MX2010012435A - Anti-cxcr4 antibodies. - Google Patents

Abstract

The present invention provides antibodies that bind human CXCR4 and are characterized as having high affinity and strong neutralizing properties. The antibodies of the invention are useful in the treatment of tumorigenesis, including tumor growth, invasion, angiogenesis, or metastasis.

Description

ANTIBODIES ANTI-CXCR4 The present invention relates to monoclonal antibodies against CXCR4 and its use in the treatment of diseases in which the pathogenesis is mediated by CXCR4 and SDF-1.

CXCR4, a chemokine receptor, is a seven transmembrane receptor, coupled to the G protein. Like other chemokine receptors, CXCR4 plays an important role in immune and inflammatory responses mediating directional migration and leukocyte activation. CXCR4 is expressed or overexpressed in a variety of cancer and tissue cell lines including breast, prostate, lung, ovaries, colon, pancreatic, kidney, and brain, as well as non-Hodgkin's lymphoma and chronic lymphocytic leukemia. The only known ligand for CXCR4 is stromal cell-derived factor-1 (SDF-1, or CXCL12). The interaction of CXCR4 and SDF-1 plays an important role in multiple phases of tumorigenesis, including tumor growth, invasion, angiogenesis, and metastasis.

In view of the involvement of CXCR4 in several serious diseases, CXCR4 has been studied as a therapeutic target. For example, AMD3100, a bicyclam CXCR4 antagonist, is available for patients with multiple myeloma and non-Hodgkin's lymphoma. CTCE9908, a peptide CXCR4 antagonist, is currently in Phase Ib / II clinical trials for cancer. In addition, antibodies directed to CXCR4 are described in the art (WO 06/089141, US 07/0059308, and Carnee et al [Carnee X, Quan L, Olson W, Hazan U, Dragic T. (2005) Anti-CXCR4 Monoclonal Antibodies Recognizing Overlapping Epitopes Differ Significantly in Their Ability to Inhibit Entry of Human Immunodeficiency and Virus Type I. Journal of Virology, Feb.2005: 1930-1933]).

Although there are several agents under development that target CXCR4, there is still a need for additional therapeutic agents directed to CXCR. The antibodies of the present invention are therapeutically useful CXCR4 antagonists that process a number of desirable properties. The antibodies of the present invention have increased chemical and physical stability, and solubility. The present invention provides CXCR4 antibodies that bind human CXCR4 with high affinity and inhibit the binding of human CXCR4 to SDF-1. The high power allows the use of low doses in therapeutic regimens. In addition, these antibodies interfere with the interaction of SDF-1 to CXCR4, and consequently reduce tumorigenesis, including tumor growth, invasion, angiogenesis, and metastasis. In addition, the antibodies of the present invention induce apoptosis of tumor cells.

The present invention includes a genetically modified human antibody or a binding fragment thereof, which binds human CXCR4, and which: to. inhibits the binding of human SDF-lx (SEQ ID NO: 33) to CXCR4 with an IC50 for human CXCR4 between 10 nM and 0.05 nM in a CXCR4 / 125I-SDF-human binding inhibition assay as described in I presented; b. inhibits the migration of cells carrying CXCR4 on their surface with an IC50 between 30 nM and 0.3 nM in the chemotaxis assay as described herein; Y c. exhibits an affinity, KD between 15 nM and 0.05 nM in the Surface Plasmon Resonance (BIAcore) assay as described herein.

The present invention preferably provides a genetically modified human antibody or a binding fragment thereof, which binds human CXCR4, and which inhibits the binding of human SDF-? A (SEQ ID NO: 33) to CXCR4 with an IC50 for human CXCR4 between 0.5 nM and 0.05 nM in a binding inhibition assay of human CXCR4 / 125I-SDF-l as described herein.

The present invention preferably provides a genetically modified human antibody or a binding fragment thereof, which binds human CXCR4, and which inhibits the migration of cells carrying CXCR4 on its surface with an IC50 between 3.0 nM and 0.3 nM in the assay of chemotaxis as described herein.

The present invention preferably provides a genetically modified human antibody or a binding fragment thereof, which binds human CXCR4, and which exhibits a KD affinity between 1.0 nM and 0.05 nM in the Surface Plasmon Resonance (BIAcore) assay as shown in FIG. describes in the present.

The present invention preferably provides a genetically modified human antibody or a binding fragment thereof, which binds human CXCR4, and which exhibits anti-tumorigenic activity by preventing tumor growth in a tumor xenograft model as described herein. it is administered at 1 mg / kg.

The present invention preferably provides a genetically modified human antibody or a binding fragment thereof, which binds human CXCR4, and which induces apoptosis of tumor cells in an apoptosis assay as described herein when administered between 2 pg. / mL and 10 pg / mL.

The present invention includes a genetically modified human antibody or a binding fragment thereof, comprising a light chain comprising a light chain variable region comprising framework regions, CDRL1 having the amino acid sequence of SEQ ID NO: 8, CDRL2 having the amino acid sequence of SEQ ID NO: 9, and CDRL3 having the amino acid sequence of SEQ ID NO: 10, and a heavy chain comprising a heavy chain variable region comprising framework regions, CDRH1 having the amino acid sequence of SEQ ID NO: l, CDRH3 having the amino acid sequence of SEQ ID NO: 3, and CDRH2 having an amino sequence selected from the group consisting of SEQ ID NO: 2, SEQ ID NO: 4 , SEQ ID NO: 5, SEQ ID NO: 6, and SEQ ID NO: 7, wherein the antibody binds to human CXCR4.

In addition, the present invention includes an antibody binding to human CXCR4, wherein a light chain comprises an amino acid sequence of SEQ ID NO: 16, and a heavy chain comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, and SEQ ID NO: 15.

In addition, the present invention includes an antibody binding to human CXCR4, wherein a light chain comprises an amino acid sequence of SEQ ID NO: 22, and a heavy chain comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 20, and SEQ ID NO: 21.

The present invention also includes an antibody binding to human CXCR4, wherein an antibody is selected from the group consisting of an antibody comprising SEQ ID NO: 17 and SEQ ID NO: 22, an antibody comprising SEQ ID NO: 18 and SEQ ID NO: 22, an antibody comprising SEQ ID NO: 19 and SEQ ID NO: 22, an antibody comprising SEQ ID NO: 20 and SEQ ID NO: 22, and an antibody comprising SEQ. NO: 21 and SEQ ID NO: 22.

The antibodies of the present invention as defined herein are characterized to have an IC50 of 10 nM or less in a CXCR / 125I-SDF-human binding inhibition assay as described herein. Preferred antibodies of the invention have a binding affinity for human CXCR4 of 5.0 nM or less. The most preferred antibodies of the invention have a binding affinity for human CXCR4 of 0.5 nM or less. The additionally preferred antibodies of the present invention have an IC50 for human CXCR4 between 10 nM and 0.05 nM in a binding inhibition assay of CXCR4 / 125I-SDF-human as described herein. The additionally preferred antibodies of the present invention have an IC50 for human CXCR4 between 0.5 nM and 0.05 nM in a binding inhibition assay of CXCR4 / 125I-SDF-human as described herein.

The antibodies of the present invention as defined herein are characterized as having an IC50 of 30 nM or less in a chemotaxis assay as described at the moment. Preferred antibodies of the invention have an IC 50 of 15 n or less in the chemotaxis assay. The most preferred antibodies of the invention have an IC50 of 3.0 nM or less in the chemotaxis assay. The additionally preferred antibodies of the present invention have an IC 50 between 30 nM and 0.3 nM in the chemotaxis assay as described herein. The additionally preferred antibodies of the present invention have an IC50 between 3.0 nM and 0.3 nM in the chemotaxis assay as described herein.

The antibodies of the present invention as defined herein are characterized by having a D of 15 nM or less in an assay that evaluates the binding activities of antibodies by Surface Plasmon Resonance (BIAcore) as described herein. . The most preferred antibodies of the invention have KD of 10 nM or less in the BIAcore assay. The most preferred antibodies of the invention have KD of 1.0 nM or less in the BIAcore assay. The additionally preferred antibodies of the present invention have KD between 1.0 nM and 0.05 nM in the BIAcore assay as described herein.

The antibodies of the present invention as defined herein are characterized by having anti-tumorigenic activity by preventing tumor growth in a tumor xenograft model using human Namalwa cells from non-Hodgkin's lymphoma and from NOD / SCID mice as described in the present when administered at 10 mg / kg. The most preferred antibodies of the invention should have anti-tumorigenesis activity preventing tumor growth when administered at 1 mg / kg.

The antibodies of the present invention as defined herein are characterized by inducing apoptosis of tumor cells in an apoptosis assay as described herein. The most preferred antibodies of the invention induce nuclear fragmentation and activation of caspase 3, seals of apoptosis, in multiple tumor cells which include Namalwa and CEM cells when administered between 2 μg / mL and 10 μg / mL.

The present invention includes a pharmaceutical composition comprising an antibody as described herein in combination with one or more pharmaceutically acceptable carriers, diluents, or excipients. In addition, the present invention includes a pharmaceutical composition for the treatment of tumorigenesis, including tumor growth, invasion, angiogenesis, or metastasis, comprising an antibody as variously described herein, in combination with one or more carriers, diluents , or pharmaceutically acceptable excipients. In addition, the present invention includes a pharmaceutical composition for the treatment of a cancer selected from the group consisting of breast cancer, pancreatic cancer, melanoma, prostate cancer, kidney cancer, neuroblastoma, non-Hodgkin's lymphoma, lung cancer, cancer of ovaries, colorectal cancer, multiple myeloma, glioblastoma multiforme, and leukemia comprising an antibody as variously described herein, in combination with one or more carriers, diluents, or excipients.

The present invention includes the use of an antibody as described herein for the preparation of a medicament for the treatment of tumorigenesis, including tumor growth, invasion, angiogenesis, or metastasis. In addition, the present invention includes the use of an antibody as described herein for the preparation of a medicament for the treatment of a cancer selected from the group consisting of breast cancer, pancreatic cancer, melanoma, prostate cancer, cancer of the kidney, neuroblastoma, non-Hodgkin's lymphoma, lung cancer, ovarian cancer, colorectal cancer, multiple myeloma, glioblastoma multiforme, and leukemia.

The present invention includes a method for treating tumorigenesis, including tumor growth, invasion, angiogenesis, or metastasis, comprising administering to a patient in need an antibody as described herein. In addition, the present invention includes a method for treating a cancer selected from the group consisting of breast cancer, pancreatic cancer, melanoma, prostate cancer, kidney cancer, neuroblastoma, non-Hodgkin's lymphopruse, lung cancer, ovarian cancer, colorectal cancer, multiple myeloma, glioblastoma multiforme, and leukemia, which comprises administering to a patient in need an antibody as described herein.

The general structure of an "antibody" is very well known in the art. For an IgG-type antibody, there are four amino acid chains (two "heavy" chains and two "light" chains) that are crosslinked via intra- and inter-chain disulfide bonds. When expressed in certain biological systems, the antibodies having unmodified human Fe sequences are glycosylated in the Fe region. The antibodies can be glycosylated at other positions as well. Subunit structures and three-dimensional configurations of the antibodies are well known in the art. Each heavy chain is comprised of one N-terminal heavy chain variable region ("HCVR") and a heavy chain constant region ("HCCR"). The heavy chain constant region is comprised of three domains (CHI, CH2, and CH3) for IgG, IgD, and IgA; and 4 domains (CH1, CH2, CH3, and CH4) for IgM and IgE. Each light chain is comprised of a light chain variable region (here "LCVR") and a heavy chain constant region ("LCCR").

The variable regions of each light / heavy chain pair form the antibody binding site. The HCVR and LCVR regions can be further subdivided into regions of hypersensitivity, called complementarity determining regions (CDRs), interposed with regions that are more conserved, called framework regions (FR). Each HCVR and LCVR is composed of three CDRs and four FRs, arranged from amino-terminus to carboxy-terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. Here, the 3 CDRs of the heavy chain are referred to as "CDRH1, CDRH2, and CDRH3" and the 3 CDRs of the light chain are referred to as "CDRL1, CDRL2 and CDRL3." CDRs contain most of the residues that form interactions specific with the antigen. The assignment of amino acids to each domain is in accordance with well-known conventions [eg, Kabat, "Sequences of Proteins of Immunological Interest," National Institutes of Health, Bethesda, Md. (1991)].

The antibodies of the present invention may have a heavy chain constant region selected from any of the immunoglobulin classes (IgA, IgD, IgG, IgM, and IgE). In addition, the antibodies of the present invention contain an Fe moiety which is derived from a Fe region of human IgG4 due to its reduced ability to bind complement factors compared to other subtypes of IgG.

An antibody can be derived from a single copy or clone, including, for example, any eukaryotic, prokaryotic, or phage clone. Preferably, there is an antibody of the invention in a homogeneous or substantially homogeneous population. An antibody may be intact, comprising full-length or whole-length constant regions, including the Fe region, or a portion or fragment of such an antibody so long as any shortened form comprises the antigen binding portion and retains the antigen binding ability. Such shortened forms include, for example, a Fab fragment, Fab 'fragment or F (ab') 2 fragment that includes the CDRs or variable regions of the anti-CXCR4 antibodies described. In addition, such shortened antibody forms can be a single chain Fv fragment that can be produced by joining the DNA encoding the LCVR and HCVR with a linker sequence. (See, Pluckthun, The Pharmacology of Monoclonal Antibodies, vol 113, Rosenburg and Moore eds., Springer-Verlag, New York, pp 269-315, 1994). Regardless of whether the fragments or portions are specified, the term "antibody" as used herein includes such fragments or portions as well as single chain forms, unless otherwise indicated.

Provided the protein retains the ability to bind specifically or preferentially to CXCR4 and includes a sequence or sequences described herein, it is included within the term "antibody." The antibodies of the invention can be produced using techniques well known in the art, for example, recombinant technologies, phage display technologies, synthetic technologies or combinations of such technologies or other technologies readily known in the art.

The term "genetically modified human antibody" refers to an antibody having frameworks, hinge regions, and constant regions of human origin that are identical or substantially identical (substantially human) with constant regions and frameworks derived from human genomic sequences. The frameworks, hinge regions, and fully human constant regions are those human germline sequences as well as naturally occurring sequences with somatic mutations. A genetically modified human antibody may comprise framework regions, hinge, or constants derived from a framework region, hinge, or fully human constant containing one or more substitutions, deletions, or amino acid additions therein. Frequently, a genetically modified human antibody is preferably substantially non-immunogenic in humans.

A variety of different human framework sequences can be used alone or in combination as a basis for the human genetically modified antibodies of the present invention. Preferably, the framework regions of the antibodies of the invention are of human or substantially human origin (at least 95%, 97% or 99% of human origin). The sequences of framework regions of human origin can be obtained from The Immunoglobulin Factsbook, by Marie-Paule Lafranc, Gerard Lefranc, Academic Press 2001, ISBN 012441351.

The framework sequence for the genetically modified human antibodies of the present invention serves as the "donor" variable framework region and can be used to create additional genetically modified human antibodies with the same CDRs specified herein using methodology known in the art. In addition, the framework sequence for the genetically modified human antibodies of the present invention can be compared to other human framework sequences to generate additional genetically modified human antibodies. Accordingly, this information can be used to "retro-mutate" another region of homologous human framework selected to the genetically modified human donor amino acid residue at these positions. In addition, any "rare" amino acid can be detected in additional human frameworks so that the genetically modified human or donor consensus residue can be used in the relevant position.

The term "inhibit" means the ability to substantially antagonize, prohibit, prevent, restrict, decrease, interrupt, eliminate, stop, reduce or reverse the biological effects of the CXCR4 receptor link.

"CXCR4" or "human CXCR4" refers to any human CXCR4, as well as functionally active mutated forms thereof. Examples include, but are not limited to, SEQ ID NO: 30, SEQ ID NO: 31, and SEQ ID NO: 32.

A "patient" is a mammal, preferably a human.

The term "treatment" (or "treat" or "treatment") means to decrease, stop, reduce, or reverse the progression or severity of a symptom, disorder, condition, or disease.

The term "prevention" (or "prevent" or "prevention") means to prohibit, restrict, or inhibit the occurrence or occurrence of a symptom, disorder, condition, or disease. Acute events and chronic conditions can be treated and prevented. In an acute event, the antibody is administered at the beginning of a symptom, disorder, condition, or disease and is interrupted when the acute event ends, while a symptom, disorder, condition, or chronic disease is treated during a longer time frame .

The term "therapeutically effective amount" refers to the amount or dose of an antibody of this invention which, in the administration of single or multiple doses to a patient, provides the desired treatment or prevention. The therapeutically effective amount may comprise an amount of about 0.001 to 20 mg / kg per single (eg, bolus), multiple or continuous administration.

The particular antibodies of this invention include: an antibody comprising amino acid sequences of SEQ ID NOs: 1, 2, 3, 8, 9, and 10; an antibody comprising amino acid sequences of SEQ ID NOs: 1, 4, 3, 8, 9, and 10; an antibody comprising amino acid sequences of SEQ ID NOs: 1, 5, 3, 8, 9, and 10; an antibody comprising amino acid sequences of SEQ ID NOs: 1, 6, 3, 8, 9, and 10; an antibody comprising amino acid sequences of SEQ ID NOs: 1, 7, 3, 8, 9, and 10. The listed sequences represent CDRH1, CDRH2, CDRH3, CDRL1, CDRL2, and CDRL3, respectively.

The particular antibodies of this invention include: an antibody comprising an LCVR having an amino acid sequence of SEQ ID NO: 16 and an HCVR having an amino acid sequence of SEQ ID NO: 11; an antibody comprising an LCVR having an amino acid sequence of SEQ ID NO: 16 and an HCVR having an amino acid sequence of SEQ ID NO: 12; an antibody comprising an LCVR having an amino acid sequence of SEQ ID NO: 16 and an HCVR having an amino acid sequence of SEQ ID NO: 13; an antibody comprising an LCVR having an amino acid sequence of SEQ ID NO: 16 and an HCVR having an amino acid sequence of SEQ ID NO: 14; an antibody comprising an LCVR having an amino acid sequence of SEQ ID NO: 16 and an HCVR having an amino acid sequence of SEQ ID NO: 15.

The present invention includes five antibodies that bind and inhibit the activity of CXCR4. In particular, the present invention includes: an antibody comprising a light chain having an amino acid sequence of SEQ ID NO: 22 and a heavy chain having an amino acid sequence of SEQ ID NO: 17; an antibody comprising a light chain having an amino acid sequence of SEQ ID NO: 22 and a heavy chain having an amino acid sequence of SEQ ID NO: 18; an antibody comprising a light chain having an amino acid sequence of SEQ ID NO: 22 and a heavy chain having an amino acid sequence of SEQ ID NO: 19; an antibody comprising a light chain having an amino acid sequence of SEQ ID NO: 22 and a heavy chain having an amino acid sequence of SEQ ID NO: 20; an antibody comprising a light chain having an amino acid sequence of SEQ ID NO: 22 and a heavy chain having an amino acid sequence of SEQ ID NO: 21.

Preferably, an antibody of the present invention wherein all six CDRs, the HCVR, the LCVR, the HCVR and the LCVR, the complete heavy chain, the complete light chain, or the heavy chain and light chain complete are limited by a sequence particular as shown by a SEQ ID NO: herein is further characterized by having an IC50 in a CXCR4 / 125I-SDF-human binding inhibition assay as described herein of approximately 10 nM or less, more preferably approximately 5.0 nM or less, and most preferably 0.5 nM or less. An additionally preferred antibody of the present invention wherein all six CDRs, the HCVR, the LCVR, the HCVR and the LCVR, the complete heavy chain, the complete light chain, or the heavy chain and light chain complete are limited by a sequence particular as shown by a SEQ ID NO: herein further characterized by having an IC50 for human CXCR4 between 10 nM and 0.05 nM in a CXCR4 / 125I-SDF-human binding inhibition assay as described herein. An additionally preferred antibody of the present invention wherein all six CDRs, the HCVR, the LCVR, the HCVR and the LCVR, the complete heavy chain, the complete light chain, or the heavy chain and light chain complete are limited by a sequence particular as shown by a SEQ ID NO: herein further characterized by having an IC50 for human CXCR4 between 0.5 nM and 0.05 nM in a CXCR / 12 I-SDF-human binding inhibition assay as described herein .

More preferably, an antibody of the present invention wherein all six CDRs, the HCVR, the LCVR, the HCVR and the LCVR, the complete heavy chain, the complete light chain, or the heavy chain and light chain complete are limited by a particular sequence as shown by a SEQ ID NO: here further characterized by having an IC50 in a chemotaxis assay as described herein of approximately 30 nM or less, more preferably approximately 15 nM or less, and even more preferably 3.0 nM or less. An additionally preferred antibody of the present invention wherein all six CDRs, the HCVR, the LCVR, the HCVR and the LCVR, the complete heavy chain, the complete light chain, or the heavy chain and light chain complete are limited by a sequence particular as shown by a SEQ ID NO: herein further characterized by having an IC50 between 30 nM and 0.3 nM in the chemotaxis assay as described herein. An additionally preferred antibody of the present invention wherein all six CDRs, the HCVR, the LCVR, the HCVR and the LCVR, the complete heavy chain, the complete light chain, or the heavy chain and light chain complete are limited by a sequence particular as shown by a SEQ ID NO: here further characterized by having an IC50 between 3.0 nM -and 0.3 nM in the chemotaxis assay as described herein.

Still more preferably, an antibody of the present invention wherein all six CDRs, the HCVR, the LCVR, the HCVR and the LCVR, the complete heavy chain, the complete light chain, or the heavy chain and light chain complete are limited by a particular sequence as shown by a SEQ ID NO: here is further characterized by having a KD in an assay that evaluates the binding activities of antibodies by Surface Plasmon Resonance (BIAcore) as described herein at about 15 nM or less, more preferably about 10 nM or less, and most preferably 1.0 nM or less. An additionally preferred antibody of the present invention wherein all six CDRs, the HCVR, the LCVR, the HCVR and the LCVR, the complete heavy chain, the complete light chain, or the heavy chain and light chain complete are limited by a sequence particular as shown by a SEQ ID NO: here is further characterized by having a KD in an assay that evaluates the binding activities of the antibodies by Surface Plasmon Resonance (BIAcore) as described herein between 15 nM and 0.05 nM in the BIAcore assay as described herein. An additionally preferred antibody of the present invention wherein all six CDRs, the HCVR, the LCVR, the HCVR and the LCVR, the complete heavy chain, the complete light chain, or the heavy chain and light chain complete are limited by a sequence particular as shown by a SEQ ID NO: here is further characterized by having a KD in an assay that evaluates the binding activities of the antibodies by Surface Plasmon Resonance (BIAcore) as described herein between 1.0 nM and 0.05 nM in the BIAcore assay as described herein.

More preferably, an antibody of the present invention wherein all six CDRs, the HCVR, the LCVR, the HCVR and the LCVR, the complete heavy chain, the complete light chain, or the heavy chain and light chain complete are limited by a Particular sequence as shown by a SEQ ID NO: here is further characterized by having an anti-tumorigenesis activity preventing tumor growth in a tumor xenograft model using human Namalwa cells from non-Hodgkin's lymphoma and from NOD / SCID mice as is described herein when administered at 10 mg / kg, and even more preferably when administered at 1 mg / kg.

Most preferably, an antibody of the present invention wherein all six CDRs, the HCVR, the LCVR, the HCVR and the LCVR, the complete heavy chain, the complete light chain, or the heavy chain and light chain complete are limited by a particular sequence as shown by a SEQ ID NO: here is further characterized for inducing apoptosis of tumor cells in an apoptosis assay as described herein. A more preferred antibody of the present invention wherein all six CDRs, the HCVR, the LCVR, the HCVR and the LCVR, the complete heavy chain, the complete light chain, or the heavy chain and light chain complete are limited by a sequence particular as shown by a SEQ ID NO: here is further characterized by inducing nuclear fragmentation and activation of caspase 3, seals of apoptosis, in multiple tumor cells including Namalwa and CEM cells when administered between 2 g / mL and 10 pg / mL.

Examples Antibodies I, II, III, IV, and V can be produced and purified as follows. An appropriate host cell, such as HEK 293 EBNA or CHO, is either temporarily or stably transfected with an expression system for secreting antibodies using an optimal predetermined HC: LC vector ratio or a single vector system encoding both HC, such as SEQ ID NO: 23, SEQ ID NO: 24, SEQ ID NO: 25, SEQ ID NO: 26, or SEQ ID NO: 27, as LC, such as SEQ ID NO: 28. The clarified medium, in which the antibody has been secreted, purified using any of many commonly used techniques. For example, the medium can be conveniently applied to a column of Protein A or G Sepharose FF that has been equilibrated with a compatible buffer, such as phosphate buffered saline (pH 7.4). The column is washed to remove non-specific binding components. The bound antibody is eluted, for example, by pH gradient (such as 0.1 M sodium phosphate buffer, pH 6.8 to 0.1 M sodium citrate buffer, pH 2.5). Antibody fractions are detected, such as by SDS-PAGE, and then pooled. Additional purification is optional, depending on the proposed use. The antibody can be concentrated and / or sterile filtered using common techniques. The soluble aggregate and multimeros can be effectively removed by common techniques, including size exclusion, hydrophobic interaction, ion exchange, or hydroxyapatite chromatography. The purity of the antibody after these chromatography steps is greater than 99%. The product can be immediately frozen at -70 ° C or can be lyophilized. The amino acid sequences for these antibodies are given below.

SEC ID NOs Link Inhibition Assay of CXCR4 / 125I-SDF-Human The binding of SDF-1 to CXCR4 is the first step in the activation of the intracellular signaling path of CXCR4. To determine if an antibody can block the interaction of SDF-1 and CXCR, human leukemia CCRF-CE cells expressing endogenous CXCR4 are used in an SDF-labeled assay with 125I. The test is carried out on an untreated polystyrene plate with a 96-well U-shaped bottom. The buffer of the binding assay is prepared with RPMI 1640 medium containing 10 mM N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid (HEPES), pH 7.5, and 0.2% bovine serum albumin (BSA). Briefly, 200 yL of reaction mixtures containing 300 pM of ligand (60 pM of 125 I-SDF-la and 240 pM of cold SDF-α), different concentrations of the test antibody in the assay buffer, 100,000 CCRF-cells Human EMFs, and 0.5 mg of scintillation proximity test beads (SPA) are incubated at room temperature for 2 hours. The plates are then counted in a liquid scintillation counter and luminescence in SPA mode. The CXCR4 antagonists decrease the binding radioactivity in this assay in a dose-dependent manner. The inhibitory potency (IC5o) of a test antibody is calculated using the GraphPad Prism software, based on the dose-dependent decrease in binding radioactivity.

The antibodies exemplified herein exhibit an IC50 value of 10 nM or less in this assay. For example, antibody III exhibits an average IC50 of 0.45 nM in this assay. The data demonstrate that the antibodies exemplified herein bind to human CXCR4 with high affinity and inhibit the binding of human CXCR4 to SDF-1.

Chemotaxis assay The CXCR4 / SDF-1 interaction regulates the migration (chemotaxis) of cells that carry CXCR4 on its surface. To determine the antagonistic and cellular activities of a test antibody, a chemotaxis assay using human histiocytic lymphoma U937 cells expressing endogenous CXCR4 is employed. Briefly, U937 cells, grown in Dulbecco's Modified Eagle Medium (D-MEM) containing 10% fetal bovine serum, 1% sodium pyruvate solution of Minimum Essential Medium (MEM), 1% non-essential amino acids of MEM, and 1% L-glutamine, are collected and washed once with chemotaxis assay buffer (lx RPMI medium containing 10 mM HEPES, pH 7.5, and 0.3% BSA). After washing, the cells are resuspended in assay buffer at a concentration of 5 x 106 cells / mL. The assay is performed on a 96-cell cell migration plate. Generally, 50 μL of the cell mixture with or without test antibody, varying from 0.5 g / mL to 50 pg / mL, are plated on the upper chamber, and 30 μL of SDF-I (10 ng / mL ) prepared in lx quimiotaxis assay buffer is added to the lower chamber. After assembly, the plate is incubated for 2.5 hours at 37 ° C under 5% carbon dioxide. After incubation, 5 μL of cell proliferation solution is added in the lower chamber. The plate is then incubated for 60 minutes at 37 ° C, and the migrated cells are detected by measuring the absorbance at 492 nm with a microplate reader. CXCR4 antagonists inhibit cell migration, reducing the absorbance reading. The inhibitory potency (IC50) of a test antibody in this assay is calculated using the GraphPad Prism software, based on the dose-dependent decrease in absorbance at 492 nm.

The antibodies exemplified herein exhibit an average IC50 value of 30 nM or less in this assay. Antibody III exhibits an average IC50 value of 5.90 nM in this assay. The data demonstrate that the antibodies exemplified herein bind to human CXCR4 with high affinity and inhibit the binding of human CXCR4 to SDF-1.

Evaluation of the binding activities of anti-CXCR4 antibodies by Surface Plasmon Resonance (BIAcore) A Biacore® 2000 instrument is used to measure binding affinity and kinetics. Biacore® uses the optical properties of surface plasmon resonance to detect the alteration in protein concentration of the interaction of molecules within a dextran biosensor matrix. Except as noted, all reagents and materials are purchased from Biacore® AB (Upsala, Sweden). All measurements are made at 4 ° C. The binding experiment is carried out essentially as described in Stenlund et al (Stenlund P, Babcock GJ, Sodroski J, Myszka DG (2003) Capture and reconstitution of G protein-coupled receptors on a biosensor surface Anal Biochem. ): 243-50) and Navratilo et al (Navratilova I, Sodroski J, Myszka DG (2005) Solubilization, stabilization, and purification of chemokine receptors using biosensor technology, Anal Biochem 339 (2): 271-81). The run buffer is 50 mM HEPES, 5 mM magnesium chloride, 1 mM calcium chloride, 150 mM sodium chloride, 2 mg / mL BSA, pH 7.5. The human CXCR4 receptor with a linear C-terminal C9 peptide tag (SEQ ID NO: 29) is cloned and overexpressed in canine thymocyte Cf2Th cells in the same manner as previously described by Mirzabekov et al (Mirzabekov, N. Bannert , M. Farzan, H. Hofmann, P. Kolchinsky, L. Wu, R. Wyatt and J. Sodroski-Enhanced expression, native purification, and characterization of CCR5, a major HIV-1 coreceptor, J. Biol. Chem. 274 ( 1999), pp. 28745-28750). The C-terminal linear C9 peptide tag is recognized by the monoclonal antibody 1D4 (DD Oprian, RS Molday, RJ Kaufman and HG Khorana, Expression of a synthetic bovine rhodopsin gene in monkey kidney cells, Proc. Nati. Acad. Sci. USA 84 (1987), pp. 8874-8878).

The link is evaluated using multiple analytical cycles as follows. Mab 1D4 (monoclonal clone 1D4, University of British Columbia) is immobilized to a CM5 chip via amine coupling (antibody of approximately 10,000-20,000 Resonance Units). The cells are resuspended in 20 mM tris (hydroxymethyl) aminomethane (pH 7.0), 0.1 M ammonium sulfate, 10% glycerol, 5 mM magnesium chloride, 1 mM calcium chloride, plus complete tablet of protease inhibitor free of ethylenediaminetetraacetic acid . 4 × 10 6 cells / mL final are used for injection on the chip (ie, 2.0 × 0.06 cells, 0.5 mL final volume). The transfected cells and the run buffer with detergent (2% cholesteryl hemisuccinate ester, 10% dodecyl maltoside, 10% 3- [(3-colamidopropyl) dimethylammonium] -1-propanesulfonate) at a ratio of 5: 1 ( ratio of cells to buffer volume) are transferred in a self-mixer. This mixture is incubated for 10 minutes. After incubation, 150 μL of solubilized receptor is injected onto the surface of 1D4 at a flow rate of 20 L / minute. The sample loop is then washed with running buffer. This is followed by an injection of 20 i of antibody at the flow rate of 100 μL / minute. The chip is then regenerated with two 10-second pulses of 10 mM sodium hydroxide + 1% n-octyl-D-glucopyranoside at 100 and L / minute. The association rate constants ("kon") and dissociation rate constants ("k0ff") for each cycle are evaluated using a "1: 1 mass transfer" link model in the BIAevaluation software. The "KD" is the dissociation constant and is calculated by the formula: k0ff / kon = D. The link parameters are summarized below. The data demonstrate that the antibodies exemplified herein bind to human CXCR4 with high affinity and inhibit the binding of human CXCR4 to SDF-1.

Anti-Tumor Activity in a Xenograft Model of SCID / Namalwa The SDF-1 / CXCR4 interaction seems to play an important role in multiple stages of tumorigenesis, including tumor growth, invasion, angiogenesis, and metastasis. To evaluate the in vivo anti-tumor activity of a test antibody in cancer, a tumor xenograft model using human Namalwa cells from non-Hodgkin's lymphoma and from NOD / SCID mice is employed. Briefly, 200,000 Namalwa cells mixed with matrigel (1: 1) are implanted subcutaneously on the hind flank of the animals. Implanted tumor cells grow as solid tumors, the dimensions of which can be continuously monitored and measured using a calibrator. To determine the in vivo efficacy of a test antibody in this model, the animals (10 / group) are treated with different doses of test antibodies dissolved in saline or phosphate-buffered saline, 48 hours post-implant cell. tumor. Antibodies are dosed subcutaneously in the range of 1 μg / mouse, 10 g / mouse, and 100 g / mouse, and tumor volume and body weight are determined every 2 or 3 days. The studies usually last 3-4 weeks, depending on the growth of the tumor. The anti-tumor growth activity of a test antibody is determined by the percentage reduction in tumor volume in the treatment groups compared to the tumor volume in the control groups treated with vehicle alone.

Antibody I inhibits tumor growth in this assay when administered at 10 μg mouse, which is approximately 0.4 mg / kg. The data demonstrate that Antibody I has tumorigenesis activity preventing tumor growth.

Hematological Lymphoma Model from SCID / Namalwa To further investigate the anti-tumor activity of the CXCR4 antibody in lymphoma, a model of hematological lymphoma was established by injecting 200,000 Namalwa cells into SCID mice via the tail vein. Generally, mice injected with tumor cells die in 5-6 weeks. To test the efficacy of the antibody in this model, the animals (10 each group) are treated with 30 pg / mouse or 100 ug / mouse of the test antibody 24 hours post-injection of the tumor cells. The antibody is dosed subcutaneously once every 4 days for 6 weeks, and animal survival is recorded on a daily basis.

The treatment groups with Antibody I have shown a statistically significant survival benefit when compared to the isotype and vehicle IgG control groups in this hematological lymphoma model.

Anti-Tumor Activity in a SCID / CEM Xenograft Model To evaluate the anti-tumor activity in vivo of a test antibody in cancer, a tumor xenograft model using CEM cells and NOD / SCID mice is employed. Briefly, 5.0x06 CEM cells mixed with matrigel (1: 1) are implanted subcutaneously in the hind flank of the animals. The implanted tumor cells grow as solid tumors which can be continuously monitored and measured by calibrator. To determine the efficacy of a test antibody in this model, animals (10 each group) are treated with 10 μ? / Mouse, 30 μg / mouse, or 100 μg / mouse test antibody 24 hours post-cell implant of tumor. The antibody is dosed subcutaneously once every 4 days, and tumor volume and body weight are measured every 2 or 3 days.

A dose-dependent inhibition of tumor growth is observed between the treatment groups with Antibody I in comparison with the control groups of isotype and vehicle IgG. Antibody I in all three doses significantly inhibits tumor growth.

Apoptosis test To investigate whether CXCR4 antibodies induce apoptosis, multiple tumor cell lines expressing high levels of CXCR4 are treated with the test antibody. The cells are treated with different concentrations of the test antibody for 2-4 days in their growth medium with 1% or 10% FBS. After the treatment, the cells are fixed with 3.7% formaldehyde and washed in D-PBS. The cells are permeabilized with 0.1% Triton X-100 in D-PBS, washed and blocked in D-PBS containing 1% BSA. The cells are then incubated for 1 hour with rabbit anti-activated Caspase 3 polyclonal antibody (Cat # 557135 BD Biosciences, NC) diluted in D-PBS with 1% BSA.

Cells are washed 2 times with D-PBS then incubated for 1 hour with Alexa Fluor 488 goat anti-rabbit IgG (Invitrogen, Carlsbad, CA) and Hoechst 33342 200ng / mL (Invitrogen, Carlsbad, CA) diluted in D -PBS with 1% BSA. Stained plates are scanned using ArrayScan Vti (Cellomics, Pittsburgh, Pennsylvania) and the Target Activation bioapplication is used for quantification of fluorescent signal.

The results demonstrate that Antibody I induces nuclear fragmentation and activation of caspase 3 in multiple tumor cells including Namalwa and CEM cells. Nuclear fragmentation and activation of caspase 3 are hallmarks of apoptosis. Therefore, the data demonstrate that Antibody I induces apoptosis of tumor cells when administered between 2 and g / mL and 10 and g / mL.

To further confirm that Antibody I induces apoptosis, changes of Annexin V are investigated by flow cytometry in Namalwa cells after treatment with the test antibody or isotype IgG control. Antibody I induces a dose-dependent increase in annexin V, while isotype IgG has no effect. In addition, Antibody I induces apoptosis, which is also observed in CEM xenograft tumors by TUNEL staining.

SEC ID Listing CDRS of Heavy Chain SEC ID N0: 1 GFTSTDYYFS SEC ID NO: 2 FIRTKSKGYTTEYSGSVKG SEC ID NO: 3 EPITTDPRDY SEC ID NO: 4 FIRSKSKGYTTEYSGSVKG SEC ID NO: 5 FIRYKSKGYTTEYSGSVKG SEC ID NO: 6 FIRNKRKGYTTEYSGSVKG SEC ID NO: 7 FIRHKSKGYTTEYSGSVKG CDRS of Light Chain SEC ID NO: 8 KSSQSLFNSRTRKKYLA SEC ID NO: 9 ASKRKS SEQ ID NO: 10 KQSRFLRA Variable Regions of Heavy Chain SEC ID NO: 11 EVQLVESGGGLVQPGGSLRLSCAASGFTSTDYYFSWVRQAPGKGLEWVGFIRTKSKGYTTE YSGSV GRFTISRDDSKNSLYLQMNSLKTEDTAVYYCAREPITTDPRDYWGQGTLVTVSS SEC ID NO: 12 EVQLVESGGGLVQPGGSLRLSCAASGFTSTDYYFS VRQAPGKGLEWVGFIRSKSKGYTTE YSGSVKGRFTISRDDSKNSLYLQMNSLKTEDTAVYYCAREPITTDPRDYWGQGTLVTVSS SEQ ID NO: 13 EVQLVESGGGLVQPGGSLRLSCAASGFTSTDYYFSWVRQAPGKGLE VGFIRYKSKGYTTE YSGSVKGRFTISRDDSKNSLYLQMNSLKTEDTAVYYCAREPITTDPRDYWGQGTLVTVSS SEQ ID NO: 14 EVQLVESGGGLVQPGGSLRLSCAASGFTSTDYYFSWVRQAPGKGLEWVGFIRNKRKGYTTE YSGSVKGRFTISRDDSKNSLYLQMNSL TEDTAVYYCAREPITTDPRDYWGQGTLVTVSS SEQ ID NO: 15 EVQLVESGGGLVQPGGSLRLSCAASGFTSTDYYFSWVRQAPGKGLEWVGFIRHKSKGYTTE YSGSVKGRFTISRDDSKNSLYLQMNSLKTEDTAVYYCAREPITTDPRDY GQGTLVTVSS Variable Regions of Light Chain SEQ ID NO: 16 DIVMTQSPDSLAVSLGERATINCKSSQSLFNSRTRKKYLAWYQQKPGQPPKLLIYWASKRK SGVPDRFSGSGSGTDFTLTISSLQAEDVAVYYCKQSRFLRAFGQGTKLEIK Complete Heavy Chains SEQ ID NO: 17 EVQLVESGGGLVQPGGSLRLSCAASGFTSTDYYFSWVRQAPGKGLEWVGFIRTKSKGYTTE YSGSVKGRFTISRDDSKNSLYLQMNSLKTEDTAVYYCAREPITTDPRDYWGQGTLVTVSSA STKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVS NSGALTSGVHTFPAVLQSSGL YSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPPCPAPEAAGGPSVFLF PP PKDTLMISRTPEVTCVVVDVSQEDPEVQFN YVDGVEVHNAKTKPREEQFNSTYRVVS VLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSL TCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCS VMHEALHNHYTQ SLSLSLG SEC ID NO: 18 EVQLVESGGGLVQPGGSLRLSCAASGFTSTDYYFSWVRQAPGKGLEWVGFIRSKSKGYTTE YSGSVKGRFTISRDDSKNSLYLQMNSLKTEDTAVYYCAREPITTDPRDYWGQGTLVTVSSA STKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVS NSGALTSGVHTFPAVLQSSGL YSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPPCPAPEAAGGPSVFLF PPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVS VLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSL TCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCS VMHEALHNHYTQKSLSLSLG SEQ ID NO: 19 EVQLVESGGGLVQPGGSLRLSCAASGFTSTDYYFSWVRQAPGKGLEWVGFIRYKSKGYTTE YSGSVKGRFTISRDDSKNSLYLQMNSLKTEDTAVYYCAREPITTDPRDYWGQGTLVTVSSA STKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGL YSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPPCPAPEAAGGPSVFLF PPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVS VLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSL TCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSR QEGNVFSCS VMHEALHNHYTQKSLSLSLG SEC ID NO: 20 EVQLVESGGGLVQPGGSLRLSCAASGFTSTDYYFSWVRQAPGKGLEWVGFIRNKRKGYTTE YSGSVKGRFTISRDDSKNSLYLQMNSLKTEDTAVYYCAREPITTDPRDYWGQGTLVTVSSA ST GPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGL YSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPPCPAPEAAGGPSVFLF PPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVS VLTVLHQDWLNGKEY CKVSN GLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSL TCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCS V HEALHNHYTQKSLSLSLG SEC ID N0: 21 EVQLVESGGGLVQPGGSLRLSCAASGFTSTDYYFSWVRQAPGKGLE VGFIRHKSKGYTTE YSGSV GRFTISRDDSKNSLYLQMNSLKTEDTAVYYCAREPITTDPRDYWGQGTLVTVSSA STKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGL YSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPPCPAPEAAGGPSVFLF PPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYR VS VLTVLHQDWLNG EY CKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSL TCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCS VMHEALHNHYTQKSLSLSLG Complete Light Chain SEQ ID NO: 22 DIVMTQSPDSLAVSLGERATINCKSSQSLFNSRTRKKYLAWYQQKPGQPPKLLIYWASKRK SGVPDRFSGSGSGTDFTLTISSLQAEDVAVYYCKQSRFLRAFGQGTKLEIKRTVAAPSVFI FPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSST LTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC Nucleotide Sequences - Heavy Chain Variable Region SEQ ID NO: 23 GAGGTGCAGCTGGTGGAGTCTGGGGGAGGCTTGGTCCAGCCTGGAGGGTCCCTGAGACTCT CCTGTGCAGCCTCTGGCTTCACCAGTACCGACTACTACTTTAGCTGGGTCCGCCAGGCTCC AGGGAAGGGGCTGGAGTGGGTTGGCTTCATCCGGACGAAGTCGAAGGGCTACACCACCGAG TACAGCGGCAGCGTGAAGGGCAGATTCACCATCTCAAGAGATGATTCAAAGAACTCACTGT ACCTGCAGATGAACAGCCTGAAAACCGAGGACACGGCCGTGTATTACTGTGCTAGAGAGCC CATCACCACCGACCCTCGGGACTACTGGGGCCAAGGGACCCTGGTCACCGTCTCCTCA SEC ID NO: 24 GAGGTGCAGCTGGTGGAGTCTGGGGGAGGCTTGGTCCAGCCTGGAGGGTCCCTGAGACTCT CCTGTGCAGCCTCTGGCTTCACCAGTACCGACTACTACTTTAGCTGGGTCCGCCAGGCTCC AGGGAAGGGGCTGGAGTGGGTTGGCTTCATCCGGTCTAAGTCGAAGGGCTACACCACCGAG TACAGCGGCAGCGTGAAGGGCAGATTCACCATCTCAAGAGATGATTCAAAGAACTCACTGT ACCTGCAGATGAACAGCCTGAAAACCGAGGACACGGCCGTGTATTACTGTGCTAGAGAGCC CATCACCACCGACCCTCGGGACTACTGGGGCCAAGGGACCCTGGTCACCGTCTCCTCA SEC ID NO: 25 GAGGTGCAGCTGGTGGAGTCTGGGGGAGGCTTGGTCCAGCCTGGAGGGTCCCTGAGACTCT CCTGTGCAGCCTCTGGCTTCACCAGTACCGACTACTACTTTAGCTGGGTCCGCCAGGCTCC AGGGAAGGGGCTGGAGTGGGTTGGCTTCATCCGGTATAAGTCGAAGGGCTACACCACCGAG TACAGCGGCAGCGTGAAGGGCAGATTCACCATCTCAAGAGATGATTCAAAGAACTCACTGT ACCTGCAGATGAACAGCCTGAAAACCGAGGACACGGCCGTGTATTACTGTGCTAGAGAGCC CATCACCACCGACCCTCGGGACTACTGGGGCCAAGGGACCCTGGTCACCGTCTCCTCA SEQ ID NO: 26 GAGGTGCAGCTGGTGGAGTCTGGGGGAGGCTTGGTCCAGCCTGGAGGGTCCCTGAGACTCT CCTGTGCAGCCTCTGGCTTCACCAGTACCGACTACTACTTTAGCTGGGTCCGCCAGGCTCC AGGGAAGGGGCTGGAGTGGGTTGGCTTCATCCGGAACAAGCGGAAGGGCTACACCACCGAG TACAGCGGCAGCGTGAAGGGCAGATTCACCATCTCAAGAGATGATTCAAAGAACTCACTGT ACCTGCAGATGAACAGCCTGAAAACCGAGGACACGGCCGTGTATTACTGTGCTAGAGAGCC CATCACCACCGACCCTCGGGACTACTGGGGCCAAGGGACCCTGGTCACCGTCTCCTCA SEC ID NO: 27 GAGGTGCAGCTGGTGGAGTCTGGGGGAGGCTTGGTCCAGCCTGGAGGGTCCCTGAGACTCT CCTGTGCAGCCTCTGGCTTCACCAGTACCGACTACTACTTTAGCTGGGTCCGCCAGGCTCC AGGGAAGGGGCTGGAGTGGGTTGGCTTCATCCGGCACAAGTCGAAGGGCTACACCACCGAG TACAGCGGCAGCGTGAAGGGCAGATTCACCATCTCAAGAGATGATTCAAAGAACTCACTGT ACCTGCAGATGAACAGCCTGAAAACCGAGGACACGGCCGTGTATTACTGTGCTAGAGAGCC CATCACCACCGACCCTCGGGACTACTGGGGCCAAGGGACCCTGGTCACCGTCTCCTCA Nucleotide Sequences - Light Chain Variable Region SE ID NO: 28 GACATCGTGATGACCCAGTCTCCAGACTCCCTGGCTGTGTCTCTGGGCGAGAGGGCCACCA TCAACTGCAAGAGCAGCCAGAGCCTGTTCAACAGCCGGACCCGGAAGAAGTACCTGGCCTG GTACCAGCAGAAACCAGGACAGCCTCCTAAGCTGCTCATTTACTGGGCCAGCAAGAGAAAG AGCGGGGTCCCTGACCGATTCAGTGGCAGCGGGTCTGGGACAGATTTCACTCTCACCATCA GCAGCCTGCAGGCTGAAGATGTGGCAGTTTATTACTGTAAGCAGAGCCGTTTTCTGAGAGC CTTTGGCCAAGGGACCAAGCTGGAGATCAAA Peptide Mark C9 Linear C-Terminal for Human CXCR4 Receiver SEC ID NO: 29 TETSQVAPA CXCR4 Human SEC ID NO: 30 MEGISSIPLPLLQIYTSDNYTEEMGSGDYDSMKEPCFREENANFNKIFLPTIYSI IFLTGI VGNGLVILVMGYQ KLRSMTDKYRLHLSVADLLFVITLPF AVDAVAN YFGNFLCKAVHV IYTVNLYSSVLILAFISLDRYLAIVHATNSQRPRKLLAEKVVYVGVWIPALLLTIPDFIFA NVSEADDRYICDRFYPNDLWVVVFQFQHIMVGLILPGIVILSCYCI I ISKLSHSKGHQKRK ALKTTVILILAFFAC LPYYIGISIDSFILLEIIKQGCEFENTVHKWISITEALAFFHCCL NPILYAFLGAKFKTSAQHALTSVSRGSSLKILSKGKRGGHSSVSTESESSSFHSS Isoform A of CXCR4 Human SEC ID NO: 31 MSIPLPLLQIYTSDNYTEEMGSGDYDSMKEPCFREENANFNKI FLPTIYSI IFLTGIVGNG LVILVMGYQKKLRSMTDKYRLHLSVADLLFVITLPFWAVDAVAN YFGNFLCKAVHVIYTV NLYSSVLILAFISLDRYLAIVHATNSQRPRKLLAEKVVYVGVWI PALLLTIPDFI FANVSE ADDRYICDRFYPNDLWVVVFQFQHIMVGLILPGIVILSCYCII IS LSHSKGHQKRKALKT TVILILAFFACWLPYYIGISIDSFILLEIIKQGCEFENTVHKWISITEALAFFHCCLNPIL YAFLGAKFKTSAQHALTSVSRGSSLKILSKGKRGGHSSVSTESESSSFHSS Isoform B of CXCR4 Human SEC ID NO: 32 MEGISIYTSDNYTEEMGSGDYDSMKEPCFREENANFNKIFLPTIYSIIFLTGIVGNGLVIL VMGYQKKLRSMTDKYRLHLSVADLLFVITLPF AVDAVANWYFGNFLCKAVHVIYTVNLYS SVLILAFISLDRYLAIVHATNSQRPRKLLAEKVVYVGV IPALLLTIPDFIFANVSEADDR YICDRFYPNDLWVVVFQFQHIMVGLILPGIVILSCYCIIISKLSHSKGHQKRKALKTTVIL ILAFFACWLPYYIGISIDSFILLEI IKQGCEFENTVHKWISITEALAFFHCCLNPILYAFL GA FKTSAQHALTSVSRGSSLKILSKGKRGGHSSVSTESESSSFHSS SDF-? A Human SEQ ID NO: 33 KPVSLSYRCPCRFFESHVARANVKHLKILNTPNCALQIVARLKNNNRQVCIDPKLKWIQEY LEKALNK

Claims (21)

1. A genetically modified human antibody or a binding fragment thereof, which binds to human CXCR4, and characterized in that: to. inhibits the binding of human SDF-? a (SEQ ID NO: 33) to CXCR4 with an IC50 for human CXCR4 between 10 n and 0.05 nM in a binding inhibition assay of CXCR4 / 125I-SDF-human as described in I presented; b. inhibits the migration of cells carrying CXCR4 on their surface with an IC50 between 30 nM and 0.3 nM in the chemotaxis assay as described herein; Y c. exhibits an affinity, KD between 15 nM and 0.05 nM in the Surface Plasmon Resonance (BIAcore) assay as described herein.

2. The antibody according to claim 1, characterized in that it inhibits the binding of SDF-? human (SEC ID NO: 33) to CXCR4 with an IC50 for human CXCR4 between 0.5 nM and 0.05 nM.

3. The antibody according to either claim 1 or claim 2, characterized in that it inhibits the migration of cells carrying CXCR4 on their surface with an IC5o between 3.0 nM and 0.3 nM.

4. The antibody according to any of claims 1 to 3, characterized in that it exhibits a KD affinity between 1.0 nM and 0.05 nM.

5. The antibody according to any of claims 1 to 4, characterized in that it exhibits anti-tumorigenic activity by preventing tumor growth in a tumor xenograft model as described herein when administered at 1 mg / kg.

6. The antibody according to any of claims 1 to 5, characterized in that it induces apoptosis of tumor cells in an apoptosis assay as described herein when administered between 2 g / mL and 10 pg / mL.

7. A genetically modified human antibody or a binding fragment thereof, characterized in that it comprises a light chain comprising a light chain variable region comprising framework regions, CDRL1 having the amino acid sequence of SEQ ID NO: 8, CDRL2 having the amino acid sequence of SEQ ID NO: 9, and CDRL3 having the amino acid sequence of SEQ ID NO: 10, and a heavy chain comprising a heavy chain variable region comprising framework regions, CDRH1 having the sequence of amino acid of SEQ ID NO: 1, CDRH3 having the amino acid sequence of SEQ ID NO: 3, and CDRH2 having an amino sequence selected from the group consisting of SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, and SEQ ID NO: 7, wherein the antibody binds to human CXCR4.

8. The antibody according to claim 7, characterized in that a light chain comprises the amino acid sequence of SEQ ID NO: 16.

9. The antibody according to either claim 7 or claim 8, characterized in that a heavy chain comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, and SEQ ID NO: 15.

10. The antibody according to any of claims 7 to 9, characterized in that a light chain comprises the amino acid sequence of SEQ ID NO: 22.

11. The antibody according to any of claims 7 to 10, characterized in that a heavy chain comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 20, and SEQ ID NO: 21.

12. The antibody according to any of claims 7 to 11, characterized in that it is selected from the group consisting of an antibody comprising SEQ ID NO: 17 and SEQ ID NO: 22, an antibody comprising SEQ ID NO: 18 and SEC ID. NO: 22, an antibody comprising SEQ ID NO: 19 and SEQ ID NO: 22, an antibody comprising SEQ ID NO: 20 and SEQ ID NO: 22, and an antibody comprising SEQ ID NO: 21 and SEC ID NO: 22

13. The antibody according to any of claims 7 to 12, characterized in that it comprises two light chains of SEQ ID NO: 22, and two heavy chains of SEQ ID NO: 17.

14. A pharmaceutical composition, characterized in that it comprises an antibody according to any of claims 1 to 13 in combination with one or more pharmaceutically acceptable carriers, diluents or excipients.

15. An antibody according to any of claims 1 to 13, characterized for use in therapy.

16. An antibody according to any of claims 1 to 13, characterized for use in the treatment of tumorigenesis, including tumor growth, invasion, angiogenesis, or metastasis.

17. The use of an antibody according to any of claims 1 to 13 in the manufacture of a medicament for the treatment of tumorigenesis, including tumor growth, invasion, angiogenesis, or metastasis.

18. A method for treating tumorigenesis, including tumor growth, invasion, angiogenesis, or metastasis, characterized in that it comprises administering to a patient in need thereof a therapeutically effective amount of an antibody according to any of claims 1 to 13.

19. An antibody according to any of claims 1 to 13, characterized for use in the treatment of a cancer selected from the group consisting of breast cancer, pancreatic cancer, melanoma, prostate cancer, kidney cancer, neuroblastoma, non-cancerous lymphoma. Hodgkin, lung cancer, ovarian cancer, colorectal cancer, multiple myeloma, glioblastoma multiforme, and leukemia.

20. The use of an antibody according to any of claims 1 to 13, in the manufacture of a medicament for the treatment of a cancer selected from the group consisting of breast cancer, pancreatic cancer, melanoma, prostate cancer, kidney cancer , neuroblastoma, non-Hodgkin's lymphoma, lung cancer, ovarian cancer, colorectal cancer, multiple myeloma, glioblastoma multiforme, and leukemia.

21. A method for treating a cancer selected from the group consisting of breast cancer, pancreatic cancer, melanoma, prostate cancer, kidney cancer, neuroblastoma, non-Hodgkin's lymphoma, lung cancer, ovarian cancer, colorectal cancer, multiple myeloma, glioblastoma multiforme, and leukemia, characterized in that it comprises administering to a patient in need thereof a therapeutically effective amount of an antibody according to any of claims 1 to 13.