comprising the amino acid sequence SEQ ID
No. 25, and in that it moreover comprises a light chain of sequence comprising the
amino acid sequence SEQ ID No. 24.
25 Another possibility, part of the present invention, is an antibody wherein the
three CDRs of the heavy chain are randomly chosen in the group comprising the CDRs
of each 13F5, 12D5 and 2D10 and wherein the three CDRs of the lignt chain are also
randomly chosen in the group comprising the CDRs of each 13F5,12D5 and 2D10.
According to another aspect, a subject of the present invention is an antibody or
3 0 one of its fictional fi-agments, according to the invention, characterized in that it does
not attach or it does not attach in a significant manner to the human insulin receptor IR.
In a preferred manner, said functional hgments according to the present
invention will be chosen from the fragments Fv, scFv, Fab, (Fablh, Fab', scFv-Fc or
diabodies, or any functional fragment whose half-life would have been increased by a
chemical modification, especially by PEGylation, or by incorporation in a liposome.
5 According to another aspect, the invention relates to murine hybridoma capable
of secreting monoclonal antibodies according to the present invention, especially
hybridoma of murine origin such as deposited at the Centre National de Culture De
Microorganisme (CNCM, National Center of Microorganism Culture) (Institut Pasteur,
Paris, France).
10 The monoclonal antibody here called 13F5, or one of its functional fragments,
characterized in that said antibody is secreted by the hybridoma deposited at the CNCM
on 25/03/2004 under the number CNCM 1-3193 is, of course, part of the present
invention. This hybridoma consists in a murine hybridoma resultkg in the cellular
fusion of immunized mouse splenocytes with a myeloma cell line (Sp20 Ag14).
15 The monoclonal antibody here called 12D5, or one of its functional fragments,
characterized in that said antibody is secreted by the hybridoma deposited at the CNCM
on 08/04/2004 under the number CNCM 1-3195 is, of course, part of the present
invention. This hybridoma consists in a murine hybridoma resulting in the cellular
hsion of immunized mouse splenocytes with a myeloma cell line (Sp20 Ag14).
20 The monoclonal antibody here called 2D10, or one of its fuactional hgments,
characterized in that said antibody is secreted by the hybridoma deposited at the CNCM
on 13 May 2004 under the number 1-3214 is, of course, part of the present invention.
This hybridoma also consists in a murine hybridoma resulting in the cellular fusion of
immunized mouse splenocytes with a myeloma cell line (Sp20 Ag14).
25 The monoclonal antibody here called 21E3, or one of its functional fragments,
characterized in that said antibody is secreted by the hybridoma deposited at the CNCM
on l* July 2004 under the number 1-3249 is, of course, part of the present invention.
This hybridoma also consists in a murine hybridoma resulting in the cellular %ion of
immunized mouse splenocytes with a myeloma cell line (Sp20 Ag14).
30 According to a likewise particular aspect, the present invention relates to a
chimeric antibody, or one of its fhctional fragments, according to the invention,
characterized in that said antibody moreover comprises the light chain and heavy chain
constant regions derived fiom an antibody of a species heterologous to the mouse,
especially man, and in a preferred manner in that the light chain and heavy chain
constant regions derived h m a human antibody are respectively the kappa and gamma-
1, gamma-2 or gamma-4 region.
5 According to a novel aspect, the present invention relates to an isolated nucleic
acid, characterized in that it is chosen fiom the following nucleic acids:
a) a nucleic acid, DNA or RNA, coding for an antibody, or one of its
hctional fragments, according to the invention;
b) a complementary nucleic acid of a nucleic acid such as defined in a).
By' nucleic acid, nucleic or nucleic acid sequence, polynucleotide,
oligonucleotide, polynucleotide sequence, nucleotide sequence, terms which will be
employed indifferently in the present invention, it is intended to indicate a precise
linkage of nucleotides, which are modified or unmodified, allowing a fiagrnent or a
region of a nucleic acid to be defined, containing or not containing unnatural
15 nucleotides, and being able to correspond just as well to a double-stranded DNA, a
single-stranded DNA as to the transcription products of said DNAs.
It must also be understood here that the present invention does not concern the
nucleotide sequences in their natural chromosomal environment, that is to say in the
natural state. It concerns sequences which have been isolated andlor purified, that is to
20 say that they have been selected directly or indirectly, for example by copy, their
environment having been at least partially modified. It is thus likewise intended to
indicate here the iidlated nucleic acids gbtained by genetic recombination by means, for
example, of host cells or obtained by chemical synthesis.
A hybridization under conditions of high stringency signifies that the
2 5 temperature conditions and ionic strength conditions are chosen in such a way that they
allow the maintenance of the hybridization between two hgments of complementary
DNA. By way of illustration, conditions of high stringency of the hybridization step for
the purposes of defrning the polynucleotide fiagments described above are
advantageously the following.
30 The DNA-DNA or DNA-RNA hybridization is canied out in two steps: (1)
prehybridization at 42OC for 3 hours in phosphate buffer (20 mM, pH 7.5) containing 5
x SSC (1 x SSC corresponds to a 0.15 M NaCl + 0.015 M sodium citrate solution), 50%
of formamide, 7% of sodium dodecyl sulfate (SDS), 10 x Denhardt's, 5% of dextran
sulfate and 1% of salmon sperm DNA; (2) actual hybridization for 20 hours at a
temperature dependent on the size of the probe (i.e.: 42"C, for a probe size > 100
nucleotides) followed by 2 washes of 20 minutes at 20°C in 2 x SSC + 2% of SDS, 1
5 wash of 20 minutes at 20°C in 0.1 x SSC + 0.1% of SDS. The last wash is carried out in
0.1 x SSC 3- 0.1% of SDS for 30 minutes at 60°C for a probe size > 100 nucleotides.
The hybridization conditions of high stringency described above for a polynucleotide of
defined size can be adapted by the person skilled in the art for oligonucleotides of
greater or smaller size, according to the teaching of Sambrook et al. (1989, Molecular
1 0 cloning: a laboratory manual. 2nd Ed. Cold Spring Harbor).
The invention likewise relates to a vector comprising a nucleic acid according to
the present invention.
The invention aims especially at cloning and/or expression vectors which
contain a nucleotide sequence according to the invention.
15 The vectors according to the invention preferably contain elements which allow
the expression and/or the secretion of the nucleotide sequences in a determined host
cell. The vector must therefore contain a promoter, signals of initiation and termination
of translation, as well as appropriate regions of regulation of transcription. It must be
able to be maintained in a stable manner in the host cell and can optionally have
2 0 particular signals which specify the secretion of the translated protein. These different
elements are chosen and optimized by the person skilled in the art as a fiinction of the
host cell used. To this effect, the nucleotide sequences according to the invention can be
inserted into autonomous replication vectors in the chosen host, or be integrative vectors
of the chosen host.
25 Such vectors are prepared by methods currently used by the person skilled in the
art, and the resulting clones can be introduced into an appropriate host by standard
methods, such as lipofection, electroporation, thermal shock, or chemical methods.
The vectors according to the invention are, for example, vectors of plasmidic or
viral origin. They are usem for transforming host cells in order to clone or to express
3 0 the nucleotide sequences according to the invention.
The invention likewise comprises the host cells transformed by or comprising a
vector according to the invention.
The host cell can be chosen fiom prokaryotic or eukaryotic systems, for example
bacterial cells but likewise yeast cells or animal cells, in particular mammalian cells. It
is likewise possible to use insect cells or plant cells.
The invention Uewise concerns animals, except man, which comprise at least
5 one cell transformed according to the invention.
According to another aspect, a subject of the invention is a process for
production of an antibody, or one of its functional fragments according to the invention,
characterized in that it comprises the following stages:
a) culture in a medium and appropriate culture conditions of a host cell
1 0 according to the invention; and .
b) the recovery of said antibodies, or one of their functional fragments, thus
produced starting from the culture medium or said cultured cells.
The cells transformed according to the invention can be used in processes for
preparation of recombinant polypeptides according to the invention. The processes for
15 preparation of a polypeptide according to the invention in recombinant form,
characterized in that they employ a vector andlor a cell transformed by a vector
according to the invention, are themselves comprised in the present invention.
Preferably, a cell transformed by a vector according to the invention is cultured under
conditions which allow the expression of said polypeptide and said recombinant peptide
2 o is recovered.
As has been said, the host cell can be chosen fiom prokaryotic or eukaryotic
systems. In particular, 6 is possible to identify nucleotide sequences according to the
invention, facilitating secretion in such a prokaryotic or eukaryotic system. A vector
according to the invention carrying such a sequence can therefore advantageously be
25 used for the production of recombinant proteins, intended to be secreted. In effect, the
purification of these recombinant proteins of interest will be facilitated by the fact that
they are present in the supernatant of the cell culture rather than in the interior of the
host cells.
It is likewise possible to prepare the polypeptides according to the invention by
3 0 chemical synthesis. Such a preparation process is likewise a subject of the invention.
The personskilled in the art knows the processes of chemical synthesis, for example the
techniques employing solid phases (see especially Steward et al., 1984, Solid phase
peptide synthesis, Pierce Chem. Company, Rockford, 11 1,2nd ed.) or techniques using
partial solid phases, by condensation of fiagments or by a classical synthesis in solution.
The polypeptides obtained by chemical synthesis and being able to contain
corresponding unnatural amino acids are likewise comprised in the invention.
5 The antibodies, or one of their functional fragments, capable of being obtained
by a process according to the invention are likewise comprised in the present invention.
According to a second embodiment, the present invention concerns an antibody
according to the invention such as described further above, characterized in that it is,
moreover, capable of binding specifically to the human epidermal growth factor
10 receptor EGFR andlor capable of specifically inhibiting the tyrosine kinase activity of
said EGFR.
The invention likewise concerns a pharmaceutical composition comprising by
way of active principle a compound consisting of an antibody, or one of its functional
fiagments according to the invention, preferably mixed with an excipient and/or a
15 pharmaceutically acceptable vehicle.
Another complementary embodiment of the invention consists in a composition
such as described above which comprises, moreover, as a combination product for
simultaneous, separate or sequential use, a cytotoxic/cytostatic agent andlor an inhibitor
of the tyrosine kinase activity respectively of the receptors for IGF-I and/or for EGF.
20 "Simultaneous use" is understood as meaning the administration of the two
compounds of the composition according to the invention in a single and identical
pharmaceutical form.
"Separate use" is understood as meaning the administration, at the same time, of
the two compounds of the composition according to the invention in distinct
2 5 pharmaceutical fonns.
"Sequential use" is understood as meaning the successive administration of the
two compounds of the composition according to the invention, each in a distinct
pharmaceutical form.
In a general fashion, the composition according to the invention considerably
3 0 increases the efficacy of the treatment of cancer. In other words, the therapeutic effect
of the anti-IGF-IR antibodies according to the invention is potentiated in an unexpected
manner by the administration of a cytotoxic agent. Another major subsequent advantage
produced by a composition according to the invention concerns the possibility of using
- lower efficacious doses of active principle, which allows the risks of appearance of
secondary effects to be avoided or to be reduced, in particular the effects of the
cytotoxic agent.
5 In addition, this composition according to the invention would allow the
expected therapeutic effect to be attained more rapidly.
In a particularly preferred embodiment, said composition as a combination
product according to the invention is characterized in that said cytotoxic/cytostatic agent
is chosen from the agents interacting with DNA, the antimetabolites, the topoisomerase
10 I or I1 inhibitors, or else the spindle inhibitor or stabilizer agents or else any agent
capable of being used in chemotherapy. Such cytotoxic/cytostatic agents, for each of the
aforesaid classes of cytotoxic agents, are, for example, cited in the 2001 edition of
VIDAL, on the page devoted to the compounds attached to the cancerology and
hematology column "Cytotoxics", these cytotoxic compounds cited with reference to
15 this document are cited here as preferred cytotoxic agents.
In a particularly preferred embodiment, said composition as a combination
product according to the invention is characterized in that said cytotoxic agent is
coupled chemically to said antibody for simultaneous use.
In a particularly preferred embodiment, said composition according to the
20 invention is characterized in that said cytotoxiclcytostatic agent is chosen &om the
spindle inhibitor or stabilizer agents, preferably vinorelbine andlor vinflunine andlor
vincristine.
In order to facilitate the coupling between said cytotoxic agent and said antibody
according to the invention, it is especially possible to introduce spacer molecules
25 between the two compounds to be coupled, such as poly(alky1ene) glycols like
polyethylene glycol, or else amino acids, or, in another embodiment, to use active
derivatives of said cytotoxic agents into which would have been introduced functions
capable of reacting with said antibody according to the invention. These coupling
techniques are well known to the person skilled in the art and will not be expanded upon
3 0 in the present description.
In another preferred embodiment, said inhibitor of the tyrosine kinase activity of
the receptors for IGF-I is selected from the group consisting of derived natural agents,
dianilinophthalirnides, pyrazolo- or pyrrolopyridopyrimidines or else quinazilines. Such
inhibitory agents are well known to the person skilled in the art and described in the
literature (Ciardiello F., Drugs 2000, Suppl. 1,2532).
According to yet another embodiment of the invention, the composition such as
5 described above can likewise comprise another antibody compound directed against the
extracellular domain of the HER21neu receptor, as a combination product for
. simultaneous, separate or sequential use, intended for the prevention and for the
treatment of cancer, especially the cancers overexpressing said HERuneu receptor and
the receptor IGF-IR, such as especially cancer of the breast.
10 Reference can be made especiaIly to the publications of Albanell et al. (J. of the
National Cancer Institute, 93(24):1830-183 1, 2001) and of Lu et al. (J. of the National
Cancer Institute, 93(24):1852-1857, 2001) justifling the unexpected interest in
combining an anti-HEWneu antibody with an anti-IGF-IR antibody according to the
present invention.
15 In a particular manner, said anti-HER2/neu antibody of the composition
according to the invention is the antibody called Trastuzurnab (also called Herceptin).
The invention relates, in another aspect, to a composition characterized in that
one, at least, of said antibodies, or one of their functional fi-agments, is conjugated with
a cell toxin and/or a radioelement.
20 Preferably, said toxin or said radioelement is capable of inhibiting at least one
cell activity of cells expressing the IGF-IR, in.a more preferred manner capable of
preventing the growth or the proliferation of said cell, especially of totally inactivating
said cell.
Preferably also, said toxin is an enterobacterial toxin, especially Pseudomonas
25 exotoxinA.
The radioelements (or radioisotopes) preferably conjugated to the antibodies
employed for the therapy are radioisotopes which emit gamma rays and preferabIy
iodinel3l, y t t r i d , gold'99, palladium100, copper6', bismuth2" and antimong". The
radioisotopes which emit beta and alpha rays can likewise be used for the therapy.
30 By toxin or radioelement conjugated to at least one antibody, or one of its
functional fragments, according to the invention, it is intended to indicate any means
allowing said toxin or said radioelement to bind to said at least one antibody, especially
by covalent coupling between the two compounds, with or without introduction of a
linking molecule.
Among the agents allowing binding in a chemical (covalent), electrostatic or
noncovalent manner of all or part of the components of the conjugate, mention may
5 particularly be made of benzoquinone, carbodiimide and more particularly EDC (1-
ethyl-3-[3-dimethylaminopropyl]-carbodiimide hydrochloride), diigleimide, dithiobisnitrobenzoic
acid (DTNB), N-succinimidyl S-acetyl thio-acetate (SATA), the bridging
agents having one or more phenylazide groups reacting with the ultraviolets (U.V.) and
preferably N-[-4-(azidosalicylamino)butyl]-3'-(2'-pyridyldithio)-propionamide (APDP),
1 0 N-succinimid-yl 3-(2-pyridy1dithio)propionate (SPDP), 6-hydrazino-nicotinamide
(HYNIC).
Another form of coupling, especially for the radioelements, can consist in the
use of a bihctional ion chelator.
Among these chelates, it is possible to mention the chelates derived from EDTA
15 (ethylenediaminetetraacetic acid) or fiom DTPA (diethylenetriaminepentaacetic acid)
which have been developed for binding metals, especially radioactive metals, and
immunoglobulins. Thus, DTPA and its derivatives can be substituted by different
groups on the carbon chain in order to increase the stability and the rigidity of the
ligand-metal complex (Krejcarek et al., 1977; Brechbiel et al., 1991; Gansow, 1991; US
2 0 patent 4,83 1,175).
For example diethylenetriaminepentaacetic acid (DTPA) and its derivatives,
which have been widely used in medicine and in biology for a long time either in their
fiee form, or in the forni of a complex with a metallic ion, have the remarkable
characteristic of forming stable chelates with metallic ions and of being coupled with
25 proteins of therapeutic or diagnostic interest such as antibodies for the development of
radioimmunoconjugates in cancer therapy @leases et al., 1984; Gansow et al., 1990).
Likewise preferably, said at least one antibody forming said conjugate according
to the invention is chosen from its functional hgments, especially the fiagrnents
amputated of their Fc component such as the scFv fkagments.
30 The present invention moreover comprises the use of the composition according
to the invention for the preparation of a medicament.
More particularly, according to another embodiment, the invention concerns the
use of an antibody, or one of its hctional fragments, andlor of a composition for the
preparation of a medicament intended for the prevention or for the treatment of an
illness induced by an overexpression and/or an abnormal activation of the IGF-I
5 receptor, and/or connected with a hyperactivation of the transduction pathway of the
signal mediated by the interaction of the 1-IGF1 or IGF2 with IGF-IR.
Preferably, said use according to the invention is characterized in that the
administration of said medicament does not induce or induces only slightly secondary
effects connected with inhibition of the insulin receptor IR, that is to say inhibition of
10 the interaction of the IR with its natural ligands due to the presence of said medicament,
especially by a competitive inhibition connected with the attachment of said
medicament to the IR.
The present invention moreover comprises the use of an antibody, or one of its
functional fi-agments, preferably humanized, andfor of a composition according to the
15 invention for the preparation of a medicament intended to inhibit the transformation of
normal cells into cells with tumoral character, preferably IGF-dependent, especially
IGF1- and/or IGF2-dependent.
The present invention likewise relates to the use of an antibody, or one of its
functional fragments, preferably humanized, andlor of a composition according to the
2 0 invention for the preparation of a medicament intended to inhibit the growth and/or the
proliferation of tumor cells, preferably IGF-dependent, especially IGFl- andlor IGF2-
dependent.
In a general manner, a subject of the present invention is the use of an antibody,
or one of its functional fragments, preferably humanized, and/or of a composition
25 according to the invention, for the preparation of a medicament intended for the
prevention or for the treatment of cancer preferably expressing IGF-IR andor of cancer
preferably having a hyperactivation of the transduction pathway of the signal mediated
by the interaction of IGFl or IGF2 with IGF-IR, such as, for example, the
overexpression of IRS 1.
30 The subject of the present invention is likewise the use of an antibody, or one of
its functional eagnents, preferably humanized, andlor of a composition according to
the invention, for the preparation of a medicament intended for the prevention or for the
treatment of psoriasis, psoriasis whose epidermal hyperproliferation can be connected
with the expression or the overexpression of IGF-IR, andlor with the hyperactivation of
the transduction pathway of the signal mediated by the interaction of IGF-IR with its
natural ligands (Wraight C.J. et al., Nat. Biotechnol., 2000, 18(5):521-526. Reversal of
5 epidermal hyperproliferation in psoriasis by insulin-lie growth factor I receptor
antisense oligonucleotides). In another embodiment, an object of the invention is the use -
of an antibody, or one of its functional fiagrnents, preferably humanized, andfor of a
composition according to the invention, for the preparation of a medicament intended
for the prevention or for the treatment of atherosclerosis.
10 Among the cancers which can be prevented and/or treated, prostate cancer,
osteosarcomas, lung cancer, breast cancer, endometrial cancer or colon cancer or any
other cancer overexpressing IGF-IR is preferred
According to yet another aspect, a subject of the present invention is a method of
diagnosis, preferably in vitro, of illnesses connected with an overexpression or an
15 underexpression, preferably an overexpression, of the IGF-I receptor starting h r n a
biological sample in, which the abnormal presence of IGF-I receptor is suspected,
characterized in that said biological sample is contacted with an antibody, or one of its
functional fragments, according to the invention, it being possible for said antibody to
be, if necessary, labeled.
20 Preferably, said illnesses connected with the overexpression of the IGF-I
receptor in said diagnosis method will be cancers:
' In another particular embodiment, antibodies according to the invention can also
be used for the treatment, prevention and/or diagnostic of illness connected with non
only the overexpression of the IGF-IR but also the overexpression of Hybrid-R.
25 More particularly, antibody according to the invention is characterized in that it
is also capable of binding to the hybrid-R, isofonn(s) A andlor B, and inhibiting the
binding of its native ligands, preferably designated herein as IGFl and/or IGF2 and/or
insulin, and/or capable of specifically inhibiting the tyrosine kinase activity of said
hybrid-R.
30 Said antibody, or one of its functional fkqpents, can be present in the foxm of
an immunoconjugate or of a labeled antibody so as to obtain a detectable and/or
quantifiable signal.
The antibodies labeled according to the invention or their functional fragments
include, for example, antibodies called irnmunoconjugates which can be conjugated, for
example, with enzymes such as peroxidase, alkaline phosphatase, a-D-galactosidase,
glucose oxydase, glucose aniylase, carbonic anhydrase, acetylcholinesterase, lysozyme,
5 malate dehydrogenase or glucose 6-phosphate dehydrogenase or by a molecule such as
biotin, digoxygenin or 5-bromodeoxyuridine. Fluorescent labels can be likewise
conjugated to the antibodies or to their functional fragments according to the invention
and especially include fluorescein and its derivatives, fluorochrome, rhodamine and its
derivatives, GFP (GFP for "Green Fluorescent Protein"), dansyl, umbelliferone etc.. In
10 such conjugates, the antibodies of the invention or their hctional hgrnents can be
prepared by methods known to the person skilled in the art. They can be coupled to the
enzymes or to the fluorescent labels directly or by the intermediary of a spacer group or
of a linking group such as a polyaldehyde, like glutaraldehyde,
ethylenediaminetetraacetic acid (EDTA), diethylene-triaminepentaacetic acid (DPTA),
15 or iri the presence of coupling agents such as those mentioned above for the therapeutic
conjugates. The conjugates containing labels of fluorescein type can be prepared by
reaction with an isothiocyanate.
Other conjugates can likewise include chemoluminescent labels such as luminol
and the dioxetanes, bio-luminescent labels such as luciferase and luciferin, or else
20 radioactive labels such as iodine'23, iodine12', iodine'26, iodine'33, bromine77,
technetiumggm, indium1", indium1 13m , gallium67, gallium68, rutheniumgs, rutheniumg7,
rutheniumlo3, mercurylo7,r nercur~?~rh~e,n iumggmr,h eniumlO', rheniumlos,
scandium47, tellurium'21m, tellurium'um, tellurium125m, thulium'65, thulium'67,
thulium168, fluorine1', yttrium199, iodine131. The methods known to the person skilled in
25 the art existing for coupling the therapeutic radioisotopes to the antibodies either
directly or via a chelating agent such as EDTA, DTPA mentioned above can be used for
the radioelements which can be used in diagnosis. It is likewise possible to mention
labeling with N ~ [ I ' ~by~ t]h e chloramine T method [Hunter W.M.a nd Greenwood F.C.,
1962, Nature 194:495] or else with technetiudgm by the technique of Crockford et al.
30 (US patent 4,424,200) or attached via DTPA as described by Hnatowich (US patent
4,479,930).
Thus, the antibodies, or their functional fiagments, according to the invention
can be employed in a process for the detection and/or the quantification of an
overexpression or of an underexpression, preferably an overexpression, of the IGF-I
receptor in a biological sample, characterized in that it comprises the following steps:
5 a) the contacting of the biological sample with an antibody, or one of its
bctional hgments, according to the invention; and
b) the demonstration of the IGF-Wantibody complex possibly formed.
In a particular embodiment, the antibodies, or their functional fi-agrnents,
according to the invention, can be employed in a process for the detection andlor the
10 quantification of the IGF-I receptor ~aI Ib iological sample, for the monitoring of the
efficacy of a prophylactic and/or therapeutic treatment of IGF-dependent cancer or else
of psoriasis or atherosclerosis.
More generally, the antibodies, or their functional fiagments, according to the
invention can be advantageously employed in any situation where the expression of the
15 IGF-I receptor must be observed in a qualitative andor quantitative manner.
Preferably, the biological sample is formed by a biological fluid, such as serum,
whole blood, cells, a tissue sample or biopsies of human origin.
Any procedure or conventional test can be employed in order to carry out such a .
detection and/or dosage. Said test can be a competition or sandwich test, or any test
20 known to the person skilled in the art dependent on the formation of an immune
complex of antibody-antigen type. Following the applications according to the
invention, the antibody or one of its functional fiagments can be immobilized or
labeled. This immobilization can be carried out on numerous supports known to the
person skilled in the art. These supports can especially include glass, polystyrene, poly-
2 5 propylene, polyethylene, dextran, nylon, or natural or modified cells. These supports
can be either soluble or insoluble.
By way of example, a prefenred method brings into play immunoenzymatic
processes according to the ELISA technique, by immunofluorescence, or radioimmunoassay
(RIA) technique or equivalent.
30 Thus, the present invention likewise comprises the kits or sets necessary for
carrying out a method of diagnosis of illnesses induced by an overexpression or an
underexpression of the IGF-I receptor or for carrying out a process for the detection
and/or the quantification of an overexpression or of an underexpression of the IGF-I
receptor in a biological sample, preferably an overexpression of said receptor,
characterized in that said kit or set comprises the following elements:
a) an antibody, or one of its functional fragments, according to the invention;
5 b) optionally, the reagents for the formation of the medium favorable to the
immunological reaction;
c) optionally, the reagents allowing the demonstration of IGF-IRIantibody
complexes produced by the immunological reaction.
The invention moreover relates to the use of a composition as a combination
10 product according to the invention, for the preparation of a medicament intended for the
prevention or for the treatment of cancer, especially cancers for which said cytotoxic
agent or said anti-HER2/neu antibody is generally prescribed and, especially, for which
cancers the tumor cells express or overexpress the IGF-I receptor.
A subject of the invention is likewise the use of an antibody according to the
15 invention for the preparation of a medicament intended for the specific targeting of a
biologically active compound to cells expressing or overexpressing the IGF-I receptor.
It is intended here by biologically active compound to indicate any compound
capable of modulating, especially of inhibiting, cell activity, in particular their growth,
their proliferation, transcription or gene translation.
20 A subject of the invention is also an in vivo diagnostic reagent comprising an
antibody according to the invention, or one of its functional fragments, preferably
labeled, especially radiolabeled, and its use in medical imaging, in particular for the
detection of cancer connected with the expression or the overexpression by a cell of the
IGF-I receptor.
The invention likewise relates to a composition as a combination product or to
an anti-IGF-Wtoxin conjugate or radioelement, according to the invention, as a
medicament.
Preferably, said composition as a combination product or said conjugate
according to the invention will be mixed with an excipient andlor a pharmaceutically
3 0 acceptable vehicle.
In the present description, pharmaceutically acceptable vehicle is intended to
indicate a compound or a combination of compounds entering into a pharmaceutical
composition not provoking secondary reactions and which allows, for example,
facilitation of the administration of the active compound(s), an increase in its lifespan
andlor in its efficacy in the body, an increase in its solubility in solution or else an
improvement in its conservation. These pharmaceutically acceptable vehicles are well
5 known and will be adapted by the person skilled in the art as a hction of the nature
and of the mode of administration of the active compound(s) chosen.
Preferably, these compounds will be administered by the systemic route, in
particular by the intravenous route, by the intramuscular, intrademal, intraperitoneal or
subcutaneous route, or by the oral route. In a more preferred manner, the composition
10 comprising the antibodies according to the invention will be administered several times,
in a sequential manner.
Their modes of administration, dosages and optimum pharmaceutical forms can
be determined according to the criteria generally taken into account in the establishment
of a treatment adapted to a patient such as, for example, the age or the body weight of
15 the patient, the seriousness of hidher general condition, the tolerance to the treatment
and the secondary effects noted.
Other characteristics and advantages of the invention appear in the continuation
of the description with the examples and the figures wherein:
20 - Figure 1 represents the in vitro evaluation of anti IGF-1R antibodies in the
MCF-7 model,
- Figures 2 and 3 represent in vivo evaluation of anti IGF-1R antibodies on
DU145,
- Figure 4 represents displacement of [lZT]-~~Fo-nl intact cells expressing IGF-
25 IR,
- Figure 5 represents displacement of ['ZIJ-~~on~ i-mlr nunocaptured HR-A,
- Figure 6 represents displacement of [l3I'j-1~~-o1n immunocaphrred HR-B,
- Figure 7 represents displacement of ['25T]-IN~o n intact cells expressing IR-A,
and
30 - Figure 8 represents displacement of [lxIl-IN~o n intact cells expressing IR-B.
Exemple 1: Generation of monoclonal antibodies against IGF-1R
Hybridomas were generated by hion of splenocytes fiom BALBIc mice
im&unized with a soluble a2432 heterotetrameric recombinant human IGF-1R (R&D
System, Minneapolis, USA) and the SP2/0-Ag14 myeloma cell line. The resulting
murine antibodies were first screened by ELISA and FACS analysis on MCF-7 cells.
Then, a final screen on SB-IGF-lr versus SfP-IR cells was performed to eliminate
antibodies recognizing both IGF-1 and IR. The selected MAbs (positives in ELISA and
recognizing the wild type receptor on MCF-7 cells) were produced as ascitic fluids and
purified by protein A chromatography before testing either in vitro andlor in vivo as
summarized in table 1.
15B9
16A12
9D5
14H1
+
+.
+
-
+
-
Nd '
Nd
-
+
-
+
IgGl K
IgGl K
IgGl K
IgGl K
+/-
+INd
Nd
-
-
Nd
Nd
TABLE 1: Selection of anti-IGF-1R monoclonal antibodies
Exemple 2: In virro activity of anti-IGF-1R antibodies
5 Method
MCF-7 cells from ATCC were routinely cultured in phenol red free-RPMI
medium (Invitrogen Corporation, Scotland, UK), 10% FCS (Invitrogen Corporation),
1% L-Glutamine (Invitrogen Corporation). MCF-7 cells were plated in 96-well tissue
culture plates at a density of 5 x lo4 cells/well in serum-fiee medium. After 24 hours a
10 dose range of IGFl from 1 to 50 ng/ml was added to the medium either in absence or in
presence at a final concentration of 5 pg/rnl of each antibody to be tested. After 3 days,
cells were pulsed with 0.5 pCi of [3~]thymidin(eA mersham Biosciences AB, Uppsala,
Sweden) for 16 hours. The magnitude of [3HJthymidine incorporated into trichloroacetic
acid-insoluble DNA was quantified by liquid scintillation counting. Results are
15 expressed as a proliferative index (cpm of cells plus IGFl plus antibody I cpm of cells
plus antibody alone).
IgG1 K
Nd
IgGl K
Nd
Nd
Nd
IgGl K
IgGl K
IgGl K
IgGl K
15N1
18B5
20D1
21 B3
13F'lo
p~
14A1
2B10
3A9
3C9
4G4
Nd
Nd -
Nd
Nd
Nd
Nd
-
Nd
-
i-
Nd
Nd
-
Nd
Nd
Nd
-
Nd
-
Nd
Nd
Nd
Nd
-
Nd
Nd
-
-
-
-
-
Nd
Nd
-
Nd
-
+
+
+
+
Nd
Nd
Nd
-
Nd
Nd
-
+I-
-t
-IResults
The in vitro evaluation was the first screening of Mabs in tenns of mitogenic
activity. For these assays, the generated antibodies, produced as ascitic fluids, were
added to MCF-7 cells at the same time as IGFl and compared to the commercially
5 available aIR3 Mab to select antibodies at least as efficacious as this latter antibody.
The positive Mabs (5 Mabs) described as (+) in Table 2* of the previous reply are the
one giving proliferative indexes <5 when cells were stimulated with the highest dose of
IGFl (50 nglml). Figure 1 shows the in vitro activity of four out of the 6 strong in vitro
inhibitors (2D10, 12D5, 12B1, 13F5). 2F2 and 21E3 Mabs have been considered as a
10 (st)* Mab (5~Proliferativein dex45 for the highest concentration of IGF1) and 7G3 and
2B10 were considered as non-neutralizing antibodies (proliferative index>l5). It is
interesting to notice that the 21E3 is the only Mab of IgG2 isotype.
Exemple 3: In vivo activity of anti-IGF-1R antibodies
15 Method
DU145 cells from ATCC were routinely cultured in DMEM medium (Invitrogen
Corporation, Scotland, UK), 10% FCS (Invitrogen Corporation), 1% LGlutamine
(Invitrogen Corporation). Cells were split two days before engrahent so that they were
in exponential phase of growth. Two million DU145 cells were engrafted in PBS to
20 Swiss nude mice. One day after implantation, animals were divided into groups of 6
mice. Mice were treated S.C. at the opposite of the tumor with 200 pg of each tib body
to be tested, 3-times a week. The control group was either treated with a rnurine isotype
control (EC2) in the first screening or PBS for subsequent screenings as it has been
shown in the h t experiment that no difference in tumor growth was observed between
25 these 2 groups of mice. Tumor volume was measured once a week and calculated by the
formula: d6 X length X width X height.
Results
Three in vivo experiments were performed to test a panel of Mabs. Figures 2 and
3 show that 13F5, 2D10 and 6E5 significantly inhibit the in vivo growth of DU 145
3 0 cells. Statistical analysis (Mann and Whitney test) are shown in Table 2.
First screening
Mann-Whitney
PBSl2F2 Mannewhihey
Table 2: Statistical analysis of in vivo data
Dl6
Third screening
Mann-Whitney
pBs116A12 (Wilcoxon)
5 Example 4: Evaluation of 2D10, 12D5, 13F5 ability to bind to IGF-IR and
hybrid-R
The used cells for this study are listed thereafter:
- Rt: R- fibroblasts stably transfected with the IGF-I receptor (IGF-IR)c DNA
- R-/IR-A: R- fibroblasts stably transfected with the insulin receptor isoform A (IR-A)
10 cDNA
. - R-/IR-B: R- fibroblasts stably transfgted with the insulin receptor isofom B (IR-B)
cDNA
- RtAR-A: R- fibroblasts stably co-transfected with the IGF-I and the insulin receptor
isoform A cDNA and, therefore, expressing hybrid receptors A (Hybrid-RsA)
15 - RthR-B: R- fibroblasts stably co-transfected with the IGF-I and the insulin receptor
isoform B cDNA and, therefore, expressing hybrid receptors A (Hybrid-RsB).
Example 4-1: Displacement analvsis of T'*'?IGP~ on IGF-IR by 2D10, 12D5,
13F5 and aIR-3
["$IGF-1 (20,000 cpm) was allowed to bind to R+ intact cells for 16 hours at
pBs113F5 (~ilcoxon) ~ , 4 7
(Wilcoxon) p-o, 18 -
D23
Dl2
~ 0 , 0 6 3
p=0,086 ()10 ,046
p=0,067
D29
D20 D26
p=O,087
p=O,O 15
@,050
D33
p=o,ll
~ 4 , 0 2 3
D36
p=0,14
D43
4"C, in the absence or presence of increasing concentrations of unlabeled ligand (IGF1,
IGF2 or insulin) or antibodies (2D10, 12D5, 13F5). Results are plotted as percent of
maximal specific binding and are represented on Figure 4.
Both 2D10 and 13F5 efficaciously and Illy displaced IGFl with sub-nanomolar
5 affinities and in this example with an ICso of 0.15 and 0.20 nM, respectively, as
compared to the reference antibody gIR3 (ICSo: 0.05 nM). The affinities are higher than
those of the natural IGF-IR ligands IGFl (2.2 nM in this example) and IGF2 (15 nM in
this example).
Example 4-2: Dimlacement analysis of r'2511~~on~ 1H vbrid-RsA by 2D10,
1 0 12D5.13F5 and 47-9
Hybrid-RsA from R+/IR-A cell lysates were immunocaptured in Maxisorb
plates coated with anti IR antibody 83-7.
[ 1 2 5 ~ -pi~g.~ 5~) w1as then allowed to bind to immunocaptured receptors in the
absence or the presence of increasing concentrations of unlabeled ligand (IGFl, IGF2 or
15 insulin) or antibodies (2D10, 12D5, 13F5,47-9, 9G4). Results are plotted as percent of
maximal specific binding and are represented in figure 5.
2D10 and 13F5 displaced efficaciously and fully labeled IGFl with very similar
subnanomolar affinities, and in this example of 0.2 and 0.35 nM respectively. By
comparaison, 47-9 yielded an ICso value of 0.18 nM (Fig. 5).
20 These afkities are higher than those of the natural Hybrid-RsA ligands IGFl
(2.0 nM in this example) and IGF2 (12 nM in this example).
Example 4-3: Displacement analysis of F ' ~ ~ ~ ~oInG HFyIb rid-RsB bv 2D10,
12D5,13F5 and 47-9
Hybrid-RsB from RtAR-B cell lysates were immunocaptured in Maxisorb plates
2 5 coated with 83-7 antibody.
[lBfl-1~(F~igl. 6) was then allowed to bind to immunocaptured receptors in the
absence or the presence of increasing concentrations of IGF1, IGm, insulin or
antibodies (2D10, 12D5, 13F5, 47-9, 9G4). Results are plotted as percent of maximal
binding.
30 2D 10 and 13F5 displaced efficaciously and hlly labeled IGF 1 with very similar
subnanomolar affinities, and in this example of 0.04 and 0.15 reiPectively. By.
comparison, 47-9 was less effective with an ICso value of 0.40 nM (Fig. 6).
Example 4-4: Displacement analysis of f'2sllinsulin on insulin receptor A (IR-A)
and B (IR-B) isofoms bv 2D 10,12D5,13F5 and MA-10
['25~insuli(n4 0,000 cpm) was allowed to bind to R-AR-A or R-/IR-B intact cells
for 16 hours at 4OC, in the absence or presence of increasing concentrations of unlabeled
5 ligand (IGF1, IGF;! or insulin) or antibodies (2D10, 12D5, 13F5). Results are plotted as
percent of m&al specific binding anr are represented on figures 7 and 8, respectively
for IR-A and IR-B.
Neither 2D10, nor 12D5 nor 13F5 displaced insulin, in contrast to the reference
antibody MA-10 (IC50: 0.90 and 1.5 nM for IR-A (Fig. 7) and IR-B (Fig. 8),
10 respectively).

We Claim:
1. An isolated antibody, or one of its functional fragments, said antibody
or one of its said fragments being capable of binding to the human insulin-like
growth factor I receptor IGF-IR and inhibiting the natural attachment of its ligands
IGFl and/or IGF2 and/or capable of specifically inhibiting the tyrosine kinase activity
of said IGF-IR, characterized in that it is capable of binding to the hybrid-R,
isoform(s) A and/or B, and/or inhibiting the binding of its native ligands, preferably
designated herein as IGFl and/or IGF2 and/or insulin, and/or capable of specifically
inhibiting the tyrosine kinase activity of said hybrid-R, isoform(s) A and/or B, said
antibody being selected in the group consisting of :
a) the antibody comprising a light chain comprising at least one
complementarity determining region CDR chosen from the CDRs of sequence SEQ ID
No. 7, 8 or 9, or at least one CDR whose sequence has at least 80% identity after
optimum alignment with the sequence SEQ ID No. 7, 8 or 9, and a heavy chain
comprising at least one CDR chosen from the CDRs'of sequence SEQ ID Nos. 10, 11
and 12, or at least one CDR whose sequence has at least 80% identity after optimum
alignment with the sequence SEQ ID Nos. 10,ll and 12; and
b) the antibody comprising a light chain comprising at least one
complementarity determining region CDR chosen from the CDRs of sequence SEQ ID
No. 13, 14 or 15, or at least one CDR whose sequence has at least 80% identity after
optimum alignment with the sequence SEQ ID No. 13, 14 or 15, and a heavy chain
comprising at least one CDR chosen from the CDRs of sequence SEQ ID Nos. 16, 17
and 18, or at least one CDR whose sequence has at least 80% identity after optimum
alignment with the sequence SEQ ID Nos. 16,17 and 18.
2. The antibody as claimed in claim 1, characterized in that it is selected in
the group consisting of :
a) the antibody called 12D5 comprising a heavy chain of sequence
comprising the amino acid sequence SEQ ID No. 22 or 23 and a light chain of
sequence comprising the amino acid sequence SEQ ID No. 21; and
b) the antibody called 2D10 comprising a heavy chain of sequence
comprising the amino acid sequence SEQ ID No. 25 and a light chain of sequence
comprising the amino acid sequence SEQ ID No. 24.
3. A murine hybridoma capable of secreting an antibody as claimed in
one of claims 1 or 2.
4. A murine hybridoma characterized in that it is selected in the group
consisting of :
a) the murine hybridoma deposited at the CNCM, Institut Pasteur, Paris,
on April 8,2004 under the number 1-3195;
b) the murine hybridoma deposited at the CNCM, Institut Pasteur, Paris,
on May 13,2004 under the number 1-3214; and
c) the murine hybridoma deposited at the CNCM, Institut Pasteur, Paris,
on July 19 2004 under the number 1-3249.
5. An antibody, or one of its functional fragments, characterized in that it
is capable of binding to the hybrid-R, isoform(s) A and/or B, and/or inhibiting the
binding of its native ligands, preferably designated herein as IGFl and/or IGF2
and/or insulin, and/or capable of specifically inhibiting the tyrosine kinase activity of
said hybrid-R, isoform(s) A and/or B, said antibody is secreted by the hybridoma as
claimed in claim 4.
6. The antibody or one of its functional fragments, as claimed in claim 1 or
5, characterized in that said antibody is a chimeric antibody and moreover comprises
the light chain and heavy chain constant regions derived from an antibody of a
species heterologous to the mouse.
7. The chimeric antibody, or one of its functional fragments, as claimed in
claim 6, characterized in that said heterologous species is man.
Dated this theadday of July 2013.
(Ritu Gand hi)
of Subramaniam & Associates
Attorneys for the Applicants