TW201311726A - Use of the antibody I-3859 for the detection and diagnosis of cancers - Google Patents

Abstract

The present invention relates to the use of a novel, isolated anti-CXCR4 antibody in the diagnosis of cancer. In particular, methods for diagnosing and/or prognosing an oncogenic disorder associated with CXCR4 expression, are disclosed.

Description

Antibody I-3859 for cancer detection and diagnosis

The present invention relates to the field of monitoring the prognosis and/or diagnosis and/or treatment of proliferative diseases in a patient. More particularly, the present invention relates to an antibody capable of specifically binding CXCR4, and the use of the antibody, and a corresponding method for detecting pathological hyperproliferative carcinogenic disorders associated with diagnosis and expression of CXCR4. In some embodiments, the disorders are carcinogenic disorders associated with increased CXCR4-related performance or any other pathology associated with overexpression of CXCR4. The invention finally comprises products and/or compositions or kits comprising at least such antibodies for monitoring the prognosis and/or diagnosis and/or treatment of certain cancers.

Chemokines are small, secreted peptides that control the movement of white blood cells along the chemical gradient of a ligand, known as a chemotactic gradient, especially during the entire immune response (Zlotnick A. et al. , 2000). They are divided into two major subfamilies, CC and CXC, and receptors that bind to G protein coupling according to the position of their NH ₂ terminal cysteine residues, and the two major subfamilies are called CCR and CXCR. More than 50 human chemokines and 18 chemokine receptors have been discovered to date.

Many cancers have complex network of chemokines that affect the infiltration of immune cells of tumors, as well as the growth, survival, movement, and angiogenesis of tumor cells. Immune cells, endothelial cells, and tumor cells, among themselves, exhibit chemokine receptors and can respond to chemokine gradients. Studies of biopsy samples of human cancer and cancer patterns in mice have shown the expression and increase of chemokine receptors in cancer cells The transfer ability is related. Malignant cells from different cancer types have different chemokine receptor profiles, but chemokine receptor 4 (CXCR4) is the most commonly found. Cells from at least 23 different types of human cancers of epithelial, mesenchymal and hematopoietic origin express the CXCR4 receptor (Balkwill F. et al., 2004).

Chemokine receptor 4 (also known as fusin, CD184, LESTR or HUMSTR) is present as two isoforms, which contain 352 or 360 amino acids. The homology a has the amino acid sequence described by Genbank accession number NP_001008540, while the homologous b has the amino acid sequence described by Genbank accession number NP_003458. The residue Asn11 is saccharified, the residue Tyr21 is modified by the addition of a sulfuric acid group, and Cys 109 and 186 are externally bound to the receptor by a disulfide bridge (Juarez J. et al., 2004). ).

This receptor consists of different kinds of normal tissues, naïve, non-memory T-cells, regulatory T cells, B-cells, neutrophils, endothelial cells, primary monocytes, dendritic cells. Natural killer cells, CD34+ hematopoietic stem cells, and low levels of the heart, colon, liver, kidneys, and brain. CXCR4 plays a key role in the transport of white blood cells, lymphocyte formation of B cells, and bone marrow cell formation.

The CXCR4 receptor is overexpressed in numerous cancers including, but not limited to, lymphoma, leukemia, multiple myeloma, colon (Ottaiano A. et al., 2004), breast (Kato M. et al., 2003), prostate (Sun YX). Et al., 2003), lung [small cell carcinoma and non-small cell carcinoma (Phillips RJ et al., 2003)], ovary (Scotton CJ et al., 2002), pancreas (Koshiba T. et al. Human, 2000), kidney, brain (Barbero S et al, 2002), glioblastoma, and lymphoma.

The unique ligand of the CXCR4 receptor described so far is stromal cell-derived factor-1 (SDF-1) or CXCL12. SDF-1 is secreted in a large amount in the lymph nodes, bone marrow, liver, lungs, and to a lesser extent by the kidneys, brain, and skin. CXCR4 is also recognized by an antagonizing chemokine, a viral macrophage inflammatory protein II (vMIP-II) encoded by a human herpesvirus type III.

The CXCR4/SDF-1 axis plays a key role in cancer and is directly implicated in movement, leading to the invasion of metastasis. More specifically, cancer cells express the CXCR4 receptor, which moves and enters the systemic circulation. The vascular bed in the organ that produces high levels of SDF-1 then blocks cancer cells, which proliferate, induce angiogenesis, and form metastatic tumors (Murphy PM., 2001). This axis also relates to cell proliferation via activation of the extracellular message-regulated kinase (ERK) pathway (Barbero S. et al., 2003) and angiogenesis (Romagnani P., 2004). More specifically, the CXCR4 receptor and its coordinator SDF-1 clearly promote angiogenesis by stimulating VEGF-A expression, which in turn increases the performance of CXCR4/SDF-1 (Bachelder R. E. et al., 2002). It is also known that tumor-associated macrophages (TAM) accumulate in hypoxic regions of tumors and stimulate cooperation with tumor cells and promote angiogenesis. It has been observed that hypoxia up-regulation of CXCR4 is selectively expressed in a wide variety of cell types, including TAM (Mantovani A. et al., 2004). It has recently been demonstrated that the CXCR4/SDF-1 axis regulates the transport/homing of CXCR4+ hematopoietic stem/precursor cells (HSC) and plays a role in angiogenesis. Evidence indicates In addition to HSC, functional CXCR4 is also expressed in stem cells from other tissues (tissue-committed stem cells = TCSCs) so SDF-1 can play a role in chemical chemottracting CXCR4+TCSCs essential for organ/tissue regeneration. The role of the central, but these TCSCs can also be the cell origin of cancer development (the stem cell theory of cancer). The stem cell origin of cancer is demonstrated in human leukemia and in recent years in several solid tumors, such as the brain and breast. There are several examples of CXCR4+ tumors that can be derived from normal CXCR4+ tissue/organ-specific stem cells, such as leukemia, brain tumors, small cell lung cancer, breast cancer, hepatoblastoma, ovarian cancer, and cervical cancer (Kucia M. et al. 2005).

In vivo use of a single antibody against the CXCR4 receptor demonstrates metastasis through a target cancer that interferes with the CXCR4 receptor (Muller A. et al., 2001). Briefly, it was shown that a single antibody against the CXCR4 receptor (Mab 173 R&D Systems) significantly reduced the number of lymph node metastases in the in situ breast cancer pattern (MDA-MB231) in SCID mice. Another study (Phillips RJ et al., 2003) also showed a key role for the SDF-1/CXCR4 axis in the metastasis of the orthotopic lung cancer model (A549), using multiple antibodies against SDF-1 but in this study Tumor growth or angiogenesis has no effect. Several other studies have also described inhibition, whether it is inhibition of in vivo metastasis, using CXCR4 dual siRNAs (Liang Z. et al., 2005) Biosafety CXCR4 peptide antagonist (Tamamura H. et al., 2003). Or inhibition of tumor growth in vivo using a small molecule antagonist of CXCR4 like AMD 3100 (Rubin JB et al, 2003; De Falco V. et al, 2007) or Mab (patent WO 2004/059285 A2). Thus, CXCR4 is an effective cancer therapeutic target.

Chemokine receptor 2 (CXCR2), another chemokine receptor, is also described as an interesting oncology target. Rather, CXCR2 transmits growth messages from back-secretory cells to several types of tumor cells and can also indirectly affect tumor growth by promoting angiogenesis (Tanaka T. et al. 2005).

The CXCR2 chemokine receptor contains 360 amino acids. It is mainly manifested in endothelial cells and especially throughout the angiogenesis. Several chemokines bind to the CXCR2 receptors: CXCL5, -6, -7, IL-8, GRO-α, -β, and γ, which belong to the ERL+ pro-angiogenic chemokine. The CXCR2 receptor shares sequence homogeneity with the CXCR4 receptor: 37% sequence identity and 48% sequence homogeneity. The CXCR2/coordinator axis is involved in several tumor growth mechanisms, such as metastasis (Singh RK. et al., 1994) cell proliferation (Owen JD et al., 1997) and ERL+chemokine-mediated angiogenesis (Strieter RM et al, 2004). Finally, tumor-associated macrophages and neutrophils are key elements of inflammatory-induced tumor growth and chemokines such as CXCL5, IL-8 and GRO-α initiate neutrophil supplementation.

Dimerization has emerged as a common mechanism for regulating the function of G-protein-coupled receptors, among which are chemokine receptors (Wang J. and Norcross M., 2008). Homogeneous dimerization and heterodimerization of the chemokine binding reaction have been shown to be necessary for the initiation and alteration of the transmission of messages through some of the chemokine receptors. Growth evidence supports that receptor dimers or oligomers are likely to be the concept of a basic functional unit of a chemokine receptor. Chemokine receptor dimers exist in the absence of a ligand and chemokines The conformational change of the acceptor dimer is induced. It is known that CXCR4 forms homodimers as well as heterodimers, for example with delta-opioid receptors (DOR) (Hereld D., 2008) or CCR2 (Percherancier Y. et al., 2005). In the latter example, the peptide in the transmembrane domain derived from CXCR4 inhibits activation by blocking the ligand-induced dimer conformational transition (Percherancier Y. et al., 2005). Another study showed that CXCR4-TM4 peptide, a synthetic peptide of the transmembrane region of CXCR4, reduces energy transfer between the CXCR4 homodimer promoter and inhibits SDF-1-induced movement and muscle in malignant cells. Kinetic protein polymerization (Wang J. et al., 2006). More recently, CXCR7 and CXCR4 have also been described to form functional heterodimers and to enhance SDF-1-induced message transmission (Sierro F. et al., 2007). Other examples of constitutive heterodimers include studies showing the interaction of CXCR1 and CXCR2 and the formation of separate homodimers. It is mentioned that none of them interacts with another GPCR (α(1A)-adrenergic receptor), indicating the specificity of the interaction between CXCR1 and CXCR2 (Wilson S. et al., 2005).

As mentioned previously, the CXCR4 and CXCR2 receptors are of interest for tumors of interest. Interfering with these receptors should inhibit tumor growth and metastasis in a very efficient manner by reducing tumor cell proliferation, angiogenesis, tumor cell migration and invasion, due to tumor neutrophil and macrophage supplementation, and Through stem cells that inhibit CXCR4 cancer.

Two monoclonal antibodies that bind to and induce a conformational change in both CXCR4 homodimers and CXCR4/CXCR2 heterodimers, and have strong antitumor activity, have been previously characterized, referred to as 515H7 and 414H5 (see WO 2010/037831). And, the applicant has already proved this one CXCR4/CXCR2 The presence of a heterodimer.

The present invention is directed to providing at least one agent lacking any in vivo activity in a cancer mode, which can be used as a diagnostic or prognostic tool for carcinogenic disorders, particularly characterized by the manifestation of CXCR4 or by abnormalities The CXCR4 represents the medium of this.

Published patent application WO 2010/125162 discloses two anti-CXCR4 monoclonal antibodies, referred to as 515H7 and 301aE5, and their use in the therapeutic field of HIV.

Surprisingly, the inventors have now demonstrated that the antibody 301aE5 (also referred to herein as 301E5 or better in this specification, with reference to a deposited fusion tumor, I-3859: for the purposes of this application, these terms are approximate In contrast to the other antibody 515H7, which exhibits potent anti-tumor activity (as described in WO 2010/037831), it does not have any in vivo activity in the field of cancer treatment. In particular, I-3859 does not prevent the CXCR4 ligand from binding to the receptor.

Moreover, the Applicant has found that the antibody I-3859 can: i) recognize CXCR4 such as monomer; ii) recognize CXCR4 such as CXCR4/CXCR4 homodimer; iii) recognize CXCR4 such as CXCR4/CXCR2 heterodimer; Iv) immunoprecipitation of CXCR4 from cell lysates; v) identification of CXCR4 at the surface of cells expressing CXCR4 by fluorescence activated cell sorting (FACS); and vi) identification of CXCR4 by immunohistochemistry.

Because the antibody I-3859 has not previously revealed such novel properties, The present inventors have found that the antibody can be used to recognize cells expressing CXCR4 and, in particular, tumor cells expressing CXCR4.

Thus, the invention relates to the use of the antibody for detecting the presence and/or location of a disease indicative of CXCR4. The invention can then be used for diagnosis and/or prognosis, preferably in vitro , a disease that exhibits CXCR4. The disease exhibiting CXCR4 is preferably a cancer.

Another advantageous property of the antibody I-3859 of the invention is that it recognizes an epitope that is close to the epitope of the therapeutic monoclonal antibody 515H7. More specifically, as demonstrated in the experimental examples, I-3859 is able to compete with the therapeutic antibody 515H7 for binding to its epitope. The I-3859 antibody can thus be used, for example, to select a patient to be treated with 515H7 Mab. In particular, the antibody I-3859 of the present invention enables the configuration of CXCR4 used to examine the surface of a cell presented to a patient to be similar to that recognized by the antibody 515H7, indicating that the patient is predominantly 515H7 antibody. The therapy is amenable.

A first aspect of the invention pertains to an isolated antibody, or antigen-binding fragment or derivative thereof, which specifically binds CXCR4 with high affinity, which antibody lacks any in vivo activity. The isolated antibody, or antigen-binding fragment or derivative thereof, can be used in a method of diagnosing or predicting a pathological hyperproliferative carcinogenic disorder mediated by the expression of CXCR4. In particular, the isolated antibody can be used for in vivo imaging. Preferably, the ligated antibody binds to human CXCR4.

In a preferred embodiment, an isolated antibody, or an antigenic junction thereof A fragment or derivative is provided for detecting the presence of a tumor exhibiting CXCR4, wherein the antibody comprises at least one complementarity determining region (CDR) selected from CDRs comprising amino acid sequence sequence number 1 to 6 Among them, or at least one CDR, the sequence has at least 80%, preferably 85%, 90%, 95% and 98% identity with the sequences 1 to 6 after optimal alignment.

More preferably, the invention encompasses antigen-binding fragments or derivatives of the invention, such as those obtained by genetic recombination or chemical synthesis.

According to a preferred embodiment, the antibody according to the invention, or a derivative thereof or antigen-binding fragment thereof, is characterized in that it consists of a monoclonal antibody.

As used herein, "monoclonal antibody" means an antibody produced by a population of closely homologous antibodies. More particularly, the individual antibodies of a population are identical except for some mutations that may naturally occur, and such naturally occurring mutations can be found in a minimal proportion. In other words, a monoclonal antibody is composed of a single cell pure strain (for example, a fusion tumor, a eukaryotic host cell transfected with a DNA molecule encoding the homologous antibody, and a DNA encoding the homologous antibody). A homologous antibody produced by the growth of a prokaryotic host cell to which the molecule is transfected, and the like, is generally characterized by one type and only one type and sub-classified heavy chain, and only one type of light chain. Individual antibodies are highly specific and target a single antigen. In addition, multi-strain antibodies typically include a wide variety of antibodies directed against a variety of loci, or epitopes, as compared to the preparation of multiple antibodies, each monoclonal antibody being determined against a single antigen of the antigen. Bit.

A typical IgG anti-system consists of a glycoprotein, which consists of Two identical heavy chains connected by sulfur bonds and two identical light chains. Each heavy chain and light chain contains a conserved region and a variable region. Each variability region comprises three segments called "complementarity determining regions" ("CDRs") or "highly variable regions", which are primarily responsible for antigen binding epitopes. These are commonly referred to as CDR1, CDR2, and CDR3, and are numbered consecutively from the N-terminus. The higher conservation area of the variability region is referred to as the "framework region."

There are 3 heavy chain CDRs and 3 light chain CDRs. The term CDR or CDRs is used herein to indicate one or several of these regions, or even the entirety of such regions, depending on the condition, and such regions include the binding affinity responsible for the antigen or antigenic epitope of the antibody. Most of the amino acid residues.

According to the invention, the CDRs of the antibody will be defined in accordance with the IMGT numbering system. It is evident from the CDRs based on IMGTt that the CDRs according to Kabat are familiar to those skilled in the art. The CDRs according to Kabat must be considered part of the scope of the invention.

IMGT's unique number has been defined to compare variants, regardless of antigen receptor, chain type, or species [Lefranc M.-P., Immunology Today 18, 509 (1997) / Lefranc M.-P., The Immunologist, 7, 132-136 (1999) / Lefranc, M.-P., Pommié, C., Ruiz, M., Giudicelli, V., Foulquier, E., Truong, L., Thouvenin-Contet, V. and Lefranc , Dev. Comp. Immunol., 27, 55-77 (2003)]. In terms of IMGT's unique numbering, conserved amino acids always have the same position, for example, cysteine 23 (1st-CYS), tryptophan 41 (CONSERVED-TRP), hydrophobic amino acid 89, half Cysteine 104 (2nd-CYS), phenylalanine or tryptophan 118 (J-PHE or J-TRP). The IMGT unique numbering provides the frame area (FR1-IMGT: positions 1 to 26, FR2-IMGT: 39 to 55, FR3-IMGT: 66 to 104 and FR4-IMGT: 118 to 128) and the complementarity decision area: CDR1-IMGT : 27 to 38, CDR2-IMGT: 56 to 65 and CDR3-IMGT: 105 to 117, the standardization demarcation. Since the gap indicates an unoccupied position, the length of the CDR-IMGT (shown between the brackets and separated by dots, such as [8.8.13]) becomes decisive information. The unique numbering of IMGT is used in 2D graphical representation, called 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 the 3D structure of the IMGT/3D structure-DB [Kaas, Q., Ruiz, M. and Lefranc, M.-P., T Cell acceptor and MHC structural data. Nucl. Acids. Res., 32, D208-D210 (2004)].

More particularly, the invention relates to an antibody, or antigen-binding fragment or derivative thereof, capable of specifically binding CXCR4, comprising i) a heavy chain containing the following definitions according to the IMGT numbering system; At least one of CDR-H1, CDR-H2 and CDR-H3, wherein CDR-H1 comprises SEQ ID NO: 1, CDR-H2 comprises SEQ ID NO: 2, and CDR-H3 comprises SEQ ID NO: 3; and/or Ii) a light chain comprising at least one of the following CDR-L1, CDR-L2 and CDR-L3 as defined by the IMGT numbering system, wherein CDR-L1 comprises sequence sequence identification number 4 and CDR-L2 comprises sequence sequence identification No. 5 and CDR-L3 contain sequence number identification number 6.

In still another embodiment, the invention is also described as an antibody, or an antigen-binding fragment or derivative thereof, comprising: - a heavy chain comprising the following three CDRs as defined by IMGT: each having a sequence CDR-H1 of sequence identification number 1, CDR-H2 having sequence number identification number 2, and CDR-H3 having sequence sequence number 3, or sequence number identification number 1, 2 or 3 after optimal alignment a sequence having at least 80%, preferably 85%, 90%, 95% and 98% identity; and - a light chain comprising the following three CDRs as defined by IMGT: sequence identification CDR-L1 of No. 4, CDR-L2 having sequence sequence identification number 5, and CDR-L3 having sequence sequence identification number 6, or at least after sequence alignment with sequence sequence identification number 4, 5 or 6 respectively A sequence of 80%, preferably 85%, 90%, 95% and 98% identity.

For the purposes of the present invention, "percent identity" between two sequences of a nucleic acid or amino acid means the same nucleotide obtained after optimal alignment between the two sequences to be compared. Or the percentage of amino acid residues, which is only statistically and the difference between the two sequences is randomly distributed along its length. Comparison of two nucleic acid or amino acid sequences has traditionally been carried out by comparing the sequences after optimal alignment, and the comparison can be carried out in segments or by using an "arrangement window". The best alignment of the sequences used for comparison can be done by manual comparison, but also by Smith and Waterman's local homology algorithm (1981) [Ad.App.Math. 2:482], by Neddleman. Local homology with Wunsch Algorithm (1970) [J. Mol. Biol. 48: 443], by Pearson and Lipman's similarity search method (1988) [Proc. Natl. Acad. Sci. USA 85: 2444] or by using this The computer software of the algorithm (Wisconsin Genetics Software Package, Genetics Computer Group, 575 Science Dr., Madison, GAP, BESTFIT, FASTA, and TFASTA of WI, or by comparison software BLAST NR or BLAST P).

The percent identity between two nucleic acid or amino acid sequences is determined by comparing the two optimally aligned sequences, wherein the nucleic acid or amino acid sequence to be compared is optimally aligned with respect to the two sequences. There may be additions or censoring compared to the reference sequence. Percent identity is calculated by determining the number of amino acid or nucleotide residues between the two sequences at the same position, dividing the number of identical positions by the total number of positions in the alignment window, and The result is multiplied by 100 to obtain the percent identity between the two sequences.

For example, the BLAST program available at http://www.ncbi.nlm.nih.gov/gorf/bl2.html, "BLAST 2 Sequence" (Tatusova et al., "Blast 2 sequences-a new tool" For comparing protein and nucleotide sequences", FEMS Microbiol., 1999, Lett. 174: 247-250), can be used with preset parameters (especially with regard to the parameter "open space penalty": 5, and "extended space penalty" : 2; the selected matrix is, for example, the "BLOSUM 62" matrix proposed by the program); the percentage identity between the two sequences to be compared is directly calculated by the program.

About at least 80%, preferably 85%, with respect to a reference amino acid sequence, Amino acid sequences of 90%, 95% and 98% identity, preferred examples include the inclusion of a reference sequence, some modifications, in particular the deletion, addition or substitution, truncation or extension of at least one amino acid, Wait. In the case of substituting one or more contiguous amino acids or discontinuous amino acids, the substituted amino acid is preferably replaced by an "equal" amino acid. Here, the expression "equal amino acid" is intended to indicate any amino acid that is likely to be substituted for one of the structural amino acids, however, it does not modify the biological activity of the corresponding antibody and The biological activities of these particular examples are defined below.

The equivalent amino acid can be determined by the structural homology of its amino acid to be substituted, or by the comparison of biological activities between various antibodies that are likely to be produced. determination.

As a non-limiting example, Table 1 below summarizes possible substitutions that are likely to proceed without causing significant modification of the biological activity of the corresponding modified antibody; it is of course possible that the reverse substitution is under the same conditions.

According to still another embodiment, the invention relates to one of antibody I-3859, or an antigen-binding fragment or derivative thereof, comprising a heavy chain variable domain sequence comprising an amino acid sequence sequence identification number 7 or a sequence of at least 80%, preferably 85%, 90%, 95% and 98% identity with sequence sequence identification number 7 after optimal alignment; and/or because it comprises a light chain Variant domain sequence, which contains amino acid sequence sequence recognition No. 8, or a sequence of at least 80%, preferably 85%, 90%, 95% and 98% identity with sequence sequence identification number 8 after optimal alignment.

In particular, the antigen-binding derivative is composed of a binding protein comprising a peptide scaffold, and at least one CDR system is grafted onto the peptide scaffold, and the CDR is grafted in such a manner as to preserve the starting antibody. The antibody binding site (paratope) identifies all or part of the property. In a preferred embodiment, the antigen-binding derivative is a fusion protein of a peptide scaffold and the at least one CDR.

One or more of the six CDR sequences described in the present invention may also be presented on various immunoglobulin scaffolds. In this case, in this case, the protein sequence makes it possible to modify the peptide backbone to fit the correct folding of the grafted CDRs so that they can maintain their antigen binding properties of the antibody binding site.

Those skilled in the art will have knowledge of the means selected for CDR grafting. More specifically, it has been known that such scaffolds to be selected must conform to as many of the following features as possible (Skerra A., J. Mol. Recogn., 2000, 13: 167-187): - good phylogenetic conservation; - Known 3-dimensional structures (e.g., by crystallography, NMR spectroscopy, or any other technique known to those skilled in the art); - small size; - little or no post-transcriptional modification; And / or - easy to produce, perform and purify.

The source of such protein scaffolds may be, but is not limited to, a structure selected from the group consisting of fibrin and preferential fibrin type III Domain 10, lipid-carrying protein, anti-anionicin (Skerra A., J. Biotechnol., 2001, 74(4): 257-75), protein produced by Domain B of Protein A of S. aureus Z, thioredoxin A or any protein with a repeating domain, eg "ankyrin repeat" (Kohl et al, PNAS, 2003, vol. 100, No. 4, 1700-1705), "犰狳(armadillo) repeats, "rich in leucine-rich" and "tri-tetrapeptide repeats". All such protein moieties have been extensively characterized in the art and are thus well known to those skilled in the art.

As explained above, these peptide scaffolds comprise from 1 to 6 CDRs produced by the original antibody. Preferably, but not a requirement, those skilled in the art will select at least one CDR from the heavy chain that is known to be primarily responsible for the specificity of the antibody. Selection of one or more related CDRs will be apparent to those skilled in the art, and those skilled in the art will then select suitable known techniques (BES et al, FEBS letters 508, 2001, 67-74).

It is necessary to know the antigen-binding fragment of the antibody according to the invention, for example, the fragment Fv, scFv (sc=single chain), Fab, F(ab') ₂ , Fab', scFv-Fc or divalent antibody (diabodies) ), or any fragment that has been modified by chemical modification to increase half-life, such as the addition of polyalkylene glycols, such as polyethylene glycol (PEGylation) (PEGylated fragments called Fv-PEG, scFv) - PEG, Fab-PEG, F(ab') ₂ -PEG or Fab'-PEG), or by incorporating liposomes, microspheres or PLGA, the fragments possessing at least one of the characteristic CDRs of the invention, It is particularly capable of exerting the activity of the antibody (which is produced by the antibody), or even part of its activity, in a general manner.

Preferably, the antigen-binding fragments will comprise or include derivatives thereof, etc. a portion of a sequence of a variable heavy or light chain of the antibody sufficient to retain the same binding specificity and sufficient affinity as the antibody producing the portion of the sequence, preferably an antibody producing the portion of the sequence The affinity of the antibody is at least equal to 1/100, more preferably at least 1/10.

The antigen-binding fragment will comprise at least 5 amino acids of the sequence of the antibody producing the antigen-binding fragment, preferably 6, 7, 8, 10, 15, 25, 50 or 100. A continuous amino acid.

Preferably, such antigen-binding fragments will belong to the class Fv, scFv, Fab, F(ab') ₂ , Fab', scFv-Fc or bivalent antibodies, which generally have the same binding specificity as the antibody from which they are produced. Sex. According to the present invention, the antigen-binding fragment of the antibody of the present invention can be obtained from the above-described antibody by a method such as enzymatic digestion, including pepsin or papain, and/or by splitting of a chemically reduced disulfide bridge. . Such antibody fragments can also be obtained by recombinant genetic techniques well known to those skilled in the art or by automated peptide synthesizers, for example, such as those sold by Applied BioSystems. Obtained by synthesis.

A fusion tumor of a murine capable of secreting a monoclonal antibody according to the present invention has been deposited with CNCM, Institut Pasteur, Paris, France on October 22, 2007 under the number I-3859. The fusion tumor was obtained by fusion of Balb/C-immunized mouse spleen cells and myeloma Sp 2/O-Ag 14 cells.

The monoclonal antibody, referred to herein as 301aE5 or I-3859, or an antigen-binding fragment or derivative thereof, is characterized in that it is secreted by the fusion tumor.

The antibody I-3859 can also be illustrated by its nucleic sequence, that is, it comprises a heavy chain containing the sequence of the sequence a CDR-H1 encoded by the number 9; a CDR-H2 encoded by the sequence identification number 10; and a CDR-H3 encoded by the sequence identification number 11; and/or a light chain containing the sequence of the sequence The CDR-L1 encoded by the identification number 12, the CDR-L2 encoded by the sequence identification number 13, and the CDR-L3 encoded by the sequence identification number 14 are identified.

The antibody I-3859 comprises a heavy chain which is encoded by nuclear sequence sequence number 15 or which, after optimal alignment, exhibits at least 80%, preferably 85%, 90%, with sequence identification number 15. 95% and 98% percent identity encoded by a nuclear sequence; and/or a light chain encoded by nuclear sequence sequence number 16, or at least after sequence alignment with sequence identification number 16 A nuclear sequence of 80%, preferably 85%, 90%, 95%, and 98% percent identity is encoded.

The terms "nucleic acid", "nucleic sequence", "nucleic acid sequence", "polynucleotide", "oligonucleotide", "polynucleotide sequence" and "nucleotide sequence" are used in this description Used interchangeably, meaning the exact sequence of a nucleotide, modified or not, defines a fragment or region of a nucleic acid, including unnatural nucleotides, and is a double strand of DNA, a single strand of DNA, or Transcription products of such DNAs.

"A nuclear sequence that exhibits at least 80%, preferably 85%, 90%, 95%, and 98% percent identity with a preferred sequence after optimal alignment" means that the reference nuclear sequence is presented Certain modifications are, for example, particularly censored, truncated, extended, chimeric fusions, and/or substituted, especially precise, nuclear sequences. Preferably, these are sequences encoding the same amino acid sequence as the reference sequence, which is related to the degeneracy of the genetic code, or is likely to be preferably Complementary sequences that hybridize specifically to a reference sequence under highly stringent conditions, especially as defined below.

Hybridization under highly stringent conditions means that conditions relating to temperature and ionic strength are selected in such a manner as to permit hybridization between two complementary DNA fragments. Based on the mere exemplification, the highly stringent conditions of the hybridization step are advantageously as follows for the purpose of defining the polynucleotide fragments described above.

DNA-DNA or DNA-RNA hybridization was performed in two steps: (1) in a solution containing 5X SSC (1X SSC corresponding to 0.15 M NaCl + 0.015 M sodium citrate), 50% formamide, 7% Pre-hybridization of sodium dodecyl sulfate (SDS), 10X Denhardt's, 5% dextran sulfate, and 1% salmon sperm DNA in phosphate buffer (20 mM, pH 7.5) at 42 °C 3 hours; (2) the main hybridization is at a temperature depending on the length of the probe (ie: 42 ° C for a probe of length > 100 nucleotides) for 20 hours followed by 2X SSC + 2% SDS within 20 Wash at 2 °C for 20 minutes, and wash once in 0.1X SSC + 0.1% SDS at 20 ° C for 20 minutes. The final wash was performed for a probe line of >100 nucleotides in length at 60 ° C in 0.1X SSC + 0.1% SDS for 30 minutes. The highly stringent conditions described above for a polynucleotide of a defined size can be engineered by the skilled artisan to be used for longer or shorter oligonucleotides, as described in Sambrook, et al. Procedure (Molecular cloning: a laboratory manual, Cold Spring Harbor Laboratory; 3rd version, 2001).

In another aspect, the invention relates to an antibody of the invention, or an antigen-binding fragment or derivative thereof, for use in ex vivo or ex vivo diagnosis or prognosis and carcinogenicity associated with CXCR4 expression obstacle.

The invention thus relates to a method of in vitro or ex vivo diagnosis or prognosis and carcinogenic disorder associated with the expression of CXCR4 comprising the steps of: testing an antibody of the invention, or an antigen binding fragment or derivative thereof, and CXCR4 The combination.

In particular, the invention provides the use of antibody I-3859, or an antigen binding fragment or derivative thereof, for in vitro diagnostic or prognostic and carcinogenic disorders associated with the performance of CXCR4.

Importantly, the antibody, or antigen-binding fragment or derivative thereof, does not have any in vivo anti-tumor activity. This property is undoubtedly advantageous for diagnostic applications because it allows screening of patients or monitoring the progress of treatment with antibodies that do not have any impact or effect on the patient. This property makes the antibody of the present invention a preferred tool for screening patients to be treated since it does not have deleterious effects on the patient. The antibodies, or antigen-binding fragments or derivatives thereof, of the invention will find utility for a variety of medical or research purposes, including detection, diagnosis and staging of various pathologies associated with the performance of CXCR4.

When used in the context of "diagnosing" a disease, the detection or detection of a pathological hyperproliferative carcinogenic disorder associated with the expression of CXCR4 or the presence of a pathological hyperproliferative carcinogenic disorder mediated by the manifestation of CXCR4 is monitored. Progression of the disease, and methods of identifying or detecting cells or samples indicative of a disorder associated with the performance of CXCR4.

As used herein, "predicting" means the likelihood of recovery from a disease or the prediction of a likely development or outcome of a disease. For example, if If the stain from the one body is negative for staining with the antibody of the present invention, then the "predicted" of the individual is better than if the sample was stain positive for CXCR4. The sample can be scored on the appropriate scale for the performance level of CXCR4 as will be explained in more detail below.

The antibody can be present as an immune complex or as a calibrated antibody to obtain a detectable/quantitative signal. The anti-system of the invention is particularly useful for both in vitro and in vivo diagnostic and prognostic applications when used with suitable markers or other suitable detectable biomolecules or chemicals.

Marks for use in immunoassays are commonly known to those skilled in the art and include enzymes, radioisotopes, and fluorescent, luminescent and chromogenic materials, including colored particles such as colloidal gold or latex beads. Suitable immunoassays include enzyme linked immunoassays (ELISA). A wide variety of types of markers, as well as methods for joining the markers to the antibodies of the invention, are well known to those skilled in the art, such as those set forth below.

As used herein, the term "a carcinogenic disorder associated with the expression of CXCR4" is intended to include diseases and other disorders in which a high level of CXCR4 (abnormal) is present in an individual suffering from the disorder, already The cause of the pathophysiology of the disorder or the factor contributing to the deterioration of the disorder or the cause of the pathophysiology of the disorder or a factor contributing to the deterioration of the disorder. Optionally, such disorders may be evidenced by, for example, an increase in the level of CXCR4 on the surface of cells in an affected cell or tissue affected by an individual suffering from the disorder. An increase in the CXCR4 level can be detected using the antibody I-3859 of the present invention.

In some specific examples, when "increased performance" relates to CXCR4, the expression level of the protein or gene is mentioned, showing a statistically significant increase in performance relative to a control (as by RNA expression or protein). Performance is measured).

A preferred aspect of the invention is a method for detecting the presence of a CXCR4-expressing tumor in vitro or ex vivo, the method comprising the steps of: (a) contacting a biological sample from the individual with the present invention An antibody of the invention, or an antigen binding fragment or derivative thereof, and (b) detecting binding of the antibody to the biological sample.

The binding of the antibodies of the invention can be detected by a variety of assays available to those skilled in the art. While the invention encompasses any suitable means for performing the assay, FACS, ELISA, Western blots and immunohistochemistry (IHC) may be specifically mentioned.

In another embodiment, the invention relates to a method for detecting the location of a tumor exhibiting CXCR4 in vivo or ex vivo, the method comprising the steps of: (a) subjecting a biological sample from the individual Contacting an antibody of the invention, or an antigen binding fragment or derivative thereof, and (b) detecting binding of the antibody, or antigen-binding fragment or derivative thereof, to the sample.

As for the detection of the presence of a tumor, many techniques known to those skilled in the art can be used. Preferred methods include IHC and FACS.

The invention also relates to detecting an internal body surface in vitro or ex vivo A method of percentage of cells of CXCR4, the method comprising the steps of: (a) contacting a biological sample from the individual with an antibody of the invention, or an antigen binding fragment or derivative thereof, and (b) quantifying the organism The percentage of cells expressing CXCR4 in the sample.

Another aspect of the invention relates to a method for determining the level of expression of CXCR4 in a tumor exhibiting CXCR4 from a body in vitro or ex vivo, the method comprising the steps of: (a) from the individual A biological sample is contacted with an antibody of the invention, or an antigen binding fragment or derivative thereof, and (b) quantifies the level of an antibody that binds to CXCR4 within the biological sample.

As will be apparent to those skilled in the art, antibody levels bound to CXCR4 can be quantified by any means known to those skilled in the art. Preferred methods involve the use of immunoenzyme methods, such as ELISA assays, immunofluorescence, IHC, radioimmunoassay (RIA), or FACS.

Preferably, the biological sample is a biological fluid, such as serum of human origin, whole blood cells, a tissue sample or a biopsy. The sample may, for example, include a biopsy tissue that can conveniently analyze the presence of a pathological hyperproliferative carcinogenic disorder associated with the performance of CXCR4.

Still another aspect of the present invention relates to a method for determining the expression level of CXCR4 in a tumor from a body in vitro or ex vivo, the method comprising the steps of: (a) making the same from the individual Contacting an antibody, or antigen-binding fragment or derivative thereof, of the invention, and (b) quantifying the binding level of the antibody, or antigen-binding fragment or derivative thereof, to CXCR4 in the sample.

Once the amount of CXCR4 present in the test sample is determined, the results can be compared to the results of the control sample, which is similar to the test sample but never has hyperproliferative carcinogenicity associated with CXCR4 performance. Obtained by the individual of the disorder. Given that the level of CXCR4 within the test sample is significantly elevated, it can be concluded that there is an increased likelihood that the individual (the disorder is derived from the individual) has or will develop the disorder.

More particularly, the present invention relates to a method for diagnosing or prognosing a tumor exhibiting CXCR4 in vitro or ex vivo, wherein the method comprises the steps of: (i) determining the level of expression of CXCR4 as described above, and (ii) Comparing the performance level of step (i) with the reference performance level of CXCR4 from normal tissue or a non-expressing CXCR4 tissue.

With regard to the development of targeted anti-tumor therapies, the diagnosis of immunohistochemical techniques provides in-situ information about the level of expression of the receptor and, thus, the ability to select a patient who is sensitive to the treatment and then requires the performance of the recipient of the treatment. .

The judgment of the period has potential prognostic value as well as providing guidelines for designing the best therapy. Simpson et al, J. Clin. Oncology 18: 2059 (2000). For example, the treatment options for solid tumors are based on tumor staging, which is typically performed using tumor/nodule/metastasis (TNM) from the American Joint Committee on Cancer (AJCC). It is generally accepted that although this test and staging system provides some valuable information about the period of solid tumors that have been diagnosed in patients, it is inaccurate and insufficient. In particular, it cannot distinguish between swollen The earliest period of tumor progression.

The present invention relates to a method for determining a score of a tumor in a body ex vivo or ex vivo, the method comprising the steps of: (a) contacting a biological sample from the individual to specifically bind one of CXCR4 An antibody, or antigen-binding fragment or derivative thereof; (b) quantifying the binding level of the antibody, or antigen-binding fragment or derivative thereof, to CXCR4 in the biological sample; and (c) by comparing the appropriate size The quantified binding level of the antibody, or antigen-binding fragment or derivative thereof, to score the tumor from the individual, characterized by the antibody, or antigen-binding fragment or derivative thereof, comprising i) a heavy chain, It comprises the following three CDRs, respectively CDR-H1 having sequence identification number 1, CDR-H2 having sequence identification number 2, and CDR-H3 having sequence number identification number 3; and, ii) a light chain, It contains the following three CDRs, which are CDR-L1 having SEQ ID NO: 4, CDR-L2 having SEQ ID NO: 5, and CDR-L3 having SEQ ID NO: 6.

In a preferred embodiment, the antibody for diagnosis is used when the tissue sample is formalin-fixed, formaldehyde-substituted, Glyco-fixx-fixed (Glyco-fixx fixed-), paraffin-encapsulated, and/or frozen. Ability to bind to the target receptor.

Any conventional risk analysis method can be used to estimate the prognostic value of CXCR4. Representative analytical methods include Cox's regression analysis, a semiparametric method for modeling survival or time-to-event data in the case of review (Hosmer and Lemeshow, 1999; Cox, 1972). Instead of His survival analysis, such as Life Tables or Kaplan-Meyer, allows Cox to include predictive variables (covariates) in the model. Using a routine analytical method, such as Cox, one can test the performance of CXCR4 on a primary tumor for the time-to-onset of the disease (no disease survival, or metastasis) The time of the disease, or the hypothesis of the relevance of the disease until the time of death (all survival time). Cox regression analysis is also known as the Cox proportional hazard analysis. This method is a standard for testing the predictive value of a tumor index for the survival time of a patient. When used in a multivariate mode, the effects of several covariates are tested in parallel so that individual covariates with independent predictive value, ie, the most useful indicators, can be identified. The term negative or positive "CXCR4 state" may also be referred to as [CXCR4(-)] or [CXCR4(+)].

A sample can be "scoreed" throughout the period of cancer diagnosis or monitoring. In its simplest form, when judged by visual inspection of a sample that passes through immunohistochemistry, the score can be negative or positive. More quantitative scoring involves determining the intensity of staining of the two parameters and the proportion of stained ("positive") cells sampled.

In the meaning of the present invention, the "CXCR4 state" is related to the classification of tumors into a CXCR4-positive [CXCR4(+)] or CXCR4-negative [CXCR4(-)] category, CXCR4, based on the determination of the expression level of CXCR4. The level of performance is measured by any method, such as immunohistochemistry (IHC), FACS, or other methods known to those skilled in the art.

In a specific example of the present invention, in order to ensure standardization, the sample may The performance level of CXCR4 was scored on a different scale, and most of them were evaluated based on the intensity of the reaction product and the percentage of positive cells (Payne et al., Predictive markers in breast cancer-the present, Histopathology 2008, 52, 82). -90).

In a more specific embodiment of one of the methods according to the invention, the scoring comprises the use of an appropriate scale according to two parameters, the intensity of the staining and the percentage of positive cells.

As a first example, the sample can be scored from 0 to 8 for the intensity of reactivity and the proportion of stained cells in terms of the level of performance of CXCR4 through the Quick Allred score of the IHC assessment of the progesterone receptor and the progesterone receptor. The overall size of the combined score is scored (Harvey JM, Clarck GM, Osborne CK, Allred DC; J. Clin. Oncol. 1999; 17; 1474-1481). The first criterion of reactivity strength is scored on a scale from 0 to 3, with 0 corresponding to "non-reactive" and 3 corresponding to "strong reactivity". The criterion for the ratio of the second reaction is scored on a scale of 0 to 5, with 0 corresponding to "non-reactive" and 5 corresponding to "proportion of 67-100% reaction". The sum of the scores of the reactive strength and the fraction of the proportion of the reaction is then calculated to produce a total score of 0 to 8.

The total score of 0-2 is considered negative and the total score of 3-8 is considered positive.

Based on this scale, the term negative or positive "CXCR4 status" as used in this specification refers to the performance level of CXCR4 corresponding to 0-2 or 3-8 of the Allred's scale, respectively.

Table 2 below sets out guidelines for interpreting IHC results based on the Orred law.

In a preferred embodiment, the method according to the invention refers to a suitable scale, which is a scale of 0 to 8, wherein the non-reactivity score is 0, and is highly reactive in a ratio of 67-100%. The score is 8.

In another embodiment of the invention, a method for determining the state of a tumor from a body in vitro or ex vivo is described, wherein the method comprises the following steps: (a) based on the size of Orred Dividing a tumor from the individual; and (b) determining that the state of the tumor having an Orred score of 3 to 8 is [CXCR4(+)]; or (c) determining that Ollie has 0 to 2 The state of the tumor of the score is [CXCR4(-)].

In a particular aspect of the invention, the tumor having one of the Orred scores of 3 is [CXCR4(+)].

In a particular aspect of the invention, one of the Orred scores having a tumor is [CXCR4(+)].

In a particular aspect of the invention, the tumor having one of the Orred scores of 5 is [CXCR4(+)].

In a particular aspect of the invention, one of the Orred scores having a tumor is [CXCR4(+)].

In a particular aspect of the invention, one of the Orred scores having a tumor is [CXCR4(+)].

In a particular aspect of the invention, one of the Orred scores having a tumor is [CXCR4(+)].

In another specific aspect of the invention, one of the Orred scores having a score of 3 to 8 is [CXCR4(+)].

As a second example, by analogy with conventional scores such as the IHC evaluation of the HER-2 receptor, the sample can be scored on a slightly simpler scoring method for the performance level of CXCR4, which is combined by the scoring method. The intensity of staining (preferably membrane staining) and the ratio of cells showing staining became a scale of a combination of 0 to 3 ⁺ .

As mentioned herein, in the scale of the simplified scale, 0 and 1 ⁺ are negative and 2 ⁺ and 3 ^{+ are} indicated as positive staining. However, the recordable score 1 ⁺ -3 ⁺ is positive because when compared to the score 0 (negative), each positive score may be associated with a significantly higher risk of recurrent and fatal disease, but a positive score The increased strength can provide additional risk reduction.

In general, the term negative or positive "CXCR4 status" as used in this specification refers to the level of expression of CXCR4 corresponding to a fraction of 0-1 ⁺ or 2 ⁺ -3 ⁺ in a simplified scale, respectively. Only the peripheral membranous reactivity of the invasive tumor should be considered and is generally similar to the appearance of a "small mesh". Under the current guidelines, samples of the CXCR4 score divided into boundaries (2 ⁺ or 3 ⁺ scores) must be further evaluated. If, as a non-limiting example, the control is not as expected, the artifacts involve most of the sample and the sample has a strong membranous positive of the normal breast duct (internal control) suggesting excessive antigen retrieval, and the IHC analysis should be rejected. And redo or test by FISH or any other method.

For the sake of clarity, these parameters are summarized in Table 3 below.

In a preferred embodiment, the method according to the invention refers to a suitable scale which is on the scale of 0 to 3+, wherein the tumor cells have no membrane-like reactivity score of 0, and more than 10% of tumors The score for strong complete reactivity among the cells was 3+.

In more detail, as explained above, the appropriate scale is a scale of 0 to 3, wherein the tumor cells have no membrane-like reactivity score of 0; in more than 10% of the tumor cells, weakly perceptible membrane-like The reactivity score was 1+; the weak to moderate complete membranous reactivity score was 2+ in more than 10% of tumor cells; and the strong complete reactivity score was 3 in more than 10% of tumor cells. +.

In another embodiment of the present invention, a method for determining a state of a tumor from a body in vitro or ex vivo is described, wherein the method package The method comprises the steps of: (a) scoring a tumor from the individual according to the simplified scale as described above; and (b) determining that the state of the tumor having a score of 2+ or 3+ is [CXCR4(+)]; Or (c) determining the state of the tumor having a score of 0 or 1+ as [CXCR4(-)].

In a particular aspect of the invention, one of the fractions having a score of 2+ is [CXCR4(+)].

In a particular aspect of the invention, one of the tumors having a score of 3+ is [CXCR4(+)].

In another specific aspect of the invention, the tumor having a fraction of 2+ or 3+ is [CXCR4(+)].

In general, the test or analysis results in accordance with one of the present invention can be presented in any of a variety of formats. The results can be presented in a qualitative manner. For example, the test report may only indicate whether a particular polypeptide is detected, perhaps accompanied by a restriction indicating detection. The results can be shown as semi-quantitative. For example, a wide variety of ranges can be defined, and such ranges can be specified to provide a certain degree of quantitative information (eg, 0 to 3 ⁺ or 0 to 8 depending on the scale used). This score can reflect a variety of factors, such as the number of cells detecting CXCR4, the intensity of the signal (which can indicate the level of expression of CXCR4 or cells with CXCR4), and the like. The results can be expressed in a quantitative manner, for example, as a percentage of cells in which the polypeptide (CXCR4) is detected, a concentration of the protein, and the like.

As will be appreciated by those skilled in the art, the type of output data provided by a test will depend on the technical limitations of the test and the detection of the polypeptide. The significance of the associated creature changes. For example, in certain polypeptide instances, a qualitative output (eg, whether certain peptides are detected at a certain level) provides significant information. In other instances, more quantitative output data (eg, the ratio of the performance level of the polypeptide within the sample being tested to the normal level) is necessary.

The present invention also provides a method for determining whether a cancerous disorder is sensitive to treatment with a primary anti-CXCR4 antibody, or a fragment or derivative thereof, wherein the method comprises the steps of: (a) living according to the method of the present invention Determining the state of CXCR4 of a tumor of a body externally or ex vivo, and (b) if the state is CXCR4(+), determining that the oncogenic disorder is for treatment with a primary antibody CXCR4 antibody, or a fragment or derivative thereof Be sensitive. In another aspect, the invention relates to a method of diagnosing a pathological hyperproliferative carcinogenic disorder associated with the expression of CXCR4 in vivo or a susceptibility to a pathological condition associated with the manifestation of CXCR4, the method comprising the following Steps: (a) determining the presence or absence of cells bearing CXCR4 (CXCR4-carrying), and (b) diagnosing a pathological condition based on the presence or absence of cells having CXCR4 (CXCR4 bearing) or Sensitivity to a pathological condition.

In the method of the present invention, an increase in the level of cells expressing CXCR4 or CXCR4 is generally indicative of a patient with or suspected of presentation. A barrier to the CXCR4 media.

The present invention thus provides a method of predicting the risk of developing a cancer in a body comprising detecting the level of expression of CXCR4 in a tissue sample, wherein a high level of expression of CXCR4 indicates a high risk of developing a cancer. .

It has been observed that the CXCR4 lineage is significantly associated with the onset of tumors in several cancers (Schimanski et al, J Clin Oncol, ASCO Annual Meeting Proceedings Part I., 24(18S): 14018, 2006; Lee et al., Int J Oncol ., 34(2): 473-480, 2009; Pagano, Tesi di dottorato, Università degli Studi di Napoli Federico II, 2008). The invention thus provides a method for assessing tumor aggressiveness as well. As used herein, "tumor aggressiveness" refers to a tumor that grows rapidly and is prone to rapid spread.

In one embodiment, the method comprises the steps of: (a) determining the level of the CXCR4 expressed by the cells in a tumor sample, and (b) determining an equal organization obtained from the same individual at a later time. The level of the CXCR4 expressed in the sample, (c) the ratio between the performance level obtained in step (a) and the ratio obtained in step (b), wherein the performance of CXCR4 in the tumor sample during the time period The ratio provides information on the risk of cancer progression.

In a preferred embodiment, the ratio of the level obtained in step (a) to the level obtained in step (b) of more than 1 indicates invasiveness. Less than or equal to 1 The ratio indicates non-invasiveness.

Another aspect of the invention is the monitoring of the response of CXCR4 expression to the therapy of the CXCR4 target. This monitoring is very useful when the therapy triggers a down regulation and/or degradation of CXCR4.

In particular, monitoring CXCR4 expression on the cell surface can be a critical tool for assessing the efficacy of treatment throughout clinical trials and "individualized" therapies.

The present application thus provides a method of determining a suitable therapeutic regimen for a body.

An increase or decrease in CXCR4 levels indicates the evolution of cancer associated with CXCR4. Thus, by measuring an increase in the number of cells expressing CXCR4 or a change in the concentration of CXCR4 present in various tissues or cells, it may be possible to determine whether a specific therapeutic regimen aimed at improving the malignancy associated with CXCR4 is To be effective.

Accordingly, the present invention is also directed to a method of determining the efficacy of a therapeutic regimen that is designed to alleviate the CXCR4-related carcinogenic disorder in an individual suffering from a CXCR4-related carcinogenic disorder, the method The method comprises the steps of: (a) determining the first performance level of CXCR4 in the first biological sample, the biological sample corresponding to the first time point of the treatment; (b) determining the CXCR4 in the second biological sample a second performance level, the second biological sample corresponding to the second, later time point of the treatment; (c) calculating the first performance level pair step obtained in step (a) ( b) a ratio of the second performance level obtained in the middle; and (d) determining that the efficacy of the therapeutic regimen is high when the ratio of step (c) is greater than one; or (e) at step (c) When the ratio is lower than or equal to 1, it is judged that the efficacy of the therapeutic regimen is low.

In a preferred embodiment, the therapeutic regimen of the CXCR4-related carcinogenic disorder in an individual designed to alleviate the oncogenic disorder associated with CXCR4 comprises administering a CXCR4 inhibitor to the individual.

Another preferred embodiment of the invention relates to a method for selecting a cancer patient predicted to benefit from the administration of a therapeutic amount of a CXCR4 inhibitor, or which does not benefit, the method comprising the steps of: (a Determining the performance level of CXCR4 according to the method of the present invention; (b) comparing the performance level obtained in step (a) with the reference performance level; and (c) setting the performance level pair obtained in (a) If the ratio of the reference performance level is greater than 1, the patient is selected to benefit from the CXCR4 inhibitor that is predicted to be administered from the drug; or (d) if the performance level obtained in (a) is the reference The ratio of performance levels is equal to or less than 1, and the patient is selected to be non-predicted to benefit from the administration of a therapeutic amount of CXCR4 inhibitor.

For the purposes of this specification, the phrase "CXCR4 inhibitor" is intended to include any compound or molecule that binds to CXCR4 and inhibits the binding of a ligand. As a non-limiting example, CXCR4 inhibitors include AMD3100 and AMD3465. Other CXCR4 inhibitors that may be used include, but are not limited to CTCE-0214; CTCE-9908; CP-1221 (linear peptide, cyclic peptide, natural amino acid, unnatural amino acid, and peptidomimetic compound); T140 and analogues; 4F-benzylidene-TN24003 KRH-1120; KRH-1636; KRH-2731; polyphemusin analogue; ALX40-4C. Still other CXCR4 inhibitors are disclosed in WO 01/85196; WO 99/50461; WO 01/94420; and WO 03/090512, each of which is incorporated herein by reference.

In a preferred embodiment, the CXCR4 inhibitor consists of monoclonal antibody 515H7.

In a preferred embodiment, the CXCR4 inhibitor is the monoclonal antibody 515H7 (WO 2010/037831).

Another object of the present invention is also to provide a method of in vivo imaging of carcinogenic disorders associated with the performance of CXCR4 such as monomeric and/or homodimers. This method is useful for locating the tumor in vivo and monitoring its invasiveness. As such, the method is useful for monitoring progress in a patient's body and/or response to treatment, the patient having previously been diagnosed with a cancer having a monomeric and/or homodimeric CXCR4 vector.

In one embodiment, the invention relates to a method for detecting the location of a tumor exhibiting CXCR4 in an individual, the method comprising the steps of: a) administering the antibody I-3859, or an antigen binding fragment or derivative thereof, to The individual; and b) detecting the binding of the antibody, wherein the binding indicates the presence of the tumor.

In another specific example, the present invention relates to detecting an individual A method of displaying the location of a tumor of CXCR4, the method comprising the steps of: (a) administering the antibody I-3859, or an antigen-binding fragment or derivative thereof, to the individual; and (b) detecting the binding of the antibody, Wherein the binding represents the location of the tumor.

As for the detection of a manifestation of a tumor, many techniques known to those skilled in the art can be used. However, preferred means are IHC and FACS.

In another aspect, the invention provides an in vivo imaging agent comprising an antibody, or an antigen binding fragment or derivative thereof, according to the invention, preferably an antibody or fragment or derivative thereof More preferably, it is radioactively calibrated. The agent can be administered in combination with a pharmaceutically effective carrier to a patient suffering from a CXCR4-mediated cancer.

The invention also contemplates the use of the agent for medical imaging in a patient suffering from CXCR4-mediated cancer.

The method of the invention comprises the steps of: (a) administering an imaging effective amount of imaging agent to the patient and (b) detecting the agent.

In one embodiment, the method of the invention allows for the detection of the presence of a tumor exhibiting CXCR4 in the patient. In another embodiment, the method of the invention allows for detecting the location of a tumor exhibiting CXCR4 in the patient.

In a preferred embodiment, the imaging agent comprises a target portion and an active portion.

As used herein, the term "target portion" refers to a specific identification and Binding to the preparation of CXCR4 on the cell surface. In a specific embodiment, the target moiety is an antibody or fragment or derivative thereof that specifically binds to CXCR4. In particular, the target moiety is an antibody or fragment or derivative thereof as described above. Preferably, the target moiety is an I-3859 antibody.

As used herein, "active moiety" refers to a formulation that allows for the detection of the imaging agent in vivo. The active moiety according to the invention comprises, inter alia, radioactive elements such as yttrium-99m (99mTc), copper-67 (Cu-67), strontium-47 (Sc-47), ruthenium (Luthetium)-77 (Lu-177) copper- 64 (Cu-64), 钇-86 (Y-86) or iodine-124 (I-124).

The imaging agent is administered in an amount effective for diagnostic use in a mammal (e.g., a human) and, in turn, detects the localization and accumulation of the imaging agent. The imaging agent can be localized and accumulated by means of radionuclide imaging, radioactive scintigraphy, magnetic resonance imaging, computed tomography, positron emission tomography, computerized tomography, X-ray or magnetic resonance imaging, Fluorescence detection and chemiluminescence detection are detected.

With regard to the development of targeted anti-tumor therapies, the diagnosis of immunohistochemical techniques provides in-situ information about the level of expression of the receptor, for example, regarding the size and/or location of the tumor. The diagnosis thus enables selection of a patient who is sensitive to the treatment and then requires the performance level of the recipient of the treatment.

More specifically, the CXCR4 expression level is preferentially measured via fluorescence activated cell sorting (FACS) or immunohistochemistry (IHC).

FACS analysis is widely used in immunology and hematology to assess the presence of different cell populations within a heterogeneous cell suspension. FACS analysis available The number of monoclonal antibodies is very large, and their coupling to different fluorescent dyes allows for easy multiple antigen staining. The immunophenotype is a necessary parameter for diagnosing a blood malignant disease. FACS analysis was used for analysis of bone marrow, peripheral blood samples, and tissue biopsy samples with suspected hematological malignancies (Martinez A. Cytometry Part B (Clinical Cytometry) 2003 56B 8-15). For example, Fiedler W et al. (Fiedler W. Blood 2003 102 2763-2767) reported the use of FACS analysis to screen for c-kit expression in AML patients prior to treatment with SU5416, a suppressor of VEGF receptors 1 and 2, c- Kit, SCF receptor and small molecule phosphorylated by tyrosine kinase-3 (FLT3) like fms.

A "biological sample" can be any sample taken from a body. This must have been allowed to determine the performance level of the biological indicator of the present invention. The nature of the sample thus depends on the nature of the tumor. If the cancer is a liquid tumor, the biological sample for determining the performance level of the biological indicator by detecting the activated Akt and/or Erk protein is a blood sample, a plasma sample, or a lymph sample. According to "liquid tumor", it refers to tumors of blood or bone marrow, that is, malignant blood diseases such as leukemia and multiple myeloma. The biological sample is preferably a blood sample. Rather, such a blood sample can be obtained through a completely harmless phenotype CXCR4 inhibitor or a non-responsive phenotype CXCR4 inhibitor obtained from the patient's blood collection and thus allowing a non-invasive diagnosis.

As used herein, when a cancer is a solid cancer, the "biological sample" also includes a patient's solid cancer sample to be tested. This solid cancer sample allows any type of measurement that is familiar to the artist to perform the level of the biological indicator of the present invention. the amount. In some cases, the method of the invention may further comprise the preliminary step of obtaining a solid cancer sample from the patient. A tumor tissue sample is referred to as a "solid cancer sample". Even in patients with cancer, the tissue at the location of the tumor still contains non-tumor healthy tissue. The "cancer sample" should therefore be limited to tumor samples taken from the patient. The "cancer sample" can be a biopsy sample or a sample taken from a surgical resection therapy.

According to one aspect, the sample from the patient is a cancer cell or a cancer tissue.

This sample can be obtained according to methods known to those skilled in the art and must be prepared according to methods known to those skilled in the art.

The cancer cell or cancer tissue in the present invention is not particularly limited.

As used herein, the term "cancer" refers to or describes a physiological condition of a mammal, which is typically characterized by unregulated cell proliferation. As used herein, the term "cancer" or "cancer" is intended to encompass all periods of the disease. Thus, as used herein, the term "cancer" can include both benign and malignant tumors. Examples of cancer include, but are not limited to, carcinoma, lymphoma, blastoma, sarcoma, and leukemia or lymphoid malignancies. More particularly, the cancer according to the present invention is selected from the group consisting of squamous cell carcinoma (eg, squamous cell carcinoma), lung cancer, including small cell lung cancer, non-small cell lung cancer, adenocarcinoma of the lung Squamous cell carcinoma of the lung, cancer of the peritoneum, hepatocellular carcinoma, gastric cancer or stomach cancer, including gastrointestinal cancer and gastrointestinal matrix cancer, pancreatic cancer, glioblastoma, cervical cancer, Ovarian cancer, liver cancer, bladder cancer, urinary cancer, liver cancer, breast cancer, colon cancer, rectal cancer, colorectal cancer, Endometrial or uterine cancer, salivary gland cancer, kidney or renal cancer, prostate cancer, genital cancer, thyroid cancer, hepatic carcinoma, anal cancer, penile cancer, melanoma, surface dissemination Melanoma, malignant freckle melanoma, extremity freckle melanoma, nodular melanoma, B-cell lymphoma (including low malignancy/follicular non-Hodgkin's lymphoma (NHL); small lymphocytes (SL) NHL; moderate malignancy/follicular NHL; moderate malignant diffuse NHL; high malignant immune blastocytosis NHL; high malignant lymphocytic cytogenetic NHL; high malignancy small non-fissure cellular NHL; giant tumor (bulky disease) NHL; mantle cell lymphoma; AID S-related lymphoma; Waldenstrom's Macroglobulinemia; chronic lymphocytic leukemia (CLL); acute lymphoblastic cell Leukemia (ALL); hairy cell leukemia; chronic myeloblastic leukemia (CML); acute myeloid leukemia (AML); and post-transplant lymphoproliferative disorder (PTLD), and abnormalities associated with phakomatoses Angiogenesis, edema (such as edema associated with brain tumors), Meigs' syndrome, brain and head and neck cancer, and associated metastases.

In a preferred embodiment, the cancer is selected from the group consisting of prostate cancer, osteosarcoma, lung cancer, breast cancer, endometrial cancer, leukemia, lymphoma, multiple myeloma, ovarian cancer, pancreatic cancer and colon cancer. . In a more preferred embodiment, the cancer comprises lymphoma cells, leukemia cells or multiple myeloma cells.

The performance level of CXCR4 is advantageously compared to the level of a control cell or sample or compared to the level of a control cell or sample. The amount, the level within a control cell or sample is also referred to as the "reference level" or "reference performance level." "Reference level", "reference performance level", "control level", and "control" are used interchangeably within this specification. By "control level" is meant an independent baseline level measured in comparable control cells that are typically free of disease or cancer. The control cells can be from the same individual, because even in a cancer patient, the tissue at the tumor site still contains non-tumor healthy tissue. It can also be derived from another individual who has acquired an unsound sample or test sample that is normal or does not present the same disease. Within the context of the present invention, the term "reference level" refers to the "control level" of the expression of CXCR4, which is used to assess the test level of CXCR4 expression in a patient's cancer cell containing sample. For example, when the CXCR4 level in a patient's biological sample is higher than the reference level of CXCR4, the cells will be considered to have a high performance level of CXCR4 or an excessive performance of CXCR4. The reference level can be determined by a number of methods. The level of performance can thus define the level of expression of cells with CXCR4 or optionally CXCR4 independent of the number of cells expressing CXCR4. Thus, the reference level for each patient can be prescribed by the ratio referenced by CXCR4, wherein the ratio of the reference can be determined by any of the methods of determining the reference level as described herein.

For example, the control can be a predetermined value, which can take a wide variety of forms. It can be a single cutoff value, such as a median or average. The "reference level" can be a single number, and can be applied individually to each patient, or the reference level can vary depending on the particular subpopulation of the patient. So, for example, older people can be about the same cancer With a different reference level than a younger person, and a woman with the same cancer can have a different reference level than a man. Optionally, the "reference level" can be determined by measuring the level of expression of CXCR4 in non-carcinogenic cancer cells from the same tissue as the tissue of the neoplastic cells to be tested. Similarly, the "reference level" can be a ratio of CXCR4 in a patient's twin cells to a level of CXCR4 in a non-tumor cell in the same patient. The “reference level” can also be the level of CXCR4 in in vitro cultured cells. Cells cultured in vitro can be manipulated to mimic tumor cells, or can be manipulated in any other way that produces performance levels, which are accurately determined. Reference level. On the other hand, the "reference level" can be established based on the comparison group, such as a group that does not have an elevated CXCR4 level and a group that has an elevated CXCR4 level. Another example of a comparison group can be a group with a particular disease, condition or symptom and a group without the disease. The predetermined value can be arranged, for example, when a tested population is divided evenly (or unevenly) into groups, such as low risk groups, intermediate risk groups, and high risk groups.

This reference level can also be determined by comparison of the levels of CXCR4 in the patient population with the same cancer. This can be done, for example, by histogram analysis, in which the entire patient group of the same attribute group is represented by a graph, where the first axis represents the level of CXCR4 and the second axis represents the same attribute group. The number of patients, such as tumor cells, exhibited a putative level of CXCR4. Two or more independent groups of patients can be determined by the identification of sub-aggregate groups of the same attribute group having the same or similar CXCR4 levels. The reference level can then be determined according to the best The area is estimated by one of these independent groups. A reference level may also represent the level of two or more indicators, one of which is CXCR4. Two or more indicators may, for example, be represented by a ratio of values of the respective indicator levels.

Similarly, an apparently healthy population would have a different 'normal' range than a group known to have a condition associated with the performance of CXCR4. Thus, the selected predetermined value can take into account the category to which a body belongs. The appropriate range and type can be selected by those skilled in the art using no more than routine experimentation. By "boosted", "increased", it is meant to be high in relation to the selected control. Typically, the control will be based on a healthy, normal individual in an appropriate age group.

It is also understood that the control according to the present invention, in addition to the predetermined value, may be a sample of the material tested in parallel with the experimental material. Examples include tissues or cells obtained from the same individual at the same time, for example, from a single biopsy portion of the individual, or a portion of a single cell sample.

In another embodiment, the invention relates to a pharmaceutical composition for in vivo imaging of a carcinogenic disorder associated with the expression of CXCR4, comprising the above monoclonal antibody or fragment thereof, which is calibrated and It binds CXCR4 in vivo; and a pharmaceutically acceptable carrier.

In another aspect of the invention, a kit for use in such a diagnostic or prognostic method is provided, the kit comprising an antibody of the invention, or a fragment or derivative thereof.

One for detecting the presence and/or location of a tumor that exhibits CXCR4 The kit may comprise at least one of: a) an antibody, or an antigen-binding fragment or derivative thereof, comprising: i) a heavy chain comprising the following three CDRs: having sequence identification number 1 CDR-H1, CDR-H2 having SEQ ID NO: 2 and CDR-H3 having SEQ ID NO: 3; and, ii) a light chain comprising the following three CDRs, respectively having sequence identification number 4 CDR-L1, CDR-L2 having SEQ ID NO: 5 and CDR-L3 having SEQ ID NO: 6, b) an antibody having a heavy chain comprising the following three CDRs: having sequence identification number 1 CDR-H1, CDR-H2 having SEQ ID NO: 2 and CDR-H3 having SEQ ID NO: 3; and, a light chain variability field, comprising sequence identification number 8; c) an antibody, a heavy chain variability field comprising sequence sequence identification number 7; and a light chain comprising the following three CDRs, CDR-L1 having sequence sequence number 4, CDR-L2 having sequence sequence number 5, and CDR-L3 with sequence sequence identification number 6 d) an antibody having a heavy chain variant 7 of the art can recognize sequence of the sequence ID; 8 light chain and the variant sequence of the sequence may be the field identification number.

The packaged material, which contains a predetermined amount of reagent combination plus instructions for performing a diagnostic assay, such as a kit, is also within the scope of the invention. The kit includes the antibody for detecting and quantifying CXCR4 in vitro, for example in an ELISA. The antibodies of the present invention can be provided in a set for detecting and quantifying CXCR4 in vitro, for example, in an ELISA. The anti-system is calibrated with an enzyme that will include the matrix and enzymes needed. Cofactor (eg, a precursor that provides a detectable chromophore or matrix of fluorophores). In addition, other additives such as a stabilizer, a buffer (for example, a blocking buffer or a dissolution buffer), and the like may be included. The set can include a socket that is divided to accept one or more containers, such as vials, tubes, and the like, which contain the individual elements of the present invention. For example, a container can include a first antibody that binds to an insoluble or partially soluble carrier. The second container can comprise a soluble, detectable-calibrated second antibody in lyophilized form or in solution. The socket may also include a third container containing a detectably calibrated third antibody in a lyophilized form or in solution. This essential set can be used in the sandwich analysis of the present invention. The label or package insert can provide a description of the composition as well as an indication of the intended in vitro or diagnostic use.

The relative amounts of the various reagents can vary widely to provide the concentration of the solution of the reagent, which substantially optimizes the sensitivity of the assay. In particular, such agents may be provided as a dry powder, typically freeze-dried, which includes excipients which, when dissolved, will provide a solution of the reagent in a suitable concentration.

In still another aspect of the invention, a monoclonal antibody or a binding fragment thereof, which is labeled with a detectable moiety as described in detail herein, is provided such that it can be packaged and used, for example, Within the kit, a cell having the aforementioned antigen is diagnosed or identified. Non-limiting examples of such markers include fluorophores such as fluorescent isothiocyanates; chromophores, radionuclides, biotins or enzymes. Such calibrated antibodies or binding fragments can be used for tissue localization of antigens, ELISA, cell sorting, And for detecting or quantifying CXCR4, and cells having the antigen, for example, other immunological techniques.

Kits are also provided which have been used as controls for purification of cells from cells or immunoprecipitation of CXCR4. In terms of isolating and purifying CXCR4, the kit can include such antibodies or antigen-binding fragments thereof as described herein coupled to beads (e.g., sepharose beads). Kits can be provided which include antibodies for detecting and quantifying CXCR4 for use in vitro, such as in ELISA. The kit includes a container and a logo or package insert on or with the container. The container contains a composition comprising at least antibody I-3859 of the invention, or an antigen binding fragment or derivative thereof. Additional containers may be included which contain, for example, diluents and buffers, control antibodies. The logo or package insert can provide a description of the composition as well as the intended in vitro indication or diagnostic use.

More particularly, the present invention relates to a kit for determining the CXCR4 status of a tumor by the method of the present invention. In a preferred embodiment, as will be explained in the examples, the present invention relates to a kit for determining the CXCR4 status of a tumor by IHC and/or FACS methods.

In a particular embodiment, the invention resides in a kit comprising the antibody I-3859, or an antigen binding fragment or derivative thereof, as described above, the anti-system being calibrated.

In a preferred embodiment, the kit according to the invention further comprises an agent for detecting the degree of binding between the antibody I-3859 and CXCR4.

In another preferred embodiment, the invention is useful for use in vitro or A kit for determining the expression level of CXCR4 within a tumor exhibiting CXCR4 ex vivo, further comprising an agent for quantifying the binding level between the calibrated antibody and CXCR4.

In still another embodiment, the kit according to the present invention further comprises: i) an agent having a degree of binding between the labeled antibody and CXCR4; and ii) having a score for scoring the CXCR4 A control sample that is positive for the level and a negative control sample.

The kit may further comprise a plurality of antibodies specific for the mouse antibody, the plurality of antibodies specific for the mouse antibody being preferably calibrated. A method for selecting a cancer patient predicted to benefit from a therapeutically effective amount of a CXCR4 inhibitor, or which does not benefit, the method comprising the steps of:

According to a particular embodiment of the invention, the kit for selecting a cancer patient predicted to benefit from a therapeutic administration of a CXCR4 inhibitor, or which does not benefit, may comprise: i) a method for detecting An agent for the degree of binding between the antibody and CXCR4; and ii) a control level that has been associated with sensitivity to a CXCR4 inhibitor and/or iii) a control position that has been associated with resistance to a CXCR4 inhibitor quasi.

The invention also relates to a diagnostic reagent in vivo or ex vivo, which consists of an antibody according to the invention, or an antigen-binding fragment or derivative thereof, preferably labeled, in particular radiolabeled, And its use in medical imaging, especially for detecting cancer associated with cellular performance or excessive performance of CXCR4.

Other features and advantages of the present invention are disclosed in the continuation of the description and the examples and drawings.

Simple illustration

Figure 1 shows both I-3859 Mab immunoprecipitated CXCR4 monomer and dimer; Figures 2A and 2B show I-3859 Mab adjustment of CXCR4 homodimer (A) and CXCR4/CXCR2 heterodimer ( B) Both; Figure 3 shows CXCR4 identified on the cell membrane by I-3859 Mab by FACS; Figures 4A and 4B show that I-3859 Mab was added to compete with anti-CXCR4 515H7 therapeutic Mab by FACS analysis to CXCR4 on the cell membrane; Figure 5 shows that I-3859 Mab has no effect on MDA-MB-231 xenograft tumor growth pattern in athymic nude mice; Figure 6 illustrates the use of a) I-3859 on RAMOS xenograft tumors IHC staining with b) mIgG1; Figure 7 illustrates IHC staining with a) I-3859 and b) mIgG1 on KARPAS299 xenograft tumors.

Example 1: Production of anti-CXCR4 I-3859 monoclonal antibody (Mab)

To generate monoclonal antibodies to CXCR4, BALB/c mice were immunized with recombinant NIH3T3-CXCR4 cells and/or peptides corresponding to CXCR4 extracellular N-terms and loops. 6-16 week old mice were immunized subcutaneously (sc) once with the antigen of Freund adjuvant immediately after the first immunization, followed by sc in combination with incomplete Fondszo The antigen of the agent is immunized 2 to 6 times. Immune response through blood collection in the orbit (retroorbital Bleeds) to monitor. Serum lines were screened by ELISA (described below) and fused using anti-CXCR4 antibodies with higher potency. Three days prior to cell fusion, the mice were boosted intravenously with antigen before sacrifice and removal of the spleen.

- ELISA

To select mice producing anti-CXCR4 antibodies, sera from immunized mice were tested via ELISA. Briefly, a microtiter plate was coated with 5 μg of equivalent peptide/mL, 100 μL/well of BSA-purified [1-41] N-terminal peptide at 4 ° C overnight, followed by Blocked with 250 μL/well of 0.5% gelatin solution in PBS. Dilutions of plasma from CXCR4-immunized mice were added to each well and incubated at 37 °C for 2 hours. The plate was washed with PBS and then incubated with HRP-conjugated goat anti-mouse IgG antibody (Jackson Laboratories) for 1 hour at 37 °C. After cleaning, the flat disk was developed with TMB, and the reaction was stopped after adding 100 μL/well of 1 M H ₂ SO ₄ . Mice that developed the highest potency anti-CXCR4 antibody were used for antibody production.

- Will produce a fusion tumor of Mabs of CXCR4

A mouse spleen cell line isolated from BALB/c mice developing the highest potency anti-CXCR4 antibody was fused to a mouse myeloma cell line Sp2/O with PEG. The cell line was plated in a microtiter plate at approximately 1×10 ⁵ /well and then in a selective medium containing ultra culture medium + 2 mM L-glutamic acid +1 mM sodium pyruvate + 1 x HAT. Incubate for 2 weeks. The anti-CXCR4 monoclonal IgG antibody well was then screened by ELISA. The antibody secreting fusion tumor is then subcloned at least 2 times by limiting dilution and cultured in vitro to produce antibodies for further analysis.

Example 2: I-3859 Mab immunoprecipitates both CXCR4 monomer and dimer

The NIH3T3-CXCR4 cell pellet was washed with 20 mM TrisHCl containing 100 mM (NH ₄ ) ₂ SO ₄ at pH 8.5 and suspended in a lysis buffer (containing 100 mM (NH ₄ ) ₂ SO ₄ , 10%). Glycerin, 1% CHAPSO and 10 μL/mL protease inhibitor cocktail pH 8.5 within 20 mM TrisHCl). The cells were disrupted using a Potter Elvehjem homogenizer. The dissolved membrane was collected by centrifugation at 105,000 g for 1 h at 4 ° C, then incubated overnight at 4 ° C with I3859 Mab coupled to Sepharose 4B beads and the mixture was poured into a glass column with lysis buffer Wash it. The proteins captured by the I3859 Mab were eluted and analyzed using Western blotting using anti-CXCR4 Mab as the primary antibody. Fractions of interest were pooled, concentrated and used for both WB analysis and preparative SDS-PAGE analysis (4-12% Bis-Tris gel).

After silver staining, the electrophoresis bands of interest were removed from the gel and the intragel ingestion was performed using an automated protein digestion system, MassPREP Workstation (Waters, Milford, MA, USA). Gel plaques were washed twice with 50 μL of 25 mM NH ₄ HCO ₃ (Sigma, Steinheim, Germany) with 50 μL of acetonitrile (Carlo Erba Reactifs-SDS, Val de Reuil, France). The cysteine residue was reduced by 50 μL of 10 mM DTT prepared in 25 mM NH ₄ HCO ₃ at 60 ° C for 1 hour and by 50 μL of 55 prepared in 25 mM NH ₄ HCO ₃ Alkylation was carried out for mM iodoacetamide (Sigma) for 20 minutes. After the gel plaque was dehydrated with acetonitrile, the protein was added by adding 10 μL of 12.5 ng/μl modified pig trypsin (Promega, Madison, WI, USA) in 25 mM NH ₄ HCO ₃ . Digest overnight in the gel at room temperature. The resulting peptide was extracted with 35 μL of 60% acetonitrile containing 5% formic acid (Riedel-de Haën, Seelze, Denmark) followed by removal of excess acetonitrile and acceptance of nano-LC-MS/MS. Department of processing large amounts of data to be collected during and transformed into the nano LC-MS / MS analysis * .mgf be carried out MASCOT ^TM file search engine. The search is performed in MS and MS/MS modes with a tolerance of 0.25 Da measurement.

Figure 1 shows Western blot analysis of the eluted concentrated fraction after immunoprecipitation using I-3859 Mab coupled with Sepharose beads. Two electrophoretic bands at 43 and 75 kDa apparent molecular weight were stained with anti-CXCR4 Mab as a primary antibody (antiboby).

The eluted concentrated fraction was also resolved by SDS-PAGE and visualized by silver staining after immunoprecipitation using I-3859 Mab coupled with Sepharose beads. The electrophoresis bands at 43 and 75 KDa were excised from the gel, digested with trypsin and analyzed by LC-MS/MS as described above. The collected peak list is Mascot for the peptide sequence database search. CXCR4 is recognized in both electrophoresis bands:

Five CXCR4 peptides were identified in the 75-kDa electrophoresis band by the MASCOTTM search engine: 31-38 peptide EENANFNK, included in the N-terminal CXCR4; 71-77 peptide SMTDKYR, included in the intracellular loop 1 ;311-322 peptide TSAQHALTSVSR, 312-322 peptide SAQHALTSVSR, 313-322 peptide AQHALTSVSR included in the C-end Inside.

The 43-kDa electrophoresis band identified 9 CXCR4 peptides via the MASCOTTM search engine: 27-30 peptide PCFR, 31-38 peptide EENANFNK, included in the N-terminus; 71-77 peptide SMTDKYR, included in the cell Within inner loop 1; 135-143 peptide YLAIVHATN and 135-146 peptide YLAIVHATNSQR, included in intracellular loop 2; 311-319 peptide TSAQHALTS, 311-322 peptide TSAQHALTSVSR, 312-322 peptide SAQHALTSVSR The 313-322 peptide AQHALTSVSR is included in the C-terminus.

The results obtained in this study clearly show that I-3859 Mab is capable of immunoprecipitating CXCR4. I-3859 Mab is identified as CXCR4 for both monomer and dimer.

Example 3: I-3859 Mab adjusts both CXCR4 homodimer and CXCR4/CXCR2 heterodimer by BRET analysis

This functional assay allows for the assessment of the conformational changes induced by the binding of SDF-1 and/or I-3859 Mab to the CXCR4 receptor at the level of the CXCR4 homodimer and the CXCR2/CXCR4 heterodimer.

The performance vector of each of the interacting partners investigated was constructed with the corresponding dyes ( Neuilla reniformis luciferase, Rluc and yellow fluorescent protein, YFP) by applying conventional molecular biology techniques. Fusion protein. Two days before the BRET experiment, the HEK293 cell line was transiently transformed with the expression vector encoding the corresponding BRET partner: [CXCR4/Rluc+CXCR4/YFP] to study CXCR4 homodimerization and [CXCR4-Rluc+CXCR2-YFP] To study the heterodimerization of CXCR4 and CXCR2. After that day, cells were distributed in white 96 MW plates pre-coated with polylysine in complete medium [DMEM supplemented with 10% FBS]. First, cells following in CO ₂ 5% at 37 ℃ serve to allow the cells to attach to the flat plate. The cells were then starved overnight in 200 μl DMEM/well. Immediately prior to the BRET experiment, DMEM was removed and the cells were washed rapidly with PBS. The cells were incubated for 15 min at 37 °C in PBS with or without antibody, before adding 50 μl of final volume of 5 μM of coelenterazine H with or without SDF-1. Incubation at 37 ° C for 5 minutes and further incubation at room temperature for 20 min, light-emission acquisition at 485 nm and 530 nm using the Mithras LB940 Multilabel Reader (Berthold) (at room temperature) 1s/wavelength/well repeat 15 times) start.

The calculation of the BRET ratio is performed as previously explained (Angers et al., 2000): [(emission _{530 nm} )-(emission _{485 nm} )X Cf]/(emission _{485 nm} ) where Cf=(emission _{530 nm)} ) / (emission _{485 nm} ) The cells exhibited only the Rluc fusion protein under the same experimental conditions. Simplifying this equation shows that when there are 2 BRET partners, the BRET ratio corresponds to the ratio obtained 530/485 nm, when only the partner fused to Rluc is present in the analysis, the ratio obtained under the same experimental conditions 530/485 nm to correct. For readability, the results are expressed as a percentage of the base signal.

The addition of SDF1 (300 nM) triggers an increase in the BRET signal due to the proximity of the adaptor fused to the CXCR4 receptor to the space of the acceptor protein, up to approximately 20%. This increase is likely to indicate the formation of a CXCR4/CXCR4 homodimer or a conformational change in an already existing dimer (Fig. 2A). I-3859 Mab was able to modulate the SDF-1-induced conformational change of the CXCR4 homodimer (69% inhibition of SDF-1-induced BRET increase). The I-3859 Mab can also be adjusted by the proximity of its own CXCR4/CXCR4 space, indicating the effect of the I-3859 Mab on the CXCR4/CXCR4 homogeneous dimer configuration (Fig. 2A).

The BRET signal for the reaction to SDF1 (300 nM) due to the proximity of the CXCR4 to the CXCR2 receptor is reduced by approximately 20%. This result suggests a CXCR4/CXCR2 heterodimer formation or a conformational change in the already existing dimer (Fig. 2B). I-3859 Mab is able to modulate the conformational change of SDF-1-induced CXCR2/CXCR4 heterodimer with approximately 100% inhibition of BDF-induced BRET reduction and is also able to borrow its own CXCR4/CXCR2 The proximity of the space was adjusted to indicate the effect of I-3859 Mab on the configuration of the CXCR4/CXCR2 heterodimer (Fig. 2B).

Example 4: Analysis of I-3859 by FACS Mab identifies CXCR4 present on the cell surface

In this experiment, the I-3859 Mab-specific CXCR4 line that binds to humans was examined by FACS analysis.

NIH3T3, NIH3T3-hCXCR4 transfected cells, MDA-MB-231, Hela, and U937 cancer cell lines were incubated with I-3859 monoclonal antibody. The cells were then washed with 1% BSA/PBS/0.01% NaN3. Alexa-labeled secondary antibodies were then added to the cells and allowed to incubate at 4 °C for 20 min. The cells were then washed twice more. After the second wash, FACS analysis was performed. The results of these binding studies are provided in Figure 3; they show that anti-CXCR4 Mab I-3859 binds to humans CXCR4-NIH3T3 transfected cell line [mean fluorescence intensity (MFI)], but not parental NIH3T3 cells (not shown). This Mab is also capable of identifying human cancer cell lines, for example, MDA-MB-231 breast cancer cells (MFI=59 at a concentration of 10 μg/ml), and pre-U937 pre-myeloid cell carcinoma cells (at a concentration of 10 μg/ml) MFI = 246) and Hela cervical cancer cells (MFI = 633 at a concentration of 10 μg/ml), indicating that these cell lines are naturally overexpressing CXCR4.

Example 5: Analysis by FACS I-3859 Mab was added to compete with anti-CXCR4 515H7 therapeutic Mab for binding to CXCR4 on the cell membrane

In this experiment, the binding of I-3859 and 515H7 Mabs to the human CXCR4 line was examined by FACS analysis.

NIH3T3-hCXCR4 transfected cell lines were incubated with biotinylated 515H7 Mab (5 μg/ml) [identifying NIH3T3-CXCR4 cells (Fig. 4A)] and I-3859 Mab or 515H7 Mab (0-1 mg) /mL) was incubated at 4 ° C for 1 hour. The cells were then washed with 1% BSA/PBS/0.01% NaN3. The calibrated streptavidin was then added to the cells and allowed to incubate at 4 °C for 20 min. The cells were then washed twice more. After the second wash, FACS analysis was performed. The results of these combined studies are provided in Figure 4B. It shows that [-average fluorescence intensity (MFI)] of anti-CXCR4 Mab I-3859 competes with anti-CXCR4 515H7 therapeutic Mab for binding to human CXCR4-NIH3T3 transformed cells. As expected, the uncalibrated 515H7 Mab also inhibited the binding of biotinylated 515H7 Mab to CXCR4.

Example 6: Evaluation of I-3859 Mab activity in MDA-MB-231 xenograft tumor growth pattern in athymic nude mice

The goal of this experiment was to evaluate the ability of anti-CXCR4 Mab I-3859 to inhibit MDB-MB-231 xenograft growth in athymic nude mice.

MDA-MB-231 cells from ECACC were routinely cultured in DMEM medium (Invitrogen Corporation, Scotland, UK), 10% FCS (Sigma, St Louis MD, USA). The cells were separated 48 hours prior to transplantation such that they were in the growth index phase. One million MDA-MB-231 cells in PBS were transplanted into 7-week-old athymic nude mice (Harlan, France). Five days after implantation, the tumors were measurable (34 mm ³ <V ³ <40 mm ³ ) and the animals were divided into groups of 12 mice with comparable tumor sizes. The mice were treated with ip at a loading dose of 2 mg/mouse of I-3859 Mab. Subsequently, the mice were injected twice a week at 1 mg/dose/mouse of I-3859 Mab. The PBS group was used as a control group in this experiment. Tumor volume was measured twice a week and the following formula was calculated: π/6 X length X width X height. Statistical analysis of each measurement was performed using the Mann-Whitney test.

In this experiment, no mortality was observed throughout the treatment period. D31 at I-3859 Mab (1 mg/dose) did not significantly inhibit tumor growth compared to the PBS group. The mean tumor volume after 4 weeks of treatment was not reduced relative to PBS for I-3859 Mab (Figure 5).

Example 7: Immunohistochemistry study (IHC)

Sections were deparaffinized, rehydrated, and placed in prewarmed Protease 1 buffer (Ventana Medical system) at 37 °C for 10 minutes for heat-induced epitope-repairing. Endogenous peroxidase after 3 washes in Tris buffer saline-0.05% tween 20 (TBS-T) (Dako S3006) The activity was blocked with a peroxidase blocking reagent (Dako K4007) for 5 minutes. The sections were washed with TBS-T and with mouse IgG1/κ (15 μg/ml, X0931, Dako) at I-3859 (15 μg/ml, strain I-3859, Pierre Fabre) or as an isotype control. Incubate in blocking reagent (UltraV block-TA-125UB-LabVision) for 5 minutes before incubation at 4 °C overnight. Sections were washed with TBS-T and incubated with SignalStain Boost IHC Detection Reagent (HRP, M) for 30 minutes at room temperature. The diamine benzidine was used to develop the brown reaction product (Dako K3468). Slides were immersed in hematoxylin for 4 minutes for contrast staining (Dako S3309) and washed in PBS prior to fixation with Faramount mounting medium plus coverslips. In this immunohistochemistry procedure, the brown reaction product is associated with positive staining of the cell membrane and the lack of a brown reaction product associated with negative staining and no cell membrane imaging.

I-3859 Mab, differentially stains cell membranes of a variety of tumor types. Panels 6 and 7 illustrate staining performed in two xenograft modes, illustrating the antitumor activity of the therapeutic anti-CXCR-4 hz515H7 antibody: RAMOS and KARPAS299. As shown in Figures 6 and 7, the detected xenotransplantation in KARPAS299 (Fig. 7) is lower than RAMOS (Fig. 6). This data correlates well with studies of CXCR-4 performance by flow cytometry. More specifically, RAMOS cells exhibited approximately 5 levels of CXCR-4 (antibody binding: RAMOS of 200 000 and KARPAS 29940 000) compared to KARPAS299 one. Membrane staining was weaker in KARPAS299 (Fig. 7), whereas membrane staining was significantly higher in RAMOS (Fig. 6).

Figure 1 shows both I-3859 Mab immunoprecipitated CXCR4 monomer and dimer; Figures 2A and 2B show I-3859 Mab adjustment of CXCR4 homodimer (A) and CXCR4/CXCR2 heterodimer ( B) Both; Figure 3 shows CXCR4 identified on the cell membrane by I-3859 Mab by FACS; Figures 4A and 4B show that I-3859 Mab was added to compete with anti-CXCR4 515H7 therapeutic Mab by FACS analysis to CXCR4 on the cell membrane; Figure 5 shows that I-3859 Mab has no effect on MDA-MB-231 xenograft tumor growth pattern in athymic nude mice; Figure 6 illustrates the use of a) I-3859 on RAMOS xenograft tumors IHC staining with b) mIgG1; Figure 7 illustrates IHC staining with a) I-3859 and b) mIgG1 on KARPAS299 xenograft tumors.

<110> Pierre Faber Pharmaceuticals

<120> Antibody I-3859 for cancer detection and diagnosis

<130> 361280D29941

<150> EP11306000.8

<151> 2011-07-29

<150> US61/513,345

<151> 2011-07-29

<160> 16

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Claims (26)

An antibody, or antigen-binding fragment or derivative thereof, for use in detecting the presence and/or location of a tumor exhibiting CXCR4, the antibody comprising i) a heavy chain comprising the following three complementarity determining regions (CDRs), respectively CDR-H1 having SEQ ID NO: 1, CDR-H2 having SEQ ID NO: 2, and CDR-H3 having SEQ ID NO: 3; and ii) a light chain comprising the following three CDRs, respectively CDR-L1 of SEQ ID NO: 4, CDR-L2 having SEQ ID NO: 5, and CDR-L3 having SEQ ID NO: 6. An antibody, or an antigen-binding fragment or derivative thereof, according to claim 1, wherein the antibody is selected from the group consisting of: a) an antibody having a heavy chain comprising the following three CDRs, each having CDR-H1 of SEQ ID NO: 1, CDR-H2 having SEQ ID NO: 2, and CDR-H3 having SEQ ID NO: 3; and a light chain variability field containing sequence identification number 8; b) An antibody having a heavy chain variable region comprising sequence sequence identification number 7; and a light chain comprising the following three CDRs, each having the sequence identification number 4, CDR-L1, having the sequence identification number 5 CDR-L2 and CDR-L3 having SEQ ID NO: 6, or c) an antibody having a heavy chain variability field comprising SEQ ID NO: 7; and a light chain variability field comprising SEQ ID NO: 8. . An antibody, or antigen-binding fragment or derivative thereof, according to claim 1 or 2, for use in a carcinogenic disorder associated with in vitro or ex vivo diagnosis or prognosis and performance of CXCR4. The antibody, or an antigen-binding fragment or derivative thereof, according to any one of claims 1 to 3, wherein the antibody does not have in vivo antitumor activity. A method for detecting the presence and/or location of a tumor exhibiting CXCR4 in vivo or ex vivo, the method comprising the steps of: (a) allowing a biological sample contact from the individual to specifically bind to CXCR4 An antibody, or antigen-binding fragment or derivative thereof; and (b) detecting binding of the antibody, or antigen-binding fragment or derivative thereof, to the biological sample, wherein the antibody, or antigen-binding fragment or derivative thereof, comprises i) a heavy chain comprising the following three CDRs, CDR-H1 having sequence identification number 1, CDR-H2 having sequence identification number 2, and CDR-H3 having sequence identification number 3; and ii) light The strand contains the following three CDRs, which are CDR-L1 having SEQ ID NO: 4, CDR-L2 having SEQ ID NO: 5, and CDR-L3 having SEQ ID NO: 6. A method for detecting the percentage of cells expressing CXCR4 in vivo or ex vivo, comprising the steps of: (a) contacting a biological sample from the individual with an antibody capable of specifically binding to CXCR4, or an antigen thereof Binding fragments or derivatives; and (b) quantifying the percentage of cells expressing CXCR4 in the biological sample More preferably, the antibody, or antigen-binding fragment or derivative thereof, comprises i) a heavy chain comprising the following three CDRs, respectively CDR-H1 having SEQ ID NO: 1, and CDR having SEQ ID NO: 2 -H2 and CDR-H3 having sequence number identification number 3; and ii) a light chain comprising the following three CDRs, respectively CDR-L1 having sequence number identification number 4, CDR- having sequence number identification number 5 L2 and CDR-L3 having sequence number identification number 6. A method for determining the expression level of CXCR4 in a tumor exhibiting CXCR4 from a body in vitro or ex vivo, the method comprising the steps of: (a) allowing a biological sample contact from the individual to be specifically bound to An antibody, or antigen-binding fragment or derivative thereof, of CXCR4; and (b) quantifying the binding level of the antibody, or antigen-binding fragment or derivative thereof, to CXCR4 in the biological sample, characterized in that the antibody, or antigen thereof The binding fragment or derivative comprises i) a heavy chain comprising the following three CDRs, respectively CDR-H1 having sequence number 1 and CDR-H2 having sequence number 2 and CDR having sequence number 3 -H3; and ii) a light chain comprising the following three CDRs, CDR-L1 having sequence number 4, CDR-L2 having sequence number 5, and CDR having sequence number 6 L3. The method of claim 7, wherein the antibody, or an antigen-binding fragment or derivative thereof, and the binding level of CXCR4 are preferentially passed through Photoactivated cell sorting (FACS) or immunohistochemistry (IHC). A method for diagnosing or prognosing a tumor exhibiting CXCR4 in vitro or ex vivo, the method comprising the steps of: (a) determining the performance level of CXCR4 according to the method of claim 7 or 8 and (b) The performance level of the comparison step (a) is compared with the reference performance level of CXCR4 from normal tissue or non-expressing CXCR4 tissue. A method for determining the score of a tumor of a body in vitro or ex vivo, the method comprising the steps of: (a) contacting a biological sample from the individual with an antibody that specifically binds to CXCR4, or antigen binding thereof a fragment or derivative; (b) quantifying the binding level of the antibody, or antigen-binding fragment or derivative thereof, to CXCR4 in the biological sample; and (c) comparing the antibody, or antigen binding thereof, to an appropriate scale A quantified binding level of a fragment or derivative to score the tumor from the individual, characterized in that the antibody, or antigen-binding fragment or derivative thereof, comprises i) a heavy chain comprising the following three CDRs, respectively CDR-H1 having sequence identification number 1, CDR-H2 having sequence identification number 2, and CDR-H3 having sequence number identification number 3; and ii) a light chain comprising the following three CDRs, respectively CDR-L1 having sequence number identification number 4, CDR-L2 having sequence sequence identification number 5, and CDR-L3 having sequence sequence identification number 6. The method of claim 10, wherein the appropriate scale is based on two parameters, the intensity of the staining and the percentage of positive cells. The method of claim 10 or 11, wherein the appropriate scale is a scale of 0 to 8, wherein the non-reactivity score is 0, and the strong reactivity score is 8 in the proportion of 67-100%. A method for determining the state of a tumor from a body in vitro or ex vivo, the method comprising the steps of: (a) the method of claim 10, 11 or 12, wherein the score is from the The tumor of the individual; and (b) the state of the tumor having a score of 3 to 8 is determined to be [CXCR4(+)]; or (c) the state of the tumor having a score of 0 to 2 is determined to be [CXCR4(- )]. The method of claim 10 or 11, wherein the appropriate scale is 0 to 3 ⁺ scale, wherein the tumor cells have no membrane-like reactivity score of 0 and are strong among more than 10% of tumor cells. The score for complete reactivity is 3 ⁺ . A method for determining the state of a tumor from a body in vitro or ex vivo, the method comprising the steps of: (a) the method of claim 10, 11 or 14 to score from The tumor of the individual; and (b) the state of the tumor having a score of 2 ⁺ or 3 ⁺ is determined to be [CXCR4(+)]; or (c) the state of the tumor having a score of 0 or 1 ⁺ is determined as [ CXCR4(-)]. A method for determining whether a cancerous disorder is compatible with treatment with a primary anti-CXCR4 antibody, or a fragment or derivative thereof, the method comprising the steps of: (a) living a living body according to the method of claim 13 or 15 Determining the state of CXCR4 of a tumor of a body externally or ex vivo, and (b) if the state is CXCR4(+), determining that the oncogenic disorder is sensitive to treatment with an anti-CXCR4 antibody, or a fragment or derivative thereof . A method for selecting a cancer patient predicted to benefit from the administration of a therapeutic amount of a CXCR4 inhibitor, or a method that does not benefit, the method comprising the steps of: (a) the method of claim 7 or 8 To determine the performance level of CXCR4; (b) compare the performance level and the reference performance level of the previous step a); and (c) set the performance level obtained in (a) to the reference performance level. If the ratio is greater than 1, the patient is selected to benefit from therapeutic administration of the CXCR4 inhibitor; or (d) the ratio of the performance level obtained in (a) to the reference performance level is Below or equal to 1, the patient is selected to be non-predicted to benefit from therapeutic administration of a CXCR4 inhibitor. A method for determining the efficacy of a therapeutic regimen in vitro or ex vivo, which is designed to alleviate carcinogenicity associated with CXCR4 The CXCR4-related carcinogenic disorder in an individual suffering from a disorder, the method comprising the steps of: (a) determining the first of CXCR4 in the first biological sample as in the method of claim 7 or 8 Performance level, the first biological sample corresponds to the first time point of the treatment; (b) the method of claim 7 or 8 determines the second performance of CXCR4 in the second biological sample Level, the second biological sample corresponds to the second, later time point of the treatment; (c) calculating the first performance level obtained in step (a) for the step (b) a ratio of the second performance level; and (d) determining that the efficacy of the therapeutic regimen is high when the ratio of step (c) is greater than one; or (e) the ratio of step (c) If the ratio of step (c)is inferior or equal to second expression level is statistically similar to or is determined, the efficacy of the therapeutic regimen is determined to be low. The method of claim 18, wherein the therapeutic regimen of the CXCR4-related carcinogenic disorder in an individual designed to alleviate the oncogenic disorder associated with CXCR4 comprises administering a CXCR4 inhibitor to the individual . A kit for detecting the presence and/or location of a tumor exhibiting CXCR4, the kit comprising at least one of: a) an antibody, or an antigen-binding fragment or derivative thereof, comprising: i) a heavy a strand comprising the following three CDRs, each having a sequence of sequences CDR-H1 of SEQ ID NO: 1, CDR-H2 having SEQ ID NO: 2, and CDR-H3 having SEQ ID NO: 3; and ii) a light chain comprising the following three CDRs, each having sequence identification CDR-L1 of No. 4, CDR-L2 having SEQ ID NO: 5, and CDR-L3 having SEQ ID NO: 6, b) an antibody having a heavy chain comprising the following three CDRs, each having a sequence Identification number 1 CDR-H1, CDR-H2 having sequence identification number 2, and CDR-H3 having sequence sequence number 3; and a light chain variable region containing sequence sequence number 8; c) an antibody , having a heavy chain variability field comprising sequence sequence identification number 7; and a light chain comprising the following three CDRs, respectively CDR-L1 having sequence number identification number 4, CDR having sequence sequence identification number 5 L2 and CDR-L3 having sequence number identification number 6; d) an antibody having a heavy chain variability field comprising SEQ ID NO: 7; and a light chain variability field comprising SEQ ID NO: 8. For example, the kit of claim 20, wherein the antibody is calibrated. A kit according to claim 20 or 21 further comprising an agent for detecting the degree of binding between the antibody and CXCR4. A kit according to claim 20 or 21 further comprising an agent for quantifying the binding level between the antibody and CXCR4. A kit according to claim 20 or 21, further comprising: i) an agent for detecting the degree of binding between the antibody and CXCR4; and ii) having a positive for scoring the CXCR4 expression level. Control sample and negative control sample. The kit of claim 24, further comprising a plurality of antibodies specific for the mouse antibody, the plurality of antibodies preferably being calibrated. The kit of claim 20 or 21 further comprising: i) an agent for detecting the degree of binding between the antibody and CXCR4; and ii) a control associated with sensitivity to the CXCR4 inhibitor. Levels and/or iii) control levels that have been associated with resistance to CXCR4 inhibitors.