NZ618655B2 - Anti-cxcr4 antibody with effector functions and its use for the treatment of cancer. - Google Patents

Abstract

Disclosed is the use of a humanised antibody binding to CXCR4, or a CH2-containing binding fragment thereof, said humanised antibody comprising a heavy chain variable domain selected from the sequences SEQ ID No.7 to 10 and a light chain variable domain selected from the sequences SEQ ID No. 11 to 17; for preparing a medicament for treating cancer by killing a CXCR4 expressing cancer cell by induction of at least one effector function, in the presence of effector cells or complement components, wherein around 92% of the carbohydrate chains borne by said antibody comprise a fucose residue, wherein the sequences are as defined in the complete specification. Further disclosed is a method for screening such antibodies. 7; for preparing a medicament for treating cancer by killing a CXCR4 expressing cancer cell by induction of at least one effector function, in the presence of effector cells or complement components, wherein around 92% of the carbohydrate chains borne by said antibody comprise a fucose residue, wherein the sequences are as defined in the complete specification. Further disclosed is a method for screening such antibodies.

Description

ANTI-CXCR4 ANTIBODY WITH EFFECTOR FUNCTIONS AND ITS USE FOR THE TREATMENT OF CANCER.

The present application relates to a method of treating cancer by administering an anti-CXCR4 monoclonal antibody capable of inducing effector on(s).

Chemokines are small, secreted peptides that control the migration of leukocytes along a al gradient of ligand, known as chemokine gradient, especially during immune reactions (Zlotnick A . et al., 2000). They are divided into two major subfamilies, CC and CXC, based on the position of their NH2-terminal cysteine residues, and bind to G protein coupled receptors, whose two major sub families are designated CCR and CXCR. More than 50 human chemokines and 18 chemokine receptors have been discovered so far.

Many cancers have a complex chemokine network that influences the immunecell infiltration of tumor, as well as tumor cell growth, survival, migration and angiogenesis. Immune cells, elial cells and tumor cells themselves express chemokine receptors and can respond to chemokine gradients. s of human cancer biopsy s and mouse cancer models show that cancer cell chemokine-receptor expression is associated with increase metastatic ty. Malignant cells from different cancer types have different profiles of chemokine-receptor expression, but Chemokine receptor 4 (CXCR4) is most commonly found. Cells from at least 23 different types of human cancers of epithelial, mesenchymal and haematopoietic origin s CXCR4 receptor (Balkwill F. et al., 2004). ine receptor 4 (also known as fusin, CD 184, LESTR or HUMSTR) exists as two isoforms comprising 352 or 360 amino acids. Residue Asnl l i s glycosylated, residue Tyr21 is modified by the addition of a sulfate group and Cys 109 and 186 are bond with a disulfide bridge on the extracellular part of the receptor z J . et al., 2004).

This receptor is expressed by different kind of normal tissues, naive, nonmemory T-cells, regulatory T cells, B-cells, neutrophils, endothelial cells, primary monocytes, dendritic cells, Natural Killer (NK) cells, CD34+ hematopoietic stem cells and at a low level in heart, colon, liver, kidneys and brain. CXCR4 plays a key role in leukocytes cking, B cell poiesis and myelopoiesis.

CXCR4 receptor is over-expressed in a large number of cancers including but not limited to ma, leukemia, multiple myeloma, colon (Ottaiano A . et al., 2004), breast (Kato M . et al., 2003), prostate (Sun Y.X. et al., 2003), lung [small-cell- and non- small-cell- carcinoma (Phillips R.J. et al., 2003)], ovary (Scotton C.J. et al., 2002), pancreas (Koshiba T. et al., 2000), kidneys, brain (Barbero S et al., 2002), glioblastoma and lymphomas.

The unique ligand of CXCR4 receptor described so far is the Stromal-cell- Derived Factor- 1 (SDF-1) or CXCL12. SDF-1 is secreted in large amount in lymph nodes, bone marrow, liver, lungs and to a less extent by kidneys, brain and skin.

CXCR4 i s also recognized by an antagonistic ine, the viral macrophage inflammatory protein II (vMIP-II) encoded by human herpesvirus type III.

CXCR4/SDF-1 axis plays a key role in cancer and is implicated directly in migration, invasion leading to metastases. Indeed, cancer cells s CXCR4 receptor, they migrate and enter the ic circulation. Then cancer cells are arrested in vascular beds in organs that produce high levels of SDF-1 where they proliferate, induce angiogenesis and form metastatic tumors (Murphy PM., 2001). This axis is also involved in cell proliferation via activation of Extracellular-signal-Regulated Kinase (ERK) pathway ro S . et al, 2003) and angiogenesis nani P., 2004).

Indeed, CXCR4 receptor and its ligand SDF-1 clearly promote angiogenesis by stimulating VEGF-A expression which in turns increases expression of CXCR4/SDF-1 (Bachelder R.E. et al., 2002). It is also known that tumor associated macrophages (TAM) lated in hypoxic areas of tumors and are stimulated to co-operate with tumor cells and promote angiogenesis. It was observed that a up-regulated selectively expression of CXCR4 in various cell types including TAM (Mantovani A . et al., 2004). It has been recently demonstrated that CXCR4/SDF-1 axis regulates the trafficking/homing of CXCR4+ hematopoietic rogenitor cells (HSC) and could play a role in neovascularization. Evidence indicates that besides HSC, functional CXCR4 is also expressed on stem cells from other tissues (tissue-committed stem cells = TCSCs) so SDF-1 may play a pivotal role in chemottracting CXCR4+ TCSCs necessary for organ/tissue regeneration but these TCSC may also be a ar origin of cancer development (cancer stem cells theory). A stem cell origin of cancer was demonstrated for human leukemia and recently for several solid tumors such as brain and breast. There are several es of CXCR4+ tumors that may derive from the normal CXCR4+ tissue/organ-specific stem cells such as leukemias, brain tumors, small cell lung cancer, breast cancer, hepatoblastoma, ovarian and cervical cancers (Kucia M . et al., 2005).

Targeting cancer metastases by interfering with CXCR4 receptor was demonstrated in vivo using a monoclonal antibody directed against CXCR4 receptor (Muller A . et al., 2001). y, it was shown that a monoclonal antibody directed t CXCR4 receptor (Mab 173 R&D Systems) decreased significantly the number of lymph node metastases in an orthotopic breast cancer model (MDA-MB231) in SCID mice. Another study (Phillips R.J et al., 2003) also showed the critical role of SDF- 1/CXCR4 axis in metastases in an orthotopic lung carcinoma model (A549) using polyclonal antibodies t SDF-1 but in this study there was no effect r on tumor growth nor on angiogenesis. Several other studies described also the inhibition of either metastasis in vivo using siRNAs duplexes of CXCR4 (Liang Z . et al., 2005) biostable CXCR4 peptide nists (Tamamura H . et al., 2003) or tumor growth in vivo using small molecule antagonist of CXCR4 like AMD 3100 (Rubin J.B. et al., 2003; De Falco V. et al., 2007) or Mab (patent WO2004/059285 A2). Thus, CXCR4 is a ted therapeutic target for cancers.

Murine monoclonal antibodies capable of direct interaction with CXCR4, and thus of inhibiting CXCR4 activation, have also been described. Such an inhibition can occur by interfering with: i) the specific g at cellular membranes of the ligand SDF-1 to the receptor CXCR4, ii) the specific binding at cellular membranes of the GTPyS to the receptor CXCR4, iii) the CXCR4-mediated modulation of cAMP production, and iv) the CXCR4 receptor-mediated mobilization of intracellular m stores modulation (see 1).

However, none of these antibodies or siRNA lead to the killing of the CXCR4- expressing cancerous cells. There is therefore still a risk of resumption of the cancer, should the CXCR4-targeted treatment be stopped. There is thus a need for agents which directly target CXCR4-expressing cells, and which are capable of killing said CXCR4- sing cells.

The present invention relates to a novel property which has never been fied in relation with an antibody targeting CXCR4. indeed, the: inventors have f0und than human or humanized antibodies directed against CXC R4 are capable of inducing effector functions againsr a CXCR4-expressing cell, thus leading to cytotoxic effects t the said coils.

More particuiariy‘ the invention s to a method for the induction of effector function(s) against a CXCRA cxprcssing canccr cell.

As described in the prior art, treatment of mctasiatic CKCR4«EXDI‘635ing tumors involved inhibition of migration, invasion, proliferation or angiogenesis, but no direct hiliing of the CXCRébcxprcssing cclis. in striking st, the present invention relates to the induction of cytotoxic effects which tend to the death of the. CXCR—‘i'exprcssing target cell In particular, theinvention provides a human or zed monoclonai antibody which is capable of inducing, one or more ef’l‘ccror functioni’s) against a (Tl-(CR4 (Expressing cancer cell, thus ing ihc killing ofthc said cell Thus the invention provides a method of ent ofcnncer through induction of one or more effector l‘unctimns) against a CXCR4 expressing, cancer cell by a human or humanized monoclonal antibodyr Reference to any prior art in the specification is not, and should not be taken as, an acknowledgment or any form of suggestion that this prior art forms part of the common l knowledge in New anland or any other jurisdiction. in a lirst aspect, the present irwention relates to a method of killing a CXCR4— expressing cancer cell with a human or humanized antibody binding to L or a CHE—containing binding fragment: thcrc-ofi said human or humanized dy sing a heavy chain le domain comprising CDR regions CDR—H l, CUR—HZ and CUR—HE comprising scqucnccs SEQ ID Nos. l, ‘2 and 3‘ rcspcctiveiy; and a light chain variahlc domain comprising CDR regions CDRJJ’, CDR-LZ and CUR—L3 comprising sequences SEQ ID N034, 5 and 6:, respectively; wherein said method comprises the step of inducing at least once effector function of" the said human or zed antibody in the presence ol'el‘fector salts or complement components. in another aspect the invention relates to the use of a human or humanized antibody binding to CXCR—‘i, or a CHZ—containing binding fragment thereof; wherein said human or zed antibody comprises a heavy chain variabie domain comprising COR regions CDR-H E, COR—HZ and CDR~H3 comprising sequences SEQ H) Nos. 3, 2 and 3, tively; and a light chain variable domain comprising CDR regions (DR-Ll. (TOR-L2 and CDR—L3 compn‘ sing sequences SEQ ID N054, 5 and 6, respectively; for preparing a composition for kiliing a CXCRfil expressing cancer cell by 1001180216 induction of at least one or function, in the presence of effector cells or complement components.

In still another aspect, the invention also relates to a human or humanized antibody binding to CXCR4, or a CH2-containing binding nt thereof; said human or humanized antibody comprising a heavy chain variable domain comprising CDR regions CDR-H1, CDR-H2 and CDR-H3 comprising sequences SEQ ID Nos. 1, 2 and 3, respectively; and a light chain variable domain comprising CDR regions CDR-L1, CDR-L2 and CDR-L3 comprising sequences SEQ ID Nos. 4, 5 and 6, respectively; for use in g a CXCR4 expressing cancer cell by induction of at least one effector function, in the presence of effector cells or complement components.

More specifically, the present invention relates to a method of treating cancer by killing a CXCR4-expressing cancer cell with a human or humanized dy binding to CXCR4, or a CH2-containing binding fragment thereof; said human or humanized antibody comprising a heavy chain variable domain comprising CDR regions CDR-H1, CDR-H2 and CDR-H3 comprising sequences SEQ ID Nos. 1, 2 and 3, respectively; and a light chain variable domain comprising CDR s CDR-L1, CDR-L2 and CDR-L3 comprising sequences SEQ ID Nos.4, 5 and 6, respectively; wherein said method comprises the step of inducing at least one effector function of the said human or humanized antibody in the presence of effector cells or complement components.

In another aspect, the invention relates to the use of a human or humanized antibody binding to CXCR4, or a CH2-containing binding fragment thereof; n said human or humanized antibody comprises a heavy chain variable domain comprising CDR regions CDR-H1, CDR-H2 and CDR-H3 comprising sequences SEQ ID Nos. 1, 2 and 3, respectively; and a light chain variable domain comprising CDR regions CDR-L1, CDR-L2 and CDR-L3 comprising sequences SEQ ID Nos.4, 5 and 6, respectively; for preparing a composition for ng cancer by killing a CXCR4 expressing cancer cell by induction of at least one or function, in the presence of effector cells or complement ents.

In still another aspect, the invention also relates to a human or humanized dy binding to CXCR4, or a CH2-containing binding fragment thereof; said human or humanized antibody comprising a heavy chain variable domain comprising CDR s , CDR-H2 and CDR-H3 comprising sequences SEQ ID Nos. 1, 2 and 3, respectively; and a light chain le domain comprising, CDR regions COR-Lt, CDR‘LE and CDR—LB comprising sequences SEQ 1D N054, ii and 6, tiveiy; for use in treating cancer by hitting a (IXCRIL expressing cancer eelt by induction of at least one effector funetien, in the presence of effector cells or complement cramponents.

As used herein, the term "comprise" and variations of the term, such as "comprising", "comprises" and "comprised”, are not intended to exclude other additives, components, integers or steps.

The term “antibody?" is used herein in the hroaricst sense and specifically covers monocional antibodies {including firtl Eength monoclonat antibodies) otany isotype such as 1’ng light, lgA, tgrx and IgE polyctnnal antibodies, nmltispecitic antihndiea chimeric nnt’iboriicn and antibody nts An antibody reactive with a Specific antigen can he generated h}: recombinant s such as selection rriries nt‘ recombinant antibodies in phage or similar vectors, or by immunizing; an animal with the antigen or an antigeirencoding nucleic acid.

A “pnlyclonal aniline-ti)?” is an antibody which was produced among or in the ce “tone or more other, nctin~identicnl antibodies, in general, polyoltmni antibodies are ed from n BAlyrngilincytc in the. presence ol‘ Several other B— lymphocytes producing non—identical antibodies Llsnnliy polyeltinal antibodies are obtained directly from. an immunized animal '\ “momyelonnl antibody”, as used herein. is an antibody obtained From a population ot‘subntnntialiy limntigenemis dies“ ie the antibodies forming this population are esscritinliy identical except for possible naturally occurring mutations which might be present in minor amounts In Other worrist a momticlnnzil nntibotigt' consists of a homogeneous antibody arising from the growth of a Single cell clone (for example a hyhridinnai a enhar’yotic hegt ceti transfected with a DNA fitG-lettttie ending. for the nenns antibody, 21 pmkaryotic hast cell itiected with a, DNA molecule coding Frn' the heritage-news antibody etc}. These antibodies are directed against a single epitope and are therefore highly specific, An ohe” is the site on the antigen to which an antibody binds. it can be formed by contiguous residues or by ntiguous residues brought into close proximity by the folding of an antigenic protein. es formed by contiguous amino acids are typicalty retained on re to denaturing solvents, s epitopes formed by nen—eontigunus amino acids are typicatly lost under said exposure Preferably, the antibody of the invention is a mtmoclonal antibody 1001180216 A typical antibody is comprised of two cal heavy chains and two identical light chains that are joined by disulfide bonds. Each heavy and light chain contains a constant region and a variable region. Each variable region contains three segments called "complementarity-determining regions" ("CDRs") or "hypervariable regions", which are primarily responsible for binding an epitope of an antigen. They are y referred to as CDRl, CDR2, and CDR3, ed sequentially from the N-terminus (see c M.-P., Immunology Today 18, 509 ( 1997) / Lefranc M.-P., The Immunologist, 7, 132-136 (1999) / Lefranc, M.-P., Pommie, C , Ruiz, M., Giudicelli, V., Foulquier, E., Truong, L., Thouvenin-Contet, V . and Lefranc, Dev. Comp.

Immunol, 27, 55-77 (2003)). The more highly conserved portions of the variable regions are called the "framework regions".

A s used herein, "VH" or "VH" refers t o the variable region of an immunoglobulin heavy chain of an dy, including the heavy chain of an Fv, scFv, dsFv, Fab, Fab', or F(ab')2 fragment. Reference to "VL" or "VL" refers to the variable region of the immunoglobulin light chain of an antibody, including the light chain of an Fv, scFv, dsFv, Fab, Fab', or F(ab')2 fragment.

Antibody constant s are not involved ly in binding an antibody to an antigen, but exhibit various effector functions. The heavy chain constant regions that correspond to the ent classes of immunoglobulins are called a, d, e, g , and m, respectively. ing on the amino acid sequence of the constant region of their heavy chains, antibodies or immunoglobulins can be assigned to different classes, i.e., IgA, IgD, IgE, IgG, and IgM, and several of these may be further divided into subclasses (isotypes), e.g., IgGl, IgG2, IgG3, and IgG4; IgAl and IgA2 (see, W. E . Paul, ed., 1993, Fundamental Immunology, Raven Press, New York, New York).

Papain digestion of antibodies es two identical antigen binding fragments, called Fab fragments, each with a single antigen binding site, and a residual "Fc" fragment. Although the boundaries of the Fc domain of an immunoglobulin heavy chain might vary, the human IgG heavy chain Fc domain is usually defined to stretch from an amino acid residue at position, ing to the EU index, Cys226 or Pro230 in the hinge region, to the carboxyl-terminus thereof containing the CH2 and CH3 domain of the heavy chain (Edelman et al, The covalent structure of an entire gammaG immunoglobulin molecule, PNAS 1969; 63:78-85). For the sake of clarity, it should be stated here that the Cys226/Pro230 es according to the EU index correspond to the Cys239/Pro243 residues in the Kabat numbering system and to the hinge residues Cysl 1/Pro 15 according to IMGT.

The term "hinge region" is generally defined as stretching from Glu216 to Pro230 of human IgGl (Burton, Mol Immunol, 22: 161-206, 1985). Hinge regions of other IgG isotypes may be aligned with the IgGl sequence by placing the first and last cysteine residues g heavy chain S-S bonds in the same positions. The "CH2 domain" of a human IgG Fc portion (also referred to as "Cy2" ) usually extends from about amino acid 231 to about amino acid 340. The CH2 domain is unique in that it is not closely paired with another domain. Rather, two N-linked branched ydrate chains are interposed between the two CH2 domains of an intact native IgG le. It has been speculated that the carbohydrate may provide a substitute for the domain-domain pairing and help stabilize the CH2 domain (Burton, Mol Immunol, 22: 161-206, 1985). The "CH3 domain" comprises the stretch of residues C- terminal to a CH2 domain in an Fc portion (i.e., from about amino acid residue 341 to about amino acid residue 447 of an IgG).

IgG immunoglobulins, including monoclonal antibodies have been shown to be N-glycosylated in the constant region of each heavy chain. They contain a single, N- linked glycan at Asn 297 in the CH2 domain on each of its two heavy chains. As used herein, the term "N-glycan" refers to an N-linked oligosaccharide, e.g., one that i s attached by an asparagine-N-acetylglucosamine e to an asparagine residue of a polypeptide. N-glycans have a common pentasaccharide core of Man3GlcNAc2 ("Man" refers to mannose; "Glc" refers to glucose; and "NAc" refers to N-acetyl; GlcNAc refers to N-acetylglucosamine).

N-glycans differ with respect to the number and the nature of branches (antennae) sing peripheral sugars (e.g., GlcNAc, galactose, fucose, and sialic acid) that are attached to the Man3 core structure. N-glycans are classified according to their ed constituents (e.g., high e, complex or hybrid). A "complex, bi- antennary" type an typically has at least one GlcNAc attached to the 1,3 mannose branch and at least one GlcNAc attached to the 1,6 mannose branch of the trimannose core. Complex bi-antennary N-glycans may also have intrachain substitutions comprising ting" GlcNAc and core fucose ("Fuc"). A "bisecting GlcNAc" is a GlcNAc residue attached to the P-l,4-mannose of the mature core ydrate structure.

Complex ennary N-glycans may also have galactose ("Gal") residues that are optionally modified with sialic acid. Sialic acid addition to the oligosaccharide chain i s catalysed by a sialyltransferase, but requires previous attachment of one or more galactose es by a gal actosy transferase to terminal N-acetylglucosamines. "Sialic acids" ing to the invention encompass both 5-N-acetylneuraminic acid (NeuNAc) and 5-glycolylneuraminic acid (NeuNGc).

Oligosaccharides may contain zero (GO), one (Gl) or two (G2) galactose residues, as well as one fucose attached to the first GlcNac or not. These forms are noted as GO/GOF, G2/G2F, Gl/GIF, respectively (see Figure 1 of Theillaud, Expert Opin Biol Ther., Suppl 1 : S15-S27, 2005). In other words, when both arms of the oligosaccharide chain comprise galactose residues, the maximum moles galactose per mole heavy chain i s two and the structure i s referred t o a s G2F when the core is fucosylated and G2 when it is not. When one arm has terminal galactose, the structure is referred t o as GIF or Gl, depending on whether it i s lated or not, while the structure is referred to as GOF or GO, respectively, when there is no terminal galactose.

A secreted IgG immunoglobulin is thus a heterogeneous mixture of glycoforms exhibiting le addition of the sugar residues fucose, galactose, sialic acid, and bisecting N-acetylglucosamine.

The F c domains are l in determining the biological functions of the immunoglobulin and these biological functions are termed "effector functions". These F c domain-mediated activities are mediated via logical effector cells, such as killer cells, l killer cells, and activated macrophages, or various complement ents. These effector functions involve activation of receptors on the surface of said effector cells, through the binding of the F c domain of an antibody t o the said receptor or to ment component(s).

The expression "Antibody-dependent cell-mediated cytotoxicity", "Antibodydependent cellular cytotoxicity" or "ADCC" refers to a form of cytotoxicity in which Ig bound onto Fc receptors (FcRs) present on certain cytotoxic effector cells enables these cytotoxic effector cells t o bind ically t o an antigen-bearing target cell and subsequently kill the target cell with cytotoxins. Lysis of the target cell is extracellular, requires direct cell-to-cell contact, and does not involve ment.

Cell destruction can occur, for example, by lysis or phagocytosis. "Cytotoxic effector cells" are leukocytes which express one or more FcRs and perform or ons. Preferably, the cells express at least FCYRIII and perform ADCC effector on. Examples of human leukocytes which mediate ADCC include eral blood mononuclear cells (PBMC), l killer (NK) cells, monocytes, cytotoxic T cells and neutrophils; with PBMCs and NK cells being preferred. The effector cells may be isolated from a native source thereof, e.g. from blood or PBMCs. Cytotoxic effector cells which are capable of cell ction by lytic means include for, e, natural killer (NK) cells, eosinophils, macrophages and neutrophils.

Of the various human immunoglobulin classes, human IgGl and IgG3 mediate ADCC more effectively than IgG2 and IgG4.

Advantageously, the human or humanized antibody of the invention, or the CH2-containing fragment thereof, is capable of killing a CXCR4-expressing cancer cell by inducing antibody-dependent cell cytotoxicity (ADCC).

Therefore, in a first preferred embodiment of the method of the invention, the said effector function consists of the antibody-dependent cell cytotoxicity (ADCC).

In other words, the use according the ion is characterized in that said effector function consists of the antibody-dependent cell cytotoxicity (ADCC).

Still in other words, the human or humanized dy according to the invention i s characterized in that said effector function consists of the antibodydependent cell xicity (ADCC).

As non limitative examples, the following methods for assessing or quantifying in vitro ADCC can be ned: Cytometry using propidium iodide (PI) or calcein, 1Cr or fluorescent dyes such as calcein-AM, carboxyfluorescein succinimidyl ester (CFSE), 2',7'-bis-(2carboxyethyl)(and-6)-carboxyfluorescein (BCECF) or Europium, or by measuring the release of cytosolic enzymes such as lactate dehydrogenase (LDH) or ATP. These methods are well-known to the person of skills in the art [see e;g. Jiang et al, "Advances in the assessment and l of the effector functions of therapeutic antibodies", Nat Rev Drug Discov., 10: 101-1 10, 201 1, and references therein] and need not be further detailed here.

By "complement-dependent cytotoxicity" or "CDC", it is herein refered a mechanism whereby complement activation triggered by specific antibody binding to an antigen on a cell surface causes the lysis of the target cell, through a series of es ement activation pathways) containing complement-related protein groups in blood. In on, protein fragments ted by the activation of a complement can induce the migration and tion of immune cells. The first step of complementdependent cytotoxicity (CDC) activation consists in the g of Clq protein to at least two Fc domains of the antibody. "Clq" is a polypeptide that includes a binding site for the Fc region of an immunoglobulin. Clq together with two serine proteases, Clr and Cls, forms the complex CI, the first component of the complement-dependent cytotoxicity (CDC) pathway.

In another advantageous embodiment of the invention, the human or zed antibody, or the CH2-containing fragment thereof, is capable of killing a CXCR4- expressing cancer cell by inducing complement dependent cytotoxicity (CDC).

Therefore, the present invention also relates to a method as described above, wherein the effector function consists of the complement dependent cytotoxicity (CDC).

In other words, the use according to the invention is characterized in that said effector function consists of the complement dependent cytotoxicity (CDC).

Still in other words, the human or humanized antibody according to the invention is characterized in that said effector function consists of the complement ent cytotoxicity (CDC).

The cytotoxicity of nucleated cells by CDC can be quantitated in vitro by several methods, such as: Trypan blue exclusion, flow cytometry using propidium iodide (PI), 1Cr release, reduction of tetrazolium salt MTT, redox dye Alamar blue, loss of intracellular ATP, CellTiter-Glo, LDH release or calcein-AM release. These s are well know to the person of skills in the art [see e;g. Jiang et al., "Advances in the assessment and control of the effector functions od therapeutic antibodies", Nat Rev Drug Discuv., 10: 101-1 10, 201 1, and references therein] and need not be r detailed here.

In a further advantageous ment of the invention, both antibodydependent cell cytotoxicity (ADCC) and the complement ent xicity (CDC) are induced, i.e. the human or humanized antibody, or the CH2-containing fragment f, i s capable of killing a expressing cancer cell by inducing both antibody-dependent cell cytotoxicity (ADCC) and the complement dependent cytotoxicity (CDC).

In a preferred embodiment, the effector ons of the method of the invention consist of the antibody-dependent cell cytotoxicity (ADCC) and the ment dependent cytotoxicity (CDC).

In other words, the use according to the invention is characterized in that said effector functions consist of the antibody-dependent cell cytotoxicity (ADCC) and the complement dependent cytotoxicity (CDC).

Still in other words, the human or humanized antibody according to the invention is characterized in that said effector functions consist of the antibodydependent cell cytotoxicity (ADCC) and the antibody-dependent cell cytotoxicity (ADCC).

These two effector functions of an antibody are directly associated with the binding of the antibody Fc portion to specific ors on the surface of immune cells - essentially FcyRIIIa (also referred as FcyRIIIA) and a (also referred as FcyRIIA) expressed on NK cells, macrophages, monocytes for ADCC and the complement cascade protein Clq for CDC.

The precise ctions between the Fc portion of an antibody and Fc Rs and Clq have been mapped precisely and the major Fc domain ed in this interaction corresponds to the CH2 domain. The affinity between the Fc portion and the Fc receptors is directly linked to the extent of immune responses that are triggered.

As described above, the human or humanized antibodies directed against CXCR4 are capable of inducing or ons against a CXCR4-expressing cell, thus leading to xic effects against the said cells. It is clear that the higher the effector function induction, the greater the cytotoxic effects against the CXCR4- expressing cells. Although some dies may display naturally elevated ADCC and/or CDC activity, it may be necessary in other cases to engineer a human or humanized antibody ed against CXCR4 in order to enhance the antibody immune responses and, more particularly, ADCC and/or CDC. Such engineered antibodies are also encompassed by the scope of the present invention.

There are several ways to engineer and to enhance antibody immune responses and, more particularly, ADCC and/or CDC, most of them being based on the direct increase of binding of the Fc portion to the e Fc R for ADCC. The major goal is to increase binding to human Fc Ra and human FcyRIIa and decrease binding to human FcyRIIb (an inhibitory receptor decreasing immune responses). This can be achieved either by mutating individual amino acid residues within the Fc portion or by modifying the glycan moiety linked to asparagine 297 of the CH2 domain to se the afucosylated glycan portion. Glycoengineering can be achieved, for example, either by shutting-down (siRNA, KO, etc; de Shinkawa et al., The e of fucose but not the presence of galactose or bisecting N-acetylglucosamine of human IgGl x-type oligosaccharides shows the critical role of enhancing antibody-dependent cellular cytotoxicity. J . Biol. Chem 2003; 278: 473) the FUT8 gene in the host cell producing the antibody, or overexpressing a GlcNAc III transferase in the antibody- producing cell (see e.g. Pablo Umana et al. Engineered glycoforms of an antineuroblastoma IgGl with zed antibody-dependent cellular cytotoxicity activity. Nat Biotechnol 1999;17:176-180).

Such antibodies may be obtained by making single or multiple substitutions in the constant domain of the antibody, thus increasing its interaction with the Fc receptors. Methods for designing such mutants can be found for example in Lazar et al. (2006, PNAS, 103(1 1): 4005-4010) and Okazaki et al. (2004, J . MoT Biol. 336(5): 1239-49).

It is also le to use cell lines specifically ered for production of improved antibodies. In particular, these lines have altered regulation of the glycosylation pathway, resulting in antibodies which are poorly fucosylated or even totally defucosylated. Such cell lines and methods for engineering them are disclosed in e.g. wa et al. (2003, J . Biol. Chem. 278(5): 3466-3473), Ferrara et al.

(Biotechnol. Bioeng. 93(5): 851-61).

Other methods of increasing ADCC have also been described by Li et al. (2006, Nat Biotechnol. 24(2):210-5), Stavenhagen et al. (2008, Advan. Enzyme Regul. 48:152- 164), Shields et al. (2001, J . Biol. Chem., 276(9):6591-6604); and .

Methods of increasing CDC have been described by Idusogie et al. (2001, J Immunol. 166(4):257 1-5), Dall'Acqua et al. (2006, J Immunol , 177(2): 1129-38), Michaelsen et al. (1990, Scand J Immunol, 32(5) :5 , Brekke et al. (1993, Mol Immunol, 30(16) :1419-25), Tan et al. (1990, Proc ; Natl. Acad. Sci. USA, 87:162-166) and Norderhaug et al. (1991, Eur J Immunol, 21(10):2379-84).

A well described technology is the Complement technology developed by Kyowa which consists in making a chimeric human IgGl/IgG3 Fc portion with enhanced CDC (see e.g. 1041).

References describing methods of increasing ADCC and CDC include Natsume et al. (2008, Cancer Res. 68(10): 872). The disclosure of each of these references is included herein by cross nce.

It will be clear for the man skilled in the art that, enhancing ADCC and/or CDC is of a particular interest in the field of the treatment of cancers as it will lead to the killing of the CXCR4-expressing cancerous cells and, as such, will y limit the risk of resumption of the cancer, should the CXCR4-targeted treatment be stopped.

By the expression "CH2-containing binding fragment", it must be understood any fragment or part of an antibody comprising the 6 CDRs of the parental antibody and at least the CH2 domain, which is known as being responsible of ng an effector function. In a most preferred embodiment, the CH2 must be dimeric, that is to say that it comprises two copies of the CH2.

In another embodiment, the ntaining g fragment comprises the 6 CDRs of the parental dy and at least the CH2 and the hinge domains.

In another embodiment, the CH2-containing binding fragment comprises the 6 CDRs of the parental antibody and at least the CHI, the hinge and the CH2 domains.

In another embodiment, the CH2-containing binding fragment comprises the 6 CDRs of the parental antibody and at least the CHI and the CH2 domains.

In another ment, the CH2-containing binding fragment comprises the 6 CDRs of the parental antibody and at least the CHI, the CH2 and the CH3 domains.

In still another ment, the CH2-containing binding fragment comprises the 6 CDRs of the parental antibody and at least the CHI, the hinge, the CH2 and the CH3 s, i.e. the full length Fc.

More preferably, the invention ses the humanized antibodies, their CH2- containing binding fragments, obtained by genetic recombination or chemical synthesis.

According to a preferred embodiment, the human or humanized antibody according to the ion is terized in that it consists of a monoclonal antibody.

In other words, the method of the invention comprises the use of a human or humanized antibodies, or a ntaining binding fragment, which comprises, according to IMGT, a heavy chain sing the following three CDRs, respectively CDR-H1, CDR-H2 and CDR-H3, wherein: - CDR-H1 comprises the sequence SEQ ID No. 1, or a sequence with at least 80%, preferably 85%, 90%, 95% and 98%>, identity after optimal alignment with sequence SEQ ID No. 1; - CDR-H2 comprises the sequence SEQ ID No. 2, or a ce with at least 80%, preferably 85%, 90%, 95% and 98%, identity after optimal alignment with sequence SEQ ID No. 2; and - CDR-H3 comprises the sequence SEQ ID No. 3, or a sequence with at least 80%, preferably 85%, 90%, 95% and 98%, ty after optimal ent with sequence SEQ ID No. 3 .

Even more preferably, the method of the invention comprises the use of a human or humanized antibodies, or a CH2-containing binding fragment, which ses, according to IMGT, a light chain comprising the following three CDRs, respectively CDR-L1, CDR-L2 and CDR-L3, wherein: - CDR-L1 comprises the sequence SEQ ID No. 4, or a sequence with at least 80%, preferably 85%, 90%, 95% and 98%, ty after optimal alignment with sequence SEQ ID No. 4; - CDR-L2 comprises the sequence SEQ ID No. 5, or a sequence with at least 80%, preferably 85%, 90%, 95% and 98%, identity after optimal alignment with sequence SEQ ID No. 5; and - CDR-L3 comprises the sequence SEQ ID No. 6, or a sequence with at least 80%, preferably 85%, 90%, 95% and 98%, identity after optimal alignment with ce SEQ ID No. 6 .

In the present description, the terms "polypeptides", "polypeptide sequences", "peptides" are interchangeable.

The IMGT unique numbering has been defined to compare the variable domains whatever the antigen receptor, the chain type, or the species [Lefranc M.-P., Immunology Today 18, 509 (1997) / Lefranc M.-P., The Immunologist, 7, 132-136 (1999) / Lefranc, M.-P., , C , Ruiz, M., Giudicelli, V., Foulquier, E., Truong, L., Thouvenin-Contet, V. and Lefranc, Dev. Comp. Immunol, 27, 55-77 (2003)]. In the IMGT unique numbering, the conserved amino acids always have the same position, for instance cystein 23 (lst-CYS), tryptophan 4 1 (CONSERVED-TRP), hydrophobic amino acid 89, cystein 104 (2nd-CYS), alanine or tryptophan 118 (J-PHE or J- TRP). The IMGT unique numbering provides a standardized delimitation of the framework regions (FR1-IMGT: positions 1 to 26, FR2-IMGT: 39 to 55, FR3-FMGT: 66 to 104 and FR4-IMGT: 118 to 128) and of the complementarity determining regions: CDR1-IMGT: 27 to 38, CDR2-IMGT: 56 to 65 and CDR3-IMGT: 105 to 117. As gaps ent unoccupied ons, the CDR-IMGT lengths (shown between brackets and separated by dots, e.g. [8.8. 13]) become l information. The IMGT unique numbering is used in 2D graphical representations, designated as IMGT Colliers de Pedes [Ruiz, M . and Lefranc, M.-P., Immunogenetics, 53, 857-883 (2002) / Kaas, Q . and Lefranc, M.-P., Current Bioinformatics, 2, 21-30 (2007)], and in 3D structures in IMGT/3Dstructure-DB [Kaas, Q., Ruiz, M . and Lefranc, M.-P., T cell receptor and MHC structural data. Nucl. Acids. Res., 32, D208-D210 (2004)].

In the sense of the present invention, the "percentage identity" between two sequences of nucleic acids or amino acids means the percentage of identical nucleotides or amino acid residues between the two sequences to be compared, obtained after optimal alignment, this tage being purely tical and the differences between the two sequences being distributed randomly along their length. The comparison of two nucleic acid or amino acid ces is traditionally carried out by comparing the sequences after having optimally aligned them, said comparison being able to be conducted by segment or by using an "alignment ". l alignment of the sequences for comparison can be carried out, in addition to comparison by hand, by means of the local homology algorithm of Smith and Waterman (1981) [Ad. App. Math. 2:482], by means of the local homology algorithm of man and Wunsch (1970) [J.

Mol. Biol. 48:443], by means of the similarity search method of Pearson and Lipman (1988) [Proc. Natl. Acad. Sci. USA 85:2444] or by means of computer software using these algorithms (GAP, BESTFIT, FASTA and TFASTA in the Wisconsin Genetics Software Package, Genetics Computer Group, 575 Science Dr., Madison, WI, or by the comparison software BLAST or BLAST P).

The percentage identity between two nucleic acid or amino acid sequences is ined by comparing the two optimally-aligned sequences in which the nucleic acid or amino acid sequence to compare can have additions or deletions compared to the reference sequence for optimal alignment between the two sequences. Percentage identity is calculated by determining the number of positions at which the amino acid nucleotide or residue is identical between the two sequences, ably between the two complete sequences, dividing the number of identical positions by the total number of ons in the alignment window and multiplying the result by 100 to obtain the percentage ty between the two sequences.

For example, the BLAST program, "BLAST 2 sequences" ova et al., "Blast 2 sequences - a new tool for comparing protein and nucleotide sequences", FEMS Microbiol, 1999, Lett. 7-2 5 0 ) a v a i l a b l e o n t h e s i t e http://www.ncbi.nlm.nih.gov/gorf/bl2.html, can be used with the t parameters (notably for the parameters "open gap penalty": 5, and "extension gap penalty": 2; the selected matrix being for example the "BLOSUM 62" matrix proposed by the program); the percentage identity between the two sequences to compare is calculated directly by the program.

For the amino acid sequence exhibiting at least 80%, ably 85%, 90%, 95% and 9 8% identity with a reference amino acid sequence, preferred examples include those containing the reference sequence, certain modifications, notably a deletion, addition or substitution of at least one amino acid, truncation or extension. In the case of substitution of one or more consecutive or non-consecutive amino acids, substitutions are preferred in which the substituted amino acids are replaced by "equivalent" amino acids. Here, the expression "equivalent amino acids" is meant to indicate any amino acids likely to be substituted for one of the structural amino acids without r modifying the biological activities of the corresponding antibodies and of those specific es defined below.

Equivalent amino acids can be ined either on their structural homology with the amino acids for which they are substituted or on the results of comparative tests of biological activity n the various antibodies likely to be generated.

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

Table 1 It is known by those skilled in the art that in the current state of the art the greatest variability (length and composition) between the six CDRs is found at the three heavy-chain CDRs and, more particularly, at CDR-H3 of this heavy chain.

Consequently, it will be evident that the preferred characteristic CDRs of the antibodies of the invention, or of one of their derived compounds or functional fragments, will be the three CDRs of the heavy chain.

Another embodiment of the ion discloses the use of a human or humanised antibody, or a CH2-containing binding fragment, which comprises: a heavy chain comprising the following three CDRs: CDR-H1 of the ce SEQ ID No. 1 or of a sequence with at least 80%, preferably 85%, 90%, 95% and 98% identity after optimal alignment with ce SEQ ID No. 1; CDR-H2 of the sequence SEQ ID No. 2 or of a sequence with at least 80%, preferably 85%, 90%, 95% and 98% identity after optimal ent with sequence SEQ ID No. 2; CDR-H3 of the sequence SEQ ID No. 3 or of a sequence with at least 80%, ably 85%, 90%, 95% and 98% identity after optimal alignment with sequence SEQ ID No 3; and a light chain comprising the following three CDRs: CDR-L1 of the sequence SEQ ID No. 4 or of a sequence with at least 80%, preferably 85%, 90%, 95% and 98% identity after optimal alignment with ce SEQ ID No. 4; CDR-L2 of the sequence SEQ ID No. 5 or of a sequence with at least 80%, preferably 85%, 90%, 95% and 98% ty after optimal alignment with sequence SEQ ID No. 5; CDR-L3 of the sequence SEQ ID No. 6 or of a sequence with at least 80%, preferably 85%, 90%, 95% and 98% identity after optimal ent with sequence SEQ ID No. 6 .

For more clarity, table 2a below summarizes the various amino acid sequences corresponding to the CDRs of the antibody hz515H7 of the invention; whereas table 2b summarizes the various amino acid sequences corresponding to the variable domains and the full length sequences of the various variants of the humanized antibody of the invention.

Table 2a Antibody Heavy chain Light chain SEQ ID NO.

Hz515H7 CDR-H1 - 1 CDR-H2 - 2 CDR(s) CDR-H3 CDR-L1 4 - CDR-L2 5 - CDR-L3 6 Table 2b Antibody Heavy chain Light chain SEQ ID NO.

VH1 - 7 VH1 D76N - 8 VH1 V48L D76N - 9 VH2 VL1 11 Variable Domains - VL1 T59A E61D 12 - VL2 13 - VL2.1 14 - VL2.2 15 - VL2.3 16 - VL3 17 VH1 - 18 VH1 D76N - 19 VH1 V48L D76N - 20 VH2 - 2 1 - VL1 22 Complete Sequences - VL1 T59A E61D 23 (without signal peptide) - VL2 24 - VL2.1 25 - VL2.2 26 - VL2.3 27 - VL3 28 As an example, for the avoidance of doubt, the expression "VH1" is similar to the expressions "VH Variant 1", "VH variant 1", "VH Var 1" or "VH var 1).

It can be mentioned here that the antibody used for the invention was obtained from the humanization of the murine antibody produced by the murine oma filed with the French collection for microorganism cultures (CNCM, Institut Pasteur, Paris, France) on June 25, 2008, under number 1-4019. Said hybridoma was obtained by the fusion of Balb/C immunized mice splenocytes and cells of the myeloma Sp 2/0- Ag 14 lines.

The murine onal antibody, here referred to as 515H7 is secreted by the hybridoma filed with the CNCM on June 25, 2008, under number 1-4019.

In a red embodiment, the antibody used in the method of the invention is a humanized antibody.

As used herein, the term "humanized antibody" refers to a chimeric antibody which n l sequence derived from non-human globulin. A "chimeric antibody", as used herein, is an antibody in which the constant region, or a portion thereof, is altered, replaced, or exchanged, so that the variable region is linked to a constant region of a different species, or belonging to another antibody class or subclass. "Chimeric antibody" also refers to an antibody in which the variable region, or a portion thereof, is altered, replaced, or exchanged, so that the constant region is linked to a variable region of a different species, or belonging to another antibody class or In certain embodiments both the variable and constant regions of the antibodies, or antigen-binding fragments, variants, or derivatives thereof are fully human. Fully human antibodies can be made using techniques that are known in the art. For example, fully human antibodies t a specific antigen can be prepared by administering the n to a transgenic animal which has been modified to produce such antibodies in response to antigenic challenge, but whose endogenous loci have been disabled.

Exemplary techniques that can be used to make such antibodies are described in US patents: 6,150,584; 6,458,592; 6,420,140. Other techniques are known in the art. Fully human antibodies can likewise be produced by various display technologies, e.g., phage display or other viral display s. See also U.S. Pat. Nos. 4,444,887, 4,716,1 11, ,545,806, and 5,814,318; and ational patent application publication numbers WO 45, WO 98/50433, WO 98/24893, WO 98/16654, WO 96/34096, WO 96/33735, and WO 41 (said references orated by reference in their entireties).

A "humanized antibody" as used herein refers to an antibody that contains CDR regions derived from an antibody of nonhuman origin, the other parts of the antibody le being derived from one (or several) human antibodies. In addition, some of the on segment es (called FR) can be modified to preserve binding affinity (Jones et al., , 321 :522-525, 1986; Verhoeyen et al., Science, 239: 1534-1536, 1988; Riechmann et al., Nature, 332:323-327, 1988).

The goal of humanization is a reduction in the immunogenicity of a xenogenic antibody, such as a murine antibody, for introduction into a human, while maintaining the full antigen binding affinity and icity of the antibody. The humanized antibodies of the invention or fragments of same can be prepared by ques known to a person skilled in the art (such as, for example, those described in Singer et al., J .

Immun, 150:2844-2857, 1992; Mountain et al., Biotechnol. Genet. Eng. Rev., 10: 1- 142, 1992; and Bebbington et al., Bio/Technology, 10: 169-175, 1992). Such humanized antibodies are preferred for their use in methods involving in vitro diagnoses or preventive and/or therapeutic treatment in vivo. Other humanization techniques, also known to a person skilled in the art, such as, for example, the "CDR grafting" technique described by PDL in s EP 0 451 261, EP 0 682 040, EP 0 939 127, EP 0 566 647 or US 5,530,101, US 6,180,370, US 5,585,089 and US 5,693,761. US patents 5,639,641 or 6,054,297, 5,886,152 and 5,877,293 can also be cited.

By extension, in the case of this present specification, the ic dy cl51H7 will be comprised in the expression ized antibody". More particularly, the c515H7 is characterized in that it comprises a heavy chain of sequence SEQ ID No. 70 (corresponding to the nucleotide SEQ ID No. 72) and a light chain of sequence SEQ ID No. 7 1 (corresponding to the nucleotide SEQ ID No. 73).

The invention relates to the humanized antibodies arising from the murine antibody 515H7 described above, said antibodies being defined by the sequences of their heavy and/or light chains variable domains. Indeed, all the humanized antibodies described herein can be used in the methods of the invention, i.e. they can be used for killing CXCR4-expressing cancer cells by induction of at least one effector function, in the presence of effector cells or complement components, or they can be used for treating cancer by killing CXCR4-expressing cancer cells by induction of at least one effector on, in the presence of effector cells or complement ents.

In a preferred embodiment of the methods of the invention, the human or humanized antibody consists of a zed dy comprising a heavy chain variable domain selected from the sequences SEQ ID No. 7 to 10 and a light chain variable domain selected from the sequences SEQ ID No. 11 to 17.

In other words, the use according to the invention is characterized in that said human or humanized antibody ts of a humanized antibody comprising a heavy chain variable domain ed from the ces SEQ ID No. 7 to 10 and a light chain variable domain selected from the ces SEQ ID No. 11 to 17.

Still in other words, the human or humanized antibody according to the invention is characterized in that it consists of a humanized antibody comprising a heavy chain variable domain selected from the sequences SEQ ID No. 7 to 10 and a light chain variable domain selected from the sequences SEQ ID No. 11 to 17.

A preferred humanized antibody according to the invention consists of a humanized antibody comprising a heavy chain variable domain of sequence SEQ ID No. 8 and a light chain variable domain selected from the sequences SEQ ID No. 11 to Still a preferred humanized antibody ing to the invention consists of a humanized antibody sing a heavy chain variable domain selected from the sequences SEQ ID No. 7 to 10 and a light chain variable domain of sequence SEQ ID No. 13.

The invention also relates to the humanized antibodies arising from the murine antibody 515H7 described above, said antibodies being defined by the sequences of their full length heavy and/or light chains.

In a preferred embodiment of the method of the invention, the human or humanized antibody consists of a humanized antibody comprising a heavy chain selected from the sequences SEQ ID No. 18 to 2 1 and a light chain selected from the sequences SEQ ID No. 22 to 28.

In other words, the use according to the invention is characterized in that said human or humanized antibody consists of a humanized antibody comprising a heavy chain selected from the sequences SEQ ID No. 18 to 2 1 and a light chain selected from the sequences SEQ ID No. 22 to 28.

Still in other words, the human or humanized antibody according to the invention is characterized in that it consists of a humanized dy sing a heavy chain selected from the sequences SEQ ID No. 18 to 2 1 and a light chain selected from the sequences SEQ ID No. 22 to 28.

A preferred humanized antibody according to the invention consists of a zed antibody comprising a heavy chain of sequence SEQ ID No. 19 and/or a light chain selected from the sequences SEQ ID No. 22 to 28. r preferred humanized antibody ing to the invention consists of a humanized dy comprising a heavy chain selected from the sequences SEQ ID No. 18 to 2 1 and/or a light chain of sequence SEQ ID No. 24.

In a preferred embodiment, the invention relates to the humanized antibody Hz515H7 VHl D76N VL2, or a derived compound or functional fragment of same, comprising a heavy chain variable region of sequence SEQ ID No. 8, and a light chain variable region of sequence SEQ ID No. 13.

In another preferred embodiment, the invention relates to the humanized antibody Hz515H7 VHl D76N VL2, or a derived compound or functional fragment of same, comprising a heavy chain of sequence SEQ ID No. 19, and a light chain of sequence SEQ ID No. 24.

In r red embodiment, the invention relates to the humanized antibody Hz515H7 VHl D76N VL2.1, or a derived compound or functional fragment of same, comprising a heavy chain variable region of sequence SEQ ID No. 8, and a light chain variable region of sequence SEQ ID No. 14.

In another preferred embodiment, the invention relates to the humanized antibody Hz515H7 VHl D76N VL2.1, or a derived compound or functional nt of same, sing a heavy chain of sequence SEQ ID No. 19, and a light chain of sequence SEQ ID No. 25.

In another preferred embodiment, the invention relates to the humanized antibody Hz515H7 VHl D76N VL2.2, or a derived nd or functional fragment of same, comprising a heavy chain variable region of sequence SEQ ID No. 8, and a light chain variable region of sequence SEQ ID No. 15.

In another preferred embodiment, the invention relates to the humanized antibody Hz515H7 VHl D76N VL2.2, or a derived compound or functional fragment of same, comprising a heavy chain of sequence SEQ ID No. 19, and a light chain of ce SEQ ID No. 26.

In another preferred embodiment, the invention relates to the humanized antibody Hz515H7 VHl D76N VL2.3, or a derived compound or functional fragment of same, comprising a heavy chain variable region of sequence SEQ ID No. 8, and a light chain variable region of sequence SEQ ID No. 16.

In another preferred ment, the invention relates to the zed antibody Hz515H7 VHl D76N VL2.3, or a derived nd or functional fragment of same, comprising a heavy chain of sequence SEQ ID No. 19, and a light chain of sequence SEQ ID No. 27.

In another preferred ment, the invention relates to the humanized antibody Hz5 15H7 VHl V48L D76N VLl, or a derived compound or functional fragment of same, comprising a heavy chain variable region of sequence SEQ ID No. 9, and a light chain variable region of sequence SEQ ID No. 11.

In another preferred embodiment, the invention relates to the zed antibody Hz5 15H7 VHl V48L D76N VLl, or a derived compound or functional fragment of same, comprising a heavy chain of sequence SEQ ID No. 20, and a light chain of sequence SEQ ID No. 22.

In r preferred embodiment, the ion relates to the humanized antibody Hz515H7 VHl V48L D76N VLl T59A E61D, or a derived compound or functional nt of same, comprising a heavy chain variable region of sequence SEQ ID No. 9, and a light chain variable region of sequence SEQ ID No. 12.

In another preferred embodiment, the ion relates to the humanized antibody Hz515H7 VHl V48L D76N VLl T59A E61D, or a derived compound or onal fragment of same, comprising a heavy chain of sequence SEQ ID No. 20, and a light chain of sequence SEQ ID No. 23.

In another preferred embodiment, the invention relates to the humanized antibody Hz5 15H7 VHl VLl, or a derived compound or functional fragment of same, comprising a heavy chain le region of sequence SEQ ID No. 7, and a light chain variable region of sequence SEQ ID No. 11.

In another preferred embodiment, the invention relates to the humanized antibody Hz515H7 VH1 VL1, or a derived compound or functional fragment of same, comprising a heavy chain of sequence SEQ ID No. 18, and a light chain of sequence SEQ ID No. 22.

It must be understood that the above exemplified VH / VL combinations are not limitative. The man skilled in the art could of course, without undue burden and without applying inventive skill, nge all the VH and VL disclosed in the present specification.

In a more preferred embodiment of the method of the invention, said human or humanized antibody consists of a humanized antibody comprising a heavy chain le domain of sequence SEQ ID No. 8 and a light chain variable domain of sequence SEQ ID No. 13.

In other words, the use according to the invention is characterized in that said human or humanized antibody consists of a humanized antibody comprising a heavy chain variable domain of sequence SEQ ID No. 8 and a light chain variable domain of sequence SEQ ID No. 13.

Still in other words, the human or zed antibody according to the invention is characterized in that it consists of a zed antibody comprising a heavy chain variable domain of sequence SEQ ID No. 8 and a light chain variable domain of sequence SEQ ID No. 13.

As for the different sequences above described, the preferred antibody (but not exclusive one) will also be described by the ces of its full length heavy and light chain sequences.

In a more red ment of the method of the invention, the human or humanized antibody consists of a humanized antibody comprising a heavy chain of sequence SEQ ID No. 19 and a light chain of sequence SEQ ID No. 24.

In other words, the use according to the invention is characterized in that said human or humanized antibody consists of a humanized antibody comprising a heavy chain of sequence SEQ ID No. 19 and a light chain of sequence SEQ ID No. 24.

Still in other words, the human or humanized antibody ing to the invention is, characterized in that it ts of a humanized antibody sing a heavy chain of ce SEQ ID No. 19 and a light chain of sequence SEQ ID No. 24.

It will be obvious for the man skilled in the art that the antibody of the invention must present structural ts necessary for presenting effector functions. More particularly, the antibody must be of a suitable e to allow ADCC and/or CDC. For e, it is known that, of the various human globulin classes, human IgGl and IgG3 mediate ADCC more effectively than IgG2 and IgG4. On the other hand, the order of potency for CDC is IgG3 > IgGl » IgG2 ~ IgG4 (Niwa et al, J Immunol s, 306: 151-160, 2005).

In a preferred embodiment of the method of the invention, the said human or humanized antibody is of the IgGl isotype.

In other words, the use according to the invention is characterized in that said human or humanized antibody is of the IgGl isotype.

Still in other words, the human or zed antibody according to the invention is characterized in that it is of the IgGl isotype.

In a particular embodiment, the invention relates to a CH2-containing binding fragment of a preferred antibody of the invention consisting of the IgGl Hz515H7 VH1 D76N VL2.

More particularly, a preferred CH2-containing binding fragment ts of a fragment comprising i) a heavy chain variable domain comprising CDR regions CDR- Hl, CDR-H2 and CDR-H3 comprising ces SEQ ID Nos. 1, 2 and 3, respectively; ii) a light chain variable domain comprising CDR s CDR-L1, CDR-L2 and CDR- L3 comprising sequences SEQ ID Nos. 4, 5 and 6, respectively; and iii) a CH2 domain comprising at least the sequence SEQ ID No. 60.

In a preferred embodiment of the method of the invention, the CH2-containing g fragment consists of a fragment comprising the 3 heavy chain CDR-H1, CDR- H2 and CDR-H3 comprising SEQ ID Nos. 1, 2 and 3, respectively; the 3 light chain CDR-L1, CDR-L2 and CDR-L3 comprising SEQ ID Nos.4, 5 and 6, respectively; and at least the CH2 domain comprising SEQ ID No. 60.

In other words, the use according to the ion is characterized in that said CH2-containing binding fragment consists of a fragment comprising the 3 heavy chain CDR-H1, CDR-H2 and CDR-H3 comprising SEQ ID Nos. 1, 2 and 3, respectively; the 3 light chain CDR-L1, CDR-L2 and CDR-L3 comprising SEQ ID Nos.4, 5 and 6, respectively; and at least the CH2 domain comprising SEQ ID No. 60.

Still in other words, the human or humanized antibody ing to the invention is characterized in that said ntaining binding fragment consists of a fragment comprising the 3 heavy chain CDR-H1, CDR-H2 and CDR-H3 comprising SEQ ID Nos. 1, 2 and 3, respectively; the 3 light chain CDR-L1, CDR-L2 and CDR-L3 sing SEQ ID Nos. 4, 5 and 6, respectively; and at least the CH2 domain comprising SEQ ID No. 60.

Another preferred CH2-containing g fragment consists of a fragment comprising i) a heavy chain variable domain comprising CDR regions , CDR- H2 and CDR-H3 comprising sequences SEQ ID Nos. 1, 2 and 3, respectively; ii) a light chain variable domain comprising CDR regions CDR-L1, CDR-L2 and CDR-L3 comprising sequences SEQ ID Nos. 4, 5 and 6, respectively; iii) a CH2 domain comprising at least the sequence SEQ ID No. 60; and iv) a hinge domain comprising at least the ce SEQ ID No. 61.

Still another red CH2-containing binding fragment consists of a fragment comprising i) a heavy chain variable domain comprising CDR regions CDR-H1, CDR- H2 and CDR-H3 comprising sequences SEQ ID Nos. 1, 2 and 3, respectively; ii) a light chain variable domain sing CDR regions CDR-L1, CDR-L2 and CDR-L3 comprising sequences SEQ ID Nos. 4, 5 and 6, tively; iii) a CH2 domain comprising at least the sequence SEQ ID No. 60; iv) a hinge domain comprising at least the sequence SEQ ID No. 61; and v) a CHI domain comprising at least the sequence SEQ ID No. 62.

Still another preferred CH2-containing binding fragment consists of a fragment comprising i) a heavy chain variable domain comprising CDR regions CDR-H1, CDR- H2 and CDR-H3 comprising sequences SEQ ID Nos. 1, 2 and 3, respectively; ii) a light chain variable domain comprising CDR regions CDR-L1, CDR-L2 and CDR-L3 comprising sequences SEQ ID Nos. 4, 5 and 6, respectively; iii) a CH2 domain comprising at least the sequence SEQ ID No. 60; iv) a hinge domain comprising at least the sequence SEQ ID No. 61; v) a CHI domain comprising at least the ce SEQ ID No. 62; and vi) a CH3 domain comprising at least the sequence SEQ ID No. 63.

In still another preferred embodiment of the invention, a preferred CH2- containing binding fragment consists of a fragment comprising i) a heavy chain variable domain comprising CDR regions CDR-H1, CDR-H2 and CDR-H3 comprising sequences SEQ ID Nos. 1, 2 and 3, respectively; ii) a light chain variable domain sing CDR regions CDR-L1, CDR-L2 and CDR-L3 sing ces SEQ ID Nos. 4, 5 and 6, respectively; and iii) a full length Fc domain comprising at least the sequence SEQ ID No. 64.

For more clarity, the following table 3 illustrates the sequences for each domain for the antibody Hz515H7 VH1 D76N VL2.

Table 3 Based on these elements, it will be clear for the man skilled in the art to generate any CH2-containing binding fragment, derived from any sequence described in the present application, t any undue experiment. As a consequence, any other CH2- containing binding fragment must be considered as part of the scope of the present ation.

Table 4a below summarizes the optimized nucleotide sequences corresponding to the CDRs of the antibody hz515H7 of the invention; whereas table 4b summarizes the various optimized nucleotide sequences corresponding to the variable domains and the full length sequences of the various ts of the humanized antibody of the invention.

Table 4a Antibody Heavy chain Light chain SEQ ID NO.

Hz515H7 optimized CDR-H1 - 29 CDR(s) CDR-H2 - 30 CDR-H3 - 3 1 - CDR-L1 32 CDR-L1 (bis) 57 - CDR-L2 33 CDR-L2 (bis) 58 - CDR-L3 34 CDR-L3 (bis) 59 Table 4b Antibody Heavy chain Light chain SEQ ID NO.

Hz515H7 VHl - 35 VHl D76N - 36 VHl V48L D76N - 37 VH2 VLl 39 le - VLl T59A E61D 40 Domains - VL2 4 1 - VL2.1 42 - VL2.2 43 - VL2.3 44 - VL3 45 VHl - 46 VHl D76N - 47 Complete VHl V48L D76N - 48 Sequences VH2 - 49 (without signal - VLl 50 peptide) - VLl T59A E61D 5 1 - VL2 52 - VL2.1 53 - VL2.2 54 - VL2.3 55 - VL3 56 The expression "optimized sequence" means that the codons ng the amino acids constitutive of the protein of interest (herein the antibody variable domains) have been modified for a better recognition by the translation machinery in a dedicated cell type, herewith mammalian cells. Indeed, it is known to the person of skills in the art that, depending on the source of the gene and of the cell used for expression, a codon optimization may be helpful to increase the expression of the encoded polypeptides of the invention. By "codon optimization", it is referred to the tions to the coding ces for the polypeptides of the invention which improve the ces for codon usage in the host cell. Codon usage tables are known in the art for mamalian cells, such as e.g. CHO cells, as well as for a variety of other organisms. In addition, zation can also be achieved by alterations of the polynucleotide sequences which include G/C content adaptation and prevention of stable RNA secondary structure (see as example Kim et al., 1997 Genel99(l-2):293-301).

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

The nucleic sequences of the present invention have all been isolated and/or purified, i.e., they were sampled directly or ctly, for example by a copy, their environment having been at least partially ed. "Nucleic sequences exhibiting a percentage identity of at least 80%, preferably 85%, 90%, 95% and 98%, after optimal alignment with a preferred sequence" means nucleic sequences exhibiting, with respect to the nce nucleic sequence, certain modifications such as, in particular, a deletion, a truncation, an extension, a chimeric fusion and/or a substitution, notably al.

Preferably, these are sequences which code for the same amino acid sequences as the reference sequence, this being related to the degeneration of the genetic code, or complementarity sequences that are likely to hybridize specifically with the nce sequences, preferably under highly stringent conditions, notably those defined below.

Hybridization under highly stringent conditions means that conditions related to temperature and ionic strength are selected in such a way that they allow hybridization to be maintained between two complementarity DNA fragments. On a purely illustrative basis, the highly stringent conditions of the ization step for the purpose of defining the polynucleotide fragments described above are advantageously as follows.

DNA-DNA or A hybridization i s carried out in two steps: (1) prehybridization at 42°C for three hours in phosphate buffer (20 mM, pH 7.5) containing 5X SSC (IX SSC corresponds to a solution of 0 .15 M NaCl + 0.015 M sodium citrate), 50% formamide, 7% sodium dodecyl e (SDS), 10X Denhardt's, % dextran sulfate and 1% salmon sperm DNA; (2) primary hybridization for 20 hours at a temperature depending on the length of the probe (i.e.: 42°C for a probe >100 nucleotides in length) followed by two 20-minute washings at 20°C in 2X SSC + 2% SDS, one 20-minute washing at 20°C in 0 .1X SSC + 0.1% SDS. The last washing is carried out in 0 .1X SSC + 0 .1% SDS for 30 minutes at 60°C for a probe >100 nucleotides in length. The highly stringent hybridization ions described above for a polynucleotide of defined size can be adapted by a person skilled in the art for longer or shorter oligonucleotides, according to the procedures described in Sambrook, et al.

(Molecular cloning: a laboratory manual, Cold Spring Harbor Laboratory; 3rd edition, 2001).

In order to express the heavy and/or light chain of the human or humanized dy, or CH2-containing binding fragment thereof, of the invention, the polynucleotides encoding said heavy and/or light chains are inserted into expression s such that the genes are operatively linked to transcriptional and translational control sequences.

"Operably " ces include both sion control sequences that are contiguous with the gene of st and expression l sequences that act in trans or at a distance to control the gene of interest. The term "expression l sequence" as used herein refers to polynucleotide sequences which are necessary to effect the expression and processing of coding sequences to which they are ligated. Expression control sequences include appropriate transcription initiation, termination, promoter and enhancer sequences; ent RNA processing signals such as splicing and polyadenylation signals; sequences that ize cytoplasmic mRNA ; sequences that enhance translation efficiency (i. e., Kozak sus sequence); sequences that enhance protein ity; and when desired, sequences that enhance protein secretion.

The nature of such control sequences s depending upon the host organism; in prokaryotes, such control sequences generally include promoter, ribosomal binding site, and transcription termination ce; in eukaryotes, generally, such control sequences include promoters and transcription termination sequence. The term "control sequences" is intended to include, at a minimum, all ents whose presence is essential for expression and processing, and can also include additional components whose presence is ageous, for example, leader sequences and fusion r sequences.

The term "vector", as used herein, is intended to refer to a nucleic acid le capable of transporting another nucleic acid to which it has been linked. One type of vector is a "plasmid", which refers to a circular double stranded DNA loop into which additional DNA segments may be ligated. Another type of vector is a viral vector, wherein additional DNA segments may be ligated into the viral genome. Certain vectors are capable of autonomous replication in a host cell into which they are introduced (e. g., bacterial vectors having a bacterial origin of replication and episomal mammalian vectors). Other vectors (e. g., non- episomal mammalian vectors) can be ated into the genome of a host cell upon uction into the host cell, and y are replicated along with the host .

Certain vectors are capable of directing the expression of genes to which they are operatively linked. Such vectors are referred to herein as "recombinant expression vectors" (or simply, "expression vectors"). In l, expression vectors of utility in recombinant DNA techniques are in the form of plasmids. In the present specification, id" and "vector" may be used interchangeably as the plasmid is the most commonly used form of vector. However, the invention is intended to include such forms of expression vectors, such as bacterial plasmids, YACs, cosmids, retrovirus, EBV-derived episomes, and all the other vectors that the skilled man will know to be convenient for ensuring the expression of the heavy and/or light chains of the antibodies of the ion. The skilled man will realize that the polynucleotides encoding the heavy and the light chains can be cloned into different vectors or in the same vector. In a preferred embodiment, said cleotides are cloned in the same vector.

In addition to the antibody chain genes and regulatory sequences, the recombinant expression vectors of the invention may carry additional sequences, such as sequences that regulate replication of the vector in host cells (e.g., origins of replication) and selectable marker genes. The selectable marker gene facilitates selection of host cells into which the vector has been introduced (see e.g., U.S. s Nos. 4,399,216, 4,634.665 and 017). For example, typically the selectable marker gene confers resistance to drugs, such as G418, ycin or methotrexate, on a host cell into which the vector has been introduced. Preferred able marker genes include the dihydrofolate reductase (DHFR) gene (for use in dhfr- host cells with methotrexate selection/amplification) and the neo gene (for G4 18 selection). A number of selection systems may be used according to the invention, including but not d to the Herpes simplex virus thymidine kinase (Wigler et al., Cell 11:223, 1977), hypoxanthine-guanine phosphoribosyltransferase lska et al., Proc Natl Acad Sci USA 48: 202, 1992), glutamate synthase selection in the presence of nine sulfoximide (Adv Drug Del Rev, 58: 671, 2006, and website or literature of Lonza Group Ltd.) and adenine phosphoribosyltransferase (Lowy et al., Cell 22: 817, 1980) genes in tk, hgprt or aprt cells, respectively. Also, antimetabolite resistance can be used as the basis of selection for the following genes: dhfr, which confers resistance to methotrexate (Wigler et al., Proc Natl Acad Sci USA 77: 357, 1980); gpt, which confers resistance to mycophenolic acid (Mulligan et al., Proc Natl Acad Sci USA 78: 2072, 1981); neo, which s resistance to the aminoglycoside, G-4 18 (Wu et al., Biotherapy 3 : 87, 1991); and hygro, which confers resistance to hygromycin (Santerre et al, Gene 30: 147, 1984). Methods known in the art of recombinant DNA technology may be routinely applied to select the desired recombinant clone, and such methods are described, for e, in Ausubel et al., eds., Current Protocols in lar Biology, John Wiley & Sons (1993). The expression levels of an antibody can be increased by vector amplification. When a marker in the vector system expressing an dy is amplifiable, an increase in the level of inhibitor present in the culture will increase the number of copies of the marker gene. Since the amplified region is associated with the gene encoding the IgG antibody of the invention, production of said antibody will also increase (Crouse et al., Mol Cell Biol 3 : 257, 1983).

Polynucleotides of the invention and vectors comprising these molecules can be used for the transformation of a suitable mammalian host cell, or any other type of host cell known to the skilled person. The term binant host cell" (or simply "host cell"), as used herein, is intended to refer to a cell into which a recombinant expression vector has been introduced. It should be understood that such terms are intended to refer not only to the particular subject cell but also to the progeny of such a cell. Because certain modifications may occur in succeeding generations due to either mutation or environmental influences, such progeny may not, in fact, be identical to the parent cell, but are still included within the scope of the term "host cell" as used herein.

Transformation can be by any known method for ucing polynucleotides into a host cell. Such methods are well known of the man skilled in the art and include dextran-mediated transformation, calcium ate precipitation, polybrene-mediated ection, protoplast fusion, oporation, encapsulation of the polynucleotide into liposomes, biolistic ion and direct microinjection of DNA into nuclei. Preferred mammalian host cells for expressing the inant antibodies of the invention e Chinese Hamster Ovary (CHO cells), NSO myeloma cells, COS cells and SP2 cells. When recombinant expression vectors encoding antibody genes are introduced into mammalian host cells, the antibodies are produced by culturing the host cells for a period of time sufficient to allow for expression of the antibody in the host cells or, more preferably, secretion of the antibody into the culture medium in which the host cells are grown.

Antibodies can be recovered from the culture medium using standard protein purification methods. Soluble forms of the antibody of the invention can be red from the culture supernatant. It may then be purified by any method known in the art for purification of an immunoglobulin molecule, for example, by chromatography (e.g., ion exchange, ty, ularly by Protein A affinity for Fc, and so on), centrifugation, differential solubility or by any other standard technique for the purification of proteins.

Suitable methods of purification will be apparent to a person of ordinary skills in the art.

The present inventors have shown that a human or zed antibody directed against CXCR4 is capable of killing a CXCR4-expressing cancer cell h induction of at least one or function of the said antibody.

By "CXCR4-expressing cancer cell", it is herein referred to a cell of a cancer showing high CXCR4 expression, relative to the CXCR 4 expression level on a normal adult cell. Such cancers include (but are not limited to) the ing: carcinomas and arcinomas, including that of the bladder, breast, colon, head-and-neck, prostate, kidney, liver, lung, ovary, pancreas, h, cervix, thyroid and skin, and including squamous cell carcinoma ; hematopoietic tumors of lymphoid lineage, including multiple myeloma, leukemia, acute and chronic lymphocytic (or lymphoid) leukemia, acute and chronic lymphoblastic leukemia, B-cell lymphoma, T-cell lymphoma, non- n lymphoma (e.g. Burkitt's lymphoma) ; hematopoietic tumors of d lineage, including acute and chronic myelogenous (myeloid or myelocytic) leukemias, and locytic leukemia; tumors of mesenchymal origin, including fibrosarcoma, osteosarcoma and rhabdomyosarcoma; tumors of the central and peripheral nervous system, including astrocytoma, neuroblastoma, glioma, and schwannomas; and other tumors, including ma, teratocarcinoma, xeroderma pigmentosum, keratoacanthoma, and seminoma, and other cancers yet to be determined in which CXCR4 is expressed.

In a preferred embodiment of the method of claim the invention, said CXCR4 expressing cancer cell consists of a malignant logical cell.

In other words, the use according to the invention is terized in that said CXCR4 expressing cancer cell consists of a malignant hematological cell.

Still in other words, the human or humanized antibody according to the invention is terized in that said CXCR4 expressing cancer cell consists of a malignant hematological cell.

More particularly, said CXCR4 malignant hematological cell is selected from the group comprising lymphoma cell, leukemia cell or multiple myeloma cell.

In other words, the use according to the invention is characterized in that said CXCR4 malignant hematological cell is selected from the group comprising lymphoma cell, leukemia cell or multiple myeloma cell.

Still in other words, the human or humanized antibody according to the invention is characterized in that said CXCR4 malignant hematological cell is selected from the group comprising lymphoma cell, leukemia cell or multiple myeloma cell.

In anther red embodiment, said malignant hematological cell consists of a lymphoma cell.

In other word, the use according to the invention is terized in that said malignant hematological cell consists of a lymphoma cell.

Still in other words, the human or humanized antibody according to the invention is terized in that said malignant hematological cell consists of a lymphoma cell.

As above ned, effector cells and/or complement components are of particular interest for the ion.

In a more preferred embodiment of the method of the invention, said effector cells comprise NK cells, macrophages, monocytes, neutrophils or eosinophils.

In other words, the use according to the invention is characterized in that said effector cells comprise NK cells, macrophages, monocytes, neutrophils or eosinophils.

Still in other words, the human or humanized antibody according to the invention is characterized in that said effector cells comprise NK cells, hages, monocytes, neutrophils or eosinophils.

Based on the following examples, particular interesting properties of the antibodies used in the inventions are bed.

In a preferred embodiment of the method of the invention, the induced ADCC level on RAMOS lymphoma cells, after an incubation period of 4 hours, is at least 40%.

In other words, the use according to the invention is characterized in that the induced ADCC level on RAMOS lymphoma cells, after an incubation period of 4 hours, is at least 40%.

Still in other words, the human or humanized antibody ing to the invention is characterized in that the induced ADCC level on RAMOS lymphoma cells, after an tion period of 4 hours, is at least 40%.

In a preferred embodiment of the method of the invention, the induced ADCC level on DAUDI ma cells, after an incubation period of 4 hours, is at least 30%, preferably at least 40%.

In other words, the use according to the invention is characterized in that the induced ADCC level on DAUDI lymphoma cells, after an incubation period of 4 hours, is at least 30%; preferably at least 40%.

Still in other words, the human or humanized dy according to the invention is terized in that the induced ADCC level on DAUDI lymphoma cells, after an incubation period of 4 hours, is at least 30%, preferably at least 40%.

In a preferred embodiment of the method of the invention, the d ADCC level on HeLa cervix cancer cells, after an incubation period of 4 hours, is at least 30%, preferably at least 40%.

In other words, the use according to the invention is characterized in that the induced ADCC level on HaLa cervix cancer cells, after an incubation period of 4 hours, is at least 30%, preferably at least 40%.

Still in other words, the human or humanized antibody ing to the invention is characterized in that the induced ADCC level on HeLa cervix cancer cells, after an incubation period of 4 hours, is at least 30%, preferably at least 40%.

Another particular important aspect of the ion relies on the specificity of induced the ADCC and CDC.

In another preferred embodiment of the method of the invention, no significant ADCC is induced on NK cells.

In other words, the use according to the invention is terized in that no significant ADCC is induced on NK cells.

Still in other words, the human or humanized antibody according to the invention is characterized in that no significant ADCC is induced on NK cells.

The complement components comprise at least the Clq.

In other words, the use according to the invention is characterized in that said complement components comprise at least the Clq.

Still in other words, the human or humanized antibody according to the invention is terized in that said complement components se at least the Clq.

In another preferred embodiment of the method of the invention, the induced CDC level on RAMOS lymphoma cells, after an incubation period of 1 hour, is at least % , preferentially at least 50% and most preferably at least 70%.

In other words, the use according to the invention is characterized in that the induced CDC level on RAMOS lymphoma cells, after an incubation period of 1 hour, is at least 30%, preferentially at least 50% and most ably at least 70%.

Still in other words, the human or humanized antibody ing to the invention is characterized in that the induced CDC level on RAMOS lymphoma cells, after an incubation period of 1 hour, is at least 30%, preferentially at least 50% and most preferably at least 70%.

Still another preferred embodiment of the method of the invention, the induced CDC level on NIH3T3 CXCR4 cells, after an incubation period of 1 hour, is at least % , entially at least 50% and most preferably at least 70%.

In other words, the use according to the invention is characterized in that the induced CDC level on NIH3T3 CXCR4 cells, after an tion period of 1 hour, is at least 30% , preferentially at least 50% and most preferably at least 70%.

Still in other words, the human or zed antibody according to the ion is characterized in that the induced CDC level on NIH3T3 CXCR4 cells, after an incubation period of 1 hour, is at least 30%, preferentially at least 50% and most preferably at least 70%.

Still another preferred ment of the method of the invention, the induced CDC level on DAUDI lymphoma cells, after an incubation period of 1 hour, is at least %, preferentially at least 40%.

In other words, the use according to the invention is characterized in that the induced CDC level on DAUDI lymphoma cells, after an incubation period of 1 hour, is at least 30%, preferentially at least 40%.

Still in other words, the human or humanized antibody according to the invention is characterized in that the induced CDC level on DAUDI lymphoma cells, after an incubation period of 1 hour, is at least 30%, preferentially at least 40%.

In the sense of the ADCC properties of the antibody of the inventions, results have been generated regarding the binding of said antibody with FcyR.

Another particular aspect of the invention relates on that the antibody of the ion, or one of its CH2-containing binding fragment, is capable of binding at least one FcyRs.

In a preferred embodiment of the method of the invention, said at least one FcyRs is FcyRI.

In other words, the use according to the invention is characterized in that said at least one FcyRs is FcyRI.

Still in other words, the human or humanized antibody according to the invention is characterized in that said at least one FcyRs is FcyRI.

In another preferred embodiment of the method of the invention, the constant of dissociation (KD) characterizing the binding of the antibody of the invention with the human Fc[gamma]RI, according to the Langmuir model, is between 1 and 10 nM.

In other words, the use ing to the invention is characterized in that the constant of dissociation (KD) characterizing the binding of the antibody of the invention with the human FcyRI , according to the Langmuir model, is between 1 and 10 nM.

Still in other words, the human or humanized antibody according to the invention is characterized in that the constant of dissociation (KD) characterizing the binding of the antibody of the invention with the human FcyRI, according to the Langmuir model, is between 1 and 10 nM.

In another preferred embodiment of the method of the invention, said at least one FcyRs is human FcyRIIIa.

In other words, the use according to the invention is terized in that said at least one FcyRs is human FcyRIIIa.

Still in other words, the human or humanized antibody according to the invention is characterized in that said at least one FcyRs is human Ia.

In a more preferred embodiment of the method of the ion, the constant of dissociation (KD) terizing the binding of the antibody of the invention with the human Ia, according to the heterogeneous ligand model, is n 200 and 1000 In other words, the use according to the invention is terized in that the constant of dissociation (KD) characterizing the binding of the antibody of the invention with the human FcyRIIIa, ing to the heterogeneous ligand model, is between 200 and 1000 nM.

Still in other words, the human or humanized antibody according to the invention is characterized in that the nt of dissociation (KD) characterizing the binding of the antibody of the invention with the human FcyRIIIa, according to the heterogeneous ligand model, is n 200 and 1000 nM.

As used herein, the term "KD" refers to the dissociation constant of a particular antibody/antigen interaction. "Binding affinity" generally refers to the strength of the sum total of non-covalent interactions n a single binding site of a molecule {e.g., an antibody) and its binding partner (e.g., an antigen). Unless indicated otherwise, as used herein, "binding affinity" refers to intrinsic g ty that reflects a 1 : 1 interaction between members of a binding pair (e.g., dy and antigen). The affinity of a le X for its partner Y can generally be represented by the KD. Affinity can be measured by common methods known in the art, including those described .

Low-affinity antibodies generally bind antigen slowly and tend to dissociate readily, whereas high- ty antibodies generally bind antigen faster and tend to remain bound longer. A variety of methods of measuring binding affinity are known in the art, any of which can be used for purposes of the present invention.

Preferably, the constant of dissociation is calculated according to the Langmuir model.

The Langmuir model is classically described as: A + B*- A B Where A is the analyte, B is the ligand, AB is the non covalent complex between the analyte and the ligand and ka and k are the association and dissociation rates, respectively of this interaction.

In the same way the heterogeneous ligand model where the ligand is considered as a e of two components is bed by the next two equation s: kai a2 A + B 1 = ® AB1 and A + B2 ® AB2 kd1 kd2 where A is the analyte, B l is the first component of the ligand, ABl is the non covalent complex between the analyte and the first component of the ligand, kai and k i are the association and dissociation rates, respectively of this interaction, B2 is the second component of the , AB2 is the non covalent complex between the e and the second component of the ligand, k a2 and k 2 are the association and dissociation rates respectively, of this interaction.

The luation version 3.1 (Biacore AB) has been used for the treatment of BIACORE data.

At last, the ion concerns also a method of treating or preventing a pathological condition ated with the presence of CXCR4 sing cancer cells comprising the step of administering an effective amount of a human or humanized antibody, or CH2-containing binding fragment thereof; said human or humanized antibody comprising a heavy chain variable domain having the 3 CDRs sequences SEQ ID Nos. 1, 2 and 3 and a light chain variable domain having the 3 CDRs sequences SEQ ID Nos.4, 5 and 6; wherein at least one effector function of the said human or humanized antibody is induced, in the presence of effector cells or complement components.

In other words, the invention relates to the use of a human or humanized antibody, or a CH2-containing binding fragment thereof, for preparing a composition for the treatment of a pathological condition associated with the presence of CXCR4 expressing cancer cells; wherein said human or humanized antibody comprises a heavy chain le domain comprising CDR regions CDR-H1, CDR-H2 and CDR-H3 sing sequences SEQ ID Nos. 1, 2 and 3, respectively; and a light chain variable domain comprising CDR s CDR-L1, CDR-L2 and CDR-L3 comprising sequences SEQ ID Nos.4, 5 and 6, respectively; wherein at least one effector function of the said human or zed antibody is induced, in the presence of effector cells or complement components.

Still in other words, the invention relates to a human or zed antibody specifically recognizing CXCR4, CH2-containing binding fragment, for use in treating a pathological condition associated with the presence of CXCR4 expressing cancer cells; said human or humanized antibody comprising a heavy chain variable domain sing CDR regions CDR-H1, CDR-H2 and CDR-H3 comprising sequences SEQ ID Nos. 1, 2 and 3, respectively; and a light chain variable domain comprising CDR regions CDR-L1, CDR-L2 and CDR-L3 comprising sequences SEQ ID Nos.4, 5 and 6, respectively; n at least one effector function of the said human or humanized antibody is induced, in the presence of or cells or complement components.

Preferably, the invention concerns also a method of treating or preventing a pathological condition associated with the presence of CXCR4 expressing cancer cells comprising the step of administering an effective amount of a human or humanized antibody, or CH2-containing binding fragment thereof; said human or humanized antibody comprising a heavy chain variable domain ed from the sequences SEQ ID No. 7 to 10 and a light chain le domain selected from the ces SEQ ID No. 11 to 17; wherein at least one effector function of the said human or humanized antibody is induced, in the presence of effector cells or complement components.

In other words, the invention relates to the use of a human or humanized antibody, or a CH2-containing binding fragment thereof, for preparing a composition for the ent of a ogical condition associated with the presence of CXCR4 expressing cancer cells; said human or zed antibody comprising a heavy chain variable domain selected from the sequences SEQ ID No. 7 to 10 and a light chain variable domain selected from the sequences SEQ ID No. 11 to 17; wherein at least one effector function of the said human or humanized antibody is induced, in the presence of effector cells or complement ents.

Still in other words, the invention relates to a human or humanized antibody specifically recognizing CXCR4, CH2-containing binding fragment, for use in treating a pathological condition associated with the presence of CXCR4 expressing cancer cells; said human or humanized antibody comprising a heavy chain variable domain selected from the sequences SEQ ID No. 7 to 10 and a light chain variable domain selected from the ces SEQ ID No. 11 to 17; wherein at least one or function of the said human or humanized antibody is induced, in the ce of effector cells or complement components.

Monoclonal antibodies are known to be N-glycosylated in the constant region of each heavy chain. Specific glycosylation variants have been shown to affect ADCC. For example, lower fucosylation of IgGls correlates with increased ADCC ds et al., J Biol Chem., 277(30): 26733-2640, 2002; Shinkawa et al., J Biol Chem., 278(5): 3466- 3473, 2003).

A significant correlation between level of galactose and CDC activity was observed. For example, the CDC ty of rituximab is decreasing with decreasing galactose content (Hodoniczky et al, Biotechnol Prog. , 2 1(6): 1644-1652, 2005) A entative glycosylation profile of hz5 15H7 Mab used for ADCC and CDC experiments is shown in the following Table 5 .

Table 5 Surprisingly, the Hz5 15H7 Mab ing to the invention induces a high tage of ADCC and CDC on cells expressing CXCR4, even though around 92 % of its carbohydrate chains comprise a fucose residue.

Thus, the present invention also relates to a method of treating cancer by killing CXCR4 expressing cancer cells, comprising the step of administering an effective amount of a human or humanized antibody, or CH2-containing binding fragment thereof; said human or humanized antibody comprising a glycan profile as follows: Glycosylation profile (FIPLC) Hz515H7 Mab % G O or GOFDGlcNac 5.0 GOF 82.5 GIF 9.1 G2F 0.5 Man5 1.8 wherein at least one effector function of the said human or humanized antibody is induced, in the presence of effector cells or complement components.

The invention also relates to a humanized antibody binding CXCR4, or a CH2- containing g fragment thereof, for use in a method of treatment of cancer by killing CXCR4 expressing cancer cells, said human or humanized antibody comprising a glycan profile as s: wherein at least one effector function of the said human or humanized antibody is induced, in the presence of effector cells or complement components.

The invention also relates to the use of a humanized antibody binding CXCR4, or a CH2-containing g fragment thereof, said human or zed antibody comprising a glycan profile as follows: for preparing a medicament for ng cancer by killing CXCR4 expressing cancer cells, wherein at least one effector function of the said human or humanized antibody is induced, in the presence of effector cells or complement components.

As shown in the Examples herein, the human or humanized antibody, or CH2- containing binding fragment thereof, of the t invention have umoral activity, at least through induction of ADCC and/or CDC responses, and are thus useful in the treatment of metastatic tumors and diseases such as cancer.

The terms ing" or "treatment" refer to administering or the administration of a composition described herein in an amount, manner, and/or mode effective to improve a condition, symptom, or parameter associated with a disorder or to prevent progression or exacerbation of the disorder ding ary damage caused by the er) to either a statistically significant degree or to a degree detectable to one skilled in the art.

Another aspect of the invention relates to pharmaceutical compositions of the human or humanized antibody, or CH2-containing binding fragment f.

The pharmaceutical composition of the invention may contain, in addition to the carrier and the human or humanized antibody, or CH2-containing binding fragment thereof, various diluents, fillers, salts, buffers, izers, solubilizers, and other materials well known in the art.

As used herein, "pharmaceutically acceptable carrier" includes any and all solvents, buffers, salt solutions, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like that are physiologically ible. The type of carrier can be selected based upon the ed route of administration. In various embodiments, the carrier i s suitable for intravenous, eritoneal, subcutaneous, intramuscular, topical, transdermal or oral administration.

Pharmaceutically acceptable rs include sterile aqueous solutions or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersion. The use of media and agents for pharmaceutically active substances is well known in the art. As detailed herebelow, additional active compounds can also be incorporated into the compositions, such as anti-cancer and/or anti-angiogenesis agents; in ular, the additional active compound can be an anti-angiogenic agent, a herapeutic agent, or a low-molecular weight agent. A typical pharmaceutical composition for intravenous infusion could be made up to contain 250 ml of e 's solution, and 100 mg of the combination. Actual s for preparing parenterally administrable compounds will be known or apparent to those skilled in the art and are described in more detail in for example, Remington's Pharmaceutical e, 17th ed., Mack Publishing Company, , Pa. (1985), and the 18 and 19 editions thereof, which are incorporated herein by reference.

The human or humanized antibody, or CH2-containing binding fragment thereof in the composition preferably is formulated in an effective amount. An "effective amount" refers to an amount effective, at dosages and for periods of time necessary, to achieve the desired , such as induction of apoptosis in tumor cells. A "therapeutically effective amount" means an amount sufficient to influence the therapeutic course of a particular disease state. A therapeutically effective amount is also one in which any toxic or detrimental effects of the agent are outweighed by the therapeutically cial effects.

For therapeutic applications, the human or humanized antibody, or CHI- ning g fragment thereof, of the invention is administered to a mammal, preferably a human, in a pharmaceutically acceptable dosage form such as those discussed above, including those that may be administered to a human intravenously as a bolus or by continuous infusion over a period of time, by intramuscular, intraperitoneal, intracerebrospinal, subcutaneous, intraarticular, intrasynovial, intrathecal, oral, l, or inhalation routes. The said human or humanized dy, or CH2-containing binding fragment thereof, i s also suitably administered by intratumoral, peritumoral, intralesional, or perilesional routes, to exert local as well as systemic therapeutic effects. The intraperitoneal route is expected to be particularly useful, for example, in the treatment of ovarian tumors.

Dosage ns may be ed to provide the optimum response. For example, a single bolus may be administered, several divided doses may be administered over time, or the dose may be proportionally reduced or increased. The compositions of the invention can be stered to a subject to effect cell growth activity in a subject. As used herein, the term ct" is intended to e living organisms in which apoptosis can be induced, and ically includes mammals, such as rabbits, dogs, cats, mice, rats, monkey transgenic species f, and preferably humans.

The human or humanized antibody of the invention, or CH2-containing binding fragment thereof, and the pharmaceutical compositions of the invention are especially useful in the treatment or prevention of several types of cancers including (but not limited to) the following: carcinomas and adenocarcinomas, including that of the bladder, breast, colon, head-and-neck, prostate, kidney, liver, lung, ovary, as, stomach, cervix, thyroid and skin, and including squamous cell carcinoma ; hematopoietic tumors of lymphoid lineage, ing le myeloma, ia, acute and c lymphocytic (or lymphoid) leukemia, acute and chronic lymphoblastic leukemia, B-cell lymphoma, T-cell lymphoma, non-Hodgkin lymphoma (e.g. Burkitt's lymphoma) ; hematopoietic tumors of myeloid lineage, including acute and chronic myelogenous (myeloid or myelocytic) leukemias, and promyelocytic leukemia; tumors of mesenchymal origin, including fibrosarcoma, osteosarcoma and rhabdomyosarcoma; tumors of the central and peripheral s system, including astrocytoma, neuroblastoma, glioma, and schwannomas; and other tumors, including melanoma, teratocarcinoma, xeroderma pigmentosum, acanthoma, and seminoma, and other cancers yet to be determined in which CXCR4 is expressed. By cancers having CXCR4 expression, it i s herein referred to cancers displaying high CXCR4 expression, relative to the CXCR 4 expression level on a normal adult cell.

The human or humanized antibody of the invention, or CH2-containing binding fragment f, and the pharmaceutical compositions of the invention are mainly useful for treating leukemia, ma and cancers resistant to the commonly used anticancer agents as the anti-CXCR4 antibodies of the ion have a unique mechanism of action.

In a preferred embodiment of the method of the invention, said pathological condition associated with the presence of CXCR4 expressing cancer cells consists of lymphoma, leukemia or multiple a, preferentially ma.

In other words, the use according to the invention is characterized in that said pathological condition associated with the ce of CXCR4 expressing cancer cells consists of ma, ia or multiple myeloma, preferentially lymphoma.

Still in other words, the human or humanized antibody according to the invention is characterized in that said pathological condition associated with the presence of CXCR4 expressing cancer cells consists of lymphoma, leukemia or multiple myeloma, preferentially lymphoma.

As a non limitative example, a process of detecting in vitro the ce and/or the location of a CXCR4 expressing tumor in a subject, comprises the steps of: (a) contacting a sample from the subject with a humanized antibody heavy chain and/or a humanized antibody light chain and/or a humanized antibody, or a derived nd or functional fragment of same, according capable of binding specifically with CXCR4; (b) detecting the binding of said antibody with the sample.

Particularly, a process of determining in vitro or ex vivo the expression level of CXCR4 in a CXCR4 expressing tumor from a subject comprises the steps of: (a') contacting a sample from the subject with a humanized antibody heavy chain and/or a humanized antibody light chain and/or a humanized antibody, or a derived compound or functional fragment of same, e of binding specifically to CXCR4; and (b') quantifying the level of antibody binding to CXCR4 in said sample.

In a preferred embodiment, the CXCR4 expression level can be measured by immunohistochemistry (IHC) or FACS analysis.

As used herein, the term "an oncogenic er associated with expression of CXCR4" or "CXCR4-expressing cancer cell" is ed to include diseases and other disorders in which the presence of high levels or abnormally low levels of CXCR4 (aberrant) in a subject suffering from the disorder has been shown to be or is suspected of being either responsible for the pathophysiology of the disorder or a factor that contributes to a worsening of the disorder. atively, such disorders may be ced, for example, by an increase in the levels of CXCR4 on the cell surface in the ed cells or s of a subject suffering from the disorder. The se in CXCR4 levels may be detected, for example, using the antibody 515H7 or hz515H7 of the invention. More, it refers to cells which t relatively autonomous , so that they exhibit an aberrant growth phenotype characterized by a icant loss of control of cell proliferation. Alternatively, the cells may express normal levels of CXCR4 but are marked by abnormal proliferation.

The invention also describes a method for the screening of humanized antibodies binding CXCR4, or CH2-containing binding fragments thereof, for use in killing a CXCR4 expressing cancer cell by induction of at least one effector on, in the presence of effector cells or complement components, wherein said method comprises at least one selection step selected from: - selecting antibodies inducing an ADCC level on RAMOS lymphoma cells, after an incubation period of 4 hours, of at least 40%; - selecting antibodies inducing a CDC level on RAMOS lymphoma cells, after an incubation period of 1 hour, of at least 30%, preferentially of at least 50% and most preferably of at least 70%; - selecting dies inducing a CDC level on NIH3T3 CXCR4 cells, after an incubation period of 1 hour, of at least 30%, preferentially of at least 50% and most preferably of at least 70%; - selecting antibodies binding FcyRI with a constant of dissociation (KD), according to the Langmuir model, n 1 and 10 nM; - ing antibodies binding FcyRIIIA with a constant of dissociation (KD), according to the heterogeneous ligand model, between 200 and 1000 nM.

The ce of the invention s, unless other otherwise indicated, conventional techniques or protein chemistry, molecular virology, microbiology, recombinant DNA technology, and cology, which are within the skill of the art.

Such techniques are explained fully in the literature. (See Ausubel e t a , Current Protocols in Molecular Biology, Eds., John Wiley & Sons, Inc. New York, 1995; Remington's Pharmaceutical Sciences, 17th ed., Mack Publishing Co., Easton, Pa., 1985; and Sambrook e t a , Molecular cloning: A tory manual 2nd edition, Cold Spring Harbor Laboratory Press - Cold Spring Harbor, NY, USA, 1989; Introduction to Glycobiology, Maureen E . Taylor, Kurt Drickamer, Oxford Univ. Press ; Worthington Enzyme Manual, Worthington Biochemical Corp. Freehold, NJ; Handbook of Biochemistry: Section A Proteins, Vol I 1976 CRC Press; Handbook of Biochemistry: Section A Proteins, Vol II 1976 CRC Press; Essentials of Glycobiology, Cold Spring Harbor Laboratory Press ). The nomenclatures used in connection with, and the laboratory ures and techniques of, molecular and cellular biology, protein biochemistry, enzymology and medicinal and pharmaceutical chemistry described herein are those well known and commonly used in the art.

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as is commonly understood by one of the skill in the art to which this invention belongs.

Other teristics and advantages of the invention appear further in the description with the examples and figures whose legends are presented below.

FIGURE LEGENDS Figure 1 shows the amino acid sequences alignment of 515H7 heavy chain variable domain with the human germline IGHV3-49*04 and IGHJ4*01. The 515H7 VH amino acid sequence is aligned with the selected human acceptor framework sequences. VHl and VH2 (VH3 i s not represented) sequences correspond to ented humanized variants of the 515H7 VH domain, with back mutated residues in bold. Variant 1 VHl carries no back mutated residue and represents a fully human variant. Variant VH2 has 8 back mutations and is the most murine variant. Variant VH3 carries 5 back mutations (not represented).

Figure 2 shows the amino acid sequences alignment of 515H7 light chain with the human germline IGKV4-1*01 and IGKJ1*01. The 515H7 VL amino acid sequence is aligned with the selected human acceptor framework sequences. VL1 to VL3 ces correspond to implemented humanized variants of the 515H7 VL domain, with back mutated residues in bold. Variant VL1 carries no back mutated residue and ents the most human variant. Variant VL2 has 13 back mutations and is the most murine variant. t VL3 carries 5 back mutations.

Figures 3A-3F show cross blocking of the biotinylated murine antibody 515H7 by the chimeric 515H7 and different variants of the zed 515H7. The activity of the humanized variants of 515H7 (hz5 15H7) to cross block the parental murine antibody 515H7 was ted by flow try using CXCR4 transfected NIH3T3 cells. The activity of the humanized variants was compared to the chimeric 515H7. The cross ng activity of the three different variants of VH (VHl - VH3) ed with the chimeric VL (cVL) were very similar (Figure 3A - Figure 3C). No reduction in the activity of VH variant 1 (VHl, the variant with no back mutations) was determined when combined with variant 1 and 2 of VL. A significant reduction of the activity was detected for the uct hz5 15H7 VHl VL3 (Figures 3D - 3F).

Figure 4 shows the BRET assay for testing the ty of the humanized antibody 515H7 variant VHl VL1. The activity of the humanized variant 515H7 VH variant 1 VL variant 1 (hz515H7 VHl VLl) was evaluated by its ty to inhibit SDF-1 mediated signal transduction. This variant showed only a minor inhibition of the SDF-1 ed signal transduction as ined by BRET. SDF-1 was used at a concentration of lOOnM.

Figures 5A-5D show comparisons of different mutants of the VHl with single or double back mutations and combinations of different VL variants with hz5 15H7 VH1D76N. Single and double back mutations were made in the VHl and combined with the VLl. These constructs were evaluated in BRET assays (Figures 5A-5C). Of these single back mutants only the construct with the back mutation D76N showed an increased inhibition of the SDF-1 mediated signal transduction. None of the double back mutant in VH had strong inhibitory activity (Figure 5C). The single back mutant D76N of the VHl was combined with different variants of VL. The SDF-1 tration was lOOnM.

Figure 6 shows ranking of different mutants of the VHl and VLl with single or double back mutations in ison to the construct VHl D76N VL2. Single and double back mutations were made in the VHl and combined with the VLl . All constructs were evaluated in BRET assays and their percent inhibition calculated. The SDF-1 concentration was lOOnM.

Figures 7A-7B show inhibition of SDF-1 binding by different constructs of the humanized 515H7 and correlation between result obtained by FACS and BRET. The different variants of the humanized antibody 515H7 with a strong activity in ng the recruitment of b-arrestin were tested in their capacity to inhibit the binding of ylated SDF-1 in flow cytometry (FACS) (A). These were compared with VHl and VLl. Results from the FACS-based assay are correlated with the results ed by BRET (B).

Figure 8 shows the amino acid sequences alignment of hz515H7 VL2 and further humanized ns 515H7 VL2.1, 515H7 VL2.2 and 515H7 VL2.3. The 515H7 VL amino acid sequence is aligned with the ed human acceptor framework sequences. VL2.1, VL2.2 and VL2.3 sequences correspond to implemented humanized variants of the humanized 515H7 VL2, with mutated residues in bold. VL2. 1 and VL2.2 carry 4 more humanized residues whereas VL2.3 contains 5 more human residues.

Figures 9A-9C show the 515H7 humanized Mabs H7 VHl D76N VL2, hz515H7 VHl D76N VL2.1, hz515H7 VHl D76N VL2.2 and hz515H7 VHl D76N VL2.3) specific binding to CXCR4 on NIH3 T3-CXCR4 (Figure 9A) U937 (Figure 9B) and Ramos cells (Figure 9C).

Figures 10 show antibody dependent cellular cytotoxicity (ADCC) effect of hz515H7VHlD76NVL2 Mab on cells expressing CXCR4, Ramos cells e 10A) and l killer cells (NK) (Figure 10B) Figures 11 show dy dependent cellular xicity (ADCC) effect of c515H7 Mab on cells expressing CXCR4, Ramos cells (Figure 11A) and Natural killer cells (NK) (Figure 1IB) Figure 12 shows complement dependent cytotoxicity (CDC) effect of hz515H7VHlD76NVL2 Mab on NIH3 R4 cell line and Ramos cells expressing CXCR4 Figure 13 shows complement dependent cytotoxicity (CDC) effect of c515H7 Mab on Ramos cells expressing CXCR4 Figures 14 show complement dependent cytotoxicity (CDC) dose effect of hz515H7VHlD76NVL2 (Figure 14A) and c515H7 (Figure 14B) Mabs on Ramos cells expressing CXCR4 Figure 15: Binding of the inant human FcyRI with hz5 15H7VHlD76NVL2 Mab immobilized on a CM4 sensorchip. 6 different concentrations of h-FcyRI were tested (200, 100, 50, 25, 12.5 and 6.25 nM).

Figure 16 : Binding o f the recombinant human FcyRIIIA with hz5 15H7VHlD76NVL2 Mab immobilized on a CM4 sensorchip. 5 different concentrations of h-FcyRIIIA were tested (1000, 500, 250, 125 and 62.5 nM).

Figure 17: nt of Dissociation determination of the h- FcYRIIIA/hz515H7VHlD76NVL2 complex by steady-state analysis using the response at the end of the association phase versus the human FcyRIIIA concentrations (1000, 500, 250, 125 and 62.5 nM) plot.

Figure 18: Binding of the recombinant mouse FcyRI with hz5 15H7VHlD76NVL2 Mab immobilized on a CM4 sensorchip. 5 different concentrations of m-FcyRI were tested (400, 200, 100, 50 and 25nM).

Figure 19 : Constant of Dissociation determination of the m- FcyW/hz515H7VHlD76NVL2 complex by steady-state analysis using the response at the end of the association phase versus the mouse FcyRI concentrations (400, 200, 100, 50 and 25nM) plot.

F i g u r e 2 0 : B i n d i n g o f the recombinant mouse FcyRIII with hz5 15H7VHlD76NVL2 Mab immobilized on a CM4 sensorchip. 5 different concentrations of m-FcyRIII were tested (400, 200, 100, 50 and 25nM).

Figure 21: Rancking of the four Fc gamma receptors g with hz- 515H7VH1D76NVL2 Mab (one component with I and m-FcyRIII and two ents with h-FcyRIIIA and m-FcyRI) on a constant of dissociation (in nMolar) plot in function of the ife (in minute) of the hz515H7VHlD76NVL2 Mab/Fc gamma receptor complexes.

Figure 22: Binding of the recombinant human FcyRI with c5 15H7 Mab immobilized on a CM4 sensorchip. 6 different concentrations of h-FcyRI were tested (200, 100, 50, 25, 12.5 and 6.25 nM).

Figure 23: Binding of the recombinant human FcyRIIIA with c5 15H7 Mab immobilized on a CM4 chip. 5 different concentrations of h-FcyRIIIA were tested (1000, 500, 250, 125 and 62.5 nM).

Figure 24: Constant of Dissociation determination of the h-FcyRIIIA/cS 15H7 complex by steady-state analysis using the response at the end of the association phase versus the human FcyRIIIA concentrations (1000, 500, 250, 125 and 62.5 nM) plot.

Figure 25: Binding of the recombinant mouse FcyRI with c5 15H7 Mab immobilized on a CM4 sensorchip. 5 different concentrations of m-FcyRI were tested (400, 200, 100, 50 and 25nM).

Figure 26: Constant of Dissociation determination of the m- FcyRI/c515H7complex by steady-state analysis using the response at the end of the ation phase versus the mouse FcyRI concentrations (400, 200, 100, 50 and 25nM) plot.

Figure 27: Binding of the recombinant mouse FcyRIII with c5 15H7 Mab immobilized on a CM4 sensorchip. 5 different concentrations of m-FcyRIII were tested (400, 200, 100, 50 and 25nM).

Figure 28: Rancking of the four Fc gamma receptors binding with c515H7 Mab (one component with h-FcyRI and III and two components with h-FcyRIIIA and m-FcyRI) on a constant of dissociation (in nMolar) plot in function of the ife (in minute) of the c515H7 Mab/Fc gamma receptor complexes.

Figure 29 shows antibody dependant cellular cytotoxicity (ADCC) effect of hz515H7VHlD76NVL2 (hz5 15H7) Mab on cells expressing CXCR4: RAMOS, DAUDI and HeLa cells.

Figure 30 shows complement dependant cytotoxicity (CDC) effect of hz515H7VHlD76NVL2 (hz515H7) Mab on cells expressing CXCR4: DAUDI and RAMOS cells.

EXAMPLES Example 1: Generation of onal antibodies (Mabs) against human CXCR4 To generate monoclonal antibodies to CXCR4, Balb/c mice were zed with recombinant NIH3T3-CXCR4 cells and/or peptides corresponding to CXCR4 ellular N-term and loops. The mice 6-1 6 weeks of age upon the first immunization, were immunized once with the n in complete Freund's adjuvant subcutaneously (s.c.) followed by 2 to 6 immunizations with antigen in incomplete Freund's adjuvant s.c. The immune response was monitored by rbital bleeds. The serum was screened by ELISA (as described bellow) and mice with the higher titers of anti-CXCR4 antibodies were used for fusions. Mice were boost intravenously with antigen two days before sacrifice and removal of the .

- ELISA To select the mice producing anti-CXCR4 antibodies, sera from immunized mice was tested by ELISA. Briefly, microtiter plates were coated with purified [1-41] N-terminal peptide conjugated to BSA at 5 g equivalent peptide/mL, IOOm II incubated at 4°C overnight, then blocked with 25C^L/well of 0.5% gelatin in PBS.

Dilutions of plasma from CXCR4-immunized mice were added to each well and ted 2 hours at 37°C. The plates were washed with PBS and then incubated with a goat anti-mouse IgG antibody conjugated to HRP (Jackson Laboratories) for 1 hour at 37°C. After washing, plates were developed with TMB substrate, the reaction was stopped 5 min later by addition of 100 m n ΐ ΐ 1M H2S0 4. Mice that developed the highest titers of anti-CXCR4 antibodies were used for antibody generation.

- Generation of hybridomas producing Mabs to CXCR4 The mouse splenocytes, isolated from a Balb/c mice that ped the highest titers of anti-CXCR4 antibodies were fused with PEG to a mouse a cell line Sp2/0. Cells were plated at approximately l x 105 /well in iter plates followed by two weeks incubation in selective medium containing ultra culture medium + 2 mM L- glutamine + 1 mM sodium pyruvate + l x HAT. Wells were then screened by ELISA for anti-CXCR4 monoclonal IgG antibodies. The antibody secreting hybridomas were then subcloned at least twice by ng dilution, cultured in vitro to generate antibody for further analysis.

Example 2 : Characterization by FACS analysis of anti-CXCR4 Mab 515H7 g specificity and cancer cell lines recognition In this experiment, specific binding to human CXCR4 of anti-CXCR4 Mab 515H7 was ed by FACS analysis.

NIH3T3, NIH3T3-hCXCR4 transfected cells, MDA-MB-23 1, Hela and U937 cancer cell lines were ted with 10 g/mL of monoclonal antibody 515H7. The cells were then washed with 1%BSA/PBS/0.01% NaN3 . Next, Alexa-labeled secondary antibodies were added to the cells and were d to incubate at 4°C for 20 min. The cells were then washed again two times. Following the second wash, FACS analysis was performed. Results of these binding studies are provided in the following Table 6 which shows [Mean Fluorescence Intensity (MFI) obtained by FACS] that anti-CXCR4 Mab 515H7 bound specifically to human CXCR4-NIH3T3 transfected cell line whereas there was no recognition on the parent NIH3T3 cells. This Mab was also able to recognize human cancer cell lines, for examples MDA-MB-23 1 breast cancer cells, U937 promyelocytic cancer cells and Hela cervix cancer cells.

Anti-CXCR4 Mab 515H7 recognized NIH3T3-hCXCR4 transfectant while there was no recognition of the parent NIH3T3 wild type cells. Mab 515H7 was also able to recognize cancer cell lines.

Table 6 Example 3: Humanization of 515H7 anti-CXCR4 murine antibody l procedure Humanization of 515H7 anti-CXCR4 antibody was performed by applying the global rules of CDR-grafting. Immunogenetic analysis and definition of CDR and framework (FR) s were performed by applying the IMGT unique numbering scheme as well as the IMGT libraries and tools (Lefranc, 1997 - www.imgt.org) .

The efficiency of the humanization process was evaluated by testing the functional activity of the engineered antibodies for their ability to t the SDF-1 - ed recruitment of b-arrestin by a Bioluminescence Resonance Energy er (BRET) assay. In this assay CXCR4 was tagged with luciferase and b-arrestin with YFP. The SDF-1 mediated recruitment of b-arrestin to CXCR4 is an ant step in the signal transduction of CXCR4. Binding of humanized variants of 515H7 was also determined on a NIH3T3 cell line stably transfected with human CXCR4. The binding activity was evaluated by a competition assay with the biotinylated mouse antibody. In a second t, humanized antibodies were ted for their ability to inhibit binding of biotinylated SDF-1 to RAMOS cells. RAMOS cells were chosen because of their high expression of CXCR4 and low expression of CXCR7 and SDF-1.

These assays were used to characterize the recombinant humanized versions of anti-CXCR4 antibodies. le domains were formatted with human IgGl/k constant domains and cloned into the mammalian expression vector pCEP. Recombinant IgGi/kderived antibodies were transiently expressed in HEK293 cells. Expression culture atants were filtered and antibodies were purified using protein A sepharose.

Purified antibodies were re-buffered in PBS and antibodies concentrations determined by ELISA.

Humanization of 515H7 variable domains In order to select an riate human germline for the CDR grafting, the human germline gene with the highest homology to the 515H7 VH murine sequence was identified. With the help of IMGT databases and tools, the human 49*04 germline gene and human IGHJ4*01 J germline gene were selected as human acceptor sequences for the murine 515H7 VH CDRs. The human V-gene IGHV3-49*04 has a homology of 80.27% to the V-gene of the le domain of the mouse 515H7 heavy chain. The homology for the human J-gene IGHJ4*01 J is 87.50%. Nineteen residues are ent between the chosen human germline genes and the VH domain of the mouse antibody 515H7. The alignment between the VH domain of the parental antibody and the germline sequences is depicted in Figure 1 .

Concerning the variable domain of the light chain, the human germline genes IGKV4-1*01 and IGKJ1*01 were selected (Figure 2). The homology with human V- gene IGKV4-1*01 is 79.12%. The 515H7 J-gene of the light chain has a homology of 84.85% to the human J-gene IGKJ1*01.

The amino acid ce of the translated human germline genes IGHV3-49*04 and IGKV4-1 *01 was used t o identify homologous dies that have been crystallized. For the heavy chain the dy with the accession number IMAM at the RCSB Protein Data Bank was chosen as a model, while for the light chain the antibody 1SBS was chosen. The two domains were led using the computer program DS visual and used as a model for the humanized antibody 515H7.

Based on the position of each residue that is different between the parental antibody and the corresponding human germline sequence, a priority rank order was given for each residue differing between the human and mouse sequences (Figures 1 and 2). These priorities were used to create three different variants of each humanized variable domain named VH1, VH2 and VH3, respectively.

In a first series of experiments, we constructed and analysed the anti-CXCR4 g activities of the three first humanized variants. The VH t 1 (VH1) was combined with the murine VL and these constructs were ted in their capacity to inhibit the binding of a biotinylated murine 515H7 parental antibody. All constructs showed similar capacity to compete with the murine antibody (Figure 3A-C). This indicates that the most human VH t has the same binding capacity as the lesser human variants. Therefore, VH1 was ed with the three different ts of VL (Figure 3D-F). Only the combination of VH1 and VL3 showed a reduced capacity to compete with the biotinylated murine antibody, while the most human variant VH1 VL1 that carries no back mutations in the frameworks showed the same cross ng activity as the chimeric dy.

This variant VH1 VL1 was r tested for its capacity to inhibit SDF-1 mediated recruitment of b-arrestin in BRET assays (Figure 4). Despite desirable binding activity to the receptor as determined by cross blocking of the parental antibody, the construct VH1 VL1 showed only a weak inhibition of the recruitment of stin. This lack of strong inhibitory activity makes substitution of human framework residues with murine es necessary. Single back ons were constructed for the VH 1 . The following es were substituted: V48L, E61D, D76N and A81L (numbering ing to the primary amino acid sequence). These single back mutants of the variant VH1 were combined with the variant VLl. Of these only the back on D76N led to an increased inhibition of the signal transduction as evaluated by BRET assay (Figure 5B).

To increase the activity of this construct and further te the importance of other residues different double back mutants were constructed for the VH 1. The inhibitory activity of these constructs was slightly improved (average inhibition of about 45-50 %), but not satisfactory (Figure 5C). The single back mutant D76N was then combined with the three different VL variants (Figure 5D). The construct hz515H7 VH D76N VL2 showed an activity of 88.2 % on average which is in the same range as the chimeric antibody.

Single and double back mutations were constructed in the variant VLl domain and compared to the activity of the construct hz515H7 VH1 D76N VL2 (Figure 6).

None of the tested combinations had a similar or better activity as this construct.

The percentage of human residues in the framework was calculated for hz515H7 VH1 D76N VL2: it contains 14 non-human residues out of 180 residues, which equals a « germinality index » of 92.2 % . By way of comparison, the humanized and marketed antibodies bevacizumab and trastuzumab contain respectively 30 and 14 non-human residues in their variable domains.

The four best humanized forms, showing the strongest efficacy to inhibit SDF-1 - mediated b-arrestin recruitment were also tested for their capacity to inhibit the binding of ylated SDF-1 (Figure 7A). A close correlation of inhibition of SDF-1 binding and b-arrestin recruitment was determined. This correlation indicates that the inhibition of SDF-1 binding is most likely the main mechanism of the inhibition of the signal transduction.

In order to further humanize the hz515H7 VL2 variant, three additional variants were designed, by using the information gained with the double and triple mutants ted in Figure 6 . Four and five additional residues were humanized in respectively t VL2.1, VL2.2 and VL2.3 (also referred as VL2-1, VL2-2 and VL2-3). They correspond to the residues D9, P49, D66, S69, S83, L84; V89. An alignment of these three variants in comparison with VL2 is shown Figure 8 .

The capacity of these VL2 variants to inhibit the SDF-1 ed recruitment of b-arrestin was evaluated. The humanized 7 VH D76N VL2, VL2.1, VL2.2 and VL2.3 ts showed an activity similar to the chimeric dy c515H7 (Figure 6).

Example 4 : Characterization by FACS analysis of anti-CXCR4 humanized Mabs 515H7 g specificity and cancer cell line recognition In this experiment, specific g to human CXCR4 of anti-CXCR4 humanized Mabs 515H7 was examined by FACS analysis.

NIH3T3, NIH3T3-hCXCR4 transfected cells and Ramos, U937 cancer cell lines were incubated with 0 to 10 g/mL of humanized Mabs 515H7 (hz515H7 VHl D76N VL2, hz5 15H7 VHl D76N VL2. 1, hz515H7 VHl D76N VL2.2 and hz5 15H7 VHl D76N VL2.3) for 20 min at 4°C in the dark in 100 mΐ Facs buffer. After 3 washing in Facs , cells were incubated with the ary antibody, a goat anti-human Alexa 488 (dilution 1/500), for 20 minutes at 4°C in the dark. After 3 washing in Facs buffer, propidium iodide was added in each well and only viable cells were analyzed by Facs.

At least 5000 viable cells were assessed to evaluate the mean value of fluorescence intensity for each condition.

Results of these binding studies are provided in Figures 9A-9C which show [Mean Fluorescence Intensity (MFI) obtained by FACS] that anti-CXCR4 humanized Mabs hz5 15H7 bound specifically to human CXCR4-NIH3T3 transfected cell line (Figure 9A) (MFI= 2.2 with N G 3T3 parent cells) and also recognize human cancer cell lines, for example U937 e 9B) and Ramos (Figure 9C).

Example 5 : Antibody dependent cellular cytotoxicity (ADCC) effect of hz515H7VHlD76NVL2 Mab on cells expressing CXCR4 ADCC was measured by a e dehydrogenase (LDH) releasing assay using the Cytotoxicity Detection Kit L S (Roche Applied Science, Indianapolis, IN, USA) according to the manufacturer's instructions. Lactate dehydrogenase is a soluble cytosolic enzyme that is released into the culture medium following loss of membrane ity resulting from either apoptosis or necrosis. LDH activity, therefore, can be used as an indicator of cell membrane integrity and serves as a l means to assess cytotoxicity, including ADCC.

Peripheral blood mononuclear cells (PBMC) were isolated from human buff coats obtained from healthy , using a Ficoll density gradient (Ficoll-Paque PLUS, GE Healthcare, Amersham, UK). Natural Killer (NK) cells were separated from the PBMC fraction according to the RoboSep® Human NK Cell Enrichment Kit cturer's protocol (StemCell Technologies). NK cells were plated in 96-well flat bottom plates at an effector-to-target ratio of 50:1 at 50 per well. 10000 Target cells (Ramos), pre-incubated with antibodies at room temperature for 10 min, were added on effector cells at 50 m II. After incubation for 4 h at 37°C, the cytotoxicity was determined by measuring the amount of LDH released. t of xicity was calculated as follows: % lysis = [experimental e - effector and target spontaneous release] / [target maximum release - target spontaneous release] x 100.

Figure 10 shows ADCC on Ramos cells expressing high level of CXCR4 and on NK cells alone [CXCR4 levels (MFI): Ramos > NK cells]. Black columns: Hz515H7VHlD76NVL2 (hz515H7VL2) (10 g/mL), white columns: isotype control hlgGl (10 g/mL). No effect was ed when cells were incubated with the hlgGl isotype control (Figures 10A and 10B). In contrast, hz515H7VHlD76NVL2 Mab was able to induce icant ADCC (47.9 % +/- 8.9) on Ramos cells (Figure 10A) whereas there was no significant ADCC (3 % +/- 3) on NK cells expressing low level of CXCR4 (Figure 10B).

Example 6 : dy dependent cellular cytotoxicity (ADCC) effect of c515H7 Mab on cells expressing CXCR4 ADCC was measured by a lactate dehydrogenase (LDH) releasing assay using the Cytotoxicity Detection Kit L S (Roche Applied Science, Indianapolis, IN, USA) according to the manufacturer's instructions.

Peripheral blood clear cells (PBMC) were isolated from human buff coats obtained from y donors, using a Ficoll density gradient (Ficoll-Paque PLUS, GE Healthcare, Amersham, UK). Natural Killer (NK) cells were separated from the PBMC fraction according to the RoboSep® Human NK Cell Enrichment Kit manufacturer's protocol ell Technologies). NK cells were plated in 96-well flat bottom plates at an effector-to-target ratio of 50:1 at 50 per well. 10000 Target cells (Ramos), pre-incubated with antibodies at room temperature for 10 min, were added on effector cells at 50 m / . After incubation for 4 h at 37°C, the cytotoxicity was determined by measuring the amount of LDH released. Percent of cytotoxicity was calculated as follows: % lysis = [experimental release - effector and target spontaneous release] / [target maximum release - target spontaneous release] x 100.

Figure 11 shows ADCC on Ramos cells expressing high level of CXCR4 and on NK cells alone [CXCR4 levels (MFI): Ramos > NK cells]. Black s: c515H7 (10 g/mL), white columns: isotype l hlgGl (10 g/mL). No effect was observed when cells were incubated with the hlgGl isotype control es 11A and 11B). In contrast, c515H7 Mab was able to induce significant ADCC (61.4 % +/- 8.1) on Ramos cells (Figure 11A) whereas there was no significant ADCC (5.4 % +/- 4.6) on NK cells expressing low level of CXCR4 (Figure 1IB).

Example 7 : Complement dependent cytotoxicity (CDC) effect of 7VHlD76NVL2 Mab on cells sing CXCR4 CDC assay was based on ATP measurement using CellTiter Glo reagent (Promega, n, WI, USA).

Briefly, 10000 target cells were plated in 96-well flat bottom plates in presence of hz515H7VHlD76NVL2. Following incubation at room temperature for 10 minutes, pooled human serum from healthy donors was added at a final concentration of 10%.

After l h at 37°C, viability was determined by measuring the amount of ATP. Percent of cytotoxicity was calculated as follows: % Cytotoxicity = 100 - [[experimental / target cell without antibody] x 100].

Figure 12 shows CDC on Ramos and NIH3T3-CXCR4 cell lines expressing high levels of CXCR4. Black columns: Hz515H7VHlD76NVL2 (hz515H7VL2) (10 g/mL), white columns: isotype control hlgGl (10 g/mL). No effect was observed when cells were incubated with the hlgGl isotype control (Figure 12). In contrast, hz515H7VHlD76NVL2 Mab was able to induce significant CDC (around 80 %) on both NIH/3 4 and RAMOS cell lines (Figure 12). e 8 : Complement dependent cytotoxicity (CDC) effect of c515H7 Mab on Ramos cells expressing high level of CXCR4 CDC assay was based on ATP measurement using CellTiter Glo reagent (Promega, Madison, WI, USA).

Briefly, 10 000 Ramos cells were plated in 96-well flat bottom plates in presence of Mabs. Following incubation at room temperature for 10 minutes, pooled human serum from y donors was added at a final concentration of 10%. After l h at 37°C, viability was ined by measuring the amount of ATP. Percent of cytotoxicity was ated as follows: % Cytotoxicity = 100 - [[experimental / target cell without antibody] x 100].

Figure 13 shows CDC on Ramos cell line expressing high level of CXCR4.

Black columns: c515H7 (10 g/mL), white columns: isotype control hlgGl (10 g/mL).

No effect was observed when cells were incubated with the hlgGl isotype control e 13). In st, c515H7 Mab was able to induce significant CDC (34%) on RAMOS cells (Figure 13) Example 9 : ment dependent cytotoxicity (CDC) dose effect of hz515H7VHlD76NVL2 and c515H7 Mabs on Ramos cells expressing high level of CXCR4 CDC assay was based on ATP measurement using CellTiter Glo reagent (Promega, Madison, WI, USA).

Briefly, 10 000 Ramos cells were plated in l flat bottom plates in presence of Mabs. Following incubation at room temperature for 10 minutes, pooled human serum from healthy donors was added at a final tration of 10%. After l h at 37°C, viability was determined by measuring the amount of ATP. Percent of cytotoxicity was calculated as follows: % Cytotoxicity = 100 - [[experimental / target cell without antibody] x 100].

Figures 14 show CDC on Ramos cell line expressing high level of CXCR4.

Black columns: either hz515H7VHlD76NVL2 (hz515H7VL2) (Figure 14A) or c515H7 (Figure 14B) (10 g/mL), white columns: isotype control hlgGl (10 g/mL). No effect was observed when cells were ted with the hlgGl isotype control (Figures 14A and 14B). In contrast, hz515H7VHlD76NVL2 (Figure 14A) and c515H7 (Figure 14B) Mabs were able to induce significant CDC on Ramos cells with CDC max of 74% and 34% , respectively, with E C o of 0.033 g/mL and 0.04 g/mL, respectively.

Example 10: Study of the interaction between hz515H7VHlD76NVL2 Mab and h-FcyRI, h-FcyRIIIA, m-FcyRI and m-FcyRIII by real time Surface Plasmon Resonance.

The experiments were d out using a Biacore X . The soluble forms of the four Fc □ gamma receptors used in this study were purchased from R&D Systems: 1-Recombinant human FcyRI [CD64] corresponds t o the Glnl 6-Pro288 fragment with a C-terminal 6-His tag [catalog number: 1257-FC]. The molecular weight of 50 kDa (specified by the supplier) used in this study ponds to the mean of the molecular weight defined by SDS-PAGE in reducing condition. 2- Recombinant human FcyRIIIA t V [CD 16a] corresponds to the Glnl7- Gln208 fragment with a C-terminal 6-His tag [catalog : 4325-FC]. The molecular weight of 45 kDa (specified by the supplier) used in this study corresponds to the mean of the molecular weight defined by SDS-PAGE in reducing condition. 3- Recombinant mouse FcyRI [CD64] corresponds to the Gln25-Pro297 fragment with a inal 6-His tag [catalog number: 2074-FC]. The molecular weight of 55 kDa (specified by the supplier) used in this study corresponds to the mean of the molecular weight defined by SDS-PAGE in ng condition. 4- Recombinant mouse FcyRIII [CD 16] corresponds to the Ala31-Thr215 fragment with a C-terminal 10-His tag [catalog number: 1960-FC]. The molecular weight of 37.5 kDa (specified by the supplier) used in this study corresponds to the mean of the molecular weight defined by SDS-PAGE in reducing condition.

The other reagents were ed by Biacore (GE Healthcare). 1964 RU of 7VHlD76NVL2 Mab were immobilized using the amine coupling kit chemistry on the second flowcell (FC2) of a CM4 chip. The first ll (FCl) activated by NHS and EDC e and tivated by ethanolamine served as the reference surface to check and subtract the non specific interaction between the analyte (Fc gamma receptors) and the sensorchip matrix.

The kinetic experiments were carried out at 25° Celsius at a flow rate of 30m1/ p h. The HBS-EP buffer was used either as the running buffer or for the preparation of analyte solutions. The analyte solutions were injected during 90 seconds iation phase) with a 90 seconds delay ciation phase). An ion of running buffer as analyte was used as a double reference. All the sensorgrams were corrected by this double reference sensorgram.

After each ion of the analyte, the sensorchip was regenerated by injection of either 20 mM NaOH solution after h-FcyRI and m-FcyRIII or 10 mM NaOH after h- FcyRIIIA and m-FcyRI.

Two mathematical models were used t o analyze the sensorgrams: the "Langmuir" and the "heterogeneous ligand" models.

Sensorgrams ed with h-FcyRI [Figure 15] were not perfectly fitted by the Langmuir model (5% < Chi2/Rmax < 20%) but the "heterogeneous ligand" model did not improve the quality of the fitting. Using the Langmui r model, the constant of dissociation was in the nanomolar range (0.9 ± 0.1 nM).

Sensorgrams obtained with h-FcyRIIIA [Figure 16] were clearly not fitted by the Langmuir model (Chi2/Rmax > 20%). The "heterogeneous ligand" model improved significantly the quality of the fitting (Chi2/Rmax < 5%). ing to this model, the hz5 15H7VHlD76NVL2 Mab Fc domain may be regarded as a mixture of two components. The major one representing 79% of the total amount showed a constant of dissociation between 300 and 350nM, the minor one (21%) showed a constant of dissociation between 27 and 32nM. According to the literature, the heterogeneity observed with h-FcyRIIIA was probably linked to the glycosylation heterogeneity on the Mab Fc domain.

A plot representing a mean of the response in RU (close to Req) at the end of the association phase versus the h-FcyRIIIA concentration (C) can be fitted with the mathematical model: Req = (KA .C.R )/KA.C.n+l), with n = 1 [Figure 17]. The constant of dissociation KD corresponding to 1/KA is the equal to 176 nM.

Sensorgrams obtained with m-FcyRI [Figure 18] may be fitted by the Langmuir model (5% < Chi2/Rmax < 10%) but the "heterogeneous ligand" model improved significantly the quality of the fitting (Chi2/Rmax < 1%). According to this model, the hz5 15H7VHlD76NVL2 Mab Fc domain may be ed as a mixture of two components. The major one enting 82% of the total amount showed a nt of iation between 75 and 80 nM, the minor one ( 18%) showed a constant of dissociation around 90 nM. Even if the constant of dissociation were close, the cs rates were significantly different (the association rate was 5.7 time better for the major component but its dissociation rate was 4.8 time quicker).

A plot representing a mean of the response in RU (close to Req) at the end of the association phase versus the m-FcyRI concentration (C) can be fitted with the mathematical model: Req = (KA.C.Rmax)/(KA.C.n+l) with n = 1 [Figure 19]. The nt of iation KD corresponding to 1/KA is the equal to 95 nM.

Sensorgrams obtained with m-FcyRIII [Figure 20] were not perfectly fitted by the Langmuir model (5% < Chi2/Rmax < 20%) but the "heterogeneous ligand" model did not improve the quality of the fitting. Using the Langmui r model the constant of dissociation was around 17 and 18 nM.

A ranking of the four Fc gamma receptors is presented in Figure 2 1 representing Kd plot in on of the half-life of the complex. In accordance with the literature, h- FcyRI binds with high affinity and h-FcyRIIIA with a lower affinity to the Fc part of a human IgGl isotype antibody. m-FcyRIII binds with an ediate affinity between the affinity of the major component of hz-515H7VHlD76NVL2 Mab for IIIA and the affinity of h-FcyRI. Both components of the hz5 15H7VH1D76NVL2 Mab interact with m-FcyRI with an intermediate affinity between the affinities of both components of hz515H7VHlD76NVL2 for h-FcyRIIIA.

These experiments y showed that the hz515H7VHlD76NVL2 Mab Fc domain interacts significantly with the four FcyR tested.

Example 11: Study of the interaction between c515H7 Mab and h-FcyRI, h- FcyRIIIA, m-FcyRI and m-FcyRIII by real time Surface Plasmon Resonance.

The experiments were carried out using a Biacore X device. The soluble forms of the four Fc □ gamma receptors used in this study were sed from R&D Systems: 1-Recombinant human FcyRI [CD64] corresponds t o the Glnl 6-Pro288 fragment with a C-terminal 6-His tag [catalog number: 1257-FC]. The molecular weight of 50 kDa (specified by the supplier) used in this study corresponds to the mean of the molecular weight defined by SDS-PAGE in reducing condition. 2- Recombinant human IA variant V [CD 16a] ponds to the Glnl7- Gln208 fragment with a C-terminal 6-His tag [catalog number: 4325-FC]. The molecular weight of 45 kDa (specified by the supplier) used in this study corresponds to the mean of the molecular weight defined by GE in reducing condition. 3- Recombinant mouse FcyRI [CD64] corresponds to the Pro297 fragment with a C-terminal 6-His tag [catalog number: 2074-FC]. The molecular weight of 55 kDa (specified by the er) used in this study corresponds to the mean of the molecular weight defined by SDS-PAGE in ng condition. 4- Recombinant mouse FcyRIII [CD 16] corresponds to the Ala31-Thr215 fragment with a C-terminal 10-His tag [catalog number: 1960-FC]. The molecular weight of 37.5 kDa (specified by the supplier) used in this study ponds to the mean of the molecular weight defined by SDS-PAGE in reducing condition.

The other reagents were supplied by Biacore (GE Healthcare). 2017 RU of c5 15H7 Mab were immobilized using the amine coupling kit chemistry on the second flowcell (FC2) of a CM4 sensorchip. The first flowcell (FC1) activated by NHS and EDC mixture and des-activated by ethanolamine served as the reference surface to check and subtract the non specific interaction between the analyte (Fc gamma receptors) and the sensorchip .

The kinetic experiments were carried out at 25° Celsius at a flow rate of 30m1/ p h. The HBS-EP buffer was used either as the running buffer or for the preparation of analyte solutions. The analyte ons were injected during 90 seconds (association phase) with a 90 seconds delay (dissociation . An injection of running buffer as analyte was used as a double reference. All the sensorgrams were corrected by this double reference sensorgram.

After each injection of the e, the sensorchip was regenerated by injection of 20 mM NaOH, 75mM NaCl solution.

Two mathematical models were used t o analyze the sensorgrams: the "Langmuir" and the "heterogeneous ligand" models.

Sensorgrams obtained with h-FcyRI [Figure 22] were not perfectly fitted by a Langmuir model (Chi2/Rmax > 10%) but the "heterogeneous ligand" model did not improve the quality of the fitting. Using the Langmui r model, the constant of dissociation was close to the nanomolar range (1.2 ± 0.1 nM).

Sensorgrams ed with h-FcyRIIIA [Figure 23] were clearly not fitted by a Langmui r model (Chi2/Rmax > 20%). The "heterogeneous " model improved icantly the quality of the fitting (Chi2/Rmax < 5%). According to this model the c515H7 Mab Fc domain may be regarded as a mixture of two components. The major one representing 81% of the total amount shows a constant of dissociation between 380 and 450nM, the minor one (19%) showed a constant of dissociation between 32 and 37nM. According to the ture the heterogeneity observed with h-FcyRIIIA was probably linked to the ylation heterogeneity on the Mab Fc domain. The end of the association phase was close to reach the steady-state. A plot representing a mean of the response in RU (close to Req) at the end of the association phase versus the h- IA tration (C) can be fitted with the mathematical model: Req= (KA.C.R )/KA.C.n+l) with n=l [Figure 24]. The constant of dissociation KD corresponding to 1/KA was 160 nM.

Sensorgrams obtained with m-FcyRI [Figure 25] may be fitted by a Langmui r model (5% < Chi2/Rmax < 10%) but the "heterogeneous " model improved significantly the quality of the fitting (Chi2/Rmax < 2%). According to this model, the c515H7 Mab Fc domain may be regarded as a mixture of two components. The major one representing 81% of the total amount showed a constant of dissociation around 380 and 450 nM, the minor one (19%) showed a constant of dissociation between 32 and 37 The end of the association phase was close to reach the steady-state. A plot representing a mean of the response in RU (close to Req) at the end of the association phase versus the the I concentration (C) can be fitted with the mathematical model: Req = (KA .C.R )/KA.C.n+l) with n=l [Figure 26]. The dissociation constant KD corresponding to 1/KA is the equal to 107nM.

Sensorgrams obtained with m-FcyRIII [Figure 27] were not perfectly fitted by a Langmuir model (10% < Chi2/Rmax < 20%) but the "heterogeneous " model did not improve the quality of the fitting. Using the Langmui r model the constant of dissociation was around 20 nM.

A ranking of the four Fc gamma receptors is presented in Figure 28 representing Kd plot in function of the half-life of the complex. In accordance with the literature, h- FcyRI binds with high affinity and h-FcyRIIIA with a lower affinity to the Fc part of a human IgGl isotype antibody. m-FcyRIII binds with an ediate affinity between the affinity of the major component of c515H7 Mab for h-FcyRIIIA and the ty for h-FcyRI. Both components of the c515H7 Mab interact with I in a similar way than with h-FcyRIIIA.

Example 12: Antibody dependant cellular cytotoxicity (ADCC) effect of hz515H7VHlD76NVL2 H7) Mab on cells sing CXCR4 ADCC was measured using the lactate dehydrogenase (LDH) release assay described above (see example 5).

Briefly, human PBMCs were isolated from volunteer healthy ' blood using a Ficoll density gradient. NK cells were purified from the PBMCs fraction according to the Human NK Cell Enrichment Kit manufacturer's ol. NK cells, used as effector cells (E), were mixed with RAMOS (lymphoma), DAUDI (lymphoma) or HeLa x cancer) tumor target cells (T) at an E : T ratio of 50: 1, said tartget cells having been usly pre-incubated for 10 minutes at room temperature with the hz515H7VHlD76NVL2 (hz515H7) antibody (10 m \). After 4 hours incubation at 37°C, specific cell lysis was determined by measuring the amount of LDH released with the Cytotoxicity Detection Kit L S according to the manufacturer's instructions.).

Percent of xicity was calculated as follows: % lysis = [experimental release - effector and target spontaneous release] / [target maximum release - target spontaneous release] x 100.

Figure 29 shows ADCC on cells expressing CXCR4: RAMOS, DAUDI and HeLa cells. No effect was observed when cells were incubated with the hlgGl isotype control (10 g/ml). In st, hz515H7 Mab (10 g/ml) was capable of inducing icant ADCC d 40%) on RAMOS, DAUDI and HeLa cells.

Example 13: Complement dependant cytotoxicity (CDC) effect of hz515H7VHlD76NVL2 (hz515H7) Mab on cells expressing CXCR4 CDC assay was based on ATP ement using CellTiter Glo reagent (Promega, Madison, WI, USA), as described in example 7 .

Briefly, 10000 target cells were plated in 96-well flat bottom plates in presence of hz515H7VHlD76NVL2 (hz515H7) Mab. Following incubation at room temperature for 10 minutes, pooled human serum from y donors was added at a final concentration of 10%. After l h at 37°C, viability was determined by measuring the amount of ATP. Percent of cytotoxicity was calculated as follows: % Cytotoxicity = 100 - [[experimental / target cell without antibody] x 100].

Figure 30 shows CDC on cell lines expressing CXCR4: RAMOS and DAUDI cells. No effect was observed when cells were incubated with the hlgGl isotype control (^g/mL). In contrast, hz515H7VHlD76NVL2 (hz515H7-l) Mab (10 g/mL) was able to induce significant CDC: around 58% for RAMOS cells and 36% for DAUDI cells.

Claims (20)

1. The use of a humanized antibody binding to CXCR4, or a CH2-containing binding fragment thereof, said zed antibody comprising a heavy chain variable domain selected from the sequences SEQ ID No. 7 to 10 and a light chain le domain selected from the sequences SEQ ID No. 11 to 17; for preparing a ment fortreating cancer by killing a CXCR4 expressing cancer cell by induction of at least one effector function, in the presence of effector cells or complement components, wherein around 92 % of the carbohydrate chains borne by said dy comprise a fucose residue.

2. The use ing to claim 1, wherein said humanized antibody comprises a glycan profile as follows: 5.0 % GO or GOFDGlcNac, 82.5 % GOF, 9.1 % GlF, and 1.8 % M21115.

3. The use according to claim 1 or 2, characterized in that said effector function consists of the antibody—dependent cell cytotoxicity (ADCC).

4. The use according to claim 1 or 2, characterized in that said effector function consists of the complement dependent cytotoxicity (CDC).

5. The use according to claim 1 or 2, characterized in that said effector on consists of the antibody—dependent cell cytotoxicity (ADCC) and the complement dependent xicity (CDC).

6. The use according to any one of claims 1 to 5, n the said humanized antibody is selected from the group consisting of: o a humanized antibody comprising a heavy chain le domain of sequence SEQ ID No. 8 and a light chain variable domain selected from the sequences SEQ ID No. 11 to 17; o a humanized antibody comprising a heavy chain variable domain selected from the sequences SEQ ID No. 7 to 10 and a light chain variable domain of sequence SEQ ID No. 13; o a humanized antibody comprising a heavy chain variable domain of sequence SEQ ID No. 8 and a light chain variable domain of sequence SEQ ID No. 13; 1001180216 o a humanized antibody comprising a heavy chain selected from the sequences SEQ ID No. 18 to 21 and/or a light chain selected from the sequences SEQ ID No. 22 to 28; o a humanized antibody sing a heavy chain of sequence SEQ ID No. 19 and/or a light chain selected from the ces SEQ ID No. 22 to 28; o a humanized antibody comprising a heavy chain selected from the sequences SEQ ID No. 18 to 21 and/or a light chain of sequence SEQ ID No. 24; and o a humanized antibody sing a heavy chain of ce SEQ ID No. 19 and/or a light chain of sequence SEQ ID No. 24.

7. The use according to any one of claims 1 to 6, characterized in that said antibody is an lgGl .

8. The use according to any one 01‘ claims 1 to 7, characterized in that said CXCR4 expressing cancer cell consists ol‘a malignant hematological cell.

9. The use according to claim 8, characterized in that said CXCR4 malignant hematological cell is selected from the group comprising lymphoma cell, leukemia cell or multiple myeloma cell.

10. The use according to any one of claims 1 to 9, characterized in that said effector cells comprise NK cells, macrophages, monocytes, neutrophils or eosinophils.

l l. The use according to any one of claims 1 to 10, characterized in that the humanized antibody, or CH2—containing binding nt f binds at least one human FcyRs.

12. The use according to claim 11, characterized in that said at least one FcyRs is human FeyRI.

13. The use according to claim 12, characterized in that it binds said FcyRI with a constant of dissociation (KD), according to the ir model, between 1 and 10 nM.

14. The use according to claim 13, characterized in that said at least one FcyRs is human FcyRIIIA. 1001180216

15. The use according to claim 14, characterized in that it binds said FcyRIIIA with a constant of dissociation (KD), according to the geneous ligand model, between 200 and 1000 nM.

16. The use of a humanized antibody binding to CXCR4, or a CH2-eontaining binding fragment thereof, for preparing a medicament for treating cancer by killing CXCR4 expressing cancer cells; said humanized antibody comprising a heavy chain variable domain selected from the sequences SEQ ID No. 7 to 10 and a light chain variable domain selected from the sequences SEQ ID No. 11 to 17; wherein at least one effector function of the said human or humanized antibody is induced, in the presence of effector cells or complement components.

17. The use according to claim 16, characterized in that said cancer is lymphoma.

18. A method for screening of zed antibodies binding to CXCR4, or CHZ— ning binding nts thereof, the antibodies being for use in killing a CXCR4 expressing cancer cell by induction of at least one effector function, in the presence of effector cells or complement components, wherein said method comprises at least one selection step selected from: 0 selecting antibodies inducing an ADCC level on RAMOS ma cells, alter an incubation period of 4 hours, of at least 40%; 0 selecting dies inducing a CDC level on RAMOS lymphoma cells, after an incubation period of 1 hour, of at least 30%, preferentially of at least 50% and most ably of at least 70%; 0 selecting antibodies inducing a CDC level on NIH3T3 CXCR4 cells, after an incubation period of 1 hour, of at least 30%, entially of at least 50% and most preferably of at least 70%; o ing antibodies binding FcyRI with a constant of dissociation (KD), according to the ir model, between 1 and 10 nM; - selecting antibodies binding FcyRIIIA with a constant of dissociation (KD), according to the heterogeneous ligand model, between 200 and 1000 nM.

19. The use according to claim 1, substantially as hereinbefore described.

20. The method according to claim 18, substantially as hereinbefore described. 1001180216