The present invention relates to a novel antibody, in particular a monoclonal antibody, capable of binding to IGF-1R, as well as the amino and nucleic acid sequences coding for said antibody.

The insulin- like growth factor 1 receptor called IGF-1R (or sometimes IGF1R) is a receptor with tyrosine kinase activity having 70% homology with the insulin receptor IR. IGF-1R is a glycoprotein of molecular weight approximately 350,000. It is a hetero-tetrameric receptor of which each half -linked by disulfide bridges- is composed of an extracellular a-subunit and of a transmembrane β-subunit. IGF-1R binds IGF1 and IGF2 with a very high affinity (Kd #1 nM) but is equally capable of binding to insulin with an affinity 100 to 1000 times lower. Conversely, the IR binds insulin with a very high affinity although the IGFs only bind to the insulin receptor with a 100 times lower affinity. The tyrosine kinase domains of IGF-1R and of IR have a very high sequence homology although the zones of weaker homology respectively concern the cysteine-rich region situated on the a-subunit and the C-terminal part of the β-subunit. The sequence differences observed in the α-subunit are situated in the binding zone of the ligands and are therefore at the origin of the relative affinities of IGF-1R and of IR for the IGFs and insulin respectively. The differences in the C-terminal part of the β-subunit result in a divergence in the signalling pathways of the two receptors; IGF-1R mediating mitogenic, differentiation and antiapoptosis effects, while the activation of the IR principally involves effects at the level of the metabolic pathways.

The role of the IGF system in carcinogenesis has become the subject of intensive research in the last 20 years. This interest followed the discovery of the fact that in addition to its mitogenic and antiapoptosis properties, IGF-1R 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-1R leads, in a great variety of cells, to a growth of the cells independent of the support in media devoid of foetal calf serum, and to the formation of tumors in nude mice. This in itself is not a unique property since a great variety of products of overexpressed genes 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 IGF-IR" cells, in which the gene coding for IGF-IR has been inactivated, are totally refractory to transformation by 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.

In such a context IGF-IR has been considered for a long time as an interesting target in oncology. A large number of projects targeting IGF-IR (humanized or human antibodies or small molecules) have been initiated to develop IGF-IR antagonists for the treatment of cancers and more than 70 clinical trials have been performed in various indications. Nevertheless, at this date, none of these projects have been successful and there are no IGF-IR antibodies on the market.

The present invention aims to provide at least one reagent that can be used as a diagnostic or prognosis biomarker for detecting and/or monitoring oncogenic disorders especially those characterized by expression of IGF-IR or those that are mediated by aberrant IGF-IR expression.

Previous attempts to develop a valuable antibody that can be used as a relevant diagnostic or prognostic tool have been reported but none of these are giving satisfaction.

As it will be apparent from the following examples, the inventors have been surprised to demonstrate that the commercial antibodies commonly used at this day for the scoring of the IGF-IR expressing tumors seem to be not relevant as they give false positive and/or false negative. This issue has lead, in part, to the failure of clinical trials with IGF-IR antibodies due to the selection of the patients rather than the real activity of the IGF-IR antibodies.

Moreover, first studies performed using commercial antibodies showed discrepancy between IGF-IR scoring and anti-tumoral activity of targeted ADC therapy.

The present invention intends to remedy this issue by providing a novel antibody which, contrary to the existing ones, is capable of straining which do correlate with the pharmacology of IGF-IR targeted therapy.

In a first aspect, a subject of the invention is an isolated antibody, or an antigen-binding fragment thereof, that binds to the IGF-IR, preferably human IGF-IR, with high affinity and can thus be useful in methods to diagnose pathological hyperproliferative oncogenic disorders mediated by IGF-IR expression.

An embodiment of the invention relates to an antibody, or an antigen-binding fragment thereof, comprising the six CDRs of sequences SEQ ID Nos. 1, 2, 3. 4, 5 and 6.

In a particular embodiment, the invention relates to an IGF-IR antibody, or an antigen-binding fragment thereof, characterized in that it comprises:

i) a heavy chain with CDR-H1 of sequence SEQ ID No. 1, CDR-H2 of sequence SEQ ID No. 2 and CDR-H3 of sequence SEQ ID No. 3; and

ii) a light chain with CDR-L1 of sequence SEQ ID No. 4, CDR-L2 of sequence SEQ ID No. 5 and CDR-L3 of sequence SEQ ID No. 6.

The terms "antibody", "antibodies" "ab" or "immunoglobulin" are used interchangeably in the broadest sense and include monoclonal antibodies, isolated, engineered, chemically synthesized, or recombinant antibodies (e.g., full length or intact monoclonal antibodies), polyclonal antibodies, multivalent antibodies or multispecific antibodies (e.g., bispecific antibodies) and also antibody fragment, so long as they exhibit the desired biological activity. In an embodiment, the invention relates to a recombinant antibody.

As used in the present specification, the expression "IGF-IR antibody" should be interpreted as similar to "anti-IGF-lR antibody" and means an antibody capable of binding to IGF- 1 R.

By "IGF-IR binding fragment" or "antigen-binding fragment" of an antibody, it is intended to indicate any peptide, polypeptide, or protein retaining the ability to bind to the IGF-1R target (also generally referred as antigen) of the antibody. In an embodiment, such "antigen binding fragments" are selected in the group consisting of Fv, scFv (sc for single chain), Fab, F(ab')2, Fab', scFv-Fc fragments or diabodies, or any fragment of which the half-life time would have been increased by chemical modification, such as the addition of poly(alkylene) glycol such as poly(ethylene) glycol ("PEGylation") (pegylated fragments called Fv-PEG, scFv-PEG, Fab-PEG, F(ab')2-PEG or Fab'-PEG) ("PEG" for Poly(Ethylene) Glycol), or by incorporation into a liposome, said fragments having at least one of the characteristic CDRs of the antibody according to the invention. Preferably, said "antigen binding fragments" will be constituted or will comprise a partial sequence of the heavy or light variable chain of the antibody from which they are derived, said partial sequence being sufficient to retain the same specificity of binding as the antibody from which it is descended and a sufficient affinity, preferably at least equal to 1/100, in a more preferred manner to at least 1/10, of the affinity of the antibody from which it is descended, with respect to the target.

Preferably, said "IGF-1R binding fragment" or "antigen-binding fragment" comprises at least:

i) the CDR-H1 of sequence SEQ ID No. 1, CDR-H2 of sequence SEQ ID No. 2 and CDR-H3 of sequence SEQ ID No. 3; and

ii) the CDR-L1 of sequence SEQ ID No. 4, CDR-L2 of sequence SEQ ID No. 5 and CDR-L3 of sequence SEQ ID No. 6.

By "binding", "binds", or the like, it is intended that the antibody, or any antigen-binding fragment thereof, forms a complex with an antigen that is relatively stable under physiologic conditions. Specific binding can be characterized by an equilibrium dissociation constant of at least about lxlO"6 M or less. Methods for determining whether two molecules bind are well known in the art and include, for example, equilibrium dialysis, surface plasmon resonance, and the like. For the avoidance of doubt, it does not mean that the said antibody could not bind or interfere, at a low level, to another antigen. Nevertheless, as an embodiment, the said antibody binds only to the said

antigen.

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 IMGT.

The IMGT unique numbering has been defined to compare the variable domains whatever the antigen receptor, the chain type, or the species [Lefranc M.-P., Immunology Today 18, 509 (1997) / Lefranc M.-P., The Immunologist, 7, 132-136 (1999) / Lefranc, M.-P., Pommie, C, Ruiz, M., Giudicelli, V., Foulquier, E., Truong, L., Thouvenin-Contet, V. and Lefranc, Dev. Comp. Immunol, 27, 55-77 (2003)]. In the IMGT unique numbering, the conserved amino acids always have the same position, for instance cystein 23 (lst-CYS), tryptophan 41 (CONSERVED-TRP), hydrophobic amino acid 89, cystein 104 (2nd-CYS), phenylalanine or tryptophan 118 (J-PHE or J-TRP). The IMGT unique numbering provides a standardized delimitation of the framework regions (FR1-IMGT: positions 1 to 26, FR2-IMGT: 39 to 55, FR3-IMGT: 66 to 104 and FR4-IMGT: 118 to 128) and of the complementarity determining regions: CDR1-IMGT: 27 to 38, CDR2-IMGT: 56 to 65 and CDR3-IMGT: 105 to 117. As gaps represent unoccupied positions, the CDR-IMGT lengths (shown between brackets and separated by dots, e.g. [8.8.13]) become crucial information. The IMGT unique numbering is used in 2D graphical representations, designated as IMGT Colliers de Perles [Ruiz, M. and Lefranc, M.-P., Immunogenetics, 53, 857-883 (2002) / Kaas, Q. and Lefranc, M.-P., Current Bioinformatics, 2, 21-30 (2007)], and in 3D structures in IMGT/3Dstructure-DB [Kaas, Q., Ruiz, M. and Lefranc, M.-P., T cell receptor and MHC structural data. Nucl. Acids. Res., 32, D208-D210 (2004)].

It must be understood that, without contradictory specification in the present specification, complementarity-determining regions or CDRs, mean the hypervariable regions of the heavy and light chains of immunoglobulins as defined according to the IMGT numbering system.

Nevertheless, CDRs can also be defined according to the Kabat numbering system (Kabat et al., Sequences of proteins of immunological interest, 5th Ed., U.S. Department of Health and Human Services, NIH, 1991, and later editions). There are three heavy-chain CDRs and three light-chain CDRs. Here, the terms "CDR" and "CDRs" are used to indicate, depending on the case, one or more, or even all, of the regions containing the majority of the amino acid residues responsible for the antibody's binding affinity for the antigen or epitope it recognizes. In order to simplify the reading of the present application, the CDRs according to Kabat are not defined. Nevertheless, it would be obvious for the person skilled in that art, using the definition of the CDRs according to IMGT, to define the CDRs according to Kabat.

In a particular embodiment, the IGF-1R antibody according to the invention is characterized in that it comprises a heavy chain variable domain of sequence SEQ ID No. 7, or any sequence with at least 90% of homology with the sequence SEQ ID No. 7.

In a particular embodiment, the IGF-1R antibody according to the invention is characterized in that it comprises a light chain variable domain of sequence SEQ ID No. 8, or any sequence with at least 90% of homology with the sequence SEQ ID No. 8.

According to still another embodiment, the antibody referred as 816C12, is characterized in that it comprises a heavy-chain variable domain sequence comprising the amino acid sequence SEQ ID No. 7 or a sequence with at least 80%, preferably 85%, 90%, 95% and 98% of homology after optimal alignment with sequence SEQ ID No. 7; and/or in that it comprises a light-chain variable domain sequence comprising the amino acid sequence SEQ ID No. 8 or a sequence with at least 80%, preferably 85%, 90%, 95% and 98% of homology after optimal alignment with sequence SEQ ID No. 8.

In the sense of the present invention, the "percentage of homology" between two sequences of nucleic acids or amino acids means the percentage of identical nucleotides or amino acid residues between the two sequences to be compared, obtained after optimal alignment, this percentage being purely statistical and the differences between the two sequences being distributed randomly along their length. The comparison of two nucleic acid or amino acid sequences is traditionally carried out by comparing the sequences after having optimally aligned them, said comparison being able to be conducted by segment or by using an "alignment window". Optimal alignment of the sequences for comparison can be carried out, in addition to comparison by hand, by means of the local homology algorithm of Smith and Waterman (1981) [Ad. App. Math. 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] or 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 by the comparison software BLAST NR or BLAST P). For the amino acid sequence exhibiting at least 80%, preferably at least 85%, 90%>, 95% and 98% of homology with a reference amino acid sequence, preferred examples include those containing the reference sequence, certain modifications, notably a deletion, addition or substitution of at least one amino acid, truncation or extension. In the case of substitution of one or more consecutive or non-consecutive amino acids, substitutions are preferred in which the substituted amino acids are replaced by "equivalent" amino acids. Here, the expression "equivalent amino acids" is meant to indicate any amino acids likely to be substituted for one of the structural amino acids without however modifying the biological activities of the corresponding antibodies and of those specific examples defined below.

Equivalent amino acids can be determined either on their structural homology with the amino acids for which they are substituted or on the results of comparative tests of biological activity between the various antigen binding proteins likely to be generated.

As a non-limiting example, table 1 below summarizes the possible substitutions likely to be carried out without resulting in a significant modification of the biological activity of the corresponding modified antigen binding protein; inverse substitutions are naturally possible under the same conditions.

Table 1

A particular aspect of the invention is that the antibody, or any antigen binding fragment thereof, does not bind to the Insulin receptor (IR).

In another embodiment, the antibody of the invention consists of a monoclonal antibody.

The term "monoclonal antibody" or "Mab" as used herein refers to an antibody obtained from a population of substantially homogeneous antibodies, i.e. the individual antibodies of the population are identical except for possible naturally occurring mutations that may be present in minor amounts. Monoclonal antibodies are highly specific, being directed against a single

epitope. Such monoclonal antibody may be produced by a single clone of B cells or hybridoma. Monoclonal antibodies may also be recombinant, i.e. produced by protein engineering. Monoclonal antibodies may also be isolated from phage antibody libraries. In addition, in contrast with preparations of polyclonal antibodies which typically include various antibodies directed against various determinants, or epitopes, each monoclonal antibody is directed against a single epitope of the antigen. The invention relates to an antibody isolated or obtained by purification from natural sources or obtained by genetic recombination or chemical synthesis.

In another embodiment, the antibody of the invention consists of a recombinant antibody. The term "recombinant antibody" refers to an antibody that results from the expression of recombinant DNA within living cells. A recombinant antibody of the invention is obtained by using laboratory methods of genetic recombination, well known by a person skilled in the art, creating DNA sequences that would not be found in biological organisms.

In another embodiment, the antibody of the invention consists of a chemically synthesized antibody.

"IGF-1R antibody" includes (without contrary specification) the murine, but also the chimeric and the humanized forms of the said IGF-1R antibody.

For more clarity, the following table 2 illustrates the sequences of the antibody 816C12, defined according to IMGT.

Table 2

CDR

Antibody Heavy chain Light chain SEQ ID NO.

numbering

CDR-H1 1

CDR-H2 2

IMGT CDR-H3 3

816C12

CDR-L1 4

1-4894 CDR-L2 5

CDR-L3 6 variable domain 7

variable domain 8

In one embodiment, the monoclonal antibody herein includes murine, chimeric and humanized antibody. The antibody can be derived from an hybridoma of murine origin filed within the French collection for microorganism cultures (CNCM, Pasteur Institute, Paris, France), said hybridoma being obtained by the fusion of Balb/C immunized mice splenocytes/lymphocytes and cells of the myeloma Sp 2/O-Ag 14 cell line.

According to another aspect, the invention relates to a murine hybridoma capable of secreting a monoclonal antibody according to the invention, notably the hybridoma of murine origin deposited at the CNCM, Institut Pasteur, Paris, France, on Septemberl7, 2014, under the number 1-4894.

The monoclonal antibody, here referred as 816C12, or any antigen-binding fragment thereof, being secreted by the said hybridoma 1-4894 obviously forms part of the present invention.

The invention relates to an IGF-1R antibody, or an antigen-binding fragment thereof, characterized in that it is secreted by the hybridoma filed at the CNCM, Institut Pasteur, Paris, on September 17, 2014, under number 1-4894.

The invention also describes the murine hybridoma filed at the CNCM, Institut Pasteur, Paris, on September 17, 2014, under number 1-4894.

A novel aspect of the present invention relates to an isolated nucleic acid, characterized in that it is chosen from the following nucleic acids:

a) a nucleic acid coding for an antibody according to the invention ; b) a nucleic acid comprising a sequence selected from the sequences SEQ ID No.9 or 10, or a sequence with at least 80%, preferably 85%, 90%, 95% and 98% of homology after optimal alignment with the sequences SEQ ID No. 9 or 10; and

e) a complementary nucleic acids of the nucleic acids as defined in a) or b).

Table 3 below summarizes the various nucleotide sequences concerning the antibody 816C 12 of the invention.

Table 3

The terms "nucleic acid", "nucleic sequence", "nucleic acid sequence", "polynucleotide", "oligonucleotide", "polynucleotide sequence" and "nucleotide sequence", used interchangeably in the present description, mean a precise sequence of nucleotides, modified or not, defining a fragment or a region of a nucleic acid, containing unnatural nucleotides or not, and being either a double- strand DNA, a single-strand DNA or transcription products of said DNAs.

It should also be included here that the present invention does not relate to nucleotide sequences in their natural chromosomal environment, i.e., in a natural state. The sequences of the present invention have been isolated and/or purified, i.e., they were sampled directly or indirectly, for example by a copy, their environment having been at least partially modified. Isolated nucleic acids obtained by recombinant genetics, by means, for example, of host cells, or obtained by chemical synthesis should also be mentioned here.

The invention also relates to a vector comprising a nucleic acid as described in the invention.

The invention notably targets cloning and/or expression vectors that contain such a nucleotide sequence.

The vectors of the invention preferably contain elements which allow the expression and/or the secretion of nucleotide sequences in a given host cell. The vector thus must contain a promoter, translation initiation and termination signals, as well as suitable transcription regulation regions. It must be able to be maintained in a stable manner in the host cell and may optionally have specific signals which specify secretion of the translated protein. These various elements are selected and optimized by a person skilled in the art according to the host cell used. For this purpose, the nucleotide sequences can be inserted in self- replicating vectors within the chosen host or be integrative vectors of the chosen host.

Such vectors are prepared by methods typically used by a person skilled in the art and the resulting clones can be introduced into a suitable host by standard methods such as lipofection, electroporation, heat shock or chemical methods.

The vectors are, for example, vectors of plasmid or viral origin. They are used to transform host cells in order to clone or express the nucleotide sequences of the invention.

The invention also comprises host cells transformed by or comprising a vector as described in the present invention.

The host cell can be selected among prokaryotic or eukaryotic systems such as bacterial cells, for example, but also yeast cells or animal cells, notably mammal cells. Insect or plant cells can also be used.

The invention also relates to animals, other than man, that have a transformed cell according to the invention.

Another aspect of the invention relates to a method for the production of an antibody according to the invention, or one of its functional fragments, characterized in that said method comprises the following steps:

a) the culture in a medium of and the suitable culture conditions for a host cell according to the invention; and

b) the recovery of said antibody, or one of its functional fragments, thus produced from the culture medium or from said cultured cells.

The transformed cells according to the invention are of use in methods for the preparation of recombinant polypeptides according to the invention.

Methods for the preparation of polypeptide according to the invention in recombinant form, characterized in that said methods use a vector and/or a cell transformed by a vector according to the invention, are also comprised in the present invention. Preferably, a cell transformed by a vector according to the invention is cultured under conditions that allow the expression of the aforesaid polypeptide and recovery of said recombinant peptide.

As already mentioned, the host cell can be selected among prokaryotic or eukaryotic systems. In particular, it is possible to identify the nucleotide sequences of the invention that facilitate secretion in such a prokaryotic or eukaryotic system. A vector according to the invention carrying such a sequence can thus be used advantageously for the production of recombinant proteins to be secreted. Indeed, the purification of these recombinant proteins of interest will be facilitated by the fact that they are present in the supernatant of the cellular culture rather than inside host cells.

The use of the antibody of the invention as bio marker is also disclosed. The methods may be used for detecting or diagnosing various hyperproliferative oncogenic disorders associated with expression of IGF-IR exemplified by, but not limited to, prostate cancer, osteosarcomas, lung cancer, breast cancer, endometrial cancer, glioblastoma, colon, cancer, gastric cancer, renal cancer, pancreas cancer, head and neck cancer or any other cancer associated with expression of IGF-IR. As would be recognized by one of ordinary skill in this art, the level of antibody expression associated with a particular disorder will vary depending on the nature and/or the severity of the pre-existing condition.

Administration of the antibodies of the present invention in any of the conventional ways known to one skilled in the art (e.g., topical, parenteral, intramuscular, etc.), will provide an extremely useful method of detecting dysplastic cells in a sample as well as allowing a clinician to monitor the therapeutic regiment of a patient undergoing treatment for a hyperproliferative disorder associated with or mediated by expression of IGF-IR.

The antibody of the invention, or an antigen-binding fragment thereof, will find use in various medical or research purposes, including the detection, diagnosis, prognosis and staging of various pathologies associated with expression of IGF-IR.

An embodiment of the invention relates to the IGF-IR antibody, or an antigen-binding fragment thereof, as above described for use as an agent for the detection of IGF-IR expressing tumoral cells.

Another embodiment of the invention is the IGF-IR antibody, or an antigen-binding fragment thereof, as above described, for use in the in vitro or ex vivo diagnosing or prognosing of an oncogenic disorder associated with

expression of IGF-IR.

"Diagnosing" a disease as used herein refers to the process of identifying or detecting the presence of a pathological hyperproliferative oncogenic disorder associated with or mediated by expression of IGF-IR, monitoring the progression of the disease, and identifying or detecting cells or samples that are indicative of a disorder associated with the expression of IGF-IR.

"Prognosis" as used herein means the likelihood of recovery from a disease or the prediction of the probable development or outcome of a disease. For example, if a sample from a subject is negative for staining with the IGF-IR antibody, then the "prognosis" for that subject is better than if the sample is positive for IGF-IR staining. Samples may be scored for IGF-IR expression levels on an appropriate scale as it will be more detailed hereinafter.

The IGF-IR antibody can be present in the form of an immunoconjugate or of a labeled-antibody to obtain a detectable/quantifiable signal. When used with suitable labels or other appropriate detectable biomolecules or chemicals, the IGF-IR antibody is particularly useful for in vitro and in vivo diagnosis and prognosis applications.

Labels for use in immunoassays are generally known to those skilled in the art and include enzymes, radioisotopes, and fluorescent, luminescent and chromogenic substances, including colored particles such as colloidal gold or latex beads. Suitable immunoassays include enzyme-linked immunosorbent assays (ELISA). Various types of labels and methods of conjugating the labels to the IGF-IR antibodies are well known to those skilled in the art, such as the ones set forth below.

As used herein, the term "an oncogenic disorder associated with expression of IGF-IR" is intended to include diseases and other disorders in which the presence of high levels of IGF-IR (aberrant) in a subject suffering from the disorder has been shown to be or is suspected of being either responsible for the pathophysiology of the disorder or a factor that contributes to a worsening of the disorder. Alternatively, such disorders may be evidenced, for example, by an increase in the levels of IGF-IR on the cell surface in the affected cells or tissues of a subject suffering from the disorder. The increase in IGF-IR levels may be detected using the IGF-IR antibody.

In certain embodiments, "increased expression" as it relates to IGF-IR refers to protein or gene expression levels that demonstrate a statistically significant increase in expression (as measured by RNA expression or protein expression) relative to a control.

An embodiment is an IGF-IR antibody, or an antigen-binding fragment thereof, as above described, for use in determining whether a patient with an oncogenic disorder is likely to benefit from treatment with an inhibitor targeting the IGF-IR pathway, preferentially an IGF-IR antibody alone, combined or conjugated.

As used in the present specification, the expression "inhibitor targeting the IGF-IR pathway" means any compound capable of decreasing or inhibiting the tyrosine kinase activity of IGF-IR, either by binding to the ligand(s) of IGF-IR or to the IGFR itself. Examples for such inhibitors are protein, peptides, antibodies or Antibody-Drug-Conjugates or any chemical compound which act as IGF-IR antagonists, antisense oligonucleotides or siRNA inhibiting expression of the IGF-IR gene or of a gene encoding one of the IGFR ligand(s), or any other drug or compound known by the person skilled in the art.

More particularly, in the sense of the present specification, the inhibitor targeting the IGF-IR pathway is intended to encompass any compound or molecule capable of binding to the IGF-IR and inhibiting the binding of its ligand(s).

Still more particularly, in the sense of the present specification, the inhibitor targeting the IGF-IR pathway is intended to encompass any monoclonal antibody which binds to the IGF-IR.

In another preferred embodiment, the inhibitor targeting the IGF-IR pathway consists of an Antibody-Drug-Conjugate (ADC) wherein the antibody moiety targets the IGF-IR and the Drug moiety can be selected from any drugs such as cytotoxic, cytostatic, toxins, etc... In an exemplified embodiment, the drug moiety can consist of an auristatin, an analog or a derivative.

It is also an object of the invention to describe a method for detecting in vitro or ex vivo the presence and/or the location of IGF-IR expressing tumoral cells in a subject, said method comprising the steps of:

(a) contacting a biological sample from the said subject with the IGF-IR antibody, or an antigen-binding fragment thereof, according to the present invention as above described; and

(b) detecting the binding of the said IGF-IR antibody, or an antigen-binding fragment thereof, with the said biological sample.

The present invention is also directed to an in vitro or ex vivo method for detecting and /or to quantify and/or to determine the level of, the expression of IGF-IR in, preferably at the surface of cells of, a subject, said method comprising the steps of:

(a) contacting a biological sample from the said subject with the IGF-IR antibody,, or an antigen-binding fragment thereof, according to the present invention as above described; and

(b) detecting, and/or quantifying, and/or determining the level of, the binding of the said IGF-IR antibody, or an antigen-binding fragment thereof, with the said biological sample.

The binding of the IGF-IR antibody may be detected and/or quantified and/or determined by various assays available to the skilled artisan. Although any suitable means for carrying out the assays are included within the invention, Fluorescence Activated Cell Sorting (FACS), ELISA, western blotting and immunohistochemistry (IHC) can be mentioned in particular. Preferred methods include IHC and FACS.

The invention also describes a method for detecting in vitro or ex vivo the percentage of tumoral cells expressing IGF-IR in a subject, said method comprising the steps of:

(a) contacting a biological sample from the said subject with the IGF-IR antibody, or an antigen-binding fragment thereof, as above described; and

(b) quantifying the percentage of cells expressing IGF-IR in the biological sample.

Another embodiment is a method for determining in vitro or ex vivo the expression level of IGF-IR in tumoral cells or in a tumor in a subject, said method comprising the steps of:

(a) contacting a biological sample from the said subject with the IGF-IR antibody, or an antigen-binding fragment thereof, as above described; and

(b) quantifying the level of binding of the said IGF-IR antibody, or an antigen-binding fragment thereof, to IGF-IR in the said biological sample.

As will be apparent to the skilled artisan, the level of IGF-IR antibody binding to IGF-IR may be quantified by any means known to the person of skills in the art. Preferred methods involve the use of immunoenzymatic processes, such as ELISA assays, immunofluorescence, IHC, radio-immunoassay (RIA), or FACS.

According to the method of the invention, the level of binding of the said IGF-IR antibody, or an antigen-binding fragment thereof, to IGF-IR is quantified by Fluorescence Activated Cell Sorting (FACS) or immunohistochemistry (IHC).

A "biological sample" may be any sample that may be taken from a subject. Such a sample must allow for the determination of the expression levels of the biomarker of the invention. The nature of the sample will thus be dependent upon the nature of the tumor.

Preferred biological samples include samples such as a blood sample, a plasma sample, or a lymph sample, if the cancer is a liquid tumor.

Preferred biological samples include samples such as a biopsy sample or a sample taken from a surgical resection therapy, if the cancer is a solid tumor.

Preferably, the biological sample is a biological fluid, such as serum, whole blood cells, a tissue sample or a biopsy of human origin. The sample may for example include, biopsied tissue, which can be conveniently assayed for the presence of a pathological oncogenic disorder associated with expression of IGF-IR.

Once a determination is made of the IGF-IR expression level in the tested biological samples, the results can be compared with those of control samples, which are obtained in a manner similar to the tested biological samples but from individuals that do not have an oncogenic disorder associated with expression of IGF-IR. If the level of IGF-IR is significantly elevated in the tested biological sample, it may be concluded that there is an increased likelihood of the subject from which it was derived has or will develop said disorder.

The invention relates to a process of in vitro or ex vivo diagnosis or prognosis of an IGF-IR expressing tumor, wherein said process comprises the steps of (i) determining the expression level of IGF-IR by the a method for determining in vitro or ex vivo the expression level of IGF-IR in tumoral cells or in a tumor in a subject according to the present invention and as above described, and (ii) comparing the expression level of step (i) with a reference expression level of IGF-IR from normal tissue or a non expressing IGF-IR tissue.

With regards to the development of targeted antitumor therapy, the diagnosis with immunohistological techniques gives in situ information on the receptor expression level and thus enables to select patients susceptible to be treated following the expression level of receptors needed for such treatment.

Stage determination has potential prognosis value and provides criteria for designing optimal therapy. Simpson et al, J. Clin. Oncology 18:2059 (2000). For example, treatment selection for solid tumors is based on tumor staging, which is usually performed using the Tumor/Node/Metastasis (TNM) test from the American Joint Committee on Cancer (AJCC). It is commonly acknowledged that, while this test and staging system provides some valuable information concerning the stage at which solid cancer has been diagnosed in the patient, it is imprecise and insufficient. In particular, it fails to identify the earliest stages of tumor progression.

Another embodiment consists of a method for determining in vitro or ex vivo the IGF-IR scoring of tumoral cells or of the tumor in a subject, said method comprising the steps of:

(a) contacting a biological sample from the said subject with the IGF-IR antibody, or an antigen-binding fragment thereof, as above described;

(b) quantifying by Fluorescence Activated Cell Sorting (FACS) or immunohistochemistry (IHC) the level of binding of the said IGF-IR antibody, or an antigen-binding fragment thereof, to IGF-IR in the said biological sample; and

(c) scoring the tumoral cells or the tumor by comparing the quantified level obtained in step (b) to an appropriate scale based on two parameters which are the intensity of the staining and the percentage of positive cells.

In an embodiment, the IGF-1R antibody is capable of binding IGF-1R when tissue samples are, formalin fixed-, formol substituted fixed-, Glyco-fixx fixed-, paraffin embedded and/or frozen.

Any conventional hazard analysis method may be used to estimate the prognostic value of IGF-1R. Representative analysis methods include Cox regression analysis, which is a semiparametric method for modeling survival or time-to-event data in the presence of censored cases (Hosmer and Lemeshow, 1999; Cox, 1972). In contrast to other survival analyses, e.g. Life Tables or Kaplan-Meyer, Cox allows the inclusion of predictor variables (covariates) in the models. Using a convention analysis method, e.g., Cox one may be able to test hypotheses regarding the correlation of IGF-1R expression status of in a primary tumor to time-to-onset of either disease relapse (disease-free survival time, or time to metastatic disease), or time to death from the disease (overall survival time). Cox regression analysis is also known as Cox proportional hazard analysis. This method is standard for testing the prognostic value of a tumor marker on patient survival time. When used in multivariate mode, the effect of several covariates are tested in parallel so that individual covariates that have independent prognostic value can be identified, i.e. the most useful markers. The term negative or positive "IGF-1R status" can also be referred as [IGF-1R (-)] or [IGF-1R (+)].

A sample may be "scored" during the diagnosis or monitoring of cancer.

CLAIMS

1. An IGF-1R antibody, or an antigen-binding fragment thereof, characterized in that it comprises:

i) a heavy chain with CDR-H1 of sequence SEQ ID No. 1, CDR-H2 of sequence SEQ ID No. 2 and CDR-H3 of sequence SEQ ID No. 3; and

ii) a light chain with CDR-L1 of sequence SEQ ID No. 4, CDR-L2 of sequence SEQ ID No. 5 and CDR-L3 of sequence SEQ ID No. 6.

2. The IGF-1R antibody according to claim 1, characterized in that it comprises a heavy chain variable domain of sequence SEQ ID No. 7, or any sequence with at least 90% of homology with the sequence SEQ ID No. 7; and/or a light chain variable domain of sequence SEQ ID No. 8, or any sequence with at least 90% of homology with the sequence SEQ ID No. 8.

3. An IGF-1R antibody, or an antigen-binding fragment thereof, characterized in that it is secreted by the hybridoma filed at the CNCM, Institut

Pasteur, Paris, on September 17, 2014, under number 1-4894.

4. The IGF-1R antibody, or an antigen-binding fragment thereof, according to anyone of claims 1 to 3 for use as an agent for the detection of IGF-1R expressing tumoral cells or for determining the expression level of IGF-1R expressing tumoral cells.

5. The IGF-1R antibody, or an antigen-binding fragment thereof, according to anyone of claims 1 to 3, for use in the in vitro or ex vivo diagnosing or prognosing of an oncogenic disorder associated with expression of IGF-1R.

6. The IGF-1R antibody, or an antigen-binding fragment thereof, according to anyone of claims 1 to 3, for use in determining whether a patient with an oncogenic disorder is likely to benefit from treatment with an inhibitor targeting the IGF-IR pathway, preferentially an IGF-IR antibody alone, combined or conjugated.

7. A method for detecting in vitro or ex vivo the presence and/or the location of IGF-IR expressing tumoral cells in a subject, said method comprising the steps of:

(a) contacting a biological sample from the said subject with the IGF-IR antibody, or an antigen-binding fragment thereof, according to anyone of claims 1 to 3; and

(b) detecting the binding of the said IGF-IR antibody, or an antigen-binding fragment thereof, with the said biological sample.

8. A method for detecting in vitro or ex vivo the percentage of tumoral cells expressing IGF-IR in a subject, said method comprising the steps of:

(a) contacting a biological sample from the said subject with the IGF-IR antibody, or an antigen-binding fragment thereof, according to anyone of claims 1 to 3; and

(b) quantifying the percentage of cells expressing IGF-IR in the biological sample.

9. A method for determining in vitro or ex vivo the expression level of IGF-IR in tumoral cells in a subject, said method comprising the steps of:

(a) contacting a biological sample from the said subject with the IGF-IR antibody, or an antigen-binding fragment thereof, according to anyone of claims 1 to 3; and

(b) quantifying the level of binding of the said IGF-IR antibody, or an antigen-binding fragment thereof, to IGF-IR in the said biological sample.

10. A method for determining in vitro or ex vivo the IGF-IR scoring of tumoral cells or of a tumor in a subject, said method comprising the steps of:

(a) contacting a biological sample from the said subject with the IGF-IR antibody, or an antigen-binding fragment thereof, according to anyone of claims 1 to 3;

(b) quantifying by Fluorescence Activated Cell Sorting (FACS) or immunohistochemistry (IHC) the level of binding of the said IGF-IR antibody, or an antigen-binding fragment thereof, to IGF-IR in the said biological sample; and

(c) scoring the tumoral cells or the tumor by comparing the quantified level obtained in step (b) to an appropriate scale based on two parameters which are the intensity of the staining and the percentage of positive cells.

11. A method for determining whether an oncogenic disorder is susceptible to treatment with an antibody drug targeting the IGF-IR pathway, said method comprising the steps of:

(a) determining in vitro or ex vivo the IGF-IR status of tumoral cells or of a tumor of a subject according to the method of claim 10, and

(b) determining that, if the IGF-IR status of tumoral cells or the tumor is

IGF-1R(+), the oncogenic disorder is susceptible to treatment with an antibody drug targeting the IGF-IR pathway.

12. A method for determining in vitro or ex vivo the efficacy of a therapeutic regimen designed to alleviate an oncogenic disorder associated with IGF-IR in a subject suffering from said disorder, said method comprising the steps of:

(a) determining a first expression level of IGF-IR according to claim 9 in a first biological sample, said first biological sample corresponding to first time point of the said treatment;

(b) determining a second expression level of IGF-IR according to claim 9 in a second biological sample, said second biological sample corresponding to a second, later time point of the said treatment;

(c) calculating the ratio of the said first expression level obtained in step (a) to the said second expression level obtained in step (b); and

(d) determining that the efficacy of said therapeutic regime is high when the ratio of step (c) is greater than 1; or determining that the efficacy of said therapeutic regime is low when the ratio of step (c) is inferior or equal to 1.

13. A method for selecting a cancer patient predicted to benefit or not from the administration of a therapeutic amount of an antibody drug targeting the IGF-IR pathway, said method comprising the steps of:

(a) determining the expression level of IGF-IR according to the method of claim 9;

(b) comparing the expression level of the previous step (a) with a reference expression level; and

(c) selecting the patient as being predicted to benefit from a treatment with an antibody drug targeting the IGF-IR pathway, if the ratio of the expression level obtained in (a) to the reference expression level is greater than 1; or

(d) selecting the patient as being not predicted to benefit from a treatment with an antibody drug targeting the IGF-IR pathway, if the ratio of the expression level obtained in (a) to the reference expression level is inferior or equal to 1.

14. A kit for the detection of IGF-IR expressing tumoral cells in a patient, characterized in that said kit comprises at least the IGF-IR antibody, or an antigen-binding fragment thereof, according to anyone of claims 1 to 3.

15. A kit for determining whether a patient with an oncogenic disorder is likely to benefit from treatment with an antibody drug targeting the IGF-IR pathway, characterized in that said kit comprises at least the IGF-IR antibody, or an antigen-binding fragment thereof, according to anyone of claims 1 to 3.