TW201311727A - Novel anti-CXCR4 antibody and its use for the detection and diagnosis of cancers - Google Patents

Abstract

The present invention provides a novel, isolated anti-CXCR4 antibody for use in the diagnosis of cancer. In particular, the antibody of the invention recognizes monomeric and homodimeric CXCR4, but not heterodimeric CXCR4.

Description

Novel anti-CXCR4 antibody and its use 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 an amino acid and nucleic acid sequence encoding the same. The invention also encompasses the use of the antibody for detecting pathological hyperproliferative carcinogenic disorders associated with the diagnosis and expression of CXCR4, and corresponding methods. 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 a product and/or composition or kit comprising at least the antibody for monitoring the prognosis or diagnosis 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. Biopsy samples of human cancer and small Studies of the cancer pattern of mice have shown that the expression of chemokine receptors in cancer cells is associated with increased metastatic potential. 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 C. J. et al., 2002), pancreas (Koshiba T. et al., 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). Recently, CXCR4/SDF-1 axis has been shown to regulate the transport/homing of CXCR4+ hematopoietic stem/progenitor cells (HSC) and to blood. The new student can play a role. Evidence indicates that in addition to HSC, functional CXCR4 is also expressed in stem cells from other tissues (tissue-committed stem cells = TCSCs) so SDF-1 can be chemottracting CXCR4+ necessary for organ/tissue regeneration. TCSCs play a central role, but these TCSCs can also be the cell origin of cancer development (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 in situ lung cancer model (A549) using multiple antibodies against SDF-1, but in this study It has no effect on tumor growth or angiogenesis. 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 (SinghRK. et al., 1994) Cell proliferation (Owen JD et al., 1997) and ERL+chemokine-mediated angiogenesis (Strieter RM et al., 2004). ;Romagnani et al., 2004). Finally, tumor-associated macrophages and neutrophils are key elements of inflammatory-induced tumor growth and complements of chemokines such as CXCL5, IL-8 and GRO-α to initiate neutrophils.

Dimerization has emerged as a versatile mechanism for regulating G-protein-coupled receptor function, 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. Tend The chemical receptor dimer is present in the absence of a ligand and the conformational change of the chemokine-induced receptor dimer. CXCR4 is known to form 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 of CXCR1 and CXCR2 (Wilson S. et al., 2005).

As mentioned previously, the CXCR4 and CXCR2 receptors are of interest for tumors of interest. Interference 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.

The Applicant has disclosed that it will bind and induce conformational changes in both CXCR4/CXCR4 homodimers and CXCR4/CXCR2 heterodimers, and Monoclonal antibodies with potent anti-tumor activity, referred to as 515H7 and 414H5 (see WO 2010/037831).

The present invention is directed to providing at least one agent that can be used as a diagnostic or prognostic tool for detecting and/or monitoring a carcinogenic disorder, particularly characterized by the performance of CXCR4 or by abnormal CXCR4 expression. The media should be such.

In particular, the present invention provides a novel, isolated antibody capable of binding to CXCR4, which can be used for diagnostic or prognostic purposes.

Surprisingly, and in contrast to prior art antibodies, the Applicant has now produced an antibody that specifically binds to CXCR4, such as a CXCR4 monomer or a CXCR4/CXCR4 homodimer. In another aspect, the antibodies of the invention do not significantly bind to any of the known CXCR4/X heterodimers and, more particularly, do not bind to, including CXCR4/CXCR2 heterodimers. As discussed in the following specification, this property has great benefits in the field of diagnosis.

Other features and advantages of the present invention will be apparent from the description and appended claims.

In a first aspect, the invention relates to one of an isolated antibody, or an antigen binding fragment or derivative thereof, which binds to CXCR4 with high affinity, preferably to human CXCR4, thus It can be used to diagnose pathological hyperproliferative carcinogenic disorders mediated by CXCR4 expression.

Other features and advantages of the present invention will be apparent from the description and appended claims.

Preferably, the present invention encompasses a functional fragment of an antibody of the present invention, a derivative thereof, or the like obtained by genetic recombination or chemical synthesis.

In the first specific example, the antibody of the present invention is a monoclonal antibody.

It is understood that "monoclonal antibody" means an antibody produced by a closely homologous population of 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 comprising two identical heavy chains joined by two disulfide 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 IMGT 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 Degrees (shown between parentheses and separated by dots, such as [8.8.13]) become decisive information. IMGT's unique numbering is used in 2D graphical representations 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 receptor and MHC structural data. Nucl. Acids. Res., 32, D208-D210 (2004) 1.

In a preferred embodiment, the antibody of the present invention, or an antigen-binding fragment or derivative thereof, comprises at least one complementarity determining region (CDR) having a selected from amino acid sequence identification number 1 to 6 The amino acid sequence in the constructed group, or at least one CDR, has a sequence which is at least 80%, preferably 85%, 90%, 95% identical to the sequence identification number 1 to 6 after optimal alignment. 98% identity.

In a more preferred embodiment, the antibody of the invention comprises i) a heavy chain comprising at least one of the following CDR-H1, CDR-H2 and CDR-H3 as defined by the IMGT numbering system, wherein the CDR-H1 comprises a sequence Sequence identification number 1, CDR-H2 comprises sequence sequence identification number 2 and CDR-H3 comprises sequence sequence identification number 3; and/or ii) a light chain comprising the following CDR-L1, CDR- as defined by the IMGT numbering system At least one of L2 and CDR-L3, wherein CDR-L1 comprises SEQ ID NO: 4, CDR-L2 comprises SEQ ID NO: 5 and CDR-L3 comprises SEQ ID NO: 6.

In another preferred embodiment, the antibody of the present invention, or an antigen-binding fragment or derivative thereof, comprises a heavy chain containing an immunological chain according to IMGT The following three CDRs are defined, namely CDR-H1, CDR-H2 and CDR-H3, wherein CDR-H1 comprises sequence identification number 1, CDR-H2 comprises sequence identification number 2 and CDR-H3 comprises sequence identification number 3.

According to another preferred embodiment, the antibody of the present invention, or an antigen-binding fragment or derivative thereof, comprises a light chain comprising the following three CDRs as defined by IMGT, respectively CDR-L1, CDR- L2 and CDR-L3, wherein CDR-L1 comprises SEQ ID NO: 4, CDR-L2 comprises SEQ ID NO: 5 and CDR-L3 comprises SEQ ID NO: 6.

In a preferred embodiment, the antibody of the present invention, or a functional fragment or derivative thereof, comprises a heavy chain comprising the following three CDRs, CDR-H1, CDR-H2 and CDR-H3, respectively. :- CDR-H1 comprises sequence sequence identification number 1, or one of at least 80%, preferably 85%, 90%, 95% and 98% identity with sequence sequence identification number 1 after optimal alignment Sequence; - CDR-H2 comprises sequence sequence identification number 2, or is at least 80%, preferably 85%, 90%, 95% and 98% identical to sequence sequence identification number 2 after optimal alignment. a sequence; and - CDR-H3 comprises sequence sequence number 3, or at least 80%, preferably 85%, 90%, 95% and 98% identical to sequence sequence number 3 after optimal alignment One of the sequences of sex.

In another preferred embodiment, the antibody of the present invention, or a functional fragment or derivative thereof, comprises a light chain comprising the following three CDRs, CDR-L1, CDR-L2 and CDR-L3, respectively ,among them: - CDR-L1 comprises sequence sequence identification number 4, or a sequence of at least 80%, preferably 85%, 90%, 95% and 98% identity with sequence sequence identification number 4 after optimal alignment ;- CDR-L2 comprises sequence sequence identification number 5, or one of at least 80%, preferably 85%, 90%, 95% and 98% identity with sequence sequence identification number 5 after optimal alignment Sequence; and - CDR-L3 comprises sequence sequence number 6 or, after optimal alignment, has at least 80%, preferably 85%, 90%, 95% and 98% identity with sequence sequence number 6. One of the sequences.

In still another embodiment, the antibody, or a functional fragment or derivative thereof, comprises: - a heavy chain comprising the following three CDRs as defined by IMGT: CDRs having sequence number 1 -H1, CDR-H2 having sequence number identification number 2 and CDR-H3 having sequence sequence number 3, or at least 80% of sequence identification number 1, 2 or 3 after optimal alignment, respectively a sequence of 85%, 90%, 95%, and 98% identity; and a light chain comprising the following three CDRs as defined by IMGT: CDR-L1 having sequence number 4 CDR-L2 having sequence identification number 5 and CDR-L3 having sequence number identification number 6, or at least 80%, preferably after sequence alignment with sequence number identification number 4, 5 or 6, respectively. A sequence of 85%, 90%, 95% and 98% identity.

For the purposes of the present invention, between two sequences of a nucleic acid or an amino acid "Percent identity" means the percentage of the same nucleotide or amino acid residue obtained after optimal alignment between the two sequences to be compared, which is only statistical and between 2 The differences between the sequences are randomly distributed along their lengths. 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 algorithm with Wunsch (1970) [J. Mol. Biol. 48: 443], by Pearson and Lipman similarity search method (1988) [Proc. Natl. Acad. Sci. USA 85: 2444] or by computer software using these algorithms (Wisconsin Genetics Software Package, Genetics Computer Group, 575 Science Dr., Madison, GA, GAP, BESTFIT, FASTA, and TFASTA, or by comparing software BLAST NR or BLAST P) to proceed.

The percent identity between two nucleic acid or amino acid sequences is determined by comparing the two optimally aligned sequences, wherein the nucleic acid to be compared or the amino acid sequence is optimal for the two sequences. Arrangements may be added or censored in comparison to the reference sequence. Percent identity is calculated by determining the number of positions where the amino acid or nucleotide residue is the same between the two sequences, dividing the number of identical positions by the total number of positions in the alignment window and Multiply the result 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.

With respect to amino acid sequences which exhibit at least 80%, preferably 85%, 90%, 95% and 98% identity with a reference amino acid sequence, preferred examples include reference sequences, certain modifications, in particular The at least one amino acid is censored, added or substituted, truncated or extended. 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.

As is known to those skilled in the art, the greatest variability (length and composition) between the six CDRs is present in the three heavy chain CDRs and, more particularly, the CDR-H3 of this heavy chain.

In a specific embodiment, the invention relates to a murine antibody, or a derivative thereof or a functional fragment thereof.

In another embodiment, the invention features an antibody, and an antigen-binding fragment or derivative thereof, comprising: a heavy chain comprising the following definitions according to "IMGT" of the CDRs 3 CDRs:- Sequence sequence identification number 1 or sequence with at least 80%, preferably 85%, 90%, 95% and 98% identity with sequence sequence identification number 1 after optimal alignment CDR-H1;- SEQ ID NO: 2 or one of at least 80%, preferably 85%, 90%, 95% and 98% identical to sequence identification number 2 after optimal alignment Sequence CDR-H2; and - sequence identification number 3 or at least 80%, preferably 85%, 90%, 95% and 98% identity with sequence sequence number 3 after optimal alignment a sequence of CDR-H3; and a light chain comprising the following three CDRs: - SEQ ID NO: 4 or at least 80% after sequence alignment with sequence number identification 4, preferably CDR-L1 of sequence of 85%, 90%, 95% and 98% identity; - sequence identification number 5 or at least 80% after sequence alignment with sequence number identification number 5 CDR-L2 of one sequence of 85%, 90%, 95% and 98% identity; and - sequence identification number 6 or at least 80% after sequence alignment with sequence number identification number 6 Compared Preferably, the CDR-L3 is one of 85%, 90%, 95% and 98% identical.

In a preferred embodiment, the invention relates to an antibody, or an antigen-binding fragment or derivative thereof, comprising i) a heavy chain comprising the following three CDRs, each having the CDR of SEQ ID NO: -H1, CDR-H2 with sequence sequence number 2 and sequence number identification number 3 CDR-H3, and ii) a light chain comprising the following three CDRs, respectively CDR-L1 having sequence number 4, CDR-L2 having sequence number 5, and sequence number 6 CDR-L3.

In another preferred embodiment of the present invention, the antibody, or an antigen-binding fragment or derivative thereof, is selected from the group consisting of: a) an antibody having a heavy chain, the heavy chain comprising the following three CDRs are CDR-H1 having sequence identification number 1, CDR-H2 having sequence identification number 2, and CDR-H3 having sequence identification number 3; and a light chain variability field containing sequence sequences Identification No. 8; b) an antibody having a heavy chain variability field comprising SEQ ID NO: 7, and a light chain comprising the following three CDRs, each having the CDR-L1 having SEQ ID NO: CDR-L2 of SEQ ID NO: 5 and CDR-L3 having SEQ ID NO: 6, and c) an antibody having a heavy chain variability field comprising SEQ ID NO: 7, and a sequence identification number of 8 Light chains can be mutated.

According to still another embodiment, the invention relates to one of antibody 427aB1, or an antigen-binding fragment or derivative thereof, comprising a heavy chain variant domain sequence comprising amino acid sequence identification number 7 or Is one of at least 80%, preferably 85%, 90%, 95%, and 98% identical in sequence to sequence sequence identification number 7 after optimal alignment; and/or because it contains a light chain that can mutate a domain sequence comprising an amino acid sequence sequence number of 8 or at least after sequence alignment with sequence number identification number 8 A sequence of 80%, preferably 85%, 90%, 95% and 98% identity.

In another preferred embodiment, the invention provides an antibody 427aB1, or an antigen-binding fragment or derivative thereof, the antibody 427aB1 comprising: a) 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; a heavy chain variable region having a sequence identification number of 7; and b) a light chain comprising: ● the following three CDRs, respectively CDR-L1 having sequence number 4 CDR-L2 having sequence number identification number 5 and CDR-L3 having sequence sequence identification number 6; and ● a light chain variability field having sequence sequence identification number 8.

The invention also relates to any compound derived from an antibody as described in the present invention.

An "antigen-binding derivative" or "derivative" of an antibody specifically means a binding protein comprising at least one of a peptide scaffold and the CDRs of the original antibody to maintain its ability to bind. Such derivative compounds are well known to those skilled in the art.

In particular, the antibody of the present invention, or a derivative thereof or antigenic junction thereof The fragment is characterized in that the derivative compound 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 initial The antibody's antibody binding site (paratope) recognizes all or part of the properties. In a preferred embodiment, the antigen binding protein 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.

Finally, as explained above, these peptide scaffolds can 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. Select one or more related CDRs pairs It will be apparent to those skilled in the art that those skilled in the art will then select suitable known techniques (BES et al., FEBS letters 508, 2001, 67-74).

The invention thus relates to an antibody, or a derivative thereof or a functional fragment thereof, wherein the peptide scaffold is selected from the group consisting of: a) phylogenetically conserved, b) robust in architecture, c) having a well-known 3-D molecular structure, d) having a small size, and/or e) containing regions that can be modified by censoring and/or insertion without modifying the stability properties.

According to a preferred embodiment, the antibody of the present invention, or a derivative thereof or a functional fragment thereof, is characterized in that the peptide scaffold is selected from the group consisting of: i) a scaffold produced by fibrin, preferably a web Protein type 3 domain 10, lipid carrier protein, anti-anionicin, protein Z produced by field B of S. aureus protein A, thioredoxin A or a protein with a repeating domain, For example: "Ankyrin Repetition" (Kohl et al, PNAS, 2003, vol. 100, No. 4, 1700-1705), "armadillo repeat", "rich leucine-rich" and "three The tetradecapeptide repeats "or iii" a neuronal NO synthase protein inhibitor (PIN).

Another aspect of the invention pertains to functional fragments of the antibodies described above.

More particularly, the invention targets an antibody, or a derivative thereof or a functional fragment thereof, wherein the functional fragment is selected from the group consisting of Fv, Fab, (Eab') ₂ , Fab', scFv, scFv- Fc and diabodies, or any fragment that has increased half-life, such as a PEGylated fragment.

Antigen-binding fragments (or "functional fragments" of the antibodies according to the invention: for the purposes of the present invention, these two terms are synonymous) are referred to herein, for example, fragments Fv, scFv (sc = single strand), Fab, F(ab') ₂ , Fab', scFv-Fc or bivalent antibody, or any fragment that has been chemically modified to increase half-life, such as the addition of polyalkylene glycols, for example Polyethylene glycol (PEGylated) (PEGylated fragments are called Fv-PEG, scFv-PEG, Fab-PEG, F(ab') ₂ -PEG or Fab'-PEG), or by In particular, the fragments according to the invention contain at least one of the characteristic CDRs of the invention, such that they retain, in particular, the binding activity and specificity of the antibody, even partial binding activity Specificity.

Preferably, the antigen-binding fragments will comprise or comprise a partial sequence of a variable heavy or light chain of the antibody from which the antibody is derived, the portion of the sequence being sufficient to retain the same binding specificity as the antibody producing the portion of the sequence Sexuality and sufficient affinity. Preferably, the affinity of the fragment is such that the affinity of the antibody producing the antibody of the fragment is at least equal to 1/100, more preferably at least 1/10.

The antigen-binding fragment comprises 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 Continuous amino acid.

According to the present invention, the antigen-binding fragment can be obtained from the antibody of the present invention by, for example, enzymatic digestion, including pepsin or papain, and/or by cleavage of a chemically reduced disulfide bridge. Such antibody fragments can also be obtained by recombinant genetic techniques or by automated peptide synthesizers.

For the sake of clarity, Table 2 below summarizes the various amino acid sequences corresponding to the antibody 427aB1 of the present invention.

According to another aspect, the present invention relates to a fusion tumor of a mouse capable of secreting a monoclonal antibody according to the present invention. In particular, the fusion tumor is a fusion tumor deposited with the CNCM of the Institut Pasteur, Paris, France on June 25, 2008, with the parameter I-4018. The fusion tumor was obtained by conjugated Balb/C-immunized mouse spleen cells and myeloma Sp 2/O-Ag 14 cells.

According to another preferred embodiment of the invention, the monoclonal antibody, herein referred to as 427aB1, or an antigen binding fragment or derivative thereof, is secreted by the fusion tumor.

A novel aspect of the invention relates to an isolated nucleic acid characterized in that it is selected from the group consisting of: a) a nucleic acid, DNA or RNA encoding an antibody or derivative thereof or a function thereof according to the invention a fragment comprising a DNA sequence comprising a sequence selected from the group consisting of sequence identification numbers 9 to 14, or at the optimal a sequence comprising at least 80%, preferably 85%, 90%, 95% and 98% identity to sequence sequence identification numbers 9 to 14; c) a nucleic acid comprising a DNA sequence comprising the sequence Sequence identification number 15 or 16, or a sequence having at least 80%, preferably 85%, 90%, 95%, and 98% identity with sequence sequence identification number 15 or 16 after optimal alignment; d) The RNA translated by the nucleic acids as defined in a), b) or c); e) the complementary nucleic acids of the nucleic acids as defined in a), b) and c); and f) at least 18 Nucleotide nucleic acid capable of being at least 80%, preferably 85%, under sequence conditions with sequence identification number 15 or 16 or after optimal alignment with sequence sequence number 15 or 16. Sequences of 90%, 95%, and 98% identity, or sequences complementary thereto, hybridize.

Table 3 below summarizes the various nucleotide sequences of the antibody 427aB1 of the present invention.

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 hybridized specifically to the reference sequence under highly stringent conditions. Complementary sequences, 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) major hybridization at a temperature dependent on the length of the probe (ie: 42 ° C for a probe >100 nucleotides in length) for 20 hours followed by 2X SSC + 2% SDS Wash twice at 20 ° 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).

The invention also relates to a vector comprising a polynucleotide of the invention.

In particular, the invention provides expression vectors for the selection and/or carrying of such a nucleotide sequence.

The vector of the present invention preferably includes an element that allows the nucleotide sequence to be expressed in a putative host cell. To express an antibody of the invention, a polynucleotide encoding the heavy and/or light chain of the antibody is inserted into an expression vector for genetical manipulation of the transcriptional and translational sequences. An "operably linked" sequence includes both expression control sequences linked to genes of interest and expression control sequences that control the genes of interest in a trans-acting manner or at a distance. As used herein, the term "expression control sequence" refers to a polynucleotide sequence which is necessary for the performance and processing of the coding sequences to which it is ligated. Expression control sequences include appropriate transcription initiation, termination, promoter sequences and enhancer sequences; efficient RNA processing messages such as cleavage and polyadenylation messages; sequences that stabilize cytoplasmic mRNA; Columns (i.e., Kozak consensus sequences); sequences that enhance protein stability; and sequences that enhance protein secretion when desired. The nature of these control sequences differs depending on the host organism; in prokaryotes, such control sequences generally include a promoter, a ribosome binding site, and a transcription termination sequence; within eukaryotes, such control sequences Promoters and transcription termination sequences are generally included. The term "control sequence" is intended to include, at a minimum, all components that must be present for performance and processing, and may also include additional components, the presence of which is advantageous, for example, a leader. Subsequences and fusion partner sequences.

As used herein, the term "vector" is intended to refer to a nucleic acid molecule capable of carrying another nucleic acid to the point where it has been ligated. One type of vector is a "plastid" which refers to a circular double stranded DNA loop that can join additional DNA segments. Another type of vector is a viral vector in which additional DNA segments can be ligated to the viral genome. Certain vectors are capable of spontaneous replication in a host cell into which they are introduced (e.g., a bacterial vector having a bacterial origin of replication and an episomal mammalian vector). Other vectors (e.g., non-episomal mammalian vectors), once introduced into a host cell, can be incorporated into the host cell genome and thereby replicated with the host genome.

Vectors according to the invention may also comprise sequences which permit stable maintenance of the vector in a host cell. It is readily understood that these sequences, referred to as origins of replication, will vary depending on the type of host cell. These various elements can be selected and optimized by the skilled artisan depending on the host cell used. For this purpose, the nucleotide sequences can be inserted into the selected sink The self-replicating vector in the host is either within the integrated vector of the selected host.

Such vectors are prepared by methods well known to those skilled in the art and the resulting pure strains can be introduced into a suitable form by standard methods such as lipofection, electroporation, heat shock or chemical methods. Within the host.

The invention is also directed to a host cell, which is transformed by a vector as described in the present invention or comprises a vector as described in the present invention.

The host cell may be selected from prokaryotic or eukaryotic systems, such as, for example, bacterial cells, but also yeast cells or animal cells, particularly mammalian cells. Insects or plant cells can also be used.

The present invention encompasses, in addition to humans, animals, or plants, which contain transformed cells in accordance with the present invention.

Another aspect of the invention relates to a method for producing an antibody, antigen-binding fragment or derivative according to the invention, characterized in that the method comprises the steps of: a) in a suitable culture medium under suitable culture conditions Host cell growth in accordance with the invention; and b) recovery of the antibody, or antigen-binding fragment or derivative thereof.

The resulting expression antibody can then be purified from the culture medium or cell extract. The soluble form of the antibody of the invention can be recovered from the culture suspension. It can then be purified by any method known in the art for purifying immunoglobulin molecules, for example, by chromatography (eg, ion exchange, affinity, specifically Fc protein A affinity, etc.) Etc.), centrifugation, differential solubility or It is standard technology by any other protein purification. A purification method suitable for those skilled in the art will be apparent.

The polypeptides of the invention may also be prepared by chemical synthesis. Such preparation methods are also within the scope of the invention. Several methods of chemical synthesis, such as solid phase techniques (see, in particular, Steward et al., 1984, Solid phase peptides synthesis, Pierce Chem. Company, Rockford, 111, 2nd ed.) or particle solid phase techniques, by condensation of fragments or It is known to those skilled in the art by conventional synthesis into a solution. Polypeptides obtained by chemical synthesis and capable of containing corresponding unnatural amino acids are also included in the present invention. The antibody obtainable by the method of the present invention, or an antigen-binding fragment or derivative thereof, is also included in the present invention.

As mentioned above, the antibodies of the invention, or antigen-binding fragments or derivatives thereof, are capable of binding to CXCR4, such as monomers and/or homodimers.

Surprisingly, the Applicant has demonstrated that the antibody, or antigen-binding fragment or derivative thereof, does not significantly bind to CXCR4, such as a heterodimer. Preferably, the antibody, or antigen-binding fragment or derivative thereof, does not significantly bind to a CXCR4/CXCR2 heterodimer.

Antibodies of the prior art, such as 515H7, are known to bind to CXCR4 such as monomers, homodimers or heterodimers (WO 2010/037831). In contrast, the antibodies of the present invention show strong specificity with respect to CXCR4 isoforms because of their ability to distinguish between homodimers and heterodimers. This property makes the antibodies of the invention a better tool for differential screening and, for example, a tool for characterizing tumors.

According to "CXCR4 such as monomer and / or homodimer", or "monomer / same The dimer CXCR4" (for the purposes of this application, these two terms are synonymous and intended to be used interchangeably), which is referred to herein as a monomeric form of CXCR4, ie, without any protein partner. Physical interaction of CXCR4, and/or homodimeric form of CXCR4, ie CXCR4, which is a complex of another molecule, CXCR4. The wording "CXCR4 such as monomeric and/or homodimer", or "monomer" /Homogeneous dimer CXCR4" is intended to specifically exclude CXCR4 heterodimers, ie dimers with any other protein partner other than CXCR4 itself. In particular, the words "such as monomeric and / or homodimerization The CXCR4" or "monomer/homogeneous dimer CXCR4" specifically excludes the CXCR4/CXCR2 heterodimer.

The invention thus relates to an antibody, or antigen-binding fragment or derivative thereof, as described above for use in in vitro or ex vivo diagnosis and/or prognosis and carcinogenicity associated with CXCR4 expression. obstacle.

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

In a preferred embodiment, the oncogenic disorder is comprised of a carcinogenic disorder associated with the performance of a CXCR4 monomer and/or a homodimer.

As used herein, "diagnosing" a disease refers to the identification or detection of pathological hyperproliferative carcinogenic disorders associated with the performance of the monomer/homomeric dimer CXCR4 or by the performance of the monomer/homomeric dimer CXCR4. The presence of a pathological hyperproliferative carcinogenic disorder in the media, monitoring the progression of the disease, and identifying or detecting the performance of the CXCR4 monomer and/or homodimer A method of correlating the cells or samples of the disorder.

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 the staining from the antibody of the present invention is negative from one body, then the "predicted" of the individual is more positive than if the sample was positive for the monomer/homogeneous dimer CXCR4. Good. The sample can be scored on the appropriate scale for the performance level of the monomer/homogeneous dimer CXCR4 as will be explained in more detail below.

The binding of an antibody of the invention, or a fragment or derivative thereof, to CXCR4, including CXCR4, CXCR4/CXCR4 homodimer, and CXCR4/CXCR2 heterodimer, can be tested in a manner known to those skilled in the art. . One such method, BRET analysis, is detailed in WO 2010/037831. The 515H7 antibody can be conveniently used as a positive control for this binding assay.

The antibody of the present invention, or an antigenic fragment or derivative thereof, may be a mouse antibody. However, since such antibodies will comprise the CDRs of the antibodies described herein, chimeric or anthropomorphic variants of such antibodies should be considered part of the scope of the invention.

Importantly, an antibody of the invention, or an antigenic fragment or derivative thereof, does not block the binding of 515H7 to CXCR4. It is therefore possible to use the antibodies of the invention during 515H7 antibody treatment without interfering with the treatment, so that the two antibodies do not compete for CXCR4. The antibodies of the invention are thus a key tool for monitoring, for example, imaging in vivo through a tumor, with the efficacy of the antibody 515H7 therapy.

In a more preferred embodiment of the invention, the antibody, or antigen-binding fragment or derivative thereof, does not have any in vivo anti-tumor activity.

This property is highly advantageous for diagnostic applications as it allows the use of antibodies to screen patients, or to track the progress of treatment with a formulation that does not have any impact or effect on the patient. This property makes antibody 427aB1 a preferred tool for screening patients to be treated because it does not have an impact on the patient. As will be recognized by those skilled in the art, the Applicant has provided a truly novel one by creating an antibody that recognizes CXCR4, such as both monomeric and protic dimers, without any in vivo antitumor activity. And inventive antibodies.

The antibody can be present as an immune complex or as a calibrated antibody to obtain a detectable and/or quantifiable 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 generally known to those skilled in the art. Such markers include, inter alia, enzymes, radioisotopes, and fluorescent, luminescent, and chromogenic materials, including colored particles such as colloidal gold or latex beads. 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 performance of a CXCR4 such as a monomeric and/or homodimer" is intended to include diseases and other disorders in which a high level of monomer/homomeric dimer is present. CXCR4 (abnormal) exists in an individual suffering from the disorder and has been shown to be the disorder The cause of pathophysiology or a factor contributing to the deterioration of the disorder is a factor that is suspected to be the pathophysiology of the disorder or a factor contributing to the deterioration of the disorder. Such disorders may be proven, for example, by CXCR4 on the surface of cells in an affected cell or tissue affected by an individual suffering from the disorder, preferably as monomeric and/or homodimeric. The level of CXCR4 is increased. An increase in the CXCR4 level of the monomeric and/or homodimer can be detected, for example, using the antibody 427aB1 of the present invention.

In some embodiments, when "increased performance" relates to CXCR4 such as monomeric and/or homodimer, the expression level of the protein or gene is mentioned, and statistically speaking relative to a control in terms of performance. Significant increase (as measured by RNA expression or protein expression).

In another embodiment, the invention relates to a method for detecting the presence of a tumor exhibiting a monomer/homomeric dimer CXCR4 in an individual, the method comprising the steps of: a) administering an antibody of the invention, or An antigen-binding fragment or derivative to the individual; and b) detecting the binding of the antibody, wherein the binding indicates the presence of the tumor.

A preferred aspect of the invention is a method for ex vivo detection of the presence of a tumor expressing a monomer/homomeric dimer CXCR4 in vivo, wherein the method comprises the steps of: (a) causing a The biological sample is contacted with an antibody of the invention, or an antigen binding fragment or derivative thereof, and (b) detects 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. Although the invention is included for analysis Any suitable means, but may specifically mention FACS, ELISA, Western blots and IHC.

In another embodiment, the invention relates to a method for detecting the location of a tumor expressing a monomer/homomeric dimer CXCR4 in vivo, comprising the steps of: a) administering an antibody according to the invention, or An antigen-binding fragment or derivative to the individual; and b) detecting the binding of the antibody, wherein the binding indicates the presence 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, the preferred means is IHC or FACS.

Another aspect of the invention relates to a method for determining, in vitro or ex vivo, the percentage of cells expressing CXCR4, such as monomeric and/or homodimer, from a tumor of a body, the method comprising the steps of: a) contacting a biological sample from the individual with an antibody, or antigen-binding fragment or derivative thereof, of the invention, and (b) quantifying CXCR4, such as a monomeric and/or homodimer, in the biological sample. The percentage of cells.

Still another aspect of the invention relates to a method for determining the performance level of a monomeric/homomeric dimer CXCR4 from a body in vitro or ex vivo, the method comprising the steps of: (a) A biological sample from the individual is contacted with an antibody of the invention, or an antigen binding fragment or derivative thereof, and (b) quantified the antibody, or an antigen binding fragment or derivative thereof, and The binding level of the monomer/homogeneous dimer CXCR4 in the biological sample.

The binding level of the antibody to the monomer/homomeric dimer CXCR4 expression level can be measured via immunohistochemistry (IHC) or FACS, preferably via IHC.

Once the amount of CXCR4, such as monomeric and/or homodimer, 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 Obtained by individuals with hyperproliferative carcinogenic disorders associated with the expression of CXCR4, such as monomeric and/or homodimers. If the level of CXCR4 of the monomer/homomeric dimer in the test sample is significantly increased, it can be concluded that there is an increased likelihood that the individual (which is derived from the individual) has or will develop the disorder.

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.

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 does not identify the earliest period of tumor progression.

The invention thus relates to a method for determining a score from 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 of the invention, or An antigen-binding fragment or derivative thereof, (b) quantifying the binding level of the antibody, or antigen-binding fragment or derivative thereof, to the monomer/homomeric dimer CXCR4 in the biological sample; and (c) The quantitative binding level of the antibody, or antigen-binding fragment or derivative thereof, is compared on an appropriate scale to score the tumor from the individual.

In a preferred embodiment, when the tissue sample is fumarin-fixed, formaldehyde-substituted, such as Glyco-fixx fixed (Glyco-fixx fixed-), paraffin-encapsulated, and/or frozen, for diagnostic purposes The antibody is capable of binding to the target receptor.

The monomer/homomeric dimer CXCR4 expression level is preferably measured via immunohistochemistry (IHC) or FACS, more preferably via IHC.

Any conventional hazard analysis method can be used to estimate the prognostic value of CXCR4, such as monomeric and/or homodimer. 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). Compared to other survival analyses, such as Life Tables or Kaplan-Meyer, Cox allows the inclusion of predictive variables (covariates) in the model. Using routine analytical methods, such as Cox, one can test the performance status of a single tumor/homogeneous dimer CXCR4 for a primary tumor for the time-to-onset of the disease (no disease) The survival time of the disease, or the time to become a metastatic disease, or the hypothesis of the correlation 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 "monomer/homomeric dimer CXCR4 state" may also be referred to as [monomer/homomeric dimer CXCR4(-)] or [monomer/homomeric dimer 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.

As used herein, "monomer/homomeric dimer CXCR4 state" refers to the classification of tumors into a monomer such as a monomer and/or a homodimer according to the determination of the performance level of the monomer/homomeric dimer CXCR4. CXCR4-positive [monomer/homomeric dimer CXCR4(+)] or CXCR4-negative [monomer/homogeneous dimer CXCR4(-)] class such as monomeric and/or homodimer. The performance of CXCR4 can be measured by any suitable method known to those skilled in the art, such as immunohistochemistry (IHC) or FACS.

In a specific example of the present invention, in order to ensure standardization, the sample can be scored on a different scale to quantify the performance level of the monomer/homomeric dimer CXCR4, and most of them are based on the intensity of the reaction product and the number of positive cells. Minute In comparison to (Payne et al, Predictive markers in breast cancer-the present, Histopathology 2008, 52, 82-90).

In a more preferred embodiment, the score includes the use of an appropriate scale based on two parameters, the intensity of staining and the percentage of positive cells.

As a first example, based on the teachings of the Quick Allred score for the IHC assessment of the eosin receptor and the progesterone receptor, the sample can be correlated with the reactivity level and staining for the monomer/homogeneous dimer CXCR4. The overall scale of the fraction of the combination of 0 to 8 of the cell ratio is scored (Harvey JM, Clarck GM, Osborne CK, Allred DC; J. Clin. Oncol. 1999; 17; 1474-1481). More specifically, the first criterion of reactivity strength is scored on a scale of 0 to 3, with 0 corresponding to "no reactivity" 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 "monomer/homogeneous dimer CXCR4 state" used in the term "tumor/homogeneous dimer CXCR4 state" as used in this specification refers to 0-2 or 3-8, respectively, corresponding to the Allred's scale. The performance level of CXCR4 such as monomer and/or homodimer.

Table 4 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 stronger in a ratio of more than 67-100%. The reactivity score was 8.

In another embodiment, a method for determining the state of a tumor from a body in vitro or ex vivo is provided, wherein the method comprises the steps of: (a) scoring from the individual according to the size of Orred And (b) determining that the state of the tumor having an Orred score of 3 to 8 is [monomer/homogeneous dimer CXCR4(+)]; or (c) determining that it has 0 to 2 The state of the tumor of the Reid score is [monomer/homogeneous dimer CXCR4(-)].

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

In a particular aspect of the invention, one of the Orred scores having 4 The tumor is [monomer/homogeneous dimer CXCR4(+)].

In a particular aspect of the invention, one of the tumors having an Orred score of 5 is [monomer/homogeneous dimer CXCR4(+)].

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

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

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

In another specific aspect of the invention, one of the tumors having an Orred fraction of 3 to 8 is [monomer/homogeneous dimer CXCR4(+)].

As a second example, the sample can be evaluated in terms of the level of performance of the monomer/homogeneous dimer CXCR4 in a slightly simpler scoring method based on the teachings of conventional scoring such as the IHC evaluation of the HER-2 receptor. In scoring, the scoring method combines the intensity of staining (preferably membrane staining) and the ratio of cells showing staining to a scale of combinations 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 "monomer/homogeneous dimer CXCR4 state" as used in this specification refers to a fraction of 0-1 ⁺ or 2 ⁺ -3 ⁺ in a simplified scale, respectively. The performance level of CXCR4 of monomeric and/or homodimer. Only the peripheral membranous reactivity of the invasive tumor should be considered and is generally similar to the appearance of a "small mesh". Under current guidelines, samples of CXCR4 such as monomeric and/or homodimers divided into boundaries (2 ⁺ or 3 ⁺ fractions) must be further evaluated.

For the sake of clarity, these parameters are summarized in Table 5 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. +.

Thus, another embodiment of the present invention provides an in vitro or ex vivo A method for determining the state of a tumor from a body, the method comprising the steps of: (a) scoring a tumor from the individual according to a simplified scale as described above; and (b) determining having 2+ or 3+ The state of the tumor is [monomer and/or homodimer CXCR4(+)]; or (c) the state of the tumor having a score of 0 or 1+ is determined as [monomer and/or homogenous Polymer CXCR4(-)].

In a particular aspect of the invention, the tumor having a fraction of 2+ is [monomer and/or homodimer CXCR4(+)].

In a particular aspect of the invention, one of the tumors having a fraction of 3+ is [monomer and/or homodimer CXCR4(+)].

In a particular aspect of the invention, the tumor having a fraction of 2+ or 3+ is [monomer and/or homodimer CXCR4(+)].

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 displayed 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 fraction can reflect a variety of factors, for example, the number of cells that detect CXCR4, such as monomeric and/or homodimer, and the strength of the signal (which can indicate the monomer/homogeneous dimer CXCR4) Performance level or cells with CXCR4), and so on. 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 vary depending on the technical limitations of the test and the significance of the organism associated with the detection of the polypeptide. 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 invention also relates to a method for determining whether a cancerous disorder is sensitive to treatment with a CXCR4 antagonist, the method comprising the steps of: (a) determining a body in vitro or ex vivo as described above The state of the tumor, and (b) if the state is [monomer/homogeneous dimer CXCR4(+)], it is determined that the oncogenic disorder is sensitive to treatment with a CXCR4 antagonist.

In a preferred embodiment, the CXCR4 antagonist is an anti-CXCR4 antibody, or a fragment or derivative thereof, as described above.

In another aspect, the invention relates to a pathological hyperproliferative carcinogenic disorder associated with the performance of a monomer/homogeneous dimer CXCR4 in vivo or in response to a monomer/homogeneous dimer CXCR4 A method for susceptibility to a pathological condition, the method comprising: (a) determining the presence or absence of the same monomer/homogeneous dimer CXCR4; Lacking, and (b) diagnosing a pathological condition or susceptibility to a pathological condition based on the presence or absence of the CXCR4, such as a monomeric and/or homodimer.

In the method of the present invention, an increase in the level of cells expressing the monomer/homogeneous dimer CXCR4 or the monomer/homogeneous dimer CXCR4 is generally indicated to indicate that a patient carries or is suspected of exhibiting a monomer. / Homogeneous dimer CXCR4 media barrier.

The invention thus provides a method of predicting the risk of a body developing a cancer comprising detecting the level of performance of a monomer/homomeric dimer CXCR4 in a biological sample, wherein high monomer/homogeneous dimerization The CXCR4 performance level 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 also relates to a method of assessing tumor aggressiveness. 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 monomer/homomeric dimer CXCR4 represented by the cells in a tumor sample of a body, and (b) determining at a later time Equal group obtained from the same individual The level of the monomer/homogeneous dimer CXCR4 represented in the woven sample, (c) the ratio between the performance level obtained in the state (a) and the performance level obtained in the step (b), wherein The performance ratio of the monomer/homomeric dimer CXCR4 within the tumor sample over time 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 less than 1 indicates invasiveness. In another embodiment, a ratio greater than or equal to 1 indicates non-aggressiveness.

Another aspect of the invention is the monitoring of the response of a CXCR4 exhibiting monomeric/homomeric dimer CXCR4 target, such as a monomeric and/or homodimer. This monitoring is very useful when the therapy triggers down-regulation and/or degradation of the monomer/homomeric dimer CXCR4.

In particular, monitoring monomer/homomeric dimer 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 the monomer/homogeneous dimer CXCR4 level indicates the evolution of the cancer associated with the monomer/homogeneous dimer CXCR4. Thus, by measuring the increase in the number of cells expressing the monomer/homogeneous dimer CXCR4 or the concentration of the monomer/homomeric dimer CXCR4 present in various tissues or cells, it may be possible to determine a Whether a specific therapeutic regimen that improves the malignancy associated with CXCR4 is effective.

Thus, the present invention is also directed to a method for determining the efficacy of a therapeutic regimen. Method, the therapeutic regimen is designed to alleviate the carcinogenic disorder associated with the monomer/homomeric dimer CXCR4 in an individual suffering from a carcinogenic disorder associated with the monomer/homogeneous dimer CXCR4, comprising The following steps: (a) determining the first performance level of the monomer/homogeneous dimer CXCR4 in the biological sample extracted from the individual at the first time point; (b) at the second later time point Determining a second performance level of the monomer/homogeneous dimer CXCR4 in the biological sample extracted from the individual; (c) determining the level obtained in (a) of the level obtained in (a) a ratio; and (d) determining that the efficacy of the therapeutic regimen is high when the ratio of step (c) is greater than 1, or (e) when the ratio of step (c) is less than or equal to 1, The efficacy of the therapeutic regimen was determined to be low.

In a preferred embodiment, the treatment of a carcinogenic disorder associated with the monomer/homomeric dimer CXCR4 in an individual designed to alleviate the oncogenic disorder associated with the monomer/homogeneous dimer CXCR4 The regimen comprises administering a CXCR4 inhibitor to the individual.

Another preferred embodiment of the present invention provides a method for selecting a cancer patient predicted to benefit from a therapeutically-treated amount of a CXCR4 inhibitor, or which does not benefit, the method comprising the steps of: (a) determining The performance level of the monomer/homomeric dimer CXCR4 in the patient; (b) the reference performance level of the monomer/homogeneous dimer CXCR4 from healthy individuals; (c) determining a ratio between the level obtained in the step (a) and the reference level obtained in the step (b); and (d) setting the ratio in the step (c) to be greater than 1, The patient is predicted to benefit from the dose of CXCR4 inhibitor; or (e) if the ratio in step (c) is equal to or less than 1, the patient is selected for non-predictive treatment The CXCR4 inhibitor benefits.

For the purposes of this specification, the phrase "CXCR4 inhibitor" or "CXCR4 inhibitor compound" refers to any compound or molecule that binds to CXCR4 and inhibits the binding of a CXCR4 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 Analog; 4F-benzimidyl-TN24003; KRH-1120; KRH-1636; KRH-2731; polyphemusin analogue; ALX40-4C; or WO 01/85196; WO 99/50461; These are described in WO 03/090512, each of which is incorporated herein by reference.

In a preferred embodiment, the inhibitor is a monoclonal antibody such as described in WO2008/060367 and WO2009/140124.

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 used to locate the tumor in vivo and to monitor its invasion. Attack. As such, the method has been used to monitor progression and/or response to treatment in a patient previously diagnosed as a cancer having a monomeric/homomeric dimeric CXCR4 vector.

In a first 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 For calibration, it is better to be radioactively calibrated. The agent can be administered in combination with a pharmaceutically effective carrier to a patient suffering from the cancer of the monomer/homomeric dimer CXCR4. The invention also contemplates the use of the agent for medical imaging in a patient suffering from cancer of the monomer/homogeneous dimer CXCR4. 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 a first embodiment, the imaging agent comprises a target moiety and an active moiety.

As used herein, the term "target portion" refers to a formulation that specifically recognizes and binds to the monomer/homomeric dimer CXCR4 on the cell surface. In a specific embodiment, the target moiety is an antibody or fragment or derivative thereof that specifically binds to the monomer/homomeric dimer CXCR4. In particular, the target moiety is an antibody or fragment or derivative thereof as described above. 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.

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 solid cancer sample of the patient 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. 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, it is swollen The tissue at the tumor site still contains non-tumor healthy tissue. The "cancer sample" should therefore be limited to the tumor tissue obtained 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 cancer or uterine cancer, salivary gland cancer, kidney cancer or kidney ( Renal cancer, prostate cancer, vaginal cancer, thyroid cancer, hepatic carcinoma, anal cancer, penile cancer, melanoma, surface-spreading black Neoplasms, malignant freckle melanoma, acral freckle melanoma, nodular melanoma, multiple myeloma and B-cell lymphoma (including low-grade/follicular non-Hodgkin's lymphoma (NHL); Small lymphocytes (SL) NHL; moderate malignancy/follicular NHL; moderate malignant diffuse NHL; high malignant immune blastocyst NHL; high malignant lymphocytic cytoplasmic NHL; high malignancy small non-fissure cell NHL; bully disease NHL; mantle cell lymphoma; AID S-associated lymphoma; Waldenstrom's Macroglobulinemia; chronic lymphocytic leukemia (CLL); Acute lymphoblastic leukemia (ALL); hairy cell leukemia; chronic myeloblastic leukemia (CML); acute myeloid blast leukemia (AML); and post-transplant lymphoproliferative disorder (PTLD), and maternal disease ( Phakomatoses) associated abnormal vascular proliferation, 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, such as monomeric and/or homodimer, is advantageously compared to the level of a control cell or sample or to the level of a control cell or sample, a control The level within a cell or sample is also referred to as a "reference level" or "reference performance level." As used herein, "control level" means comparable control cells that are generally free of disease or cancer. Independent baseline levels measured within. It may be from the same individual or 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 performance of the monomer/homomeric dimer CXCR4, which is used to assess monomer/homogeneity in a patient's cancer-containing cell sample. Test level for performance of dimer CXCR4.

For example, when the CXCR4 level of a monomer and/or a homodimer such as a monomer and/or a homodimer in a biological sample of a patient is higher, the reference level of the CXCR4 is higher. The cells are considered to have a high performance level of CXCR4 such as monomeric and/or homodimers, or an overexpression of CXCR4 such as monomeric and/or homodimers.

The reference level can be determined by a number of methods. The level of performance thus defines the number of cells in which the monomer/homomeric dimer CXCR4 is expressed. Optionally, the performance level can define the level of performance of the monomer/homomeric dimer CXCR4, independent of the number of cells expressing the monomer/homogeneous dimer CXCR4.

Thus, the reference level for each patient can be prescribed by the ratio of the monomer/homomeric dimer CXCR4 reference, 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 measured by measuring the level of CXCR4 in a non-carcinogenic cancer cell, such as a monomeric and/or homodimer, from the same tissue as the tissue of the neoplastic cell to be tested. To decide. Similarly, the "reference level" can be in a patient's twin cells such as monomeric and/or homodimeric CXCR4 associated with non-tumor cells such as monomeric and/or homodimers in the same patient. A ratio of the level of CXCR4. The "reference level" can also be the level of CXCR4 in vitro cultured cells such as monomers and/or homodimers. In vitro cultured cells can be manipulated to mimic tumor cells, or can be used to produce any level of performance. Other ways to manipulate it accurately determine the reference level. On the other hand, the "reference level" can be established based on the comparison set, for example, a group having no elevated monomer/homogeneous dimer CXCR4 and a level of elevated monomer/homogeneous dimer CXCR4. group. 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, such as monomeric and/or homodimers, in a patient population having the same cancer. This can be done, for example, by histogram analysis, in which the entire patient group of the same attribute group is presented by a graph, where the first axis represents the level of CXCR4 such as monomeric and/or homodimer. And the second axis represents the number of patients in the same attribute group, and the tumor cells present a hypothetical level Such as monomeric and / or homodimer CXCR4. Two or more independent groups of patients can be determined by identification of a subset of subgroups of the same attribute group having the same or similar levels of CXCR4, such as monomeric and/or homodimeric. The determination of the reference level can then be estimated based on optimally distinguishing one of the levels of such independent groups. A reference level may also indicate the level of two or more indicators, one of which is the monomer/homogeneous dimer CXCR4. Two or more indicators may, for example, be represented by a ratio of values of the respective indicator levels.

Likewise, an apparently healthy population will have a different 'normal' range than a population known to have a condition associated with the performance of CXCR4, such as monomeric and/or homodimeric. 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, such as a monomeric and/or homodimer, comprising a pharmaceutical composition comprising The monoclonal antibody of the invention, or an antigen-binding fragment or derivative thereof, the antibody or derivative thereof Fragments are calibrated and bound in vivo to a CXCR4 such as a monomeric and/or homodimer; and a pharmaceutically acceptable carrier. In another aspect, the invention provides a kit for use in the methods described above, the kit comprising an antibody of the invention.

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 the monomer/homomeric dimer CXCR4 in vitro, for example, in an ELISA or Western blot. The antibodies of the invention can be provided in a set for detecting and quantifying the monomer/homogeneous dimer CXCR4 in vitro, for example in ELISA or Western blots. The anti-system is calibrated with an enzyme that will include the matrix and the cofactors required for the enzyme (eg, a precursor that provides a detectable chromophore or matrix of fluorophores). In addition, other additives such as stabilizers, buffers (for example, a blocking buffer or dissolution buffer) and the like may be included. The set can include a socket that is distinguished 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 agents can be provided as a dry powder, usually freeze-dried, It includes excipients that provide a solution of the reagent at an appropriate concentration when dissolved.

In still another aspect of the invention, a monoclonal antibody, or antigen-binding fragment or derivative thereof, calibrated with a detectable moiety as described in detail herein is provided such that it can be packaged and used For example, within a 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 monomer/homomeric dimer CXCR4r, as well as cells having such antigens, for example, , other immunological techniques.

The invention also encompasses kits in which the antibody, or antigen-binding fragment or derivative thereof, is calibrated.

Kits were also provided for use as controls that were positive for purification of cells or immunoprecipitated monomer/homomeric dimer CXCR4. In terms of isolating and purifying the monomer/homomeric dimer CXCR4, the kit can include such antibodies, or antigen-binding fragments or derivatives thereof, as described herein, coupled to beads (eg, sepharose beads). Kits can be provided which include antibodies for detecting and quantifying the monomer/homomeric dimer CXCR4, either in vitro or ex vivo, such as in ELISA or Western blots. The kit includes a container and a logo or package insert on or with the container. The container holds a composition comprising at least one antibody, or a binding fragment or derivative thereof, of the invention. Can include additional containers, etc., for example, diluted Liquid and buffer, control antibody. 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 state of a monomer/homomeric dimer CXCR4 of a tumor by any method known to those skilled in the art. In a preferred embodiment, as will be explained in the Examples, the present invention relates to a kit for determining the state of a monomer/homomeric dimer CXCR4 of a tumor by the IHC method or FACS.

In a particular embodiment, the kit of the invention comprises at least one anti-monomer/homomeric dimeric CXCR4 antibody, or an antigen-binding fragment or derivative thereof, as described above, which antibody is preferably calibrated.

In a preferred embodiment, the kit for detecting the presence and/or location of a tumor exhibiting a monomer/homogeneous dimer CXCR4 in vivo further comprises a method for detecting the resistance An agent for the degree of binding between the CXCR4 antibody and the monomer/homogeneous dimer CXCR4.

The kit according to the invention may further comprise an agent for quantifying the binding level between the antibody, or an antigen-binding fragment or derivative thereof, and the monomer/homogeneous dimer CXCR4.

In still another embodiment, the kit according to the present invention further comprises a control sample and a negative control sample for counting the positive of the monomer/homomeric dimer CXCR4 expression level.

The kit may further comprise a plurality of antibodies that specifically recognize mouse antibodies. The multi-strain antibody is advantageously calibrated.

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 the CXCR4 monomer and the homodimer of the 427aB1 Mab identifying cell lysates; Figures 2A and 2B show both the 427aB1 Mab immunoprecipitated CXCR4 monomer and dimer; 3A, 3B, 3C 3D and 3E plots show that CXCR4 is recognized by the 427aB1 Mab at the cell membrane by FACS; Figures 4A and 4B are analyzed by FACS. Even though anti-CXCR4 515H7 therapeutic Mab is present, 427aB1 Mab binds to CXCR4 at the cell membrane; The figure shows that 427aB1 Mab does not adjust the CXCR4/CXCR2 heterodimer configuration; Figure 6 shows that 427aB1 Mab has no effect on MDA-MB-231 xenograft tumor growth patterns in Nod/Scid mice; Figure 7 shows IHC staining with m427aB1 and b) IHC staining with mIgG1 on RAMOS; Figure 8 shows a) staining with IHC using 427aB1 and b) IHC staining with mIgG1 on KARPAS299 xenograft tumors.

Example 1: Production of anti-CXCR4 427aB1 monoclonal antibody (Mab) (F50067-006 (5C) 427aB1 cl1B, CNCM No. I-4018)

To generate CXCR4 monoclonal antibodies, 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 immediately after the first immunization Immunization was performed subcutaneously (s.c.) with a complete Freund adjuvant (s.c.), followed by immunization 2 to 6 times with s.c with an antigen containing an incomplete Freund's adjuvant. The immune response was monitored by retroorbital bleeds. 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 purified [1-41] N-terminal peptide bound to BSA overnight at 4 ° C, and then used in PBS. The 0.5% gelatin solution was blocked with 250 μL/well. 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: 427aB1 Mab identifies both CXCR4 monomer and homodimer of cell lysate

NIH3T3-CXCR4 transfected cells, MDA-MB-231 (breast) and U937 (AML) cancer cell lines were washed twice with PBS. The following buffer was then used to dissolve 100.10 ⁶ cells/mL at 4 ° C for 30 min: 20 mM TrisHCl pH 8.5, 100 mM (NH ₄ ) ₂ SO ₄ , 10% glycerol, 1% CHAPSO and 1% protease inhibitor cocktail. Cell lysates were collected by centrifugation at 10000 g for 20 min at +4 °C and Western blotting using 427aB1 Mab as primary antibody. Figure 1 shows that Mab 427aB1 recognizes both CXCR4 monomer and homodimer in NIH3T3-CXCR4. The cancer cell lines MDA-MB-231 and U937 appear to be predominantly CXCR4 like the qualitative dimer.

Example 3: 427aB1 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 427aB1 Mab coupled to Sepharose 4B beads and the mixture was poured into a glass column with lysis buffer Wash it. The protein captured by the 427aB1 Mab was eluted and analyzed using Western blotting using 427aB1 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 2A shows Western blot analysis of the eluted concentrated fraction after immunoprecipitation with 427aB1 Mab coupled to Sepharose beads. The 427aB1 Mab was identified in three electrophoretic bands of 37-43, 75 and 150 kDa apparent molecular weight.

The eluted concentrated fraction was also resolved by SDS-PAGE and visualized by silver staining after immunoprecipitation using 427aB1 Mab coupled with Sepharose beads. The electrophoresis bands ( Fig. 2B ) at 37-43, 75 and 150 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 was identified in all electrophoresis bands: 6 CXCR4 peptides were identified in the 37-43-kDa electrophoresis band (electrophoresis band number 1) via the MASCOT ^TM search engine: 31-38 peptide EENANFNK, included in the N-terminus 135-146 peptide YLAIVHATNSQR and 135-148 peptide YLAIVHATNSQRPR, and 184-188 peptide YICDR, included in the extracellular loop 2; 272-282 peptide QGCEFENTVHK, included in the extracellular loop 3 And the 311-322 peptide TSAQHALTSVSR is included in the C-terminus.

The 75-kDa electrophoresis band (electrophoresis band number 2) contains five internal CXCR4 peptides: 31-38 peptide EENANFNK, which is contained within the N-terminal CXCR4; 135-146 peptide YLAIVHATNSQR, which is included in the intracellular loop 2 135-148 peptide YLAIVHATNSQRPR, included in the intracellular loop 2; 272-282 peptide QGCEFENTVHK, included in the intracellular loop 3 and 311-322 peptide TSAQHALTSVSR, included in the C-terminus . The peptides are identified by the MASCOT ^TM search engine

In the 150-kDa band electrophoresis (electrophoresis No. 3), identified two kinds of CXCR4 MASCOT ^TM peptide via a search engine. The 31-38 peptide EENANFNK, which is included in the N-terminus and the 311-322 peptide TSAQHALTSVSR, is included in the C-terminus.

The results obtained in this study clearly show that 427aB1 Mab can immunoprecipitate CXCR4. In addition, the 427aB1 Mab recognizes CXCR4 such as both monomer and homodimer.

Example 4: Analysis of 427aB1 by FACS Mab identifies CXCR4 located at the cell membrane

In this experiment, the 427aB1 Mab-specific CXCR4 line that binds to humans was evaluated by FACS analysis.

NIH3T3, NIH3T3-hCXCR4 transfected cells, MDA-MB-231, Hela, HT-29, and U937 cancer cell lines were incubated with 427aB1 monoclonal antibody (0-10 μg/mL). 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, which shows that 427aB1 binds to human CXCR4-NIH3T3 transfected cell lines ( Fig. 3A ) but does not bind to parental NIH3T3 cells (not shown). This Mab can also identify human cancer cell lines, such as HT-29 colon cancer cells ( Fig. 3B ), MDA-MB-231 breast cancer cells ( Fig. 3C ), and pre-U937 preclinded bone marrow cancer cells ( Fig. 3D ). As well as Hela cervical cancer cells ( Fig. 3E ), it is suggested that these cell lines naturally exhibit CXCR4 monomers and/or homodimers.

Example 5: Analysis by FACS Even in the presence of anti-CXCR4 515H7 therapeutic Mab, 427aB1 Mab binds to CXCR4 at the cell membrane

In this experiment, the binding of anti-CXCR4 Mabs 427aB1 and 515H7 to human CXCR4 line was examined by FACS analysis.

The transduced cell line of NIH3T3-hCXCR4 was first incubated with biotinylated 515H7 Mab (5 μg/ml) [identifying NIH3T3-CXCR4 cells ( Fig. 4A )], and then 427aB1 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 labeled streptavidin was then added to the cells and allowed to incubate at 4 °C for 20 min before another pair of washes. After the second wash, FACS analysis was performed. These combined results of the study are provided in Figure 4B and show that even the presence of 515H7 Mab, anti -CXCR4 Mab 427aB1 but still bind to human CXCR4-NIH3T3 cells transfected shape. In contrast, the presence of biotinylated 515H7 Mab inhibited the binding of uncalibrated 515H7 Mab to CXCR4 as expected.

Example 6: 427aB1 Mab by BRET analysis does not adjust CXCR4/CXCR2 heterodimer

This functional analysis allows for the assessment of the conformational changes induced by the binding of SDF-1 and/or 427aB1 Mab to the CXCR4 receptor at the level of 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 was performed, the HEK293 cell line was transiently transformed with the expression vector encoding the corresponding BRET partner: [CXCR4/Rluc+CXCR4-YFP]. On the next 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 obtained ratio of 530/485 nm, and when only the partner fused to Rluc is present in the analysis, the ratio 530 obtained under the same experimental conditions /485 nm to correct. For readability, the results are expressed as a percentage of the base signal.

SDF1 (300 nM) reduces the BRET signal by approximately 20% due to the proximity of the CXCR4 to the CXCR2 receptor. It is likely to indicate the formation of a CXCR4/CXCR2 heterodimer or a change in the configuration of an existing dimer ( Fig . 5 ). 427aB1 Mab does not modulate the SDF-1-induced conformational change of the CXCR2/CXCR4 heterodimer and does not adjust the spatial proximity of CXCR4/CXCR2 by itself. This indicates that 427aB1 Mab has no effect on the CXCR4/CXCR2 heterodimer configuration ( Fig . 5 ).

Example 7: Evaluation of activity of MDA-MB-231 xenograft tumor growth pattern in 427aB1 Mab in Nod/Scid mice

The goal of this experiment was to evaluate the ability of anti-CXCR4 Mab 427aB1 to inhibit MDB-MB-231 xenografts in Nod/Scid 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 Nod/Scid mice (Charles River, 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 6 mice with comparable tumor sizes. The mice were treated with ip at a loading dose of 2 mg/mouse of Mab 427aB1. Subsequently, the mice were injected twice a week at 1 mg/dose/mouse of Mab 427aB1. 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.

No mortality was observed throughout the treatment period. D40 at 1 mg/dose of 427aB1 Mab did not significantly inhibit tumor growth compared to the PBS control group (p=0.485). In addition, the mean tumor volume after 5 weeks of treatment was not reduced relative to PBS for Mab 427aB1 ( Fig . 6 ).

Example 8: 427aB1 Mab identifies CXCR4 (paraffin-encapsulated tumor IHC staining) present at the cell membrane

Sections were deparaffinized, rehydrated, and placed at 98 ° C pre-warmed at 98 ° C EDTA pH 8 for 5 minutes for heat-induced epitope replacement and for an additional 5 minutes at room temperature in warm EDTA buffer. . The slices were then rinsed with tap water for 5 minutes. After three washes in Tris buffered saline-0.05% Tween 20 (TBS-T) (Dako S3006), the endogenous peroxidase activity was blocked with a peroxidase blocking reagent (Dako K4007). 5 minutes. The sections were washed with TBS-T and in mouse anti-CXCR-4 mouse antibody (5 μg/ml, 427aB1, Pierre Fabre) or as an isotype control mouse IgG1/κ (5 μg/ml, 25 X0931, Dako) was incubated 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.

The 427aB1 Mab differentially stains cell membranes of a wide variety of tumor types. Figures 7 and 8 illustrate staining performed within two xenograft modes, The antitumor activity of the therapeutic anti-CXCR-4 hz515H7 antibody has been described: RAMOS and KARPAS299.

As shown in Figures 7 and 8, the detected xenotransplantation in KARPAS299 (Fig. 8) is lower than RAMOS (Fig. 7). This data correlates well with studies of CXCR-4 performance by flow cytometry. More specifically, RAMOS cells exhibited approximately 5 grades of CXCR-4 (antibody binding: 200,000 for RAMOS and 40,000 for KARPAS299) than KARPAS299 one. Membrane staining was weaker in KARPAS299 (Fig. 8), whereas membrane staining was significantly higher in RAMOS (Fig. 7).

Figure 1 shows both the CXCR4 monomer and the homodimer of the 427aB1 Mab identifying cell lysates; Figures 2A and 2B show both the 427aB1 Mab immunoprecipitated CXCR4 monomer and dimer; 3A, 3B, 3C 3D and 3E plots show that CXCR4 is recognized by the 427aB1 Mab at the cell membrane by FACS; Figures 4A and 4B are analyzed by FACS. Even though anti-CXCR4 515H7 therapeutic Mab is present, 427aB1 Mab binds to CXCR4 at the cell membrane; The figure shows that 427aB1 Mab does not adjust the CXCR4/CXCR2 heterodimer configuration; Figure 6 shows that 427aB1 Mab has no effect on MDA-MB-231 xenograft tumor growth patterns in Nod/Scid mice; Figure 7 shows For a) on the RAMOS, IHC dyed with m427aB1 Color and b) IHC staining with mIgG1; Figure 8 shows a) IHC staining with 427aB1 and b) IHC staining with mIgG1 on KARPAS299 xenograft tumors.

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Claims (36)

An antibody, or an antigen-binding fragment or derivative thereof, comprising i) a heavy chain comprising the following three complementarity determining regions (CDRs), respectively CDR-H1 having sequence number 1 and having a sequence identification number CDR-H2 of 2 and CDR-H3 having SEQ ID NO: 3; and ii) a light chain comprising the following three CDRs, respectively CDR-L1 having sequence identification number 4, having sequence identification number 5 CDR-L2 and CDR-L3 having sequence number identification number 6. An antibody, or an antigen-binding fragment or derivative thereof, according to claim 1, wherein the anti-system is selected from the group consisting of: a. an antibody having a heavy chain comprising the following three CDRs, each having a sequence CDR-H1 of sequence identification number 1, CDR-H2 having sequence sequence identification number 2, and CDR-H3 having sequence sequence identification number 3; and a light chain variability field, comprising sequence 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, respectively CDR-L1 having sequence number identification number 4, and CDR having sequence sequence identification number 5 -L2 and CDR-L3 having sequence number identification number 6; or c. an antibody having a heavy chain variability field comprising sequence sequence identification number 7; and a light chain variability field comprising sequence sequence identification number 8. An antibody, or antigen-binding fragment thereof, as claimed in claim 1 or 2 a derivative, wherein the antibody comprises: a) a heavy chain comprising: • the following three CDRs, respectively CDR-H1 having sequence number 1 and CDR-H2 having sequence number 2 and having sequences CDR-H3 of sequence identification number 3; and a light chain variability field, comprising sequence sequence identification number 8; and a heavy chain variability field, the heavy chain variability field having sequence sequence identification number 7; and b) a light chain comprising: ● the following three CDRs, respectively CDR-L1 having sequence identification number 4, CDR-L2 having sequence identification number 5, and CDR-L3 having sequence number identification number 6; A light chain variability field having a sequence sequence identification number of 8. The antibody, or an antigen-binding fragment or derivative thereof, according to any one of claims 1 to 3, wherein the antibody is capable of binding to a chemotactic receptor 4 such as a monomer and/or a homodimer (CXCR4) ). An antibody, or an antigen-binding fragment or derivative thereof, according to any one of claims 1 to 4, for use in in vitro or ex vivo diagnosis and carcinogenicity associated with CXCR4 expression The disorder either determines the prognosis of carcinogenic disorders associated with the performance of CXCR4 in vitro or ex vivo. An antibody, or an antigen-binding fragment or derivative thereof, according to claim 5, characterized in that the carcinogenic disorder is constituted by a carcinogenic disorder associated with the expression of CXCR4 such as a monomer and/or a homodimer. . The antibody, or an antigen-binding fragment or derivative thereof, according to any one of claims 1 to 6, wherein the antibody is a mouse antibody. The antibody, or antigen-binding fragment or derivative thereof, according to any one of claims 1 to 7, wherein the antibody does not block the binding of 515H7 to CXCR4. The antibody, or antigen-binding fragment or derivative thereof, according to any one of claims 1 to 8, wherein the antibody does not have any in vivo antitumor activity. A mouse fusion tumor which is capable of secreting an antibody, or an antigen-binding fragment or derivative thereof, according to any one of claims 1 to 9. The mouse fusion tumor, as claimed in claim 10, was deposited at the CNCM of the Institut Pasteur, Paris, France on June 25, 2008 under the number I-4018. An isolated nucleic acid, characterized in that it is selected from the group consisting of: a) a nucleic acid, DNA or RNA, encoding an antibody or a derivative compound or function thereof according to any one of claims 1 to 8. a fragment comprising: a nucleic acid comprising a sequence selected from the group consisting of sequence identification numbers 9 to 14, or after optimal alignment with sequence identification number 9 to 14 having a sequence of at least 80%, preferably 85%, 90%, 95% and 98% identity; c) a nucleic acid comprising a DNA sequence comprising sequence sequence number 15 or 16, or at least 80%, preferably 85%, 90 after sequence alignment with the sequence identification number 15 or 16. Sequences of %, 95%, and 98% identity; d) RNA translated by the nucleic acids as defined in a), b) or c); e) as in a), b) and c) a complementary nucleic acid of the nucleic acid as defined; and f) a nucleic acid of at least 18 nucleotides which is capable of recognition under sequence conditions 15 or 16 under optimal conditions or after optimal alignment and sequence identification No. 15 or 16 has a sequence of at least 80%, preferably 85%, 90%, 95% and 98% identity, or a complementary sequence thereof. A vector comprising the nucleic acid according to item 12 of the patent application. A host cell which is transformed by a carrier as claimed in claim 13 or which comprises a carrier as claimed in claim 13. A method for producing an antibody, or an antigen-binding fragment or derivative thereof, characterized in that the method comprises the steps of: a) cultivating a host cell as in claim 14 in a medium and under suitable culture conditions; b) recovering the antibody, or antigen-binding fragment or derivative thereof, from the culture medium or the cultured cells. A method for detecting the presence of a tumor exhibiting a monomer/homomeric dimer CXCR4 in vitro or ex vivo, wherein the method comprises the steps of: (a) contacting a biological sample from the individual as in the patent application form The antibody, or an antigen-binding fragment or derivative thereof, according to any one of items 1 to 9 or an antibody obtained by the method of claim 15 or an antigen-binding fragment or derivative thereof An antibody produced by the fusion tumor of claim 10 or 11 or an antigen-binding fragment or derivative thereof; and (b) detecting binding of the antibody, or antigen-binding fragment or derivative thereof, to the biological sample . A method for determining, in vitro or ex vivo, the percentage of cells expressing CXCR4, such as monomeric and/or homodimer, from a tumor of a body, the method comprising the steps of: (a) bringing the same contact from the individual An antibody, or an antigen-binding fragment or derivative thereof, according to any one of claims 1 to 9 or an antibody obtained by the method of claim 15 or an antigen-binding fragment or derivative thereof Or an antibody produced by the fusion tumor of claim 10 or 11 or an antigen-binding fragment or derivative thereof; and (b) quantifying the expression of the monomer and/or homodimerization in the sample. Percentage of cells of CXCR4. A method for determining the performance level of a monomer/homomeric dimer CXCR4, such as a monomeric and/or homodimer, from a tumor in a body, ex vivo or ex vivo, comprising the steps of: (a) A biological sample from the individual is contacted with an antibody, or an antigen-binding fragment or derivative thereof, according to any one of claims 1 to 9 or an anti-obtainment obtained by the method of claim 15 Or an antigen-binding fragment or derivative thereof, or an antibody produced by the fusion tumor of claim 10 or 11 or an antigen-binding fragment or derivative thereof; and (b) quantifying the antibody, or The binding level of the antigen-binding fragment or derivative to the monomer/homogeneous dimer CXCR4 in the biological sample. The method of claim 18, wherein the binding level of the antibody, or antigen-binding fragment or derivative thereof, to the monomer/homogeneous dimer CXCR4 is measured by immunohistochemistry (IHC) or FACS. Jia is measured by IHC. A method for determining a score from a tumor of a body in vitro or ex vivo, the method comprising the steps of: (a) contacting a biological sample from the individual as in any one of claims 1 to 9 of the patent application; An antibody, or an antigen-binding fragment or derivative thereof, or an antibody obtained by the method of claim 15 or an antigen-binding fragment or derivative thereof, or by the application of Patent No. 10 or 11 An antibody produced by the fusion tumor, or an antigen-binding fragment or derivative thereof; (b) quantifying the antibody, or an antigen-binding fragment or derivative thereof, and a monomer and/or a homodimer in the biological sample Binding level of the monomer/homomeric dimer CXCR4; and (c) scoring the quantitative binding level of the antibody, or antigen-binding fragment or derivative thereof, on an appropriate scale to score the individual from the individual Tumor. For example, the method of claim 20, wherein the appropriate scale is rooted According to two parameters, the two parameters are the intensity of staining and the percentage of positive cells. The method of claim 20 or 21, wherein the appropriate scale is a scale of 0 to 8, wherein the "non-reactivity" score is 0, and is strong in the ratio of "67-100% reaction ratio" The reactivity score was 8. 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 20, 21 or 22 to score from the individual (a subject) of the tumor; and b) determining the state of the tumor having a score of 3 to 8 as [monomer/homogeneous dimer CXCR4(+)]; or (c) determining having a score of 0 to 2 The state of the tumor is [monomer/homogeneous dimer CXCR4(-)]. The method of claim 20 or 21, wherein the appropriate scale is 0 to 3 ⁺ scale, wherein the tumor cells have no membrane-like reactivity score of 0, and are stronger among more than 10% of tumor cells. The complete reactivity score 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 20, 21 or 24 to score from the individual a tumor of (a subject); and (b) determining the state of the tumor having a score of 2 ⁺ or 3 ⁺ as [monomer/homogeneous dimer CXCR4(+)]; or (c) determining having 0 or 1 The state of the tumor with a fraction of ⁺ is [monomer/homogeneous dimer CXCR4(-)]. A method for determining whether a cancerous disorder is sensitized to treatment with a CXCR4 antagonist, the method comprising the steps of: (a) determining the in vitro or ex vivo assay as in the method of claim 23 or 25 The state of the individual's tumor, and (b) if the state is [monomer/homogeneous dimer CXCR4(+)], it is determined that the oncogenic disorder is sensitive to treatment with a CXCR4 antagonist. A method for determining the efficacy of a therapeutic regimen in vitro or ex vivo, which is designed to alleviate the monomer in an individual suffering from a carcinogenic disorder associated with the monomer/homogeneous dimer CXCR4 /Homogeneous dimer CXCR4-related carcinogenic disorder, the method comprising the steps of: (a) determining the single in the biological sample extracted from the individual at the first time point, as in the method of claim 18 or 19 The first performance level of the bulk/homogeneous dimer CXCR4; (b) at the second, later time point, as in the method of claim 18 or 19, to determine the biological sample extracted from the individual a second performance level of the monomer/homogeneous dimer CXCR4; (c) determining the ratio of the level obtained in (a) to the level obtained in (b); and (d) at step (c) When the ratio is higher than 1, it is determined that the efficacy of the therapeutic regimen is high; or (e) when the ratio of the step (c) is lower than or equal to 1, the efficacy of the therapeutic regimen is determined to be low. . The method of claim 27, wherein the monomer/homogeneous dimer CXCR4-associated carcinogenic disorder is designed to alleviate an individual suffering from a carcinogenic disorder associated with the monomer/homogeneous dimer CXCR4 The therapeutic regimen comprises administering a CXCR4 inhibitor to the individual. A method of selecting, in vitro or ex vivo, a cancer patient predicted to benefit or not benefit from administering a therapeutic amount of a CXCR4 inhibitor, the method comprising the steps of: (a) as claimed in claim 18 or 19 Method for determining the level of performance of the monomer/homomeric dimer CXCR4 in the patient; (b) determining the monomer/homomeric dimer from a healthy individual as claimed in claim 18 or 19 a reference performance level of CXCR4; and (c) determining a ratio between the level obtained in step (a) and the level obtained in step (b); and (d) setting the step in step (c) If the ratio is greater than 1, the patient is selected to benefit from the CXCR4 inhibitor that is predicted to be administered from the drug; or (e) if the ratio in step (c) is less than or equal to 1, the patient is selected Non-predicted will benefit from the administration of a therapeutic amount of CXCR4 inhibitor. The method of claim 28, wherein the CXCR4 inhibitor is monoclonal antibody 515H7. A kit comprising at least one antibody, or an antigen-binding fragment or derivative thereof, according to any one of claims 1 to 9 or an antibody obtained by the method of claim 15 of the patent application, Antigen knot A fragment or derivative or an antibody produced by the fusion tumor of claim 10 or 11 or an antigen-binding fragment or derivative thereof. A kit according to claim 31, characterized in that the antibody, or antigen-binding fragment or derivative thereof, is calibrated. A kit according to claim 31 or 32, which further comprises an agent for detecting the degree of binding between the antibody, or an antigen-binding fragment or derivative thereof, and the monomer/homogeneous dimer CXCR4. The kit of any one of claims 31 to 33, further comprising for quantifying the binding level between the antibody, or an antigen-binding fragment or derivative thereof, and the monomer/homogeneous dimer CXCR4 Reagents. The kit of any one of claims 31 to 34, further comprising a control sample and a negative control sample for counting the positive of the monomer/homomeric dimer CXCR4 expression level. For example, the kit of claim 35, further comprising a plurality of antibodies that specifically recognize mouse antibodies, the plurality of antibodies preferably being calibrated.