Anti-cxcr4 antibodies and uses thereof

Abstract

The present disclosure provides antibodies that bind to C-X-C Motif Chemokine Receptor 4 (CXCR4), and methods of use. In various embodiments of the disclosure, the antibodies are fully human antibodies that specifically bind to CXCR4. In some embodiments, the antibodies of the disclosure are useful for inhibiting activation of CXCR4 activity, thus providing a means of affecting the chemotaxis of blood cells and / or immune cells.

Description

ANTI-CXCR4 ANTIBODIES AND USES THEREOFCROSS REFERENCE TO RELATED APPLICATION

[0001] This application claims the benefit under 35 USC §1 19(e) of US Provisional Application No. 63 / 534,939, filed August 28, 2023, which is incorporated herein by reference in its entirety for all purposes.REFERENCE TO A SEQUENCE LISTING

[0002] This application incorporates by reference the Sequence Listing submitted as an Extensible Markup Language (XML) file named 11521 WO01_Sequence, created on August 26, 2024, and containing 46,062 bytes.BACKGROUND OF THE DISCLOSURE

[0003] C-X-C Motif Chemokine Receptor 4 (CXCR4) is a seven transmembrane domain G- protein coupled receptor expressed by hematopoietic, endothelial, neuron, and stem cells. The natural ligand of this receptor is stromal derived factor-1 (SDF-1 ) (also known as CXCL12), a small protein secreted by bone marrow stromal cells. Activation of the CXCR4 receptor by SDF-1 instigates chemotactic movement of hematopoietic, monocytes, macrophages, natural killer, and dendritic cells toward bone marrow.

[0004] CXCR4 is overexpressed in 75% of cancer cells including leukemia, lymphoma, pancreatic, breast, ovarian, lung, prostate, and colorectal cancers. Overexpression of CXCR4 in cancer cells is correlated with increased disease severity, increased likelihood for metastasis, and decreased survival of cancer patients. Leukemia cells, in particular, produce high levels of CXCR4 leading to homing and retention of these cancer cells in the protective microenvironment of the bone marrow. One treatment method for leukemia, and many other hematological diseases, can be the use of hematopoietic stem cell transplantation.

[0005] Hematopoietic stem cell transplantation (HSCT) can fully replace the hematopoietic system of the transplant recipient with donor-derived cells. This approach can be curative for hematologic malignancies, genetic deficiencies of blood lineages, and severe autoimmune diseases refractory to standard treatment. However, patients must first undergo treatments — termed host conditioning — that partially or fully ablate their immune system to facilitate HSCT graft uptake. Traditional approaches to transplant host conditioning employ genotoxins, such as chemotherapeutic agents and radiation, with little specificity for the desired target cells.Unfortunately, the associated risks of off-target tissue damage, secondary malignancies, and opportunistic infection have limited the application of HSCT to a treatment of last resort.SUMMARY

[0006] In one aspect, the present disclosure provides an antibody or antigen binding fragment thereof that binds specifically to an epitope of human C-X-C Motif Chemokine Receptor 4 (CXCR4), wherein the epitope comprises amino acid residues M24, E26, P27, C28, F29, R30, S178, E179, A180, D181 , D182, 1185, D187, F189, Y190, N192, and D193.

[0007] In some embodiments, the antibody or antigen binding fragment binds to an epitope of human CXCR4 comprising amino acid residues M24, K25, E26, P27, C28, F29, R30, E32, A34, S178, E179, A180, D181 , D182, R183, 1185, D187, R188, F189, Y190, N192, D193, and V196.

[0008] In some embodiments, the antibody or antigen binding fragment binds to an epitope of human CXCR4 comprising amino acid residues M24, E26, P27, C28, F29, R30, N176, S178, E179, A180, D181 , D182, 1185, D187, F189, Y190, N192, and D193, C274, and E275.

[0009] In some embodiments, the antibody or antigen-binding fragment thereof comprises three heavy chain complementarity determining regions (CDRs) (HCDR1 , HCDR2 and HCDR3) contained within a heavy chain variable region (HCVR) comprising the amino acid sequence of SEQ ID NO: 2; and three light chain CDRs (LCDR1 , LCDR2 and LCDR3) contained within a light chain variable region (LCVR) comprising the amino acid sequence of SEQ ID NO: 10.

[0010] In some embodiments, the antibody or antigen-binding fragment thereof comprises three heavy chain complementarity determining regions (CDRs) (HCDR1 , HCDR2 and HCDR3) contained within a heavy chain variable region (HCVR) comprising the amino acid sequence of SEQ ID NO: 18; and three light chain CDRs (LCDR1 , LCDR2 and LCDR3) contained within a light chain variable region (LCVR) comprising the amino acid sequence of SEQ ID NO: 26.

[0011] In some embodiments, antibody or antigen-binding fragment comprises HCDR1 - HCDR2-HCDR3-LCDR1 -LCDR2-LCDR3 that comprise the amino acid sequences of SEQ ID NOs: 4-6-8-12-14-16, respectively.

[0012] In some embodiments, the antibody or antigen-binding fragment comprises HCDR1 - HCDR2-HCDR3-LCDR1 -LCDR2-LCDR3 that comprise the amino acid sequences of SEQ ID NOs: 20-22-24-28-30-32, respectively.

[0013] In one aspect, the present disclosure provides an antibody or antigen-binding fragment thereof that binds specifically to C-X-C Motif Chemokine Receptor 4 (CXCR4), wherein the antibody or antigen binding fragment comprises three heavy chain complementarity determining regions (CDRs) and three light chain CDRs contained within a heavy chain variable region(HCVR) and light chain variable region amino acid sequence pair (HCVR / LCVR) selected from the group consisting of SEQ ID NOs: 2 / 10 and 18 / 26.

[0014] In some embodiments, the antibody or antigen-binding fragment comprises HCDR1 - HCDR2-HCDR3-LCDR1 -LCDR2-LCDR3 domains comprising the amino acid sequences, respectively, selected from the group consisting of SEQ ID NOs: 4-6-8-12-14-16 and 20-22-24- 28-30-32.

[0015] In some embodiments, the antibody or antigen binding fragment comprises a HCVR / LCVR amino acid sequence pair selected from the group consisting of: SEQ ID NOs: 2 / 10 and 18 / 26.

[0016] In some embodiments, the antibody comprises a heavy chain (HC) and a light chain (LC) paired with one another, wherein the HC / LC pair comprises the amino acid sequences selected from the group consisting of SEQ ID NOs: 34 / 36 and 38 / 40.

[0017] In one aspect, the present disclosure provides a pair of polynucleotide molecules encoding a heavy chain variable region (HCVR) and a light chain variable region (LCVR) of an antibody or antigen binding fragment thereof that binds specifically to CXCR4, wherein the pair of polynucleotide molecules encode, respectively, a HCVR and a LCVR comprising six complementarity determining regions (CDRs) of a HCVR / LCVR amino acid sequence pair selected from the group consisting of SEQ ID NOs: 2 / 10 and 18 / 26.

[0018] In some embodiments, wherein the six CDRs, HCDR1-HCDR2-HCDR3-LCDR1-LCDR2- LCDR3, comprise the amino acid sequences, respectively, selected from the group consisting of SEQ ID NOs: 4-6-8-12-14-16 and 20-22-24-28-30-32.

[0019] In one aspect, the present disclosure provides a pair of polynucleotide molecules encoding a heavy chain variable region (HCVR) and a light chain variable region (LCVR) of an antibody or antigen binding fragment thereof that binds to CXCR4, wherein the pair of polynucleotide molecules encode, respectively, a HCVR / LCVR amino acid sequence pair selected from the group consisting of SEQ ID NOs: 2 / 10 and 18 / 26.

[0020] In some embodiments, the polynucleotide molecule encoding the HCVR further encodes an immunoglobulin heavy chain constant region of the antibody.

[0021] In some embodiments, the polynucleotide molecule encoding the LCVR further encodes an immunoglobulin light chain constant region of the antibody.

[0022] In one aspect, the present disclosure provides a recombinant expression vector comprising a polynucleotide molecule encoding a heavy chain variable region (HCVR) comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 2 and 18.

[0023] In one aspect, the present disclosure provides a recombinant expression vector comprising a polynucleotide molecule encoding a light chain variable region (LCVR) comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 10 and 26.

[0024] In one aspect, the present disclosure provides a pair of polynucleotide molecules encoding a heavy chain (HC) and a light chain (LC) of an antibody or antigen-binding fragment thereof that binds specifically to C-X-C Motif Chemokine Receptor 4 (CXCR4), wherein the pair of polynucleotide molecules encode, respectively, a HC / LC amino acid sequence pair selected from the group consisting of SEQ ID NOs: 34 / 36 and 38 / 40.

[0025] In some embodiments, the pair of polynucleotides encoding the HC / LC amino acid sequence pair are polynucleotide sequences selected from the group consisting of SEQ ID NOs: 33 / 35 and 37 / 39.

[0026] In one aspect, the present disclosure provides a recombinant expression vector comprising a polynucleotide molecule encoding a heavy chain (HC) comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 34 and 38.

[0027] In one aspect, the present disclosure provides a recombinant expression vector comprising a polynucleotides molecule encoding a light chain (LC) comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 36 and 40.

[0028] In some embodiments, a recombinant expression vector comprises any of the pairs of polynucleotides described herein.

[0029] In some embodiments, an isolated host cell comprises any of the antibodies, antigen binding fragments, polynucleotides, or vectors described herein, or any combination thereof.

[0030] In one aspect, the present disclosure provides a pair of polynucleotide molecules encoding a heavy chain variable region (HCVR) and a light chain variable region (LCVR), respectively, of an antibody or antigen-binding fragment thereof that binds specifically to C-X-C Motif Chemokine Receptor 4 (CXCR4), wherein: (a) the HCVR comprises three heavy chain complementarity determining regions (HCDR1 , HCDR2 and HCDR3), wherein the HCDR1 comprises the amino acid sequence of SEQ ID NO: 4, the HCDR2 comprises the amino acid sequence of SEQ ID NO: 6, and the HCDR3 comprises the amino acid sequence of SEQ ID NO: 8, and (b) the LCVR comprises three light chain complementarity determining regions (LCDR1 , LCDR2 and LCDR3), wherein the LCDR1 comprises the amino acid sequence of SEQ ID NO: 12, the LCDR2 comprises the amino acid sequence of SEQ ID NO: 14, and the LCDR3 comprises the amino acid sequence of SEQ ID NO: 16.

[0031] In some embodiments, a pair of polynucleotide molecules comprises the HCVR comprises the amino acid sequence of SEQ ID NO: 2.

[0032] In some embodiments, the polynucleotide molecule encoding the HCVR comprises the HCDR1 nucleic acid sequence set forth in SEQ ID NO: 3, the HCDR2 nucleic acid sequence set forth in SEQ ID NO: 5, and the HCDR3 nucleic acid sequence set forth in SEQ ID NO: 7.

[0033] In some embodiments, the polynucleotide molecule encoding the HCVR comprises the nucleic acid sequence of SEQ ID NO: 1 .

[0034] In some embodiments, the polynucleotide molecule encoding the HCVR further encodes an immunoglobulin heavy chain constant region of the antibody.

[0035] In some embodiments, the immunoglobulin heavy chain constant region encoded by a polynucleotide is a human lgG1 constant region.

[0036] In some embodiments, the pair of polynucleotide molecules comprises an LCVR that comprises the amino acid sequence of SEQ ID NO: 10.

[0037] In some embodiments, the polynucleotide molecule encoding the LCVR comprises the LCDR1 nucleic acid sequence set forth in SEQ ID NO: 11 , the LCDR2 nucleic acid sequence set forth in SEQ ID NO: 13, and the LCDR3 nucleic acid sequence set forth in SEQ ID NO: 15.

[0038] In some embodiments, the polynucleotide molecule encoding the LCVR comprises the nucleic acid sequence of SEQ ID NO: 9.

[0039] In some embodiments, the polynucleotide molecule encoding the LCVR further encodes an immunoglobulin light chain constant region of the antibody.

[0040] In some embodiments, the immunoglobulin light chain constant region encoded by a disclosed by a polynucleotide is a human kappa constant region.

[0041] In one aspect of the present disclosure, a pair of polynucleotide molecules encoding a heavy chain variable region (HCVR) and a light chain variable region (LCVR), respectively, of an antibody or antigen-binding fragment thereof that binds specifically to C-X-C Motif Chemokine Receptor 4 (CXCR4), wherein: (a) the HCVR comprises three heavy chain complementarity determining regions (HCDR1 , HCDR2 and HCDR3), wherein the HCDR1 comprises the amino acid sequence of SEQ ID NO: 20, the HCDR2 comprises the amino acid sequence of SEQ ID NO: 22, and the HCDR3 comprises the amino acid sequence of SEQ ID NO: 24, and (b) the LCVR comprises three light chain complementarity determining regions (LCDR1 , LCDR2 and LCDR3), wherein the LCDR1 comprises the amino acid sequence of SEQ ID NO: 28, the LCDR2 comprises the amino acid sequence of SEQ ID NO: 30, and the LCDR3 comprises the amino acid sequence of SEQ ID NO: 32.

[0042] In some embodiments, the pair of polynucleotide molecules comprises a HCVR that comprises the amino acid sequence of SEQ ID NO: 18.

[0043] In some embodiments, wherein the polynucleotide molecule encoding the HCVR comprises the HCDR1 nucleic acid sequence set forth in SEQ ID NO: 19, the HCDR2 nucleic acid sequence set forth in SEQ ID NO: 21 , and the HCDR3 nucleic acid sequence set forth in SEQ ID NO: 23.

[0044] In some embodiments, the polynucleotide molecule encoding the HCVR comprises the nucleic acid sequence of SEQ ID NO: 17.

[0045] In some embodiments, the polynucleotide molecule encoding the HCVR further encodes an immunoglobulin heavy chain constant region of the antibody.

[0046] In some embodiments, the immunoglobulin heavy chain constant region encoded by a polynucleotide is a human lgG1 constant region.

[0047] In some embodiments, the polynucleotide molecule encoding the LCVR comprises the amino acid sequence of SEQ ID NO: 26.

[0048] In some embodiments, the polynucleotide molecule encoding the LCVR comprises the LCDR1 nucleic acid sequence set forth in SEQ ID NO: 27, the LCDR2 nucleic acid sequence set forth in SEQ ID NO: 29, and the LCDR3 nucleic acid sequence set forth in SEQ ID NO: 31 .

[0049] In some embodiments, the polynucleotide molecule encoding the LCVR comprises the nucleic acid sequence of SEQ ID NO: 25.

[0050] In some embodiments, the polynucleotide molecule encoding the LCVR further encodes an immunoglobulin light chain constant region of the antibody.

[0051] In some embodiments, the immunoglobulin light chain constant region encoded by a polynucleotide is a human kappa constant region.

[0052] In one aspect, the present disclosure provides a method of producing an anti-CXCR4 antibody or antigen-binding fragment thereof by culturing any of the isolated host cells disclosed herein under conditions permitting production of the antibody or antigen-binding fragment, and recovering the antibody or antigen-binding fragment so produced.

[0053] In some embodiments, the isolated host cell is a Chinese hamster ovary (CHO) cell.

[0054] In one aspect, the present disclosure provides a method to enhance hematopoietic stem cell engraftment, comprising administering to a subject in need thereof an antibody or antigen binding fragment thereof disclosed herein.

[0055] In one aspect, the present disclosure provides a method of treating cancer, comprising administering to a subject in need thereof, any of the antibodies or antigen binding fragments disclosed herein.

[0056] In some embodiments, the method of treating cancer includes the treatment of a leukemia or a lymphoma.

[0057] In one aspect, the present disclosure provides a method of altering B-cell localization in the bone marrow or peripheral blood supply, comprising administering to a subject in need thereof any of the antibodies or antigen binding fragments disclosed herein.

[0058] In one aspect, the present disclosure provides a method of hematopoietic stem cell transplant (HSCT) host conditioning, comprising administering to a subject in need thereof, any of the antibodies or antigen binding fragments disclosed herein. HSCT can fully replace the hematopoietic system of the transplant recipient with donor-derived cells. This approach can be curative for hematologic malignancies, genetic deficiencies of blood lineages, and severe autoimmune diseases refractory to standard treatment. However, patients must first undergo treatments — termed host conditioning — that partially or fully ablate their immune system to facilitate HSCT graft uptake. Traditional approaches to transplant host conditioning employ genotoxins, such as chemotherapeutic agents and radiation, with little specificity for the desired target cells. The associated risks of off-target tissue damage, secondary malignancies, and opportunistic infection thus limit the application of HSCT to a treatment of last resort. Anti- CXCR4 antibody or antigen-binding fragment thereof can displace hematopoietic stem cell (HSC)s from their resident niches in the bone marrow (BM) could facilitate the engraftment of donor HSCs. Blockade of CXCR4, a G-protein-coupled receptor (GPCR) that is essential for localization of hematopoietic cells to and within BM, is known to trigger mobilization of mature and progenitor immune cells from BM to peripheral sites. Anti-CXCR4 antibody or antigenbinding fragment thereof that occlude the interaction of CXCR4 with its ligand CXCL12 (aka SDF1 ) can have this effect.

[0059] In one aspect, the present disclosure provides a method of inducing cancer cell apoptosis, comprising administering to a subject in need thereof, any of the antibodies or antigen binding fragments disclosed herein.

[0060] In one aspect, the present disclosure provides a method of preventing HIV infection, comprising administering to a subject in need thereof any of the antibodies or antigen binding fragments disclosed herein.

[0061] In various embodiments, any of the features or components of embodiments discussed above or herein may be combined, and such combinations are encompassed within the scope of the present disclosure. Any specific value discussed above or herein may be combined with another related value discussed above or herein to recite a range with the values representing the upper and lower ends of the range, and such ranges and all values falling within such ranges are encompassed within the scope of the present disclosure. Each of the valuesdiscussed above or herein may be expressed with a variation of 1%, 5%, 10% or 20%. Other embodiments will become apparent from a review of the ensuing detailed description.BRIEF DESCRIPTION OF DRAWINGS

[0062] Figure 1A shows the CryoEM structure of REGN7663 Fab HC and FabLC binding to CXCR4.

[0063] Figure 1 B shows the top-down view of CXCR4 with the epitope residues labelled which interact with REGN7663.

[0064] Figure 2A shows the CryoEM structure of REGN7664 Fab HC and FabLC binding to CXCR4.

[0065] Figure 2B shows the top-down view of CXCR4 with the epitope residues labelled which interact with REGN7664.

[0066] Figure 3 shows human / mouse CXCR4 domain swap chimeric constructs designed to test binding sites for anti-human-CXCR4 antibodies.

[0067] Figure 4 shows flow cytometry data establishing that REGN7663 loses binding to all variants where the ECL2 domain is murine (pMM331 , pMM335, pMM336, pMM341 , pMM345) yet binds efficiently to murine CXCR4 with a humanized ECL2 domain (pMM346).

[0068] Figure 5 shows flow cytometry data establishing that REGN7664 loses binding to all variants where the ECL2 domain is murine (pMM331 , pMM335, pMM336, pMM341 , pMM345) yet binds efficiently to murine CXCR4 with a humanized ECL2 domain (pMM346).

[0069] Figure 6 shows staining with isotype REGN1945 antibody confirmed binding specificity.

[0070] Figure 7 shows that staining with a human-mouse cross-reactive anti-CXCR4 (m / hCXCR4 clone 2B1 1) confirmed expression of all constructs except for pMM335, which failed to express.

[0071] Figure 8A shows a bioassay using a HEK293 / CRE-luc / hCXCR4 reporter line for use in Example 7 CXCR4 / SDF-1 signaling assays where forskolin increases CRE-driven luminescence in the absence of SDF-1 binding to the CXCR4.

[0072] Figure 8B shows the basis of measuring SDF-1 / CXCR4 signaling activity with the HEK293 / CRE-luc / hCXCR4 bioassay for use in Example 7 where binding of SDF-1 to CXCR4 impedes forskolin-induced luminescence.

[0073] Figure 8C shows the basis of measuring blockade of SDF-1 / CXCR4 signaling with anti- CXCR4 with the HEK293 / CRE-luc / hCXCR4 reporter line for use in Example 7 CXCR4 / SDF-1 signaling assays where anti-CXCR4 prevents SDF-1 from binding to CXCR4 thereby restoring forskolin-induced luminescence.

[0074] Figure 9A shows a schematic of the methods used in Example 12 to determine effects of CXCR4 blockade on leukocyte mobilization in CXCR4 Humin mice.

[0075] Figure 9B shows that a single 10 mg / kg dose of anti-CXCR4 antibodies with signaling blocking function (REGN7663 and REGN7664) led to transient mobilization of BM-resident leukocytes 2 and 6 hours post-administration, as compared to the pre-administration baseline (O-hour) and isotype control antibody (REGN1945).

[0076] Figure 10A shows a schematic of the methods used in Example 12 to assess the effects of CXCR4 blockade on HSC / HSPC mobilization in CXCR4 Humin mice.

[0077] Figure 10B shows REGN7664, but not REGN7663, treatment led to a detectable elevation of rare lineage-negative cKit+ (LK) and lineage-negative Sca1 + cKit+ (LSK) hematopoietic progenitor cells in peripheral blood, as compared to isotype antibody, REGN1925, treatment.

[0078] Figure 11 A shows a schematic of the methods used in Example 12 to examine the effects of CXCR4 blockade on bone marrow B cells in CXCR4 Humin mice via single dose on Day 5 or repeated, consecutive doses from Day 1 to Day 5 of CXCR4 blocking antibodies.

[0079] Figure 11 B shows that both the single dose and the repeated, consecutive doses of REGN7664 causes profound reduction of mature B cells in bone marrow as compared to isotype (REGN1945).

[0080] Figure 11C shows that the observed decrease in mature cells was significant and consistent both for REGN7663 and REGN7664 treatment groups and was coupled with an increased proportion of early pre-B cells.DETAILED DESCRIPTION OF THE DISCLOSURE

[0081] Before the present methods are described, it is to be understood that this disclosure is not limited to particular methods, and experimental conditions described, as such methods and conditions may vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to be limiting, since the scope of the present disclosure will be limited only by the appended claims.Terms and Definitions

[0082] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs. Although any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present disclosure, preferred methods and materialsare now described. All publications mentioned herein are incorporated herein by reference in their entirety.

[0083] The term “CXCR4” refers to the C-X-C motif chemokine receptor 4. The amino acid sequence of full length human CXCR4 is provided as SEQ ID NO: 43 (accession number NP 003458.1 ). The amino acid sequence of full length Macaca mulatta is provided as SEQ ID NO: 44 (accession number NP 0010361 10.1 ). The term “CXCR4” includes recombinant CXCR4 or a fragment thereof. The term also encompasses CXCR4 or a fragment thereof coupled to, for example, histidine tag, mouse or human Fc, or a signal sequence.

[0084] The term "antibody", as used herein, is intended to refer to immunoglobulin molecules comprised of four polypeptide chains, two heavy (H) chains and two light (L) chains interconnected by disulfide bonds ( / .e., "full antibody molecules"), as well as multimers thereof (e.g. IgM) or antigen-binding fragments thereof. Each heavy chain is comprised of a heavy chain variable region (“HCVR” or “VH”) and a heavy chain constant region (comprised of domains CH1 , CH2 and CH3). Each light chain is comprised of a light chain variable region (“LCVR or “VL”) and a light chain constant region (CL). The VH and VL regions can be further subdivided into regions of hypervariability, termed complementarity determining regions (CDR), interspersed with regions that are more conserved, termed framework regions (FR). Each VH and VL is composed of three CDRs and four FRs, arranged from amino-terminus to carboxyterminus in the following order: FR1 , CDR1 , FR2, CDR2, FR3, CDR3, FR4. In certain embodiments of the disclosure, the FRs of the antibody (or antigen binding fragment thereof) may be identical to the human germline sequences, or may be naturally or artificially modified. An amino acid consensus sequence may be defined based on a side-by-side analysis of two or more CDRs.

[0085] Substitution of one or more CDR residues or omission of one or more CDRs is also possible. Antibodies have been described in the scientific literature in which one or two CDRs can be dispensed with for binding. Padlan et al. (1995 FASEB J. 9:133-139) analyzed the contact regions between antibodies and their antigens, based on published crystal structures, and concluded that only about one fifth to one third of CDR residues actually contact the antigen. Padlan also found many antibodies in which one or two CDRs had no amino acids in contact with an antigen (see also, Vajdos et al. 2002 J Mol Biol 320:415-428).

[0086] CDR residues not contacting antigen can be identified based on previous studies (for example residues H60-H65 in CDRH2 are often not required), from regions of Kabat CDRs lying outside Chothia CDRs, by molecular modeling and / or empirically. If a CDR or residue(s) thereof is omitted, it is usually substituted with an amino acid occupying the corresponding position inanother human antibody sequence or a consensus of such sequences. Positions for substitution within CDRs and amino acids to substitute can also be selected empirically. Empirical substitutions can be conservative or non-conservative substitutions.

[0087] The fully human anti-CXCR4 monoclonal antibodies disclosed herein may comprise one or more amino acid substitutions, insertions, and / or deletions in the framework and / or CDR regions of the heavy and light chain variable domains as compared to the corresponding germline sequences. Such mutations can be readily ascertained by comparing the amino acid sequences disclosed herein to germline sequences available from, for example, public antibody sequence databases. The present disclosure includes antibodies, and antigen-binding fragments thereof, which are derived from any of the amino acid sequences disclosed herein, wherein one or more amino acids within one or more framework and / or CDR regions are mutated to the corresponding residue(s) of the germline sequence from which the antibody was derived, or to the corresponding residue(s) of another human germline sequence, or to a conservative amino acid substitution of the corresponding germline residue(s) (such sequence changes are referred to herein collectively as "germline mutations"). A person of ordinary skill in the art, starting with the heavy and light chain variable region sequences disclosed herein, can easily produce numerous antibodies and antigen-binding fragments which comprise one or more individual germline mutations or combinations thereof. In certain embodiments, all of the framework and / or CDR residues within the VH and / or VL domains are mutated back to the residues found in the original germline sequence from which the antibody was derived. In other embodiments, only certain residues are mutated back to the original germline sequence, e.g., only the mutated residues found within the first 8 amino acids of FR1 or within the last 8 amino acids of FR4, or only the mutated residues found within CDR1 , CDR2 or CDR3. In other embodiments, one or more of the framework and / or CDR residue(s) are mutated to the corresponding residue(s) of a different germline sequence ( / .e., a germline sequence that is different from the germline sequence from which the antibody was originally derived). Furthermore, the antibodies of the present disclosure may contain any combination of two or more germline mutations within the framework and / or CDR regions, e.g., wherein certain individual residues are mutated to the corresponding residue of a particular germline sequence while certain other residues that differ from the original germline sequence are maintained or are mutated to the corresponding residue of a different germline sequence. Once obtained, antibodies and antigen-binding fragments that contain one or more germline mutations can be easily tested for one or more desired property such as, improved binding specificity, increased binding affinity, improved or enhanced antagonistic or agonistic biological properties (as thecase may be), reduced immunogenicity, etc. Antibodies and antigen-binding fragments obtained in this general manner are encompassed within the present disclosure.

[0088] The present disclosure also includes fully human anti-CXCR4 monoclonal antibodies comprising variants of any of the HCVR, LCVR, and / or CDR amino acid sequences disclosed herein having one or more conservative substitutions. For example, the present disclosure includes anti-CXCR4 antibodies having HCVR, LCVR, and / or CDR amino acid sequences with, e.g., 10 or fewer, 8 or fewer, 6 or fewer, 4 or fewer, etc. conservative amino acid substitutions relative to any of the HCVR, LCVR, and / or CDR amino acid sequences disclosed herein.

[0089] The terms "human antibody" and “fully human antibody,” as used herein, are intended to include antibodies having variable and constant regions derived from human germline immunoglobulin sequences. The human mAbs of the disclosure may include amino acid residues not encoded by human germline immunoglobulin sequences (e.g., mutations introduced by random or site-specific mutagenesis in vitro or by somatic mutation in vivo), for example in the CDRs and in particular CDR3. However, the terms "human antibody" and “fully human antibody,” as used herein, are not intended to include mAbs in which CDR sequences derived from the germline of another mammalian species (e.g., mouse), have been grafted onto human FR sequences. The terms include antibodies recombinantly produced in a non-human mammal, or in cells of a non-human mammal. The terms are not intended to include antibodies isolated from or generated in a human subject.

[0090] The term “recombinant”, as used herein, refers to antibodies or antigen-binding fragments thereof of the disclosure created, expressed, isolated or obtained by technologies or methods known in the art as recombinant DNA technology which include, e.g., DNA splicing and transgenic expression. The term refers to antibodies expressed in a non-human mammal (including transgenic non-human mammals, e.g., transgenic mice), or a cell (e.g., CHO cells) expression system or isolated from a recombinant combinatorial human antibody library.

[0091] The term "specifically binds," or “binds specifically to”, or the like, means that an antibody or antigen-binding fragment thereof forms a complex with an antigen that is relatively stable under physiologic conditions. Specific binding can be characterized by an equilibrium dissociation constant of at least about 1 x108M or less (e.g., a smaller KDdenotes a tighter binding). Methods for determining whether two molecules specifically bind are well known in the art and include, for example, equilibrium dialysis, surface plasmon resonance, and the like. As described herein, antibodies have been identified by surface plasmon resonance, e.g., BIACORE™, which bind specifically to CXCR4. Moreover, multi-specific antibodies that bind to one domain in CXCR4 and one or more additional antigens or a bi-specific that binds to twodifferent regions of CXCR4 are nonetheless considered antibodies that “specifically bind”, as used herein.

[0092] The term “high affinity” antibody refers to those mAbs having a binding affinity to CXCR4, expressed as KD, of at least 10'8M; preferably 10'9M; more preferably 1 O’1oM, even more preferably 10'11M, even more preferably 10'12M, as measured by surface plasmon resonance, e.g., BIACORE™ or solution-affinity ELISA.

[0093] By the term “slow off rate”, “Koff” or “kd” is meant an antibody that dissociates from CXCR4, with a rate constant of 1 x 10'3s-1or less, preferably 1 x 10-4s'1or less, as determined by surface plasmon resonance, e.g., BIACORE™.

[0094] The terms "antigen-binding portion" of an antibody, "antigen-binding fragment" of an antibody, and the like, as used herein, include any naturally occurring, enzymatically obtainable, synthetic, or genetically engineered polypeptide or glycoprotein that specifically binds an antigen to form a complex. The terms "antigen-binding fragment" of an antibody, or "antibody fragment”, as used herein, refers to one or more fragments of an antibody that retain the ability to bind to CXCR4.

[0095] In specific embodiments, antibody or antibody fragments of the disclosure may be conjugated to a moiety such a ligand or a therapeutic moiety (“immunoconjugate”), such as a cytotoxin, a second anti-CXCR4 antibody, an antibody to a tumor-specific antigen, an anticancer drug, or any other therapeutic moiety useful for treating a disease or condition including cancer or viral infection including chronic viral infection.

[0096] The phrase "an antibody that binds CXCR4" or an "anti-CXCR4 antibody" includes antibodies and antigen-binding fragments thereof that specifically recognize a single CXCR4 molecule.

[0097] An "isolated antibody", as used herein, is intended to refer to an antibody that is substantially free of other antibodies (Abs) having different antigenic specificities (e.g., an isolated antibody that specifically binds CXCR4, or a fragment thereof, is substantially free of Abs that specifically bind antigens other than CXCR4.

[0098] A “blocking antibody” or a "neutralizing antibody", as used herein (or an "antibody that neutralizes CXCR4 activation" or “antagonist antibody”), is intended to refer to an antibody whose binding to CXCR4 results in inhibition of at least one biological activity of CXCR4. For example, an antibody of this disclosure may prevent or block SDF-1 binding to CXCR4.

[0099] An “activating antibody” or an “enhancing antibody”, as used herein (or an “agonist antibody”), is intended to refer to an antibody whose binding to CXCR4 results in increasing or stimulating at least one biological activity of CXCR4.

[0100] The term "surface plasmon resonance", as used herein, refers to an optical phenomenon that allows for the analysis of real-time biomolecular interactions by detection of alterations in protein concentrations within a biosensor matrix, for example using the BIACORE™ system (Pharmacia Biosensor AB, Uppsala, Sweden and Piscataway, N.J.).

[0101] The term "KD ", as used herein, is intended to refer to the equilibrium dissociation constant of a particular antibody-antigen interaction.

[0102] The term “epitope” refers to an antigenic determinant that interacts with a specific antigen binding site in the variable region of an antibody molecule known as a paratope. A single antigen may have more than one epitope. Thus, different antibodies may bind to different areas on an antigen and may have different biological effects. The term “epitope” also refers to a site on an antigen to which B and / or T cells respond. It also refers to a region of an antigen that is bound by an antibody. Epitopes may be defined as structural or functional. Functional epitopes are generally a subset of the structural epitopes and have those residues that directly contribute to the affinity of the interaction. Epitopes may also be conformational, that is, composed of non-linear amino acids. In certain embodiments, epitopes may include determinants that are chemically active surface groupings of molecules such as amino acids, sugar side chains, phosphoryl groups, or sulfonyl groups, and, in certain embodiments, may have specific three-dimensional structural characteristics, and / or specific charge characteristics.

[0103] The term “cross-competes”, as used herein, means an antibody or antigen-binding fragment thereof binds to an antigen and inhibits or blocks the binding of another antibody or antigen-binding fragment thereof. The term also includes competition between two antibodies in both orientations, i.e., a first antibody that binds and blocks binding of second antibody and vice- versa. In certain embodiments, the first antibody and second antibody may bind to the same epitope. Alternatively, the first and second antibodies may bind to different, but overlapping epitopes such that binding of one inhibits or blocks the binding of the second antibody, e.g., via steric hindrance. Cross-competition between antibodies may be measured by methods known in the art, for example, by a real-time, label-free bio-layer interferometry assay. Crosscompetition between two antibodies may be expressed as the binding of the second antibody that is less than the background signal due to self-self binding (wherein first and second antibodies is the same antibody). Cross-competition between two antibodies may be expressed, for example, as percent binding of the second antibody that is less than the baseline self-self background binding (wherein first and second antibodies is the same antibody).

[0104] The term "substantial identity" or "substantially identical," when referring to a nucleic acid or fragment thereof, indicates that, when optimally aligned with appropriate nucleotide insertionsor deletions with another nucleic acid (or its complementary strand), there is nucleotide sequence identity in at least about 90%, and more preferably at least about 95%, 96%, 97%, 98% or 99% of the nucleotide bases, as measured by any well-known algorithm of sequence identity, as discussed below. A nucleic acid molecule having substantial identity to a reference nucleic acid molecule may, in certain instances, encode a polypeptide having the same or substantially similar amino acid sequence as the polypeptide encoded by the reference nucleic acid molecule.

[0105] Sequence identity can be calculated using an algorithm, for example, the Needleman Wunsch algorithm (Needleman and Wunsch 1970, J. Mol. Biol. 48: 443-453) for global alignment, or the Smith Waterman algorithm (Smith and Waterman 1981 , J. Mol. Biol. 147: 195- 197) for local alignment. Another preferred algorithm is described by Dufresne et al in Nature Biotechnology in 2002 (vol. 20, pp. 1269-71) and is used in the software GenePAST (GQ Life Sciences, Inc. Boston, MA).

[0106] As applied to polypeptides, the term "substantial similarity" or “substantially similar” means that two peptide sequences, when optimally aligned, such as by the programs GAP or BESTFIT using default gap weights, share at least 90% sequence identity, even more preferably at least 95%, 98% or 99% sequence identity. Preferably, residue positions, which are not identical, differ by conservative amino acid substitutions. A "conservative amino acid substitution" is one in which an amino acid residue is substituted by another amino acid residue having a side chain (R group) with similar chemical properties (e.g., charge or hydrophobicity). In general, a conservative amino acid substitution will not substantially change the functional properties of a protein. In cases where two or more amino acid sequences differ from each other by conservative substitutions, the percent or degree of similarity may be adjusted upwards to correct for the conservative nature of the substitution. Means for making this adjustment are well known to those of skill in the art. See, e.g., Pearson (1994) Methods Mol. Biol. 24: 307- 331 , which is herein incorporated by reference. Examples of groups of amino acids that have side chains with similar chemical properties include 1) aliphatic side chains: glycine, alanine, valine, leucine and isoleucine; 2) aliphatic-hydroxyl side chains: serine and threonine; 3) amide- containing side chains: asparagine and glutamine; 4) aromatic side chains: phenylalanine, tyrosine, and tryptophan; 5) basic side chains: lysine, arginine, and histidine; 6) acidic side chains: aspartate and glutamate, and 7) sulfur-containing side chains: cysteine and methionine. Preferred conservative amino acids substitution groups are: valine-leucine-isoleucine, phenylalanine-tyrosine, lysine-arginine, alanine-valine, glutamate-aspartate, and asparagineglutamine. Alternatively, a conservative replacement is any change having a positive value inthe PAM250 log-likelihood matrix disclosed in Gonnet et al. (1992) Science 256: 1443 45, herein incorporated by reference. A "moderately conservative" replacement is any change having a nonnegative value in the PAM250 log-likelihood matrix

[0107] Sequence similarity for polypeptides is typically measured using sequence analysis software. Protein analysis software matches similar sequences using measures of similarity assigned to various substitutions, deletions and other modifications, including conservative amino acid substitutions. For instance, GCG software contains programs such as GAP and BESTFIT which can be used with default parameters to determine sequence homology or sequence identity between closely related polypeptides, such as homologous polypeptides from different species of organisms or between a wild type protein and a mutein thereof. See, e.g., GCG Version 6.1 . Polypeptide sequences also can be compared using FASTA with default or recommended parameters; a program in GCG Version 6.1. FASTA {e.g., FASTA2 and FASTA3) provides alignments and percent sequence identity of the regions of the best overlap between the query and search sequences (Pearson (2000) supra). Another preferred algorithm when comparing a sequence of the disclosure to a database containing a large number of sequences from different organisms is the computer program BLAST, especially BLASTP or TBLASTN, using default parameters. See, e.g., Altschul et al. (1990) J. Mol. Biol. 215: 403-410 and (1997) Nucleic Acids Res. 25:3389-3402, each of which is herein incorporated by reference.

[0108] Polynucleotides, as discussed herein, may encode all or a portion of an antibody or antigen-binding fragment as discussed throughout the present disclosure. In some cases, a single polynucleotide may encode both a HCVR and a LCVR e.g., defined with reference to the CDRs contained within the respective amino acid sequence-defined HCVR and LCVR, defined with reference to the amino acid sequences of the CDRs of the HCVR and LCVR. respectively, or defined with reference to the amino acid sequences of the HCVR and LCVR, respectively) of an antibody or antigen-binding fragment, or the HCVR and LCVR may be encoded by separate polynucleotides {i.e., a pair of polynucleotides). In the latter case, in which the HCVR and LCVR are encoded by separate polynucleotides, the polynucleotides may be combined in a single vector or may be contained in separate vectors {i.e., a pair of vectors). In any case, a host cell used to express the polynucleotide(s) or vector(s) may contain the full complement of component parts to generate the antibody or antigen-binding fragment thereof. For example, a host cell may comprise separate vectors, each encoding a HCVR and a LCVR, respectively, of an antibody or antigen-binding fragment thereof as discussed above or herein. Similarly, the polynucleotide or polynucleotides, and the vector or vectors, may be used to express the full- length heavy chain and full-length light chain of an antibody as discussed above or herein. Forexample, a host cell may comprise a single vector with polynucleotides encoding both a heavy chain and a light chain of an antibody, or the host cell may comprise separate vectors with polynucleotides encoding, respectively, a heavy chain and a light chain of an antibody as discussed above or herein.

[0109] The term “vector”, as used herein, means any molecule or particle used to deliver a foreign nucleic acid into a cell. For example, the nucleic a can be, for example, RNA or DNA. The molecule or particle can be, for example, a plasmid, cosmid, phage, or virus.

[0110] By the phrase “therapeutically effective amount” is meant an amount that produces the desired effect for which it is administered. The exact amount will depend on the purpose of the treatment, and will be ascertainable by one skilled in the art using known techniques (see, for example, Lloyd (1999) The Art, Science and Technology of Pharmaceutical Compounding).

[0111] As used herein, the term “subject” refers to an animal, preferably a mammal, in need of amelioration, prevention and / or treatment of a disease or disorder such as viral infection, or cancer. The term includes human subjects who have or are at risk of having cancer, metastatic cancer or viral infection.

[0112] As used herein, “anti-cancer drug” means any agent useful to treat or ameliorate or inhibit cancer including, but not limited to, cytotoxins and agents such as anti metabolites, alkylating agents, anthracyclines, antibiotics, antimitotic agents, procarbazine, hydroxyurea, asparaginase, corticosteroids, cyclophosphamide, mytotane (O,P'-(DDD)), biologies (e.g. antibodies and interferons) and radioactive agents. As used herein, “a cytotoxin or cytotoxic agent”, also refers to a chemotherapeutic agent and means any agent that is detrimental to cells. Examples include Taxol® (paclitaxel), temozolamide, cytochalasin B, gramicidin D, ethidium bromide, emetine, cisplatin, mitomycin, etoposide, tenoposide, vincristine, vinbiastine, coichicin, doxorubicin, daunorubicin, dihydroxy anthracin dione, mitoxantrone, mithramycin, actinomycin D, 1 -dehydrotestosterone, glucocorticoids, procaine, tetracaine, lidocaine, propranolol, and puromycin and analogs or homologs thereof.

[0113] As used herein, the term “anti-viral drug” refers to any drug or therapy used to treat, prevent, or ameliorate a viral infection in a host subject. The term “anti-viral drug” includes, but is not limited to zidovudine, lamivudine, abacavir, ribavirin, lopinavir, efavirenz, cobicistat, tenofovir, rilpivirine, analgesics and corticosteroids. In the context of the present disclosure, the viral infections include long-term or chronic infections caused by viruses including, but not limited to, human immunodeficiency virus (HIV), hepatitis B virus (HBV), hepatitis C virus (HCV), human papilloma virus (HPV), lymphocytic choriomeningitis virus (LCMV), and simian immunodeficiency virus (SIV).Overview

[0114] The antibodies and antigen-binding fragments of the present disclosure specifically bind to CXCR4 and block CXCR4 activation. The anti-CXCR4 antibodies may bind to CXCR4 with high affinity or with low affinity. In certain embodiments, the antibodies of the present disclosure may be blocking antibodies wherein the antibodies may bind to CXCR4 and inhibit CXCR4 signaling. In some embodiments, the antibodies of the disclosure block the binding of CXCR4 to SDF-1 to prevent CXCR4 activation. In some embodiments, the antibodies bind to CXCR4 and affect the homing of cells to bone marrow. In certain embodiments, the antibodies bind to CXCR4 and affect the localization of B-cells in peripheral blood supply. In some embodiments, the antibodies may be useful for stimulating or enhancing apoptosis of cancer cells. The antibodies may reduce infection by HIV. The antibodies may also be used to inhibit the growth of tumor cells in a subject. The antibodies may also be used alone or as adjunct therapy with other therapeutic moieties or modalities known in the art for treating cancer, or viral infection.

[0115] In certain embodiments, the anti-CXCR4 antibodies may be multi-specific antigenbinding molecules, wherein they comprise a first binding specificity to CXCR4 and a second binding specificity to an antigen selected from the group consisting of another receptor inhibitor, and a different epitope of CXCR4.

[0116] An immunogen comprising any one of the following can be used to generate antibodies to CXCR4. In certain embodiments, the antibodies of the disclosure are obtained from mice immunized with a full length, native human CXCR4 (See NCBI accession number NP 003458.1 ) (SEQ ID NO: 43), native Macaca mulatta CXCR4 (See NCBI accession number NP 001036110.1 ) (SEQ ID NO: 44) or with a recombinant CXCR4 peptide. Alternatively, CXCR4 or a fragment thereof may be produced using standard biochemical techniques and used as immunogen.

[0117] In certain embodiments, the immunogen is the extracellular domain of CXCR4. In one embodiment of the disclosure, the immunogen is a fragment of the extracellular domain of CXCR4.

[0118] In some embodiments, the immunogen may be a recombinant CXCR4 peptide expressed in E. coli or in any other eukaryotic or mammalian cells such as Chinese hamster ovary (CHO) cells.

[0119] In certain embodiments, antibodies that bind specifically to CXCR4 may be prepared using fragments of the extracellular regions, or peptides that extend beyond the designated regions by about 5 to about 20 amino acid residues from either, or both, the N or C terminalends of the regions described herein. In certain embodiments, any combination of the abovenoted regions or fragments thereof may be used in the preparation of CXCR4 specific antibodies.

[0120] The peptides may be modified to include addition or substitution of certain residues for tagging or for purposes of conjugation to carrier molecules, such as, KLH. For example, a cysteine may be added at either the N terminal or C terminal end of a peptide, or a linker sequence may be added to prepare the peptide for conjugation to, for example, KLH for immunization.

[0121] Certain anti-CXCR4 antibodies of the present disclosure are able to bind to and neutralize the activity of CXCR4, as determined by in vitro or in vivo assays. The ability of the antibodies of the disclosure to bind to and neutralize the activity of CXCR4 may be measured using any standard method known to those skilled in the art, including binding assays, or activity assays, as described herein.

[0122] Non-limiting, exemplary in vitro assays for measuring binding activity are illustrated in Examples herein. In Example 3, the structural binding of disclosed antibodies to CXCR4 was assessed using cryo-electron microscopy (CryoEM). In Example 4, the structural binding of antibodies to human CXCR4 was confirmed using human-mouse CXCR4 domain swap constructs. Example 5 describes a cell binding analysis using flow cytometry. Example 6 describes an electrochemiluminescence binding of the anti-CXCR4 antibodies to cells expressing CXCR4. In Examples 6 through 9, the blocking of CXCR4 activation by disclosed antibodies was assessed using cell-based bioassays. Example 10 describes the activation of CXCR4 for p-arrestin recruitment. Example 11 describes HIV pseudo particles neutralization by the disclosed anti-CXCR4 antibodies.

[0123] In certain embodiments, the antibodies of the present disclosure are able to affect cell migration in vivo, in a subject. Example 12 demonstrates that in certain embodiments, use of disclosed anti-CXCR4 antibodies leads to the mobilization of blood and / or immune cells. In some embodiments, the disclosed anti-CXCR4 antibodies suppresses the retention of mature B cells in the bone marrow of a subject. In certain embodiments, the antibodies of the present disclosure are used in combination with a second therapeutic agent, such as another antibody, to provide a therapeutic benefit to a subject.

[0124] The antibodies specific for CXCR4 may contain no additional labels or moieties, or they may contain a label or moiety, e.g., an N-terminal or C-terminal label or moiety. In one embodiment, the label or moiety is biotin. In a binding assay, the location of a label (if any) may determine the orientation of the peptide relative to the surface upon which the peptide is bound.For example, if a surface is coated with avidin, a peptide containing an N-terminal biotin will be oriented such that the C-terminal portion of the peptide will be distal to the surface. In one embodiment, the label may be a radionuclide, a fluorescent dye or a MRI-detectable label. In certain embodiments, such labeled antibodies may be used in diagnostic assays including imaging assays.Antibodies and Antigen Binding Fragments Thereof

[0125] Unless specifically indicated otherwise, the term "antibody," as used herein, shall be understood to encompass antibody molecules comprising two immunoglobulin heavy chains and two immunoglobulin light chains ( / .e., "full antibody molecules") as well as antigen-binding fragments thereof. The terms "antigen-binding portion" of an antibody, "antigen-binding fragment" of an antibody, and the like, as used herein, include any naturally occurring, enzymatically obtainable, synthetic, or genetically engineered polypeptide or glycoprotein that specifically binds an antigen to form a complex. The terms "antigen-binding fragment" of an antibody, or "antibody fragment”, as used herein, refers to one or more fragments of an antibody that retain the ability to specifically bind to CXCR4. An antibody fragment may include a Fab fragment, a F(ab')2 fragment, a Fv fragment, a dAb fragment, a fragment containing a CDR, or an isolated CDR. In certain embodiments, the term “antigen-binding fragment” refers to a polypeptide fragment of a multi-specific antigen-binding molecule. Antigen-binding fragments of an antibody may be derived, e.g., from full antibody molecules using any suitable standard techniques such as proteolytic digestion or recombinant genetic engineering techniques involving the manipulation and expression of DNA encoding antibody variable and (optionally) constant domains. Such DNA is known and / or is readily available from, e.g., commercial sources, DNA libraries (including, e.g., phage-antibody libraries), or can be synthesized. The DNA may be sequenced and manipulated chemically or by using molecular biology techniques, for example, to arrange one or more variable and / or constant domains into a suitable configuration, or to introduce codons, create cysteine residues, modify, add or delete amino acids, etc.

[0126] In various embodiments, the disclosure provides an antibody (e.g., an isolated recombinant monoclonal antibody) or antigen-binding fragment thereof that binds to CXCR4 (e.g., human CXCR4), wherein the antibody or antigen-binding fragment thereof exhibits one or more of the following characteristics: (i) comprises a HCVR comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 2 and 18, or a substantially similar sequence thereof having at least 90%, at least 95%, at least 98% or at least 99% sequence identity to oneof the aforementioned sequences; (ii) comprises a LCVR comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 10 and 26, or a substantially similar sequence thereof having at least 90%, at least 95%, at least 98% or at least 99% sequence identity to one of the aforementioned sequences; (iii) comprises a HCDR3 domain having an amino acid sequence selected from the group consisting of SEQ ID NO: 8 and 24, or a substantially similar sequence thereof having at least 90%, at least 95%, at least 98% or at least 99% sequence identity to one of the aforementioned sequences; and a LCDR3 domain having an amino acid sequence selected from the group consisting of SEQ ID NO: 16 and 32, or a substantially similar sequence thereof having at least 90%, at least 95%, at least 98% or at least 99% sequence identity to one of the aforementioned sequences; (iv) comprises a HCDR1 domain having an amino acid sequence selected from the group consisting of SEQ ID NO: 4 and 20, or a substantially similar sequence thereof having at least 90%, at least 95%, at least 98% or at least 99% sequence identity to one of the aforementioned sequences; a HCDR2 domain having an amino acid sequence selected from the group consisting of SEQ ID NO: 6 and 22, or a substantially similar sequence thereof having at least 90%, at least 95%, at least 98% or at least 99% sequence identity to one of the aforementioned sequences; a LCDR1 domain having an amino acid sequence selected from the group consisting of SEQ ID NO: 12 and 28, or a substantially similar sequence thereof having at least 90%, at least 95%, at least 98% or at least 99% sequence identity to one of the aforementioned sequences; and a LCDR2 domain having an amino acid sequence selected from the group consisting of SEQ ID NO: 14 and 30, or a substantially similar sequence thereof having at least 90%, at least 95%, at least 98% or at least 99% sequence identity to one of the aforementioned sequences.

[0127] In some embodiments, an antibody or antigen-binding fragment thereof that binds specifically to C-X-C Motif Chemokine Receptor 4 (CXCR4), wherein the antibody or antigenbinding fragment thereof comprises: (a) three heavy chain complementarity determining regions (CDRs) (HCDR1 , HCDR2 and HCDR3) contained within a heavy chain variable region (HCVR) comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 2 and 18, and (b) three light chain CDRs (LCDR1 , LCDR2 and LCDR3) contained within a light chain variable region (LCVR) comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 10 and 26.

[0128] In some embodiments, the antibody or antigen-binding fragment comprises the heavy and light chain CDRs of a HCVR / LCVR amino acid sequence pair selected from the group consisting of SEQ ID NOs: 2 / 10 and 18 / 26.

[0129] Methods and techniques for identifying CDRs within HCVR and LCVR amino acid sequences are well known in the art and can be used to identify CDRs within the specified HCVR and / or LCVR amino acid sequences disclosed herein. Exemplary conventions that can be used to identify the boundaries of CDRs include, e.g., the Kabat definition, the Chothia definition, and the AbM definition. In general terms, the Kabat definition is based on sequence variability, the Chothia definition is based on the location of the structural loop regions, and the AbM definition is a compromise between the Kabat and Chothia approaches. See, e.g., Kabat, "Sequences of Proteins of Immunological Interest," National Institutes of Health, Bethesda, Md. (1991 ); Al-Lazikani et al., J Mol Biol 273:927-948 (1997); and Martin et al., PNAS (USA) 86:9268-9272 (1989). Public databases are also available for identifying CDR sequences within an antibody.

[0130] In some embodiments, the antibody or antigen-binding fragment thereof comprises HCDR1-HCDR2-HCDR3-LCDR1-LCDR2-LCDR3 domains comprising the amino acid sequences, respectively, selected from the group consisting of SEQ ID NOs: 4-6-8-12-14-16 or 20-22-24-28-30-32, respectively.

[0131] In some embodiments, the antibody or antigen-binding fragment thereof comprises: (a) a HCVR comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 2 and 18; and (b) a LCVR comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 10 and 26. In some cases, the antibody or antigen-binding fragment comprises a HCVR / LCVR amino acid sequence pair selected from the group consisting of SEQ ID NOs: 2 / 10 and 18 / 26.

[0132] In some embodiments, the antibody or antigen binding fragment thereof that binds specifically to an epitope of human C-X-C Motif Chemokine Receptor 4 (CXCR4), wherein the epitope comprises amino acid residues M24, E26, P27, C28, F29, R30, S178, E179, A180, D181 , D182, 1185, D187, F189, Y190, N192, and D193. In some cases, the antibody or antigen binding fragment thereof binds to an epitope of human CXCR4 comprising amino acid residues M24, K25, E26, P27, C28, F29, R30, E32, A34, S178, E179, A180, D181 , D182, R183, 1185, D187, R188, F189, Y190, N192, D193, and V196. In some cases, the antibody or antigen binding fragment thereof binds to an epitope of human CXCR4 comprising amino acid residues M24, E26, P27, C28, F29, R30, N176, S178, E179, A180, D181 , D182, 1185, D187, F189, Y190, N192, and D193, C274, and E275.

[0133] In some embodiments, the antibody or antigen binding fragment thereof comprises three heavy chain complementarity determining regions (CDRs) (HCDR1 , HCDR2 and HCDR3) contained within a heavy chain variable region (HCVR) comprising the amino acid sequence ofSEQ ID NO: 2; and three light chain CDRs (LCDR1 , LCDR2 and LCDR3) contained within a light chain variable region (LCVR) comprising the amino acid sequence of SEQ ID NO: 10, and the antibody or fragment binds to human CXCR4 within an epitope comprising amino acid residues M24, K25, E26, P27, C28, F29, R30, E32, A34, S178, E179, A180, D181 , D182, R183, 1185, D187, R188, F189, Y190, N192, D193, and V196.

[0134] In some embodiments, the antibody or antigen binding fragment thereof comprises three heavy chain complementarity determining regions (CDRs) (HCDR1 , HCDR2 and HCDR3) contained within a heavy chain variable region (HCVR) comprising the amino acid sequence of SEQ ID NO: 18; and three light chain CDRs (LCDR1 , LCDR2 and LCDR3) contained within a light chain variable region (LCVR) comprising the amino acid sequence of SEQ ID NO: 26, and the antibody or fragment binds to human CXCR4 within an epitope comprising amino acid residues M24, E26, P27, C28, F29, R30, N176, S178, E179, A180, D181 , D182, 1185, D187, F189, Y190, N192, D193, C274, and E275.

[0135] In some embodiments, the antibody or antigen-binding fragment comprises HCDR1 - HCDR2-HCDR3-LCDR1 -LCDR2-LCDR3 domains comprising the amino acid sequences of SEQ ID NOs: 4-6-8-12-14-16, respectively.

[0136] In some embodiments, the antibody or antigen-binding fragment comprises HCDR1 - HCDR2-HCDR3-LCDR1 -LCDR2-LCDR3 domains comprising the amino acid sequences of SEQ ID NOs: 20-22-24-28-30-32, respectively.

[0137] Non-limiting examples of antigen-binding fragments include: (i) Fab fragments;(ii) F(ab')2 fragments; (iii) Fd fragments; (iv) Fv fragments; (v) single-chain Fv (scFv) molecules; (vi) dAb fragments; and (vii) minimal recognition units consisting of the amino acid residues that mimic the hypervariable region of an antibody (e.g., an isolated complementarity determining region (CDR) such as a CDR3 peptide), or a constrained FR3- CDR3-FR4 peptide. Other engineered molecules, such as domain-specific antibodies, single domain antibodies, domain-deleted antibodies, chimeric antibodies, CDR-grafted antibodies, diabodies, triabodies, tetrabodies, minibodies, nanobodies e.g. monovalent nanobodies, bivalent nanobodies, etc.), small modular immunopharmaceuticals (SMIPs), and shark variable IgNAR domains, are also encompassed within the expression "antigen-binding fragment," as used herein.

[0138] An antigen-binding fragment of an antibody will typically comprise at least one variable domain. The variable domain may be of any size or amino acid composition and will generally comprise at least one CDR, which is adjacent to or in frame with one or more framework sequences. In antigen-binding fragments having a VH domain associated with a VL domain, theVHand VLdomains may be situated relative to one another in any suitable arrangement. For example, the variable region may be dimeric and contain V - V , V - VLor VL- VLdimers. Alternatively, the antigen-binding fragment of an antibody may contain a monomeric VHor VLdomain.

[0139] In certain embodiments, an antigen-binding fragment of an antibody may contain at least one variable domain covalently linked to at least one constant domain. Non-limiting, exemplary configurations of variable and constant domains that may be found within an antigen-binding fragment of an antibody of the present disclosure include: (i) V -CH1 ; (ii) V -CH2; (iii) V -CH3; (iv) VH -CH1 -CH2; (V) VH -CH1 -CH2-CH3; (vi) VH -CH2-CH3; (vii) VH -CL; (viii) VL -CH1 ; (ix) VL -CH2; (x) VL-CH3; (xi) VL-CH1 -CH2; (xii) VL-CH1 -CH2-CH3; (xiii) VL-CH2-CH3; and (xiv) VL-CL. In any configuration of variable and constant domains, including any of the exemplary configurations listed above, the variable and constant domains may be either directly linked to one another or may be linked by a full or partial hinge or linker region. A hinge region may consist of at least 2 (e.g., 5, 10, 15, 20, 40, 60 or more) amino acids, which result in a flexible or semi-flexible linkage between adjacent variable and / or constant domains in a single polypeptide molecule.Moreover, an antigen-binding fragment of an antibody of the present disclosure may comprise a homo-dimer or hetero-dimer (or other multimer) of any of the variable and constant domain configurations listed above in non-covalent association with one another and / or with one or more monomeric VH or VL domain (e.g., by disulfide bond(s)).

[0140] As with full antibody molecules, antigen-binding fragments may be mono-specific or multi-specific (e.g., bi-specific). A multi-specific antigen-binding fragment of an antibody will typically comprise at least two different variable domains, wherein each variable domain is capable of specifically binding to a separate antigen or to a different epitope on the same antigen. Any multi-specific antibody format, including the exemplary bi-specific antibody formats disclosed herein, may be adapted for use in the context of an antigen-binding fragment of an antibody of the present disclosure using routine techniques available in the art.In some embodiments, the antibody comprises a heavy chain (HC) and a light chain (LC) paired with one another, wherein the HC / LC pair comprises the amino acid sequences selected from the group consisting of SEQ ID NOs: 34 / 36 and 38 / 40. In some embodiment, the antibody comprises a HC comprising the amino acid sequence of SEQ ID NO: 34 and a LC comprising the amino acid sequence of SEQ ID NO: 36. In some embodiment, the antibody comprises a HC comprising the amino acid sequence of SEQ ID NO: 38 and a LC comprising the amino acid sequence of SEQ ID NO: 40.Polynucleotides, Vectors, and Host Cells

[0141] In various embodiments, the present disclosure provides a polynucleotide encoding a polypeptide (e.g., an antibody or part thereof) discussed herein, a vector comprising the polynucleotide, and / or a host cell comprising the antibody or antigen-binding fragment or polypeptide or polynucleotide or vector. In various embodiments, the present disclosure provides a polynucleotide that encodes a HCVR, a LCVR, or both a HCVR and a LCVR of an antibody or antigen-binding fragment thereof as discussed herein. The HCVR and / or LCVR may be defined by the CDRs contained within the HCVR sequence, the LCVR sequence, or both the HCVR sequence and the LCVR sequence, respectively, as set forth in Table 1 for the amino acid sequences and Table 2 for the nucleic acid sequences. The HCVR and / or LCVR may also be defined by the heavy chain CDR sequences, the light chain CDR sequences, or both the heavy and light chain CDR sequences, respectively, as set forth in Table 1 for the amino acid sequences and Table 2 for the nucleic acid sequences. The HCVR and / or LCVR may also be defined by the HCVR sequence, the LCVR sequence, or both the HCVR and LCVR sequences, respectively, as set forth in Table 1 for the amino acid sequences and Table 2 for the nucleic acid sequences. In various embodiments, the present disclosure provides a polynucleotide that encodes a heavy chain, a light chain, or both a heavy chain and a light chain of an antibody as discussed above or herein. The heavy chain and / or light chain may be defined by the heavy and light chain sequences, respectively, as set forth in Table 3. In various embodiments, the polynucleotide comprises a nucleic acid sequence as set forth in Table 4. In various embodiments, the present disclosure provides a vector or vectors comprising the polynucleotides discussed above, and / or a host cell comprising the polynucleotides or vectors, or HCVR or LCVR, of HC or LC, or an assembled antibody or antigen-binding fragment thereof as discussed above or herein. In various embodiments, the present disclosure provides a pair of polynucleotides, wherein (a) the first polynucleotide encodes: (i) a HCVR comprising the CDR sequences contained in a HCVR of an antibody of Table 1 , (ii) a HCVR comprising the HCDR1 , HCDR2 and HCDR3 sequences as set forth for an antibody in Table 1 , (iii) a HCVR comprising the HCVR sequence of an antibody of Table 1 , or (iv) a heavy chain (HC) comprising the HC sequence of an antibody of Table 1 , and (b) the second polynucleotide encodes: (i) a LCVR comprising the CDR sequences contained in a LCVR of an antibody of Table 1 , (ii) a LCVR comprising the LCDR1 , LCDR2 and LCDR3 sequences as set forth for an antibody in Table 1 , (iii) a LCVR comprising the LCVR sequence of an antibody of Table 1 , or (iv) a light chain (LC) comprising the LC sequence of an antibody of Table 1 . In various embodiments, the present disclosure descriptions of vectors comprising, respectively, the polynucleotides discussedabove, and / or a host cell comprising the vectors. A host cell can be, for example, a hamster ovary cell. In any of the various embodiments, the nucleic acid sequence for the corresponding component of the antibody or antigen-binding fragment thereof (e.g., the HCVR, the LCVR, or any one or all of the CDRs) may be as set forth in Table 2.

[0142] In some embodiments, the pair of polynucleotides encodes components of an antibody or antigen-binding fragment thereof, such as: (a) the first polynucleotide encodes a HCVR comprising the CDRs contained within a HCVR comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 2 and 18, and the second polynucleotide encodes a LCVR comprising the CDRs contained in a LCVR comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 10 or 26, (b) the first polynucleotide encodes a HCVR comprising the HCDR1 -HCDR2-HCDR3 amino acid sequences, respectively, selected from the group consisting of SEQ ID NOs: 4-6-8 and 20-22-24, and the second polynucleotide encodes a LCVR comprising the LCDR1 -LCDR2-LCDR3 amino acid sequences, respectively, selected from the group consisting of SEQ ID NOs: 12-14-16 and 28-30-32.

[0143] In some embodiments, a pair of polynucleotide molecules encodes a heavy chain variable region (HCVR) and a light chain variable region (LCVR), respectively, of an antibody or antigen-binding fragment thereof that binds specifically to C-X-C Motif Chemokine Receptor 4 (CXCR4), wherein: (a) a first polynucleotide encodes a heavy chain variable region (HCVR) comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 2 and 18; and (b) a second polynucleotide encodes a light chain variable region (LCVR) comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 10 and 26.

[0144] In some embodiments, a pair of polynucleotide molecules encode a HCVR / LCVR amino acid sequence pair, respectively, selected from the group consisting of SEQ ID NOs: 1 / 9 and 17 / 25.

[0145] In some embodiments, a pair of polynucleotide molecules encodes a heavy chain (HC) and a light chain (LC), respectively, of an antibody that binds specifically to C-X-C Motif Chemokine Receptor 4 (CXCR4), wherein (a) the first polynucleotide encodes a HC comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 34 and 38, and (b) the second polynucleotide encodes a LC comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 36 and 40. In some embodiments, a pair of polynucleotide molecules encode a HC / LC amino acid sequence pair, respectively, selected from the group consisting of SEQ ID NOs: 34 / 36 and 38 / 40. In some embodiments, a pair of polynucleotide molecules encodes a heavy chain (HC) and a light chain (LC), respectively, of an antibody that binds specifically to C-X-C Motif Chemokine Receptor 4 (CXCR4), wherein (a) the firstpolynucleotide comprises a HC polynucleotide sequence selected from the group consisting of SEQ ID NOs: 33 and 37, and (b) the second polynucleotide comprises a LC polynucleotide sequence selected from the group consisting of SEQ ID NOs: 35 and 39. In some embodiments, a pair of polynucleotide molecules encode a HC / LC pair and comprise the nucleic acid sequences selected from the group consisting of SEQ ID NOs: 33 / 35 and 37 / 39.Preparation of Human Antibodies

[0146] Methods for generating human antibodies in transgenic mice are known in the art. Any such known methods can be used in the context of the present disclosure to make human antibodies that specifically bind to CXCR4.

[0147] Using VELOCIMMUNE® technology (see, for example, US 6,596,541 , Regeneron Pharmaceuticals, VELOCIMMUNE®) or any other known method for generating monoclonal antibodies, high affinity chimeric antibodies to CXCR4 are initially isolated having a human variable region and a mouse constant region. The VELOCIMMUNE® technology involves generation of a transgenic mouse having a genome comprising human heavy and light chain variable regions operably linked to endogenous mouse constant region loci such that the mouse produces an antibody comprising a human variable region and a mouse constant region in response to antigenic stimulation. The DNA encoding the variable regions of the heavy and light chains of the antibody are isolated and operably linked to DNA encoding the human heavy and light chain constant regions. The DNA is then expressed in a cell capable of expressing the fully human antibody.

[0148] Generally, a VELOCIMMUNE® mouse is challenged with the antigen of interest, and lymphatic cells (such as B-cells) are recovered from the mice that express antibodies. The lymphatic cells may be fused with a myeloma cell line to prepare immortal hybridoma cell lines, and such hybridoma cell lines are screened and selected to identify hybridoma cell lines that produce antibodies specific to the antigen of interest. DNA encoding the variable regions of the heavy chain and light chain may be isolated and linked to desirable isotypic constant regions of the heavy chain and light chain. Such an antibody protein may be produced in a cell, such as a CHO cell. Alternatively, DNA encoding the antigen-specific chimeric antibodies or the variable domains of the light and heavy chains may be isolated directly from antigen-specific lymphocytes.

[0149] Initially, high affinity chimeric antibodies are isolated having a human variable region and a mouse constant region. As in the experimental section below, the antibodies are characterized and selected for desirable characteristics, including affinity, selectivity, epitope,etc. The mouse constant regions are replaced with a desired human constant region to generate the fully human antibody of the disclosure, for example wild-type or modified IgG 1 or lgG4. While the constant region selected may vary according to specific use, high affinity antigen-binding and target specificity characteristics reside in the variable region.Anti-CXCR4 Antibodies Comprising Fc Variants

[0150] According to certain embodiments of the present invention, anti-CXCR4 antibodies are provided comprising an Fc domain comprising one or more mutations which enhance or diminish antibody binding to the FcRn receptor, e.g., at acidic pH as compared to neutral pH. For example, the present invention includes anti-CXCR4 antibodies comprising a mutation in the CH2 or a CH3 region of the Fc domain, wherein the mutation(s) increases the affinity of the Fc domain to FcRn in an acidic environment (e.g., in an endosome where pH ranges from about 5.5 to about 6.0). Such mutations may result in an increase in serum half-life of the antibody when administered to an animal. Non-limiting examples of such Fc modifications include, e.g., a modification at position 250 (e.g., E or Q); 250 and 428 (e.g., L or F); 252 (e.g., L / Y / F / W or T), 254 (e.g., S or T), and 256 (e.g., S / R / Q / E / D or T); or a modification at position 428 and / or 433 (e.g., H / L / R / S / P / Q or K) and / or 434 (e.g., A, W, H, F or Y [N434A, N434W, N434H, N434F or N434Y]); or a modification at position 250 and / or 428; or a modification at position 307 or 308 (e.g., 308F, V308F), and 434. In one embodiment, the modification comprises a 428L (e.g., M428L) and 434S (e.g., N434S) modification; a 428L, 259I (e.g., V259I), and 308F (e.g., V308F) modification; a 433K (e.g., H433K) and a 434 (e.g., 434Y) modification; a 252, 254, and 256 (e.g., 252Y, 254T, and 256E) modification; a 250Q and 428L modification (e.g., T250Q and M428L); and a 307 and / or 308 modification (e.g., 308F or 308P). In yet another embodiment, the modification comprises a 265A (e.g., D265A) and / or a 297A (e.g., N297A) modification.

[0151] For example, the present invention includes anti-CXCR4 antibodies comprising an Fc domain comprising one or more pairs or groups of mutations selected from the group consisting of: 250Q and 248L (e.g., T250Q and M248L); 252Y, 254T and 256E (e.g., M252Y, S254T and T256E); 428L and 434S (e.g., M428L and N434S); 257I and 3111 (e.g., P257I and Q3111); 257I and 434H (e.g., P257I and N434H); 376V and 434H (e.g., D376V and N434H); 307A, 380A and 434A (e.g., T307A, E380A and N434A); and 433K and 434F (e.g., H433K and N434F). In one embodiment, the present invention includes anti-CXCR4 antibodies comprising an Fc domain comprising a S108P mutation in the hinge region of lgG4 to promote dimer stabilization. All possible combinations of the foregoing Fc domain mutations, and other mutations within the antibody variable domains disclosed herein, are contemplated within the scope of the presentinvention.

[0152] The present invention also includes anti-CXCR4 antibodies comprising a chimeric heavy chain constant (CH) region, wherein the chimeric CH region comprises segments derived from the CH regions of more than one immunoglobulin isotype. For example, the antibodies of the invention may comprise a chimeric CH region comprising part or all of a CH2 domain derived from a human IgG 1 , human lgG2 or human lgG4 molecule, combined with part or all of a CH3 domain derived from a human IgG 1 , human lgG2 or human lgG4 molecule. According to certain embodiments, the antibodies of the invention comprise a chimeric CH region having a chimeric hinge region. For example, a chimeric hinge may comprise an "upper hinge" amino acid sequence (amino acid residues from positions 216 to 227 according to EU numbering) derived from a human lgG1 , a human lgG2 or a human lgG4 hinge region, combined with a "lower hinge" sequence (amino acid residues from positions 228 to 236 according to EU numbering) derived from a human lgG1 , a human lgG2 or a human lgG4 hinge region.According to certain embodiments, the chimeric hinge region comprises amino acid residues derived from a human IgG 1 or a human lgG4 upper hinge and amino acid residues derived from a human lgG2 lower hinge. An antibody comprising a chimeric CH region as described herein may, in certain embodiments, exhibit modified Fc effector functions without adversely affecting the therapeutic or pharmacokinetic properties of the antibody. (See e.g., US Patent Publication No. 20140243504, the disclosure of which is hereby incorporated by reference in its entirety).Bioequivalents

[0153] The anti-CXCR4 antibodies and antibody fragments of the present disclosure encompass proteins having amino acid sequences that vary from those of the described antibodies, but that retain the ability to bind CXCR4. Such variant antibodies and antibody fragments comprise one or more additions, deletions, or substitutions of amino acids when compared to parent sequence, but exhibit biological activity that is essentially equivalent to that of the described antibodies. Likewise, the antibody-encoding DNA sequences of the present disclosure encompass sequences that comprise one or more additions, deletions, or substitutions of nucleotides when compared to the disclosed sequence, but that encode an antibody or antibody fragment that is essentially bioequivalent to an antibody or antibody fragment of the disclosure.

[0154] Two antigen-binding proteins, or antibodies, are considered bioequivalent if, for example, they are pharmaceutical equivalents or pharmaceutical alternatives whose rate and extent of absorption do not show a significant difference when administered at the same molardose under similar experimental conditions, either single dose or multiple doses. Some antibodies will be considered equivalents or pharmaceutical alternatives if they are equivalent in the extent of their absorption but not in their rate of absorption and yet may be considered bioequivalent because such differences in the rate of absorption are intentional and are reflected in the labeling, are not essential to the attainment of effective body drug concentrations on, e.g., chronic use, and are considered medically insignificant for the particular drug product studied.

[0155] In one embodiment, two antigen-binding proteins are bioequivalent if there are no clinically meaningful differences in their safety, purity, or potency.

[0156] In one embodiment, two antigen-binding proteins are bioequivalent if a patient can be switched one or more times between the reference product and the biological product without an expected increase in the risk of adverse effects, including a clinically significant change in immunogenicity, or diminished effectiveness, as compared to continued therapy without such switching.

[0157] In one embodiment, two antigen-binding proteins are bioequivalent if they both act by a common mechanism or mechanisms of action for the condition or conditions of use, to the extent that such mechanisms are known.

[0158] Bioequivalence may be demonstrated by in vivo and / or in vitro methods. Bioequivalence measures include, e.g., (a) an in vivo test in humans or other mammals, in which the concentration of the antibody or its metabolites is measured in blood, plasma, serum, or other biological fluid as a function of time; (b) an in vitro test that has been correlated with and is reasonably predictive of human in vivo bioavailability data; (c) an in vivo test in humans or other mammals in which the appropriate acute pharmacological effect of the antibody (or its target) is measured as a function of time; and (d) in a well-controlled clinical trial that establishes safety, efficacy, or bioavailability or bioequivalence of an antibody.

[0159] Bioequivalent variants of the antibodies of the disclosure may be constructed by, for example, making various substitutions of residues or sequences or deleting terminal or internal residues or sequences not needed for biological activity. For example, cysteine residues not essential for biological activity can be deleted or replaced with other amino acids to prevent formation of unnecessary or incorrect intramolecular disulfide bridges upon renaturation. In other contexts, bioequivalent antibodies may include antibody variants comprising amino acid changes, which modify the glycosylation characteristics of the antibodies, e.g., mutations that eliminate or remove glycosylation.Biological Characteristics of the Antibodies

[0160] In general, the antibodies of the present disclosure function by binding to CXCR4. The present disclosure includes anti-CXCR4 antibodies and antigen-binding fragments thereof that bind CXCR4 molecules with high affinity. For example, the present disclosure includes antibodies and antigen-binding fragments of antibodies that bind dimeric human and monkey CXCR4.

[0161] The present disclosure also includes antibodies or antigen-binding fragments thereof that block hCXCR4 binding to SDF-1 as shown in Examples 3-10 and 12.

[0162] The antibodies of the present disclosure may possess one or more of the aforementioned biological characteristics, or any combinations thereof. Other biological characteristics of the antibodies of the present disclosure will be evident to a person of ordinary skill in the art from a review of the present disclosure including the working examples herein.Species Selectivity and Species Cross-Reactivity

[0163] According to certain embodiments of the disclosure, the anti-CXCR4 antibodies bind to human CXCR4 but not to CXCR4 from other species. Alternatively, the anti-CXCR4 antibodies of the disclosure, in certain embodiments, bind to human CXCR4 and to CXCR4 from one or more non-human species. For example, the anti-CXCR4 antibodies of the disclosure may bind to human CXCR4 and may bind or not bind, as the case may be, to one or more of mouse, rat, guinea pig, hamster, gerbil, pig, cat, dog, rabbit, goat, sheep, cow, horse, camel, cynomolgus, marmoset, rhesus or chimpanzee CXCR4. In certain embodiments, the anti-CXCR4 antibodies of the disclosure may bind to human and cynomolgus CXCR4 with the same affinities or with different affinities, but do not bind to rat and mouse CXCR4.Epitope Mapping and Related Technologies

[0164] The present disclosure includes anti-CXCR4 antibodies which interact with one or more amino acids found within one or more domains of the CXCR4 molecule including, e.g., an extracellular domain, a transmembrane domain, and a cytoplasmic domain. The epitope to which the antibodies bind may consist of a single contiguous sequence of 3 or more (e.g., 3, 4, 5, 6, 7, 8, 9, 10, 11 , 12, 13, 14, 15, 16, 17, 18, 19, 20 or more) amino acids located within any of the aforementioned domains of the CXCR4 molecule (e.g. a linear epitope in a domain). Alternatively, the epitope may consist of a plurality of non-contiguous amino acids (or amino acid sequences) located within any or all of the aforementioned domains of the CXCR4 molecule (e.g. a conformational epitope).

[0165] Various techniques known to persons of ordinary skill in the art can be used to determine whether an antibody "interacts with one or more amino acids" within a polypeptide or protein. Exemplary techniques include, for example, routine cross-blocking assays, such as that described in Antibodies, Harlow and Lane (Cold Spring Harbor Press, Cold Spring Harbor, NY). Other methods include alanine scanning mutational analysis, peptide blot analysis (Reineke (2004) Methods Mol. Biol. 248: 443-63), peptide cleavage analysis crystallographic studies and NMR analysis. In addition, methods such as epitope excision, epitope extraction and chemical modification of antigens can be employed (Tomer (2000) Prot. Sci. 9: 487-496). Another method that can be used to identify the amino acids within a polypeptide with which an antibody interacts is hydrogen / deuterium exchange detected by mass spectrometry. In general terms, the hydrogen / deuterium exchange method involves deuterium-labeling the protein of interest, followed by binding the antibody to the deuterium-labeled protein. Next, the protein / antibody complex is transferred to water and exchangeable protons within amino acids that are protected by the antibody complex undergo deuterium-to-hydrogen back-exchange at a slower rate than exchangeable protons within amino acids that are not part of the interface. As a result, amino acids that form part of the protein / antibody interface may retain deuterium and therefore exhibit relatively higher mass compared to amino acids not included in the interface.After dissociation of the antibody, the target protein is subjected to protease cleavage and mass spectrometry analysis, thereby revealing the deuterium-labeled residues which correspond to the specific amino acids with which the antibody interacts. See, e.g., Ehring (1999) Analytical Biochemistry 267: 252-259; Engen and Smith (2001 ) Anal. Chem. 73: 256A-265A.

[0166] The term "epitope" refers to a site on an antigen to which B and / or T cells respond. B- cell epitopes can be formed both from contiguous amino acids or noncontiguous amino acids juxtaposed by tertiary folding of a protein. Epitopes formed from contiguous amino acids are typically retained on exposure to denaturing solvents, whereas epitopes formed by tertiary folding are typically lost on treatment with denaturing solvents. An epitope typically includes at least 3, and more usually, at least 5 or 8-10 amino acids in a unique spatial conformation.

[0167] Modification-Assisted Profiling (MAP), also known as Antigen Structure-based Antibody Profiling (ASAP) is a method that categorizes large numbers of monoclonal antibodies (mAbs) directed against the same antigen according to the similarities of the binding profile of each antibody to chemically or enzymatically modified antigen surfaces (see US 2004 / 0101920, herein specifically incorporated by reference in its entirety). Each category may reflect a unique epitope either distinctly different from or partially overlapping with epitope represented by another category. This technology allows rapid filtering of genetically identical antibodies, suchthat characterization can be focused on genetically distinct antibodies. When applied to hybridoma screening, MAP may facilitate identification of rare hybridoma clones that produce mAbs having the desired characteristics. MAP may be used to sort the antibodies of the disclosure into groups of antibodies binding different epitopes.

[0168] In certain embodiments, the anti-CXCR4 antibodies or antigen-binding fragments thereof bind an epitope within any one or more of the regions exemplified in CXCR4, either in natural form, as exemplified in SEQ ID NO: 43 or 44, or recombinantly produced, or to a fragment thereof.

[0169] The present disclosure includes anti-CXCR4 antibodies that bind to the same epitope, or a portion of the epitope, as any of the specific exemplary antibodies described herein in Table 1 , or an antibody having the CDR sequences of any of the exemplary antibodies described in Table 1 . Likewise, the present disclosure also includes anti-CXCR4 antibodies that compete for binding to CXCR4 or a CXCR4 fragment with any of the specific exemplary antibodies described herein in Table 1 , or an antibody having the CDR sequences of any of the exemplary antibodies described in Table 1. For example, the present disclosure includes anti-CXCR4 antibodies that cross-compete for binding to CXCR4 with one or more antibodies as exemplified herein (e.g., H1 M16435N, H2aM15295N, REGN7663, or REGN7664). In some embodiments, the epitope comprises amino acid residues M24, E26, P27, C28, F29, R30, S178, E179, A180, D181 , D182, 1185, D187, F189, Y190, N192, and D193 of human CXCR4. In some cases, the epitope comprises amino acid residues M24, K25, E26, P27, C28, F29, R30, E32, A34, S178, E179, A180, D181 , D182, R183, 1185, D187, R188, F189, Y190, N192, D193, and V196 of human CXCR4. In some cases, the epitope comprises amino acid residues M24, E26, P27, C28, F29, R30, N176, S178, E179, A180, D181 , D182, 1185, D187, F189, Y190, N192, and D193, C274, and E275.

[0170] One can easily determine whether an antibody binds to the same epitope as, or competes for binding with, a reference anti-CXCR4 antibody by using routine methods known in the art. For example, to determine if a test antibody binds to the same epitope as a reference anti-CXCR4 antibody of the disclosure, the reference antibody is allowed to bind to a CXCR4 protein or peptide under saturating conditions. Next, the ability of a test antibody to bind to the CXCR4 molecule is assessed. If the test antibody is able to bind to CXCR4 following saturation binding with the reference anti-CXCR4 antibody, it can be concluded that the test antibody binds to a different epitope than the reference anti-CXCR4 antibody. On the other hand, if the test antibody is not able to bind to the CXCR4 protein following saturation binding with the reference anti-CXCR4 antibody, then the test antibody may bind to the same epitope as the epitope boundby the reference anti-CXCR4 antibody of the disclosure.

[0171] To determine if an antibody competes for binding with a reference anti-CXCR4 antibody, the above-described binding methodology is performed in two orientations: In a first orientation, the reference antibody is allowed to bind to a CXCR4 protein under saturating conditions followed by assessment of binding of the test antibody to the CXCR4 molecule. In a second orientation, the test antibody is allowed to bind to a CXCR4 molecule under saturating conditions followed by assessment of binding of the reference antibody to the CXCR4 molecule. If, in both orientations, only the first (saturating) antibody is capable of binding to the CXCR4 molecule, then it is concluded that the test antibody and the reference antibody compete for binding to CXCR4. As will be appreciated by a person of ordinary skill in the art, an antibody that competes for binding with a reference antibody may not necessarily bind to the identical epitope as the reference antibody, but may sterically block binding of the reference antibody by binding an overlapping or adjacent epitope.

[0172] Two antibodies bind to the same or overlapping epitope if each competitively inhibits (blocks) binding of the other to the antigen. That is, a 1 -, 5-, 10-, 20- or 100-fold excess of one antibody inhibits binding of the other by at least 50% but preferably 75%, 90% or even 99% as measured in a competitive binding assay (see, e.g., Junghans et al., Cancer Res. 1990 50:1495-1502). Alternatively, two antibodies have the same epitope if essentially all amino acid mutations in the antigen that reduce or eliminate binding of one antibody reduce or eliminate binding of the other. Two antibodies have overlapping epitopes if some amino acid mutations that reduce or eliminate binding of one antibody reduce or eliminate binding of the other.

[0173] Additional routine experimentation e.g., peptide mutation and binding analyses) can then be carried out to confirm whether the observed lack of binding of the test antibody is in fact due to binding to the same epitope as the reference antibody or if steric blocking (or another phenomenon) is responsible for the lack of observed binding. Experiments of this sort can be performed using ELISA, RIA, surface plasmon resonance, flow cytometry or any other quantitative or qualitative antibody-binding assay available in the art.Immunoconjugates

[0174] The disclosure encompasses a human anti-CXCR4 monoclonal antibody conjugated to a therapeutic moiety (“immunoconjugate”), such as a cytotoxin or a chemotherapeutic agent to treat cancer. As used herein, the term “immunoconjugate” refers to an antibody which is chemically or biologically linked to a cytotoxin, a radioactive agent, a cytokine, an interferon, a target or reporter moiety, an enzyme, a toxin, a peptide or protein or a therapeutic agent. Theantibody may be linked to the cytotoxin, radioactive agent, cytokine, interferon, target or reporter moiety, enzyme, toxin, peptide or therapeutic agent at any location along the molecule so long as it is able to bind its target. Examples of immunoconjugates include antibody drug conjugates and antibody-toxin fusion proteins. In one embodiment, the agent may be a second different antibody to CXCR4. In certain embodiments, the antibody may be conjugated to an agent specific for a tumor cell or a virally infected cell. In one embodiment, the antibody is conjugated to an agent specific for a T-cell. The type of therapeutic moiety that may be conjugated to the anti-CXCR4 antibody and will take into account the condition to be treated and the desired therapeutic effect to be achieved. Examples of suitable agents for forming immunoconjugates are known in the art; see for example, WO 05 / 103081 .Multi-Specific Antibodies

[0175] The antibodies of the present disclosure may be mono-specific, bi-specific, or multispecific. Multi-specific antibodies may be specific for different epitopes of one target polypeptide or may contain antigen-binding domains specific for more than one target polypeptide. See, e.g., Tutt et al., 1991 , J. Immunol. 147:60-69; Kufer et aL, 2004, Trends Biotechnol. 22:238-244.

[0176] In one aspect, the present disclosure includes multi-specific antigen-binding molecules or antigen-binding fragments thereof wherein one specificity of an immunoglobulin is specific for the extracellular domain of CXCR4, or a fragment thereof, and the other specificity of the immunoglobulin is specific for binding outside the extracellular domain of CXCR4, or a second therapeutic target, or is conjugated to a therapeutic moiety.

[0177] Any of the multi-specific antigen-binding molecules of the disclosure, or variants thereof, may be constructed using standard molecular biological techniques (e.g., recombinant DNA and protein expression technology), as will be known to a person of ordinary skill in the art.

[0178] In some embodiments, CXCR4-specific antibodies are generated in a bi-specific format (a "bi-specific") in which variable regions binding to distinct domains of CXCR4 are linked together to confer dual-domain specificity within a single binding molecule. Appropriately designed bi-specifics may enhance overall CXCR4 inhibitory efficacy through increasing both specificity and binding avidity. Variable regions with specificity for individual domains, (e.g., segments of the N-terminal domain), or that can bind to different regions within one domain, are paired on a structural scaffold that allows each region to bind simultaneously to the separate epitopes, or to different regions within one domain. In one example for a bi-specific, heavy chain variable regions (VH) from a binder with specificity for one domain are recombined withlight chain variable regions (VL) from a series of binders with specificity for a second domain to identify non-cognate VLpartners that can be paired with an original V without disrupting the original specificity for that V . In this way, a single VLsegment (e.g., VL1) can be combined with two different VHdomains (e.g., VH1 and VH2) to generate a bi-specific comprised of two binding "arms" (VH1 - VL1 and VH2- VL1 ). Use of a single VL segment reduces the complexity of the system and thereby simplifies and increases efficiency in cloning, expression, and purification processes used to generate the bi-specific (See, for example, USSN13 / 022759 and US2010 / 0331527).

[0179] Alternatively, antibodies that bind more than one domains and a second target, such as, but not limited to, for example, a second different anti-CXCR4 antibody, may be prepared in a bi-specific format using techniques described herein, or other techniques known to those skilled in the art. Antibody variable regions binding to distinct regions may be linked together with variable regions that bind to relevant sites on, for example, the extracellular domain of CXCR4, to confer dual-antigen specificity within a single binding molecule. Appropriately designed bispecifics of this nature serve a dual function. Variable regions with specificity for the extracellular domain are combined with a variable region with specificity for outside the extracellular domain and are paired on a structural scaffold that allows each variable region to bind to the separate antigens.

[0180] An exemplary bi-specific antibody format that can be used in the context of the present disclosure involves the use of a first immunoglobulin (Ig) CH3 domain and a second Ig CH3 domain, wherein the first and second Ig CH3 domains differ from one another by at least one amino acid, and wherein at least one amino acid difference reduces binding of the bi-specific antibody to Protein A as compared to a bi-specific antibody lacking the amino acid difference. In one embodiment, the first Ig CH3 domain binds Protein A and the second Ig CH3 domain contains a mutation that reduces or abolishes Protein A binding such as an H95R modification (by IMGT exon numbering; H435R by EU numbering). The second CH3 may further comprise a Y96F modification (by IMGT ; Y436F by EU). Further modifications that may be found within the second C 3 include: D16E, L18M, N44S, K52N, V57M, and V82I (by IMGT; D356E, L358M, N384S, K392N, V397M, and V422I by EU) in the case of IgG 1 antibodies; N44S, K52N, and V82I (IMGT; N384S, K392N, and V422I by EU) in the case of lgG2 antibodies; and Q15R, N44S, K52N, V57M, R69K, E79Q, and V82I (by IMGT; Q355R, N384S, K392N, V397M, R409K, E419Q, and V422I by EU) in the case of lgG4 antibodies. Variations on the bi-specific antibody format described above are contemplated within the scope of the present disclosure.

[0181] Other exemplary bispecific formats that can be used in the context of the presentdisclosure include, without limitation, e.g., scFv-based or diabody bispecific formats, IgG-scFv fusions, dual variable domain (DVD)-lg, Quadroma, knobs-into-holes, common light chain (e.g., common light chain with knobs-into-holes, etc.), CrossMab, CrossFab, (SEED)body, leucine zipper, Duobody, lgG1 / lgG2, dual acting Fab (DAF)-lgG, and Mab2bispecific formats (see, e.g., Klein et al. 2012, mAbs 4:6, 1 -1 1 , and references cited therein, for a review of the foregoing formats). Bispecific antibodies can also be constructed using peptide / nucleic acid conjugation, e.g., wherein unnatural amino acids with orthogonal chemical reactivity are used to generate site-specific antibody-oligonucleotide conjugates which then self-assemble into multimeric complexes with defined composition, valency and geometry. (See, e.g., Kazane et al., J. Am. Chem. Soc. [Epub Dec. 4, 2012\).Therapeutic Administration and Formulations

[0182] The disclosure provides therapeutic compositions comprising the anti-CXCR4 antibodies or antigen-binding fragments thereof of the present disclosure. Therapeutic compositions in accordance with the disclosure will be administered with suitable carriers, excipients, and other agents that are incorporated into formulations to provide improved transfer, delivery, tolerance, and the like. A multitude of appropriate formulations can be found in the formulary known to all pharmaceutical chemists: Remington's Pharmaceutical Sciences, Mack Publishing Company, Easton, PA. These formulations include, for example, powders, pastes, ointments, jellies, waxes, oils, lipids, lipid (cationic or anionic) containing vesicles (such as LIPOFECTIN™), DNA conjugates, anhydrous absorption pastes, oil-in-water and water-in-oil emulsions, emulsions carbowax (polyethylene glycols of various molecular weights), semi-solid gels, and semi-solid mixtures containing carbowax. See also Powell et al. "Compendium of excipients for parenteral formulations" PDA (1998) J Pharm Sci Technol 52:238-311 .

[0183] The dose of antibody may vary depending upon the age and the size of a subject to be administered, target disease, conditions, route of administration, and the like. When an antibody of the present disclosure is used for treating a disease or disorder in an adult patient, or for preventing such a disease, it is advantageous to administer the antibody of the present disclosure normally at a single dose of about 0.1 to about 60 mg / kg body weight, about 5 to about 60, about 20 to about 50, about 10 to about 50, about 1 to about 10, or about 0.8 to about 11 mg / kg body weight. Depending on the severity of the condition, the frequency and the duration of the treatment can be adjusted. In certain embodiments, the antibody or antigenbinding fragment thereof can be administered as an initial dose of at least about 0.1 mg to about 800 mg, about 1 to about 500 mg, about 5 to about 300 mg, or about 10 to about 200 mg, toabout 100 mg, or to about 50 mg. In certain embodiments, the initial dose may be followed by administration of a second or a plurality of subsequent doses of the antibody or antigen-binding fragment thereof in an amount that can be approximately the same or less than that of the initial dose, wherein the subsequent doses are separated by at least 1 day to 3 days; at least one week, at least 2 weeks; at least 3 weeks; at least 4 weeks; at least 5 weeks; at least 6 weeks; at least 7 weeks; at least 8 weeks; at least 9 weeks; at least 10 weeks; at least 12 weeks; or at least 14 weeks.

[0184] Various delivery systems are known and can be used to administer the pharmaceutical composition of the present disclosure, e.g., encapsulation in liposomes, microparticles, microcapsules, recombinant cells capable of expressing the mutant viruses, receptor mediated endocytosis (see, e.g., Wu et al. (1987) J. Biol. Chem. 262:4429-4432). Methods of introduction include, but are not limited to, intradermal, transdermal, intramuscular, intraperitoneal, intravenous, subcutaneous, intranasal, epidural and oral routes. The composition may be administered by any convenient route, for example by infusion or bolus injection, by absorption through epithelial or mucocutaneous linings e.g., oral mucosa, rectal and intestinal mucosa, etc.) and may be administered together with other biologically active agents. Administration can be systemic or local. The pharmaceutical composition can be also delivered in a vesicle, in particular a liposome (see, for example, Langer (1990) Science 249:1527-1533).

[0185] The use of nanoparticles to deliver the antibodies of the present disclosure is also contemplated herein. Antibody-conjugated nanoparticles may be used both for therapeutic and diagnostic applications. Antibody-conjugated nanoparticles and methods of preparation and use are described in detail by Arruebo, M., et al. 2009 (“Antibody-conjugated nanoparticles for biomedical applications” in J. Nanomat. Volume 2009, Article ID 439389, 24 pages, doi: 10.1155 / 2009 / 439389), incorporated herein by reference. Nanoparticles may be developed and conjugated to antibodies contained in pharmaceutical compositions to target tumor cells or autoimmune tissue cells or virally infected cells. Nanoparticles for drug delivery have also been described in, for example, US 8257740, or US 8246995, each incorporated herein in its entirety.

[0186] In certain situations, the pharmaceutical composition can be delivered in a controlled release system. In one embodiment, a pump may be used. In another embodiment, polymeric materials can be used. In yet another embodiment, a controlled release system can be placed in proximity of the composition’s target, thus requiring only a fraction of the systemic dose.

[0187] The injectable preparations may include dosage forms for intravenous, subcutaneous, intratumoral, peritumoral, intracutaneous, intracranial, intraperitoneal and intramuscular injections, drip infusions, etc. These injectable preparations may be prepared by methodspublicly known. For example, the injectable preparations may be prepared, e.g., by dissolving, suspending or emulsifying the antibody or its salt described above in a sterile aqueous medium or an oily medium conventionally used for injections. As the aqueous medium for injections, there are, for example, physiological saline, an isotonic solution containing glucose and other auxiliary agents, etc., which may be used in combination with an appropriate solubilizing agent such as an alcohol (e.g., ethanol), a polyalcohol (e.g., propylene glycol, polyethylene glycol), a nonionic surfactant [e.g., polysorbate 80, HCO-50 (polyoxyethylene (50 mol) adduct of hydrogenated castor oil)], etc. As the oily medium, there are employed, e.g., sesame oil, soybean oil, etc., which may be used in combination with a solubilizing agent such as benzyl benzoate, benzyl alcohol, etc. The injection thus prepared is preferably filled in an appropriate ampoule.

[0188] A pharmaceutical composition of the present disclosure can be delivered subcutaneously or intravenously with a standard needle and syringe. In addition, with respect to subcutaneous delivery, a pen delivery device readily has applications in delivering a pharmaceutical composition of the present disclosure. Such a pen delivery device can be reusable or disposable. A reusable pen delivery device generally utilizes a replaceable cartridge that contains a pharmaceutical composition. Once all of the pharmaceutical composition within the cartridge has been administered and the cartridge is empty, the empty cartridge can readily be discarded and replaced with a new cartridge that contains the pharmaceutical composition. The pen delivery device can then be reused. In a disposable pen delivery device, there is no replaceable cartridge. Rather, the disposable pen delivery device comes prefilled with the pharmaceutical composition held in a reservoir within the device. Once the reservoir is emptied of the pharmaceutical composition, the entire device is discarded.

[0189] Numerous reusable pen and autoinjector delivery devices have applications in the subcutaneous delivery of a pharmaceutical composition of the present disclosure. Examples include, but certainly are not limited to AUTOPEN™ (Owen Mumford, Inc., Woodstock, UK), DISETRONIC™ pen (Disetronic Medical Systems, Burghdorf, Switzerland), HUMALOG MIX 75 / 25™ pen, HUMALOG™ pen, HUMALIN 70 / 30™ pen (Eli Lilly and Co., Indianapolis, IN), NOVOPEN™ I, II and III (Novo Nordisk, Copenhagen, Denmark), NOVOPEN JUNIOR™ (Novo Nordisk, Copenhagen, Denmark), BD™ pen (Becton Dickinson, Franklin Lakes, NJ), OPTIPEN™, OPTIPEN PRO™, OPTIPEN STARLET™, and OPTICLIK™ (Sanofi-Aventis, Frankfurt, Germany), to name only a few. Examples of disposable pen delivery devices having applications in subcutaneous delivery of a pharmaceutical composition of the present disclosure include, but certainly are not limited to the SOLOSTAR™ pen (Sanofi-Aventis), the FLEXPEN™(Novo Nordisk), and the KWIKPEN™ (Eli Lilly), the SURECLICKTMAutoinjector (Amgen, Thousand Oaks, CA), the PENLETTM(Haselmeier, Stuttgart, Germany), the EPIPEN (Dey, LSEQ ID NO: ) and the HUMIRA™ Pen (Abbott Labs, Abbott Park, IL), to name only a few.

[0190] Advantageously, the pharmaceutical compositions for oral or parenteral use described above are prepared into dosage forms in a unit dose suited to fit a dose of the active ingredients. Such dosage forms in a unit dose include, for example, tablets, pills, capsules, injections (ampoules), suppositories, etc. The amount of the antibody contained is generally about 5 to about 500 mg per dosage form in a unit dose; especially in the form of injection, it is preferred that the antibody is contained in about 5 to about 100 mg and in about 10 to about 250 mg for the other dosage forms.Therapeutic Uses of the Antibodies

[0191] The antibodies of the disclosure are useful, inter alia, for the treatment, prevention and / or amelioration of any disease or disorder associated with or mediated by CXCR4 expression, signaling or activity, or treatable by blocking the interaction between CXCR4 and the CXCR4 ligands, or otherwise inhibiting CXCR4 activity and / or signaling. One or more antibodies of the present disclosure may be administered to relieve or prevent or decrease the severity of one or more of the symptoms or conditions of the disease or disorder. For example, the present disclosure provides methods for improving bone marrow transplant success, treating cancer and / or viral infections by administering an anti-CXCR4 antibody (or pharmaceutical composition comprising an anti-CXCR4 antibody) as described herein to a patient in need of such treatment, and anti-CXCR4 antibodies (or pharmaceutical composition comprising an anti-CXCR4 antibody) for use in the treatment of cancer (tumor growth inhibition) and / or viral infections. The antibodies of the present disclosure are useful for the treatment, prevention, and / or amelioration of disease or disorder or condition such as cancer or a viral infection and / or for ameliorating at least one symptom associated with such disease, disorder or condition. In the context of the methods of treatment described herein, the anti-CXCR4 antibody may be administered as a monotherapy ( / .e., as the only therapeutic agent) or in combination with one or more additional therapeutic agents (examples of which are described elsewhere herein).

[0192] In some embodiments of the disclosure, the antibodies described herein are useful for host bone marrow mobilization to enhance hematopoietic stem cell transplant by leading to transient mobilization of bone marrow resident leukocytes and progenitor cells. In some embodiments, the antibodies disclosed herein cause loss of mature B cells in bone marrow.

[0193] In some embodiments of the disclosure, the antibodies described herein are useful for treating subjects suffering from primary or recurrent cancer, including, but not limited to, blood cancer, brain cancer (e.g., glioblastoma multiforme), renal cell carcinoma (e.g., clear cell renal cancer), ovarian cancer, bladder cancer, prostate cancer, breast cancer e.g., triple negative breast cancer), kidney cancer, cervical cancer, skin cancer, liver cancer, stomach cancer, pancreatic cancer, hepatic cell carcinoma, bone cancer, colon cancer, non-small-cell lung cancer, squamous cell carcinoma of head and neck, colorectal cancer, mesothelioma, and melanoma.

[0194] As used herein, the term “blood cancer” includes a hematologic malignancy that affects blood, bone marrow, lymph or lymphatic system. As such, the term includes malignancies of cells from the lymphoid and myeloid cell lineages. The myeloid cell line normally produces granulocytes, erythrocytes, thrombocytes, macrophages, and mast cells; the lymphoid cell line produces B, T, NK and plasma cells. The term, therefore, includes malignancies of the above- mentioned cells, viz. lymphomas, myelomas, lymphoid leukemias and myelogenous leukemias. Examples include, but are not limited to, acute lymphoblastic leukemia, acute myelogenous leukemia, chronic lymphocytic leukemia, chronic myelogenous leukemia, acute monocytic leukemia, Hodgkin’s lymphomas, non-Hodgkin’s lymphomas (e.g., B cell lymphoma, diffuse large B cell lymphoma), and myeloma (including multiple myeloma).

[0195] The antibodies may be used to treat early stage or late-stage symptoms of cancer. In one embodiment, an antibody or fragment thereof of the disclosure may be used to treat advanced or metastatic cancer. The antibodies are useful in reducing or inhibiting or shrinking tumor growth of both solid tumors and blood cancers. In certain embodiments, treatment with an antibody or antigen-binding fragment thereof of the disclosure leads to more than 40% regression, more than 50% regression, more than 60% regression, more than 70% regression, more than 80% regression or more than 90% regression of a tumor in a subject. In certain embodiments, the antibodies may be used to prevent relapse of a tumor. In certain embodiments, the antibodies are useful in extending progression-free survival or overall survival in a subject with cancer. In some embodiments, the antibodies are useful in reducing toxicity due to chemotherapy or radiotherapy while maintaining long-term survival in a patient suffering from cancer. In certain embodiments, one or more antibodies of the present disclosure are injected locally into one or more tumor lesions 9intratumorally or pertumorally), and lead to inhibition of tumor growth in the injected tumor as well as in one or more adjacent or distant tumors in the subject (abscopal effect).

[0196] In certain embodiments, the antibodies of the disclosure are useful to treat subjectssuffering from a chronic viral infection. In some embodiments, the antibodies of the disclosure are useful in decreasing viral titers in the host and / or rescuing exhausted T cells. In certain embodiments, an antibody or fragment thereof of the disclosure may be used to treat chronic viral infection by lymphocytic choriomeningitis virus (LCMV). In some embodiments, an antibody or antigen-binding fragment thereof may be administered at a therapeutic dose to a patient with an infection by human immunodeficiency virus (HIV) or human papilloma virus (HPV) or hepatitis B / C virus (HBV / HCV). In a related embodiment, an antibody or antigenbinding fragment thereof of the disclosure may be used to treat an infection by simian immunodeficiency virus (SIV) in a simian subject such as cynomolgus.

[0197] In certain embodiments, a blocking antibody of the present disclosure may be administered in a therapeutically effective amount to a subject suffering from a cancer or a viral infection.

[0198] It is also contemplated herein to use one or more antibodies of the present disclosure prophylactically to patients at risk for developing a disease or disorder such as cancer, and viral infection.

[0199] In a further embodiment of the disclosure, the present antibodies are used for the preparation of a pharmaceutical composition for treating patients suffering from cancer, or viral infection. In another embodiment of the disclosure, the present antibodies are used as adjunct therapy with any other agent or any other therapy known to those skilled in the art useful for treating cancer or viral infection.Combination Therapies and Formulations

[0200] Combination therapies may include an anti-CXCR4 antibody of the disclosure and any additional therapeutic agent that may be advantageously combined with an antibody of the disclosure, or with a biologically active fragment of an antibody of the disclosure.

[0201] In certain embodiments, the anti-CXCR4 antibodies of the disclosure may be administered in combination with one or more anti-viral drugs to treat chronic viral infection caused by LCMV, HIV, HPV, HBV or HCV. Examples of anti-viral drugs include, but are not limited to, zidovudine, lamivudine, abacavir, ribavirin, lopinavir, efavirenz, cobicistat, tenofovir, rilpivirine and corticosteroids.

[0202] The additional therapeutically active agent(s) / component(s) may be administered prior to, concurrent with, or after the administration of the anti-CXCR4 antibody of the present disclosure. For purposes of the present disclosure, such administration regimens are considered the administration of an anti-CXCR4 antibody “in combination with” a secondtherapeutically active component.

[0203] The additional therapeutically active component(s) may be administered to a subject prior to administration of an anti-CXCR4 antibody of the present disclosure. For example, a first component may be deemed to be administered "prior to" a second component if the first component is administered 1 week before, 72 hours before, 60 hours before, 48 hours before, 36 hours before, 24 hours before, 12 hours before, 6 hours before, 5 hours before, 4 hours before, 3 hours before, 2 hours before, 1 hour before, 30 minutes before, 15 minutes before, 10 minutes before, 5 minutes before, or less than 1 minute before administration of the second component. In other embodiments, the additional therapeutically active component(s) may be administered to a subject after administration of an anti-CXCR4 antibody of the present disclosure. For example, a first component may be deemed to be administered "after" a second component if the first component is administered 1 minute after, 5 minutes after, 10 minutes after, 15 minutes after, 30 minutes after, 1 hour after, 2 hours after, 3 hours after, 4 hours after, 5 hours after, 6 hours after, 12 hours after, 24 hours after, 36 hours after, 48 hours after, 60 hours after, 72 hours after administration of the second component. In yet other embodiments, the additional therapeutically active component(s) may be administered to a subject concurrent with administration of an anti-CXCR4 antibody of the present disclosure. "Concurrent" administration, for purposes of the present disclosure, includes, e.g., administration of an anti- CXCR4 antibody and an additional therapeutically active component to a subject in a single dosage form (e.g., co-formulated), or in separate dosage forms administered to the subject within about 30 minutes or less of each other. If administered in separate dosage forms, each dosage form may be administered via the same route (e.g., both the anti-CXCR4 antibody and the additional therapeutically active component may be administered intravenously, subcutaneously, etc.); alternatively, each dosage form may be administered via a different route (e.g., the anti-CXCR4 antibody may be administered intravenously, and the additional therapeutically active component may be administered subcutaneously). In any event, administering the components in a single dosage from, in separate dosage forms by the same route, or in separate dosage forms by different routes are all considered "concurrent administration," for purposes of the present disclosure. For purposes of the present disclosure, administration of an anti-CXCR4 antibody "prior to", "concurrent with," or "after" (as those terms are defined herein above) administration of an additional therapeutically active component is considered administration of an anti-CXCR4 antibody "in combination with" an additional therapeutically active component).

[0204] The present disclosure includes pharmaceutical compositions in which an anti-CXCR4antibody of the present disclosure is co-formulated with one or more of the additional therapeutically active component(s) as described elsewhere herein using a variety of dosage combinations.Administrative Regimens

[0205] According to certain embodiments of the present disclosure, multiple doses of an anti- CXCR4 antibody (or a pharmaceutical composition comprising a combination of an anti-CXCR4 antibody and any of the additional therapeutically active agents mentioned herein) may be administered to a subject over a defined time course. The methods according to this aspect of the disclosure comprise sequentially administering to a subject multiple doses of an anti-CXCR4 antibody of the disclosure. As used herein, "sequentially administering" means that each dose of anti-CXCR4 antibody is administered to the subject at a different point in time, e.g., on different days separated by a pre-determined interval (e.g., hours, days, weeks or months). The present disclosure includes methods which comprise sequentially administering to the patient a single initial dose of an anti-CXCR4 antibody, followed by one or more secondary doses of the anti-CXCR4 antibody, and optionally followed by one or more tertiary doses of the anti-CXCR4 antibody. The anti-CXCR4 antibody may be administered at a dose between 0.1 mg / kg to 100 mg / kg body weight of the subject.

[0206] The terms "initial dose," "secondary doses," and "tertiary doses," refer to the temporal sequence of administration of the anti-CXCR4 antibody of the disclosure. Thus, the "initial dose" is the dose which is administered at the beginning of the treatment regimen (also referred to as the "baseline dose"); the "secondary doses" are the doses which are administered after the initial dose; and the "tertiary doses" are the doses which are administered after the secondary doses. The initial, secondary, and tertiary doses may all contain the same amount of anti-CXCR4 antibody, but generally may differ from one another in terms of frequency of administration. In certain embodiments, however, the amount of anti-CXCR4 antibody contained in the initial, secondary and / or tertiary doses varies from one another (e.g., adjusted up or down as appropriate) during the course of treatment. In certain embodiments, two or more (e.g., 2, 3, 4, or 5) doses are administered at the beginning of the treatment regimen as "loading doses" followed by subsequent doses that are administered on a less frequent basis (e.g., "maintenance doses").

[0207] In certain embodiments, the amount of anti-CXCR4 antibody contained in the initial, secondary and / or tertiary doses may be sub-optimal or sub-therapeutic. As used herein, the terms “sub-therapeutic” or “sub-optimal” refer to an antibody dose administered at too low alevel to produce a therapeutic effect or below the level necessary to treat a disease such as cancer.

[0208] In certain exemplary embodiments of the present disclosure, each secondary and / or tertiary dose is administered 1 to 26 (e.g., 1 , 11 / 2, 2, 21 / s, 3, 31 / z, 4, 41 / a, 5, 51 / s, 6, 61 / s, 7, 71 / 2, 8, 81 / 2, 9, 91 / 2, 10, 101 / 2, 1 1 , 1 11 / 2, 12, 121 / 2, 13, 131Z>, 14, 141 / 2, 15, 151 / 2, 16, 161 / 2, 17, 171 / 2, 18, 181 / 2, 19, 191 / 2, 20, 201 / 2, 21 , 21 / 2, 22, 221 / 2, 23, 231Z>, 24, 241 / 2, 25, 251 / 2, 26, 2672, or more) weeks after the immediately preceding dose. The phrase "the immediately preceding dose," as used herein, means, in a sequence of multiple administrations, the dose of anti-CXCR4 antibody which is administered to a patient prior to the administration of the very next dose in the sequence with no intervening doses.

[0209] The methods according to this aspect of the disclosure may comprise administering to a patient any number of secondary and / or tertiary doses of an anti-CXCR4 antibody. For example, in certain embodiments, only a single secondary dose is administered to the patient. In other embodiments, two or more (e.g., 2, 3, 4, 5, 6, 7, 8, or more) secondary doses are administered to the patient. Likewise, in certain embodiments, only a single tertiary dose is administered to the patient. In other embodiments, two or more (e.g., 2, 3, 4, 5, 6, 7, 8, or more) tertiary doses are administered to the patient.

[0210] In embodiments involving multiple secondary doses, each secondary dose may be administered at the same frequency as the other secondary doses. For example, each secondary dose may be administered to the patient 1 to 2 weeks or 1 to 2 months after the immediately preceding dose. Similarly, in embodiments involving multiple tertiary doses, each tertiary dose may be administered at the same frequency as the other tertiary doses. For example, each tertiary dose may be administered to the patient 2 to 12 weeks after the immediately preceding dose. In certain embodiments of the disclosure, the frequency at which the secondary and / or tertiary doses are administered to a patient can vary over the course of the treatment regimen. The frequency of administration may also be adjusted during the course of treatment by a physician depending on the needs of the individual patient following clinical examination.Diagnostic Uses of the Antibodies

[0211] The anti-CXCR4 antibodies of the present disclosure may be used to detect and / or measure CXCR4 in a sample, e.g., for diagnostic purposes. Some embodiments contemplate the use of one or more antibodies of the present disclosure in assays to detect a disease or disorder such as cancer, autoimmune disease or viral infection. Exemplary diagnostic assaysfor CXCR4 may comprise, e.g., contacting a sample, obtained from a subject (e.g., a patient), with an anti-CXCR4 antibody of the disclosure, wherein the anti-CXCR4 antibody is labeled with a detectable label or reporter molecule or used as a capture ligand to selectively isolate CXCR4 from subject samples. Alternatively, an unlabeled anti-CXCR4 antibody can be used in diagnostic applications in combination with a secondary antibody which is itself delectably labeled. The detectable label or reporter molecule can be a radioisotope, such as3H,14C,32P,35S, or125l; a fluorescent or chemiluminescent moiety such as fluorescein isothiocyanate, or rhodamine; or an enzyme such as alkaline phosphatase, p-galactosidase, horseradish peroxidase, or luciferase. Specific exemplary assays that can be used to detect or measure CXCR4 in a sample include enzyme-linked immunosorbent assay (ELISA), radioimmunoassay (RIA), and fluorescence-activated cell sorting (FACS).

[0212] Samples that can be used in CXCR4 diagnostic assays according to the present disclosure include any tissue or fluid sample obtainable from a subject, which contains detectable quantities of either CXCR4 protein, or fragments thereof, under normal or pathological conditions. Generally, levels of CXCR4 in a particular sample obtained from a healthy patient (e.g., a patient not afflicted with cancer or an autoimmune disease) will be measured to initially establish a baseline, or standard, level of CXCR4. This baseline level of CXCR4 can then be compared against the levels of CXCR4 measured in samples obtained from individuals suspected of having a cancer-related condition, or symptoms associated with such condition.

[0213] The antibodies specific for CXCR4 may contain no additional labels or moieties, or they may contain an N-terminal or C-terminal label or moiety. In one embodiment, the label or moiety is biotin. In a binding assay, the location of a label (if any) may determine the orientation of the peptide relative to the surface upon which the peptide is bound. For example, if a surface is coated with avidin, a peptide containing an N-terminal biotin will be oriented such that the C- terminal portion of the peptide will be distal to the surface.

[0214] Aspects of the disclosure relate to use of the disclosed antibodies as markers for predicting prognosis of cancer or a viral infection in patients. Antibodies of the present disclosure may be used in diagnostic assays to evaluate prognosis of cancer in a patient and to predict survival.EXAMPLES

[0215] The following examples are put forth so as to provide those of ordinary skill in the art with a complete disclosure and description of how to make and use the methods andcompositions of the disclosure, and are not intended to limit the scope of what the inventors regard as their disclosure. Efforts have been made to ensure accuracy with respect to numbers used (e.g., amounts, temperature, etc.) but some experimental errors and deviations should be accounted for. Unless indicated otherwise, parts are parts by weight, molecular weight is average molecular weight, temperature is in degrees Centigrade, room temperature is about 25°C, and pressure is at or near atmospheric.Example 1 : Generation of Human Antibodies to CXCR4

[0216] Human antibodies to CXCR4 were generated using either a human CXCR4 protein, e.g., SEQ ID NO: 43, or DNA encoding hCXCR4 (Accession No: NP 003458.1 ) as the immunogen. The immunogen was administered directly, with an adjuvant to stimulate the immune response, to a VELOCIMMUNE® mouse ( / .e., an engineered mouse comprising DNA encoding human Immunoglobulin heavy and kappa light chain variable regions), as described in US 8502018 B2. The antibody immune response was monitored by a CXCR4-specific immunoassay. When a desired immune response was achieved, splenocytes were harvested and fused with mouse myeloma cells to preserve their viability and form hybridoma cell lines. The hybridoma cell lines were screened and selected to identify cell lines that produce CXCR4-specific antibodies.Using this technique, and the immunogen described above, several anti-CXCR4 chimeric antibodies ( / .e., antibodies possessing human variable domains and mouse constant domains) were obtained; exemplary antibodies generated in this manner from the VELOCIMMUNE® mice were designated as H2aM15295N, H1 M16435N, REGN7663, and REGN7664.

[0217] The biological properties of the exemplary antibodies generated in accordance with the methods of this Example are described in detail in the Examples set forth below.Example 2: Heavy and Light Chain Variable Region Amino Acid and Nucleotide Seouences

[0218] Table 1 sets forth the amino acid sequence identifiers of the heavy and light chain variable regions and CDRs of selected anti-CXCR4 antibodies of the disclosure. The corresponding nucleic acid sequence identifiers are set forth in Table 2. Table 3 sets forth the amino acid sequence identifiers of the full length heavy and light chains for selected disclosed antibodies. Table 4 sets forth the nucleic acid sequence identifiers for the full length heavy and lights of selected disclosed antibodies.Table 1 : Amino Acid Sequence IdentifiersTable 2: Nucleic Acid Sequence Identifiers

[0219] In certain embodiments, selected antibodies with a mouse IgG Fc were converted to antibodies with human lgG4 Fc. In some embodiments, the lower hinge region of human lgG4 Fc domain (amino acids CPSCPAPEFLG; SEQ ID NO: 41 ) is replaced with the lower hinge region of human lgG2 (amino acids CPPCPAPPVA; SEQ ID NO: 42) to promote dimer stabilization. For example, REGN7663 comprises the light chain and heavy chain variable regions of H1 M16435N and the constant regions of human lgG4, where the lower hinge region of human lgG4 Fc domain (amino acids CPSCPAPEFLG; SEQ ID NO: 41 ) is replaced with the lower hinge region of human lgG2 (amino acids CPPCPAPPVA; SEQ ID NO: 42). Similarly, REGN7664 comprises the light chain and heavy chain variable regions of H2aM15295N and the constant regions of human lgG4, where the lower hinge region of human lgG4 Fc domain (amino acids CPSCPAPEFLG; SEQ ID NO: 41 ) is replaced with the lower hinge region of human lgG2 (amino acids CPPCPAPPVA; SEQ ID NO: 42). Table 3 sets forth the amino acid sequence identifiers of heavy chain and light chain sequences of selected anti-CXCR4 antibodies with human lgG4 Fc.Table 3: Amino Acid Sequences Identifiers for Full Length Heavy and Light Chains of Select AntibodiesTable 4: Nucleic Acid Sequences Identifiers for Full Length Heavy and Light Chains of Select Antibodies

[0220] Antibodies are typically referred to herein according to the following nomenclature: Fc prefix (e.g. "H1 M," "H4H," etc.), followed by a numerical identifier (e.g. "15295," etc., as shown in Table 1 ), followed by a "P," "P2," or "N" suffix. Thus, according to this nomenclature, an antibody may be referred to herein as, e.g., "H2Ma15295N," "H1 M16435N," etc. The H1 H prefix on the antibody designations used herein indicates the particular Fc region isotype of the antibody. For example, an "H1 H" antibody has a human IgG 1 Fc, an "H1 M" antibody has a mouse IgG 1 Fc, and an "H2M" antibody has a mouse lgG2 Fc (all variable regions are fully human as denoted by the first 'H' in the antibody designation). As will be appreciated by a person of ordinary skill in the art, an antibody having a particular Fc isotype can be converted to an antibody with a different Fc isotype (e.g., an antibody with a mouse lgG1 Fc can be converted to an antibody with a human IgG 1 or a human lgG4, etc.), but in any event, the variable domains (including the CDRs) - which are indicated by the numerical identifiers shown in Tables 1 and 2 - will remain the same, and the binding properties to antigen are expected to be identical or substantially similar regardless of the nature of the Fc domain.

[0221] Antibody H1 M16435N contains the same human variable region as REGN7663. Antibody H2aM15295N contains the same human variable region as REGN7664. REGN7663 and REGN7664 are fully human antibodies and have both human variable regions and human constant regions while both H1 M16435N and H2aM15295N have mouse constant regions.Control Constructs

[0222] Two control constructs were included in the following experiments for comparative purposes:COMP1571 : binds to human C-C Chemokine Receptor 5 (huCCR5) receptor. COMP3547: is also known as ulocuplumab and binds to human CXCR4 (huCXCR4) receptor.Example 3: CXCR4 CrvoEM Epitope Structural Analysis Overview

[0223] To better understand the binding of REGN7663 and REGN7664 to CXCR4, a structural analysis was performed via cryo-electron microscopy (cryoEM).Methods

[0224] ExpiSf9 cells at approximately 5x106 / mL were infected with baculovirus encoding either CXCR4or Gai and Gpi / Gy2. Cells were harvested by centrifugation (3000 ref, 10 min, 4°C) after 72 hours of growth (120 rpm shaking, 27°C, 2 L flat-bottom flask, Innova 44 shaker). Cell pellets were washed in ice-cold DPBS with complete™ (EDTA-free) protease inhibitor and then subjected to freeze-thaw (-80°C). Pellets were then resuspended in lysis buffer (25 mM Tris- HCI pH 7.5, 50 mM NaCI, 2 mM MgCh. 1 x complete™ (EDTA-free) protease inhibitor, 5 mM CaCl2, 50 mll / mL Apyrase), mixed together, and stirred at 4°C. After 1 hour, an equal volume (1 mL for every 1 mL of lysis buffer) of solubilization buffer (25 mM Tris-HCI pH 7.5, 50 mM NaCI, 2 mM MgCL, 5 mM CaCL 2% Lauryl Maltose Neopentyl Glycol “LMNG”, 0.2% Cholesteryl Hemisuccinate Tris Salt “CHS”) was added to the slurry and the mixture was stirred at 4°C for 1 hr. Insoluble material was removed by centrifugation (100,000 ref, 4°C, 30 min). The supernatant was applied to Anti-FLAG M2 Affinity Gel: the protein-loaded resin was washed with SEC buffer (25 mM Tris-HCI pH 7.5, 150 mM NaCI, 2 mM MgCh, 0.01% LMNG, 0.001% CHS) and protein was eluted in SEC buffer containing 0.15 mg / ml 3X FLAG Peptide. The eluate was concentrated and subjected to SEC. A tandem column was used: a Superose 6 Increase 10 / 300 GL column was upstream of a Superdex 200 Increase 10 / 300 GL column. Fractions containing CXCR4 / G protein complex were selected, pooled, and concentrated. To produce Fab’ fragments, REGN7663 and REGN7664 were each diluted to 2 mg / mL in 20 mM HEPES pH 7.4, 150 mM NaCI. IdeS was added and the cleavage reaction was carried out at 37°C for 30 min. F(ab’)2 was reduced using approximately 88 mM cysteamine hydrochloride at 37°C for 10 min, in the presence of approximately 18 mM EDTA. Reduced Fab’ was dialyzed against 20 mM HEPES pH 7.4, 150 mM NaCI overnight at 4°C. Fab’ was further purified byIMAC (negative-pass to remove His-tagged IdeS) and CaptureSelect IgG-Fc (Multispecies) Affinity Matrix (negative-pass to remove Fc fragment). Fab’ was treated with 20 mM iodoactamide at room temperature, in the dark, for 30 min to alkylate reduced, hinge cysteines. Fab' was concentrated, purified further via SEC (HighLoad 16 / 600 Superdex 75 pg, 25 mM Tris pH 7.5, 150 mM NaCI), and concentrated again before use.

[0225] Complexes between CXCR4 / Gi and REGN7663 Fab or REGN7664 Fab were assembled by mixing the components and incubating on ice for approximately one hour. Protein solutions were deposited onto freshly plasma cleaned UltrAufoil® grids (0.6 / 1 .0, 300 mesh). Excess solution was blotted away using filter paper and plunge frozen into liquid ethane using a Vitrobot Mark IV. The cryoEM grids were inserted into a Titan Krios (Thermo Fisher) equipped with a BioQuantum K3 imaging system (Gatan). Movies were collected using EPU (Thermo Fisher) at 105,000x magnification, corresponding to a pixel size of 0.85 A. A dose rate of 15 electrons per pixel per second was used and each movie was 2 seconds, corresponding to a total dose of ~40 electrons per A2. 10,346 movies were collected for the REGN7663 Fab / CXCR4 / Gi complex and 6,137 movies were collected for the REGN7664 Fab / CXCR4 / Gi complex.

[0226] CryoEM data processing was carried out using cryoSPARC v3.3.1 . Patch motion correction and Patch CTF estimation were used to align movie frames and estimate CTF parameters, respectively. Particle images were picked using 2D template-based picker, then extracted and subjected to multiple rounds of 2D classification, Complexes between CXCR4 / Gi and REGN7663 Fab or REGN7664 Fab were assembled by mixing the components and incubating on ice for approximately one hour. Protein solutions were deposited onto freshly plasma cleaned UltrAufoil grids (0.6 / 1 .0, 300 mesh). Excess solution was blotted away using filter paper and plunge frozen into liquid ethane using a Vitrobot Mark IV. The cryoEM grids were inserted into a Titan Krios (Thermo Fisher) equipped with a BioQuantum K3 imaging system (Gatan). Movies were collected using EPU (ThermoFisher) at 105,000x magnification, corresponding to a pixel size of 0.85 A. A dose rate of 15 electrons per pixel per second was used and each movie was 2 seconds, corresponding to a total dose of ~40 electrons per A2. 10,346 movies were collected for the REGN7663 Fab / CXCR4 / Gi complex and 6,137 movies were collected for the REGN7664 Fab / CXCR4 / G complex.

[0227] Model building was aided by initial docking of published models of CXCR4 (Protein Data Bank ID 4RWS) and Gi heterotrimer (Protein Data Bank ID 7T2G), as well as a previously determined internal Fab structure. Multiple rounds of manual coordinate adjustment in Coot and real-space refinement in Phenix were conducted to obtain the model used for epitope analysis.Epitope residues were determined in Pymol by selecting CXCR4 residues within 4 A interatomic distance of Fab.Results

[0228] The CXCR4 epitope residues which REGN7663 and REGN7664 bind to were identified.The CXCR4 epitopes for REGN7663 are noted in Table 5 and can be seen in Figure 1 A and 1 B.The CXCR4 epitopes for REGN7664 are noted in Table 6 and can be seen in Figures 2A and 2B.Table 5: Epitopes on CXCR4 recognized by REGN7663.Table 6: Epitopes on CXCR4 recognized by REGN7664.Example 4: Human-Mouse CXCR4 Domain Swap Construct Confirm Antibodies Binding to Human CXCR4 Extracellular Loop 2Overview

[0229] This example confirms the epitope determinants identified in Example 3 and shows that anti-human CXCR4 mAbs REGN776 and REGN7664 bind the extracellular loop 2 (ECL2) domain of CXCR4Methods

[0230] Plasmid constructs encoding the domain-swap CXCR4 variants shown in Figure 3 were transiently transfected into 293T cells.Results

[0231] To further confirm epitope determinants for REGN7663 and REGN7664 established by cryoEM, a series of human-mouse domain swap CXCR4 constructs were developed as shown in Figure 3. Early studies established that REGN7663 and REGN7664 bind human but not mouse CXCR4, suggesting that murinization of residues important for antibody recognition should abolish binding.

[0232] Figures 4-7 show flow cytometry data establishing that REGN7663 and REGN7664 lose binding to all variants where the ECL2 domain is murine (pMM331 , pMM335, pMM336, pMM341 , pMM345). Moreover, REGN7663 and REGN7664 bind efficiently to murine CXCR4 with a humanized ECL2 domain (pMM346). Staining with isotype (REGN1945) shown in Figure 6 confirmed binding specificity, and staining with a human-mouse cross reactive anti-CXCR4 (clone 2B11 ) shown in Figure 7 confirmed expression of all constructs, except for pMM335 which failed to express. This data establishes that mAb-CXCR4 residue contacts in human ECL2 domain of established by cryoEM studies are necessary and sufficient for the interaction.Example 5: Cell Binding Analysis with NIH3T3 / hCXCR4 Cells by Flow Cytometry Overview

[0233] This example illustrates the cell of binding of the antibodies disclosed herein using flow cytometry.Methods

[0234] To test the antibodies binding to cells, a cell line was generated in NIH3T3 cells to stably express full-length human CXCR4 (hCXCR4; amino acids 1 -352 of accession number NP 003458.1 ; SEQ ID NO: 43). The stable cell lines (NIH3T3 / hCXCR4 cells) were isolated, sorted for high expression of CXCR4, and maintained in Dulbecco’s Modified Eagle Medium (DMEM) containing 10% Bovine serum, penicillin / streptomycin / L-glutamine, and 500 pg / mL G418 sulfate.

[0235] For the fluorescence-activated cell sorting (FACS) flow cytometry analysis, NIH3T3 parental cells and NIH3T3 / hCXCR4 cells were dissociated and plated onto 96-well v-bottomplates at 5 x 105cells / well in FACS buffer composed of phosphate buffered saline (PBS) with 2% fetal bovine serum (FBS). Antibodies were added to cells at 10 pg / ml and were incubated for 30 minutes at 4°C. After washing one time with FACS buffer, cells were incubated with 2 pg / mL of allophycocyanin (APC) conjugated anti-human IgG or anti-mouse IgG secondary antibodies (Jackson ImmunoResearch Laboratories Inc.) for 30 minutes at 4°C. Cells were washed one time with FACS buffer and fixed using BD CytoFix™ (Becton Dickinson, # 554655). The cells were then filtered, and analyzed on Accuri C6 ™ Cytometer (BD Biosciences). Unstained and secondary antibody alone controls were also tested for all cell lines. The results were analyzed using FlowJo version 10 software to determine the geometric means of fluorescence for viable cells. The geometric mean for each sample was then normalized to the geometric mean of unstained cells to obtain relative binding per condition referred to as “binding ratios”, and these binding ratios were recorded for each antibody tested.Results

[0236] As shown in Table 7, H2aM15295N and H1 M16435N showed specific binding to NIH3T3 / hCXCR4 cells with binding ratios of 93 and 63, respectively. These antibodies did not show significant binding to the parental NIH3T3 cells with binding ratios of 1 -2. The comparator antibody, Comp3547, showed binding to NIH3T3 / hCXCR4 cells with a binding ratio of 195 and to NIH3T3 parental cells with a binding ratio of 2. Control mAb1 , mouse IgG control, and Control mAbs 2, human IgG control, and anti-mouse and human IgG secondary antibodies alone showed binding to both cell lines with binding ratios of 1 -2.Table 7: Binding ratios of anti-CXCR4 antibodies with NIH3T3 / hCXCR4 cells analyzed by FACS flow cytometryExample 6: Electrochemiluminescence-Based Detection Cell Surface Binding Assay Overview

[0237] To investigate the ability of a panel of anti-CXCR4 monoclonal antibodies to bind cellsurface expressed CXCR4, an in vitro binding assay utilizing human and monkey expressing cell lines in an electrochemiluminescence based detection platform (MSD) was developed.Methods

[0238] HEK293 cell lines were generated to stably express full-length human CXCR4 (hCXCR4; amino acids 1 -352 of accession number NP 003458.1 , SEQ ID NO: 43) or monkey CXCR4 (Macaca mulatta, mmCXCR4; amino acids 1 -352 of accession numberNP 001036110.1 , SEQ ID NO: 44) along with a luciferase reporter cAMP response element (CRE, 4X)-luciferase-IRES-GFP). The stable cell lines (HEK293 / CRE-luc / hCXCR4 and HEK293 / CRE-luc / mfCXCR4 cells) were isolated and maintained in DMEM containing 10% FBS, non-essential amino acids (NEAA), penicillin / streptomycin / L-glutamine, and 0.5 mg / mL G418. A parental HEK293 cell line expressing just the CRE-luc reporter (293 / CRE-luc) was also included for reference. Included in the experiment are anti-Fel d human lgG4P and anti-human CD48 mouse IgG 1 antibodies as irrelevant controls for the IgG detection antibodies.

[0239] Cells from the three cell lines described above were cultured in respective growth media to 80-90% confluency before the assay. On the day of assay, cells were rinsed once in 1xPBS buffer without Ca2+ / Mg2+ followed by a 10-minute incubation at 37°C with Enzyme Free Cell Dissociation Solution. The detached cells were washed one time with 1xPBS with Ca2+ / Mg2+ and counted with a CellometerTM Auto T4 cell counter (Nexcelom Bioscience). Approximately 10,000 cells per well in the cell wash buffer were seeded into the 96-well carbon electrode plates (Mil LT I -AR RAY high bind plate, MSD) and incubated for 1 hour at 37°C to allow the cells to adhere. Nonspecific binding sites were blocked by 2% BSA (w / v) in PBS for 1 hour at room temperature. To the plate-bound cells, solutions of the anti-CXCR4 antibodies in serial dilutions ranging from 1 .7 pM to 100 nM in 1 X PBS + 0.5% BSA, and solutions without the presence of the antibody, were added in duplicate. The plates were then incubated for 1 hour at room temperature. The plates were then washed with 1X PBS to remove the unbound antibodies using a plate washer, AquaMax2000™ (MDS Analytical Technologies). The plate-bound antibodies were detected with a SULFO-TAGTM-conjugated anti-human IgG antibody (Meso Scale Development) or a SULFO-TAGTM-conjugated anti-mouse IgG antibody (Jackson Immunoresearch) for 1 hour at room temperature. After washes, the plates were developed with the Read Buffer (MSD) according to manufacturer’s recommended procedure and theluminescent signals were recorded with a SECTOR Imager 6000 (MSD) instrument. The luminescence intensity, measured in relative light units (RLU) for the three cell lines is recorded to indicate the binding intensity of each antibody. The ratio of signal detected with 1 ,2nM or 11 ,1 nM antibody binding to the HEK293 / CRE-luc / hCXCR4 or HEK293 / CRE-luc / mfCXCR4 cells compared to the HEK293 / CRE-luc parental cells is reported as an indication of specificity and potency of CXCR4 binding.Results

[0240] The ability of the anti-CXCR4 monoclonal antibodies to bind specifically to HEK293 cells engineered to express human and monkey CXCR4 compared with the parental HEK293 cells was assessed using an immunobinding assay. Antibody dose dependent binding to the immobilized cells on 96-well High Bind plates (MSD), with antibody concentrations up to 100nM, were detected using SULFO-TAG™-conjugated anti-human IgG or anti-mouse IgG antibody, and the binding signals in electrochemiluminescence were recorded on a Sector Imager 6000 (MSD). RLU values were determined for the antibody binding to CXCR4 expressing cells, and a comparison of the binding signals of the antibodies at 1 .2 and 11.1 nM to each species of CXCR4 expressing and parental cells was used to evaluate binding specificity. Specific binding is defined as antibodies having a ratio of >3-fold binding to CXCR4 expressing cells compared to parental HEK293 cells at either of the concentrations reported.

[0241] The binding results are summarized in Table 8. Ratios of antibodies binding to human and monkey expressing CXCR4 cells vs parental cells at 1 .2 and 11.1 nM concentration respectively are reported, along with the binding signal (RLU) to the parental cells for each antibody at the same concentrations for background binding reference. All test antibodies evaluated bound specifically to cells expressing the human CXCR4 receptor. High background binding was seen in the parental HEK293 / CRE-luc cells at 11.1 nM, as seen by the RLU values indicated in the table. The comparator binding was specific on both human and monkey cells with potency on HEK293 / CRE-luc / hCXCR4 cells of 14.9 and 9.9 fold higher than parental cells at 1 .2 nM and 1 1.1 nM antibody respectively, and on HEK293 / CRE-luc / mfCXCR4 cells 6.7 and 4.3 fold higher with 1 .2 nM and 11.1 nM of the antibody. Irrelevant controls binding to the HEK293 / CRE-luc / hCXCR4 and HEK293 / CRE-luc / mfCXCR4 cells were non-specific as expected, with anti-Fel d-hlgG4p, and anti-human CD48-mlgG1 binding with ratios below 3-fold binding above the HEK293 / CRE-luc parental cells.Table 8: Binding Ratios per Antibody TestedExample 7: CXCR4 Bioassay with HEK293 / CRE-luc / hCXCR4 CellsOverview

[0242] A bioassay was developed to detect activation of CXCR4 through the G-alpha subunit, subsequent regulation of cAMP levels, and transcriptional activation. Schematics of this assay are shown in Figures 8A-8C.Methods

[0243] For the bioassay, HEK293 / CRE-luc / hCXCR4 cells were seeded onto 96-well assay plates in OPTIMEM™, 0.1%FBS, pen / strep / glut, and incubated at 37°C and 5% CO2 overnight. The next morning, SDF-1 was serially diluted at 1 :3 from 100 nM to 0.002 nM, including a “no SDF-1 ” condition, mixed with 5 pM forskolin and added to cells. For inhibition of hCXCR4, antibodies were serially diluted and added to cells and pre-incubated for 30 min. Following the incubation, either 500 pM SDF-1 or no SDF-1 was added to cells. Luciferase activity was detected by adding 100 pl / well of OneGlo reagent after 5.5 hours of incubation in 37°C and 5% CO2 (Victor X, Perkin Elmer). Results were analyzed using nonlinear regression (4-parameter logistics) with Prism software (GraphPad) to obtain EC50 and IC50 values.Results

[0244] H1 M16435N, H2aM15295N, REGN7663, REGN7664 and comparator mAb, COMP3547, showed a dose dependent inhibition of 500 pM SDF-1 and basal activity of CXCR4 and the results are shown in Table 9 and Table 10. SDF-1 showed a dose dependent inhibition of forskolin activation of luciferase reporter activity. Negative control antibodies, Control mAb1 , mouse IgG control, and Control mAb2, human IgG control, were included, which did not affect CXCR4 activation or inhibition. “Max. Inh.” in Tables 9 and 10 is maximum inhibition and is determined using the range of activation achieved by maximum concentration of SDF-1 for conditions without ligand or the constant concentration of SDF-1 with ligand.Table 9: Activation of HEK293 / CRE-luc / hCXCR4 cells by anti-CXCR4 antibodies with or without SDF-1 in the presence of forskolinTable 10: Anti-CXCR4 Antibody Inhibition of Human CXCR4 in CRE Luciferase Reporter Assay Using HEK293 / CREIuc / hCXCR4 CellsExample 8: Bioassay for Detection of Activation of CXCR4 through Ga SubunitOverview

[0245] A bioassay was developed to detect activation of CXCR4 through the Ga subunit andsubsequent regulation of cAMP levels by measuring cAMP accumulation.Methods

[0246] CEM-CCRF cells are cultured in 20 mL of RPMI complete medium (RCM) containing RPMI+10% FBS+1% PSG) in T75 flask. For the migration assay, CEM-CCRF cells are activated with IL-2 the day before the assay. Every 20 mL of RCM contains 2 mL (10%) of IL-2 and 300 pL of 1 mg / mL of PHA-L and rest of the volume with 20-25 million of CEM-CCRF cell suspension in RCM and left overnight at 37°C in 5% CO2 incubator. The following day, when observed under the microscope, large clumps of cells are seen indicating that the cells are activated. Cells are washed twice with 10 mL of migration assay media (1X HBSS with 0.5% BSA and 20 mM HEPES) and re-suspended in migration assay media at 30 x 107cells / mL. 1 .5 x 106cells are incubated with 600 ng / mL of antibody for 30 minutes at 37°C in a 5% CO2 incubator. 151 nM of SDF-1 made in migration assay media is added to the lower chamber followed by addition of cells and antibody mixture to the insert of a 24 transwell plate and incubated for 2 hours at 37°C in 5% CO2 incubator. After 2 hours, cells from the lower chamber are harvested and total number of cell migrated is measured using CellTiter-Glo® luminescent cell viability kit as per manufacturer instructions on Victor X3. The percent migration is calculated by subtracting the background values of untreated cell controls from samples and comparing those values to the average of the SDF-1 only treated cells to obtain a percent of maximum migration.Results

[0247] Anti-CXCR4 antibodies, including comparator CXCR4 mAb, COMP3547, showed a dose-dependent inhibition of 500 pM SDF-1 and basal activity of CXCR4 and the results are shown in Table 11 . SDF-1 showed a dose-dependent inhibition of forskolin activation. Negative control antibodies, Control mAb, human IgG control, were included, which did not affect CXCR4 activation or inhibition.Table 11 : Anti-CXCR4 Antibody Inhibition of Human CXCR4 in cAMP Accumulation Assay Using HEK293 / CREIuc / hCXCR4 CellsExample 9: Calcium Regulation Bioassav in Jurkat CellsOverview

[0248] A bioassay was developed to detect activation of CXCR4 and subsequent regulation of calcium levels by calcium flux assay in Jurkat cells that endogenously express CXCR4.Methods

[0249] Cells were plated in a clear-bottom PDL-coated black 96-well plate (Greiner, Cat# 655948) with RPMI-1640 media (Irvine Scientific, Cat# 9160) containing 10% FBS (Seradigm, Cat #1500-500) and Penicillin / Streptomycin (Invitrogen, Cat# 10378-016) and L-Glutamine (Irvine Scientific, Cat# 9317) for overnight. The next day, calcium-sensitive dye was loaded to the cells for 30 minutes at 37°C with 5% CO2 and then 30 minute at room temperature. Serially diluted SDF-1 (1 :3 titration from 100 nM to 15 pM with a no SFD1 condition) or CXCR4 antibody or control antibody (1 :4 titration from 300 nM to 4.6 pM with a no antibody condition) induced calcium mobilization was determined by using a FLIPRTETRAinstrument (Molecular Device) to measure fluorescence intensity [excitation / emission (Ex / Em) = 490 nm / 515 nm]. For inhibition of SDF-1 -induced CXCR4 activation, serially diluted CXCR4 antibody (1 :4 titration from 300 nM to 4.6 pM with a no antibody condition) was add to cells during the last 30-minute calcium dye load step. Then, addition of 3.7 nM SDF-1 to cells and measurement of calcium mobilization were executed by using a FLIPRTETRAinstrument as described previously. The results were analyzed using nonlinear regression (4-parameter logistics) with PrismTM6 software (GraphPad) to obtain EC50 or IC50 values.Results

[0250] REGN7663 and REGN7664 showed a dose-dependent inhibition of SDF-1 in a calcium flux assay and the results are shown in Table 12. Comparator, COMP3547, showed weak inhibition. SDF-1 showed a dose-dependent activation. Negative control antibodies, Control mAb, human IgG control, were included, which did not affect CXCR4 activation or inhibition. In Table 12, “ND” represents “not determined” because a concentration-dependent increase was not observed, and “Inh” means inhibition. “Max. Inh” refers to “maximum inhibition” and isdetermined using the range of activation achieved by the constant concentration of SDF-1 .Table 12: Anti-CXCR4 Antibody Inhibition of Human CXCR4 in Calcium Flux Assay Using Jurkat CellsExample 10: Activation of CXCR4 for B-Arrestin Recruitment BioassavOverview

[0251] A bioassay was developed to detect activation of CXCR4 in [3-arrestin recruitment assay.Methods

[0252] The PathHunter [3-arrestin recruitment assay was performed according to the manufacturer’s protocol (DiscoveRx). Briefly, C2C12 / p-arrestin2 / hCXCR4 cells (DiscoverX, Cat# 93-0203C7) were plated in AssayComplete™ Cell Plating 9 Reagent (DiscoverX, Cat# 93- 0563R9B) for overnight. The next day, media were replaced with AssayComplete™ Cell Plating 4 Reagent (DiscoverX, Cat# 93-0563R4A) for 3 hours at 37°C with 5% CO2. Cells were then treated with serial titration of SDF-1 (1 :3 titration from 333 nM to 17 pM with a no SDF-1 condition) or CXCR4 antibody or control antibody (1 :3 titration from 300 nM to 5.1 pM including a no antibody condition) for measuring CXCR4 activation. For measuring inhibition of SDF-1 - induced CXCR4 activation, cells were pre-incubated with serial titration of CXCR4 antibody or control antibody for 20 minutes at 37°C with 5% CO2 followed with adding 0.37 nM SDF-1 . Cells were incubated for 90 minutes at 37°C with 5% CO2. At the conclusion of the incubations, detection reagent (DiscoverX, Cat# 93-0001 ) was added to wells for 1 hour at room temperature. Luminescence values were determined by using an EnVision Plate reader (PerkinElmer). The results were analyzed using nonlinear regression (4-parameter logistics) with PrismTM6 software (Graph Pad) to obtain EC50 or IC50 values.Results

[0253] Anti-CXCR4 antibodies, including comparator CXCR4 mAb, COMP3547, showed a dose-dependent inhibition of SDF-1 in a p-arrestin recruitment assay and the results are shown in Table 13. SDF-1 showed a dose-dependent activation. Negative control antibodies, Control mAb, human IgG control, were included, which did not affect CXCR4 activation or inhibition.Table 13: Anti-CXCR4 Antibody Inhibition of Human CXCR4 in p-arrestin Recruitment Assay Using C2C12 / p-arrestin2 / hCXCR4 CellsExample 11 : HIV Pseudo Particles Neutralization AssayOverview

[0254] CXCR4 is a coreceptor for HIV infection of cells in combination with the primary receptor CD4. Presence of viruses with tropism for CXCR4 in patients correlates with an accelerated disease progression to AIDS. This Example shows the HIV particle neutralization activity of the antibodies disclosed herein.MethodsHIV Pseudo Particles Generation

[0255] pNL4-3-deltaE-EGFP (NIH AIDS reagent program) and envelope containing plasmids, pSVIII-92HT593.1 (Dual tropic) (NIH AIDS reagent program) and pcDNA-3.1 -MN-0.3 (X4 tropic) (amino acids 1 -857 of accession ADI62634.1 ) are co-transfected in 293T-17 cells with lipofectamine 2000 as per the manufacturer instructions. An example of an amino acid sequence of an HIV envelope glycoprotein is SEQ ID NO: 45. Supernatants containing pseudoparticles were collected after 48 hours, spun, aliquoted and stored in -80 freezer until further use. Expression of [3-galactosidase upon infection by the pseudo particles in TZM-bL cells (A HELA derived cell line expressing human CD4, CCR5 and CXCR4 receptors - CD4 / CXCR4 / CCR5 / luciferase and [3-galactosidase) grown in DMEM complete medium (DCM) containing DMEM / 10% FBS / 1% PSG is measured via luminescence on Victor. Multiplicity of infection (MOI) of the pseudoparticles is measured in TZM-bl cells using [3-Galactosidase Reporter Gene Staining Kit containing a substrate X-Gal (5-bromo-4-chloro-3-indolyl [3-D- galactopyranoside) that results in staining of the infected cells indigo blue. The blue cells are counted under a microscope to determine the MOI.HIV Pseudo Particles Neutralization Assay

[0256] The HIV neutralization assay consists of blocking single stage infection of TZM-bl cells, a HELA derived cell line expressing human CD4, CCR5 and CXCR4 receptors. Infection of TZM-bl cells with HIV pseudo virus particles results in TAT induced luciferase expression. Neutralization of HIV pseudo particles results in a reduction of luciferase signal. Because TZM- bl cells express both the co-receptors for HIV, neutralization of HIV strains with different tropisms (CCR5, CXCR4, and dual tropic) can be evaluated.

[0257] To measure the neutralization potency of the anti-CXCR4 antibodies, TZM-bl cells are detached with 0.02% EDTA, neutralized with 5 mL of DCM, spun at 1500 rpm for 5 minutes to remove 0.02% EDTA and re-suspended in fresh DCM at 4x105cells / mL. A total of 20,000 TZM- bl cells are incubated with anti-CXCR4 antibodies serially diluted at 1 :3 from 1333.3 nM to 0.008 nM (upon adding the cells, 2X antibody dilutions become 1 X) and incubated for 45 minutes at 37°C in 5% CO2 incubator. Virus dilutions at 4.3x105 / mL of pseudo particles are made in DCM containing 15 pg / mL of Dextran, are added to 96 well plate followed by the addition of antibody. The cell mixtures are then incubated for 48-72 hours at 37°C in 5% CO2 incubator. After 2-3 days of incubation, supernatant is aspirated and plates are processed using Galacto-Star™ [3- Galactosidase Reporter Gene Assay kit according to manufacturer’s instructions. Total infection is measured on Victor X3 in relative light units (RLLI). The results were analyzed using nonlinear regression (3 parameter logistics) with Prism 6 software (Graphpad) to obtain IC50 values.Results

[0258] Summarized in Table 14, anti-CXCR4 antibodies neutralize X4 HIV pseudovirus withIC50s ranging from 2.35E-10 to 5.65E-10 [M], Isotype control REGN496 showed no effect.Neutralization of both X4 tropic and dual tropic pseudo particles was tested to evaluate ability of antibodies to prevent infection of two pseudo particles with different infectious properties (X4 vs R5). Results are shown in Table 14.Table 14: ICso’s of anti-CXCR4 mAbs tested in HIV neutralization assay against HIV X4 tropic pseudo particles.Example 12: Host Bone Marrow Mobilization to Enhance Hematopoietic Stem Cell Transplantation (HSCT) EngraftmentOverview

[0259] The SDF-1 / CXCR4 axis regulates localization and homing of mature leukocytes and progenitors in bone marrow. This Example analyzes how the disclosed antibodies affect bone marrow cell mobilization in vivo.Methods

[0260] CXCR4 Humin mice were injected intravenously with 10 mg / kg REGN7664, REGN7663, or REGN1945 isotype control antibody. Peripheral blood leukocyte levels were measured at baseline prior to antibody administration or at indicated time points post-administration. For blood leukocyte measurements, 30 to 50 pL of blood were collected via tail nick into K2 EDTA BD microtainer collection tubes (Beckton Dickinson, NJ, US) and complete blood counts including 6-part white blood cell differential were determined on the GENESIS veterinary hematology system (Oxford Science, CT, US). For measurement of mobilized hematopoietic stem and progenitor cells, blood was collected via terminal cardiac puncture 2 hours post-final antibody dose, cleared of red blood cells by ACK lysis, and analyzed by flow cytometry.

[0261] To isolate bone marrow cells for analysis, lower limbs were dissected, cleaned of muscle and skin, and collected in RPMI-1640 medium (ThermoFisher Scientific, US) supplemented with 10% heat-inactivated FBS (ThermoFisher Scientific, US). Both pairs of femur and tibia were then crushed with mortar and pestle, rinsed with additional RPMI-1640 medium, filtered through 40 urn cell strainers, and lysed with ACK lysing buffer (Quality Biological, MD, US).

[0262] For flow cytometry analysis, RBC-cleared single cells suspensions were suspended in PBS and stained with LIVE / DEAD fixable aqua dead cell stain (ThermoFischer Scientific) to exclude viable cells. Fc receptors were blocked using purified anti-mouse CD16 / 32 (clone 2.4G2). Cells were stained with mixtures of flurochrome-conjugated antibodies against cell surface markers, suspended in PBS containing 5% FBS. Cells were washed in PBS / FBS to remove excess antibodies then fixed in a buffered solution of 1% paraformaldehyde prior to acquisition. Flow cytometry was performed on BD FACSymphony A3 Cell Analyzer (Beckton Dickinson), data were analyzed using FlowJo software (BD Biosciences) and plotted using GraphPad Prisma (GraphPad Software, Inc.).Results

[0263] A study to examine the effects of CXCR4 blockade on leukocyte mobilization in CXCR4 Humin mice is schematized in Figure 9A. As shown in Figure 9B, a single 10 mg / kg dose of anti-CXCR4 antibodies with signaling blocking function (REGN7663 and REGN7664) led to mobilization of BM-resident leukocytes 2 and 6 hours post-administration, as compared to preadministration baseline (0 hour) and isotype control antibody (REGN1945). Significant elevation was observed for total white blood cells and major subsets, but not red blood cells.

[0264] A study to examine the effects of CXCR4 blockade on HSC / HSPC mobilization in CXCR4 Humin mice via repeated doses of CXCR4 blocking antibodies is schematized in 10A. As shown in Figure 10B, REGN7664 but not REGN7663 treatment led to a detectable elevation of rare lineage-negative cKit+ (LK) and lineage-negative Sca1 + cKit+ (LSK) hematopoietic progenitor cells in peripheral blood, as compared to isotype (REGN1945) treatment.

[0265] A study to examine the effects on CXCR4 blockade on BM B cells in CXCR4 Humin via single (black arrowhead) and repeated (arrows) doses of CXCR4 blocking antibodies is schematized in Figure 11 A. As shown in representative flow plots in Figure 11 B, single and repeated doses of REGN7664 caused profound reduction of mature B cells (bolded rectangular gate) in bone marrow as compared to isotype (REGN1945). As summarized in Figure 11 C, the observed decrease in mature B cells was significant and consistent both for REGN7663 and REGN7664 treatment groups, and was coupled with an increased proportion of early pre-B cells. A modest decrease in transitional B cells and increase in pro-B cells was also observed, only with five repeated doses with REGN7664.

[0266] In conclusion, anti-CXCR4 antibody or antigen-binding fragment thereof can displace leukocytes and their progenitors from their resident niches in the bone marrow (BM) could facilitate the engraftment of donor bone marrow cells in a transplant context.Exemplary Embodiments

[0267] Item 1 . An antibody or antigen binding fragment thereof that binds specifically to an epitope of human C-X-C Motif Chemokine Receptor 4 (CXCR4), wherein the epitope comprises amino acid residues M24, E26, P27, C28, F29, R30, S178, E179, A180, D181 , D182, 1185, D187, F189, Y190, N192, and D193.

[0268] Item 2. The antibody or antigen binding fragment thereof of item 1 that binds to an epitope of human CXCR4 comprising amino acid residues M24, K25, E26, P27, C28, F29, R30, E32, A34, S178, E179, A180, D181 , D182, R183, 1185, D187, R188, F189, Y190, N192, D193, and V196.

[0269] Item 3. The antibody or antigen binding fragment thereof of item 1 that binds to an epitope of human CXCR4 comprising amino acid residues M24, E26, P27, C28, F29, R30, N176, S178, E179, A180, D181 , D182, 1185, D187, F189, Y190, N192, and D193, C274, and E275.

[0270] Item 4. The antibody or antigen binding fragment thereof of item 2, wherein the antibody or antigen-binding fragment thereof comprises three heavy chain complementarity determining regions (CDRs) (HCDR1 , HCDR2 and HCDR3) contained within a heavy chain variable region (HCVR) comprising the amino acid sequence of SEQ ID NO: 2; and three light chain CDRs (LCDR1 , LCDR2 and LCDR3) contained within a light chain variable region (LCVR) comprising the amino acid sequence of SEQ ID NO: 10.

[0271] Item 5. The antibody or antigen binding fragment thereof of item 3, wherein the antibody or antigen-binding fragment thereof comprises three heavy chain complementarity determining regions (CDRs) (HCDR1 , HCDR2 and HCDR3) contained within a heavy chain variable region (HCVR) comprising the amino acid sequence of SEQ ID NO: 18; and three light chain CDRs (LCDR1 , LCDR2 and LCDR3) contained within a light chain variable region (LCVR) comprising the amino acid sequence of SEQ ID NO: 26.

[0272] Item 6. The antibody or antigen-binding fragment of item 4, wherein HCDR1 -HCDR2- HCDR3-LCDR1 -LCDR2-LCDR3 comprise the amino acid sequences of SEQ ID NOs: 4-6-8-12- 14-16, respectively.

[0273] Item 7. The antibody or antigen-binding fragment of item 5, wherein HCDR1 -HCDR2- HCDR3-LCDR1 -LCDR2-LCDR3 comprise the amino acid sequences of SEQ ID NOs: 20-22-24- 28-30-32, respectively.

[0274] Item 8. An antibody or antigen binding fragment thereof that binds specifically to C-X-C Motif Chemokine Receptor 4 (CXCR4), wherein the antibody or antigen-binding fragmentcomprises three heavy chain complementarity determining regions (CDRs) and three light chain CDRs contained within a heavy chain variable region (HCVR) and light chain variable region (LCVR) amino acid sequence pair selected from the group consisting of SEQ ID NOs: 2 / 10 and 18 / 26.

[0275] Item 9. The antibody or antigen-binding fragment of item 8, wherein the antibody or antigen-binding fragment comprises HCDR1 -HCDR2-HCDR3-LCDR1 -LCDR2-LCDR3 domains comprising the amino acid sequences, respectively, selected from the group consisting of SEQ ID NOs: 4-6-8-12-14-16 and 20-22-24-28-30-32.

[0276] Item 10. The antibody or antigen-binding fragment of item 9, wherein the antibody or antigen-binding fragment comprises a HCVR / LCVR amino acid sequence pair selected from the group consisting of: SEQ ID NOs: 2 / 10 and 18 / 26.

[0277] Item 1 1 . The antibody of any one of items 8 - 10, wherein the antibody comprises a heavy chain (HC) and a light chain (LC) forming a HC / LC pair, wherein the HC / LC pair comprise the amino acid sequences selected from the group consisting of SEQ ID NOs: 34 / 36 and 38 / 40.

[0278] Item 12. A pair of polynucleotide molecules encoding a heavy chain variable region (HCVR) and a light chain variable region (LCVR) of an antibody or antigen-binding fragment thereof that binds specifically to C-X-C Motif Chemokine Receptor 4 (CXCR4), wherein the pair of polynucleotide molecules encode, respectively, a HCVR and a LCVR comprising six complementarity determining regions (CDRs) of a HCVR / LCVR amino acid sequence pair selected from the group consisting of SEQ ID NOs: 2 / 10 and 18 / 26.

[0279] Item 13. The pair of polynucleotide molecules of item 12, wherein the six CDRs, HCDR1-HCDR2-HCDR3-LCDR1-LCDR2-LCDR3, comprise the amino acid sequences, respectively, selected from the group consisting of SEQ ID NOs: 4-6-8-12-14-16 and 20-22-24- 28-30-32.

[0280] Item 14. A pair of polynucleotide molecules encoding a heavy chain variable region (HCVR) and a light chain variable region (LCVR) of an antibody or antigen-binding fragment thereof that binds specifically to C-X-C Motif Chemokine Receptor 4 (CXCR4), wherein the pair of polynucleotide molecules encode, respectively, a HCVR / LCVR amino acid sequence pair selected from the group consisting of SEQ ID NOs: 2 / 10 and 18 / 26.

[0281] Item 15. A pair of polynucleotide molecules encoding a heavy chain (HC) and a light chain (LC) of an antibody that binds specifically to C-X-C Motif Chemokine Receptor 4 (CXCR4), wherein the pair of polynucleotide molecules encode, respectively, a HC / LC amino acid sequence pair selected from the group consisting of SEQ ID NOs: 34 / 36 and 38 / 40.

[0282] Item 16. The pair of polynucleotide molecules of item 15, wherein the pair of polynucleotides encoding the HC / LC amino acid sequence pair comprise polynucleotide sequences selected from the group consisting of SEQ ID NOs: 33 / 35 and 37 / 39.

[0283] Item 17. The pair of polynucleotide molecules of any one of items 12-16, further encoding an immunoglobulin heavy chain constant region of the antibody.

[0284] Item 18. The pair of polynucleotide molecules of any one of items 12-16, further encoding an immunoglobulin light chain constant region of the antibody.

[0285] Item 19. A recombinant expression vector comprising the pair of polynucleotide molecules of any one of items 12-16.

[0286] Item 20. A recombinant expression vector comprising a polynucleotide molecule encoding for a heavy chain variable region (HCVR) comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 2 and 18.

[0287] Item 21. A recombinant expression vector comprising a polynucleotide molecule encoding for a light chain variable region (LCVR) comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 10 and 26.

[0288] Item 22. A recombinant expression vector comprising a polynucleotide molecule encoding for a heavy chain (HC) comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 34 and 36.

[0289] Item 23. A recombinant expression vector comprising a polynucleotides molecule encoding for a light chain (LC) comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 36 and 40.

[0290] Item 24. An isolated host cell comprising the antibody or antigen binding fragment thereof of any one of items 1 -1 1 , the pair of polynucleotide molecules of any one of items 12-18, or the vector of any one of items 19-23, or any combination thereof.

[0291] Item 25. A pair of polynucleotide molecules encoding a heavy chain variable region (HCVR) and a light chain variable region (LCVR), respectively, of an antibody or antigen-binding fragment thereof that binds specifically to C-X-C Motif Chemokine Receptor 4 (CXCR4), wherein: (a) the polynucleotide molecule encoding the HCVR comprises three polynucleotide molecules encoding three heavy chain complementarity determining regions (HCDR1 , HCDR2 and HCDR3) within the HCVR polynucleotide sequence, wherein the HCDR1 polynucleotide sequence comprises a nucleic acid sequence encoding the amino acid sequence of SEQ ID NO: 4, the HCDR2 polynucleotide sequence comprises a nucleic acid sequence encoding the amino acid sequence of SEQ ID NO: 6, and the HCDR3 polynucleotide sequence comprises a nucleic acid sequence encoding the amino acid sequence of SEQ ID NO: 8, and (b) thepolynucleotide molecule encoding the LCVR comprises three polynucleotide molecules encoding three light chain complementarity determining regions (LCDR1 , LCDR2 and LCDR3) within the LCVR polynucleotide sequence, wherein the LCDR1 polynucleotide sequence comprises a nucleic acid sequence encoding the amino acid sequence of SEQ ID NO: 12, the LCDR2 polynucleotide sequence comprises a nucleic acid sequence encoding the amino acid sequence of SEQ ID NO: 14, and the LCDR3 polynucleotide sequence comprises a nucleic acid sequence encoding the amino acid sequence of SEQ ID NO: 16.

[0292] Item 26. The pair of polynucleotide molecules of item 25, wherein the polynucleotide molecule encoding the HCVR comprises a nucleic acid sequence encoding the amino acid sequence of SEQ ID NO: 2.

[0293] Item 27. The pair of polynucleotide molecules of item 25, wherein the polynucleotide molecule encoding the HCVR comprises the HCDR1 nucleic acid sequence set forth in SEQ ID NO: 3, the HCDR2 nucleic acid sequence set forth in SEQ ID NO: 5, and the HCDR3 nucleic acid sequence set forth in SEQ ID NO: 7.

[0294] Item 28. The pair of polynucleotide molecules of item 25, wherein the polynucleotide molecule encoding the HCVR comprises the nucleic acid sequence of SEQ ID NO: 1 .

[0295] Item 29. The pair of polynucleotide molecules of item 25, wherein the polynucleotide molecule encoding the HCVR further encodes an immunoglobulin heavy chain constant region of the antibody.

[0296] Item 30. The pair of polynucleotide molecules of item 25, wherein the immunoglobulin heavy chain constant region is a human IgG 1 constant region.

[0297] Item 31. The pair of polynucleotide molecules of item 25, wherein the LCVR comprises the amino acid sequence of SEQ ID NO: 10.

[0298] Item 32. The pair of polynucleotide molecules of item 25, wherein the polynucleotide molecule encoding the LCVR comprises the LCDR1 nucleic acid sequence set forth in SEQ ID NO: 11 , the LCDR2 nucleic acid sequence set forth in SEQ ID NO: 13, and the LCDR3 nucleic acid sequence set forth in SEQ ID NO: 15.

[0299] Item 33. The pair of polynucleotide molecules of item 25, wherein the polynucleotide molecule encoding the LCVR comprises the nucleic acid sequence of SEQ ID NO: 9.

[0300] Item 34. The pair of polynucleotide molecules of item 25, wherein the polynucleotide molecule encoding the LCVR further encodes an immunoglobulin light chain constant region of the antibody.

[0301] Item 35. The pair of polynucleotide molecules of item 34, wherein the immunoglobulin light chain constant region is a human kappa constant region.

[0302] Item 36. A pair of polynucleotide molecules encoding a heavy chain variable region (HCVR) and a light chain variable region (LCVR), respectively, of an antibody or antigen-binding fragment thereof that binds specifically to C-X-C Motif Chemokine Receptor 4 (CXCR4), wherein: (a) the HCVR comprises three heavy chain complementarity determining regions (HCDR1 , HCDR2 and HCDR3), wherein the HCDR1 comprises the amino acid sequence of SEQ ID NO: 20, the HCDR2 comprises the amino acid sequence of SEQ ID NO: 22, and the HCDR3 comprises the amino acid sequence of SEQ ID NO: 24, and (b) the LCVR comprises three light chain complementarity determining regions (LCDR1 , LCDR2 and LCDR3), wherein the LCDR1 comprises the amino acid sequence of SEQ ID NO: 28, the LCDR2 comprises the amino acid sequence of SEQ ID NO: 30, and the LCDR3 comprises the amino acid sequence of SEQ ID NO: 32.

[0303] Item 37. The pair of polynucleotide molecules of item 36, wherein the HCVR comprises the amino acid sequence of SEQ ID NO: 18.

[0304] Item 38. The pair of polynucleotide molecules of item 36, wherein the polynucleotide molecule encoding the HCVR comprises the HCDR1 nucleic acid sequence set forth in SEQ ID NO: 19, the HCDR2 nucleic acid sequence set forth in SEQ ID NO: 21 , and the HCDR3 nucleic acid sequence set forth in SEQ ID NO: 23.

[0305] Item 39. The pair of polynucleotide molecules of item 36, wherein the polynucleotide molecule encoding the HCVR comprises the nucleic acid sequence of SEQ ID NO: 17.

[0306] Item 40. The pair of polynucleotide molecules of item 36, wherein the polynucleotide molecule encoding the HCVR further encodes an immunoglobulin heavy chain constant region of the antibody.

[0307] Item 41. The pair of polynucleotide molecules of item 40, wherein the immunoglobulin heavy chain constant region is a human IgG 1 constant region.

[0308] Item 42. The pair of polynucleotide molecules of item 36, wherein the LCVR comprises the amino acid sequence of SEQ ID NO: 26.

[0309] Item 43. The pair of polynucleotide molecules of item 36, wherein the polynucleotide molecule encoding the LCVR comprises the LCDR1 nucleic acid sequence set forth in SEQ ID NO: 27, the LCDR2 nucleic acid sequence set forth in SEQ ID NO: 29, and the LCDR3 nucleic acid sequence set forth in SEQ ID NO: 31 .

[0310] Item 44. The pair of polynucleotide molecules of item 36, wherein the polynucleotide molecule encoding the LCVR comprises the nucleic acid sequence of SEQ ID NO: 25.

[0311] Item 45. The pair of polynucleotide molecules of item 36, wherein the polynucleotide molecule encoding the LCVR further encodes an immunoglobulin light chain constant region of the antibody.

[0312] Item 46. The pair of polynucleotide molecules of item 45, wherein the immunoglobulin light chain constant region is a human kappa constant region.

[0313] Item 47. A method of producing an anti-CXCR4 antibody or antigen-binding fragment thereof by culturing the isolated host cell of item 24 under conditions permitting production of the antibody or antigen-binding fragment, and recovering the antibody or antigen-binding fragment so produced.

[0314] Item 48. The method of item 47, wherein the isolated host cell is a Chinese hamster ovary (CHO) cell.

[0315] Item 49. A method to enhance hematopoietic stem cell engraftment, comprising administering to a subject in need thereof an antibody or antigen binding fragment thereof of any one of items 1 -11.

[0316] Item 50. A method of treating cancer, comprising administering to a subject in need thereof an antibody or antigen binding fragment thereof of any one of items 1 -1 1.

[0317] Item 51. The method of item 50, wherein the cancer is a leukemia or a lymphoma.

[0318] Item 52. A method of altering B-cell localization in the bone marrow or peripheral blood supply, comprising administering to a subject in need thereof an antibody or antigen binding fragment thereof of any one of items 1-11.

[0319] Item 53. A method of hematopoietic stem cell engraftment host conditioning, comprising administering to a subject in need thereof an antibody or antigen binding fragment thereof or any one of items 1 -11.

[0320] Item 54. A method of inducing cancer cell apoptosis, comprising administering to a subject in need thereof an antibody or antigen binding fragment thereof of any one of items 1 - 11.

[0321] Item 55. A method of preventing HIV infection, comprising administering to a subject in need thereof an antibody or antigen binding fragment thereof of any one of items 1 -11.

[0322] The present disclosure is not to be limited in scope by the specific embodiments described herein. Indeed, various modifications of the disclosure in addition to those described herein will become apparent to those skilled in the art from the foregoing description. Such modifications are intended to fall within the scope of the appended claims.

[0323] SequencesSEQ ID NO: 1.CAGGTTCAGCTGGTGCAGTCTGGAGCTGAGGTGAAGAAGCCTGGGGCCTCAGTGAAGGTCTCCTGCAAGGCTTCTGGTTACACCTTTACCAGCTATGGTATCAGTTGGGTGCGACAGGCCCCTGGACAAGGGATTGAGTGGATGGGATGGATCAGCACTTACAATGGAAACAGAAACTATGCACAGAAGGTCCAGGGCAGAGTCACCATGACCACAGACAGATCCACGAGCACAGCCTACATGGACCTGAGGAGTCTGAGATC TGACGACACGGCCGTGTATTACTGTGCGAGACACGGTATAACTGGAGCTAGGAATTACTACTACCAC TACGGTATGGACGTCTGGGGCCAAGGGACCACGGTCACCGTCTCCTCA;SEQ ID NO: 2.QVQLVQSGAEVKKPGASVKVSCKASGYTFTSYGISWVRQAPGQGIEWMGWISTYNGNRNYAQKVQGRVTMTTDRSTSTAYMDLRSLRSDDTAVYYCARHGITGARNYYYHYGMDVWGQGTTVTVSS;SEQ ID NO: 3.GGT TAC ACC TTT ACC AGC TAT GGT;SEQ ID NO: 4.G Y T F T S Y G;SEQ ID NO: 5.ATC AGC ACT TAC AAT GGA AAC AGA;SEQ ID NO: 6.I S T Y N G N R;SEQ ID NO: 7.GCG AGA CAC GGT ATA ACT GGA GCT AGG AAT TAC TAC TAC CAC TAC GGT ATG GAC GTC;SEQ ID NO: 8.A R H G I T G A R N Y Y Y H Y G M D V;SEQ ID NO: 9.GATGTTGTGATGACTCAGTCTCCACTCTCCCTGCCCGTCACCCTTGGACAGCCGGCCTCCATCTCCTGCAGGTCTAGTCAAAGCCTCGTATACACTGATGGAAACACCTACTTGAATTGGTTTCAGCAGAGGCCAGGCCAATCTCCAAGGCGCCTAATTTATAAGGTTTCTAACCGGGACTCTGGGGTCCCAGACAGATTCAGCGGCAGTGGGTCAGGCACTGATTTCACACTGAAAATCAGCAGGGTGGAGGCTGAGGATGTTGGG TTTTATTACTGCATGCAAAATACACACTGGCCGCTCACTTTCGGCGGAGGGACCAAGGTGGAGATCA AA;SEQ ID NO: 10.DVVMTQSPLSLPVTLGQPASISCRSSQSLVYTDGNTYLNWFQQRPGQSPRRLIYKVSNRDSGVPDRFSG SGSGTDFTLKISRVEAEDVGFYYCMQNTHWPLTFGGGTKVEIK;SEQ ID NO: 11.CAA AGC CTC GTA TAC ACT GAT GGA AAC ACC TAC;SEQ ID NO: 12.Q S L V Y T D G N T Y;SEQ ID NO: 13.AAG GTT TCT;SEQ ID NO: 14.K V S;SEQ ID NO: 15.ATG CAA AAT AC A CAC TGG CCG CTC ACT;SEQ ID NO: 16.M Q N T H W P L T;SEQ ID NO: 17.CAGGTGCAGCTGGTGGAGTCTGGGGGAGGCGTGGTCCAGCCTGGGAGGTCCCTGAGACTCTCCTGTGCAGCGTCTGGATTCACCTTCAGTTATTATGGCATGCACTGGGTCCGCCAGGCTCCAGGCAAGGGGCTGGAGTGGGTGGCATTTTTACGGTATGATGGAAATAATAAATATTATTCAGACTCCGTGAAGGGCCGATTCACCATGTCCAGAGACAATTCCAAGAACACGCTGTATCTGCAAATGAACAGCCTGAGAGCCGATGACACGGCTGTGTATTACTGTGCGAGAGGGGCCCTCTCACGTGAATATAGTTATGGTCTTGACTACTGGGGCCAGGGAACCCTGGTCACCGTCTCCTCA;SEQ ID NO: 18.QVQLVESGGGVVQPGRSLRLSCAASGFTFSYYGMHWVRQAPGKGLEWVAFLRYDGNNKYYSDSVKGRFTMSRDNSKNTLYLQMNSLRADDTAVYYCARGALSREYSYGLDYWGQGTLVTVSS;SEQ ID NO: 19.GGA TTC ACC TTC AGT TAT TAT GGC;SEQ ID NO: 20.G F T F S Y Y G;SEQ ID NO: 21.TTA CGG TAT GAT GGA AAT AAT AAA;SEQ ID NO: 22.L R Y D G N N K;SEQ ID NO: 23.GCG AGA GGG GCC CTC TCA CGT GAA TAT AGT TAT GGT CTT GAC TAG;SEQ ID NO: 24.A R G A L S R E Y S Y G L D Y;SEQ ID NO: 25.GACATCCAGATGACCCAGTCTCCATCATCCCTGTCTGCATCTGTAGGAGACAGAGTCACCATCACTTGCCGGGCAAGTCAGGGCATTAGAAATGATTTAGGCTGGTTTCAGCAGAAACCAGGGAAAGCCCCTAAGCGCCTGATCTATGCTGCATCCAGTTTGCAAAGTGGGGTCCCATCAAGGTTCAGCGGCAGTGGATCTGGGACAGATTTCACTCTCACAATCAGCAGCCTGCAGCCTGAAGATTTTGCAACTTACTACTGTCTACAGCATAATACTTACCCTCTCACTTTCGGCGGAGGGACCAAGGTGGAGATCAGA;SEQ ID NO: 26.DIQMTQSPSSLSASVGDRVTITCRASQGIRNDLGWFQQKPGKAPKRLIYAASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCLQHNTYPLTFGGGTKVEIR;SEQ ID NO: 27.CAG GGC ATT AGA AAT GAT;SEQ ID NO: 28.Q G I R N D;SEQ ID NO: 29.GCTGCA TCC;SEQ ID NO: 30.A A S;SEQ ID NO: 31.CTA GAG CAT AAT ACT TAG CCT CTC ACT;SEQ ID NO: 32.L Q H N T Y P L T;SEQ ID NO: 33.CAGGTTCAGCTGGTGCAGTCTGGAGCTGAGGTGAAGAAGCCTGGGGCCTCAGTGAAGGTCTCCTGCAAGGCTTCTGGTTACACCTTTACCAGCTATGGTATCAGTTGGGTGCGACAGGCCCCTGGACAAGGGATTGAGTGGATGGGATGGATCAGCACTTACAATGGAAACAGAAACTATGCACAGAAGGTCCAGGGCAGAGTCACCATGACCACAGACAGATCCACGAGCACAGCCTACATGGACCTGAGGAGTCTGAGATCTGACGACACGGCCGTGTATTACTGTGCGAGACACGGTATAACTGGAGCTAGGAATTACTACTACCACTACGGTATGGACGTCTGGGGCCAAGGGACCACGGTCACCGTCTCCTCAGCCTCCACCAAGGGCCCATCGGTCTTCCCCCTGGCGCCCTGCTCCAGGAGCACCTCCGAGAGCACAGCCGCCCTGGGCTGCCTGGTCAAGGACTACTTCCCCGAACCGGTGACGGTGTCGTGGAACTCAGGCGCCCTGACCAGCGGCGTGCACACCTTCCCGGCTGTCCTACAGTCCTCAGGACTCTACTCCCTCAGCAGCGTGGTGACCGTGCCCTCCAGCAGCTTGGGCACGAAGACCTACACCTGCAACGTAGATCACAAGCCCAGCAACACCAAGGTGGACAAGAGAGTTGAGTCCAAATATGGTCCCCCATGCCCACCGTGCCCAGCACCACCTGTGGCAGGACCATCAGTCTTCCTGTTCCCCCCAAAACCCAAGGACACTCTCATGATCTCCCGGACCCCTGAGGTCACGTGCGTGGTGGTGGACGTGAGCCAGGAAGACCCCGAGGTCCAGTTCAACTGGTACGTGGATGGCGTGGAGGTGCATAATGCCAAGACAAAGCCGCGGGAGGAGCAGTTCAACAGCACGTACCGTGTGGTCAGCGTCCTCACCGTCCTGCACCAGGACTGGCTGAACGGCAAGGAGTACAAGTGCAAGGTCTCCAACAAAGGCCTCCCGTCCTCCATCGAGAAAACCATCTCCAAAGCCAAAGGGCAGCCCCGAGAGCCACAGGTGTACACCCTGCCCCCATCCCAGGAGGAGATGACCAAGAACCAGGTCAGCCTGACCTGCCTGGTCAAAGGCTTCTACCCCAGCGACATCGCCGTGGAGTGGGAGAGCAATGGGCAGCCGGAGAACAACTACAAGACCACGCCTCCCGTGCTGGACTCCGACGGCTCCTTCTTCCTCTACAGCAGGCTCACCGTGGACAAGAGCAGGTGGCAGGAGGGGAATGTCTTCTCATGCTCCGTGATGCATGAGGCTCTGCACAACCACTACACACAGAAGTCCCTCTCCCTGTCTCTGGGTAAATGA;SEQ ID NO: 34.QVQLVQSGAEVKKPGASVKVSCKASGYTFTSYGISWVRQAPGQGIEWMGWISTYNGNRNYAQKVQGRVTMTTDRSTSTAYMDLRSLRSDDTAVYYCARHGITGARNYYYHYGMDVWGQGTTVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPPCPAPPVAGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK;SEQ ID NO: 35.GATGTTGTGATGACTCAGTCTCCACTCTCCCTGCCCGTCACCCTTGGACAGCCGGCCTCCATCTCCTGCAGGTCTAGTCAAAGCCTCGTATACACTGATGGAAACACCTACTTGAATTGGTTTCAGCAGAGGCCAGGCCAATCTCCAAGGCGCCTAATTTATAAGGTTTCTAACCGGGACTCTGGGGTCCCAGACAGATTCAGCGGCAGTGGGTCAGGCACTGATTTCACACTGAAAATCAGCAGGGTGGAGGCTGAGGATGTTGGGTTTTATTACTGCATGCAAAATACACACTGGCCGCTCACTTTCGGCGGAGGGACCAAGGTGGAGATCAAACGAACTGTGGCTGCACCATCTGTCTTCATCTTCCCGCCATCTGATGAGCAGTTGAAATCTGGAACTGCCTCTGTTGTGTGCCTGCTGAATAACTTCTATCCCAGAGAGGCCAAAGTACAGTGGAAGGTGGATAACGCCCTCCAATCGGGTAACTCCCAGGAGAGTGTCACAGAGCAGGACAGCAAGGACAGCACCTACAGCCTCAGCAGCACCCTGACGCTGAGCAAAGCAGACTACGAGAAACACAAAGTCTACGCCTGCGAAGTCACCCATCAGGGCCTGAGCTCGCCCGTCACAAAGAGCTTCAACAGGGGAGAGTGTTAG;SEQ ID NO: 36.DVVMTQSPLSLPVTLGQPASISCRSSQSLVYTDGNTYLNWFQQRPGQSPRRLIYKVSNRDSGVPDRFSGSGSGTDFTLKISRVEAEDVGFYYCMQNTHWPLTFGGGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGECSEQ ID NO: 37.CAGGTGCAGCTGGTGGAGTCTGGGGGAGGCGTGGTCCAGCCTGGGAGGTCCCTGAGACTCTCCTGTGCAGCGTCTGGATTCACCTTCAGTTATTATGGCATGCACTGGGTCCGCCAGGCTCCAGGCAAGGGGCTGGAGTGGGTGGCATTTTTACGGTATGATGGAAATAATAAATATTATTCAGACTCCGTGAAGGGCCGATTCACCATGTCCAGAGACAATTCCAAGAACACGCTGTATCTGCAAATGAACAGCCTGAGAGCCGATGACACGGCTGTGTATTACTGTGCGAGAGGGGCCCTCTCACGTGAATATAGTTATGGTCTTGACTACTGGGGCCAGGGAACCCTGGTCACCGTCTCCTCAGCCTCCACCAAGGGCCCATCGGTCTTCCCCCTGGCGCCCTGCTCCAGGAGCACCTCCGAGAGCACAGCCGCCCTGGGCTGCCTGGTCAAGGACTACTTCCCCGAACCGGTGACGGTGTCGTGGAACTCAGGCGCCCTGACCAGCGGCGTGCACACCTTCCCGGCTGTCCTACAGTCCTCAGGACTCTACTCCCTCAGCAGCGTGGTGACCGTGCCCTCCAGCAGCTTGGGCACGAAGACCTACACCTGCAACGTAGATCACAAGCCCAGCAACACCAAGGTGGACAAGAGAGTTGAGTCCAAATATGGTCCCCCATGCCCACCGTGCCCAGCACCACCTGTGGCAGGACCATCAGTCTTCCTGTTCCCCCCAAAACCCAAGGACACTCTCATGATCTCCCGGACCCCTGAGGTCACGTGCGTGGTGGTGGACGTGAGCCAGGAAGACCCCGAGGTCCAGTTCAACTGGTACGTGGATGGCGTGGAGGTGCATAATGCCAAGACAAAGCCGCGGGAGGAGCAGTTCAACAGCACGTACCGTGTGGTCAGCGTCCTCACCGTCCTGCACCAGGACTGGCTGAACGGCAAGGAGTACAAGTGCAAGGTCTCCAACAAAGGCCTCCCGTCCTCCATCGAGAAAACCATCTCCAAAGCCAAAGGGCAGCCCCGAGAGCCACAGGTGTACACCCTGCCCCCATCCCAGGAGGAGATGACCAAGAACCAGGTCAGCCTGACCTGCCTGGTCAAAGGCTTCTACCCCAGCGACATCGCCGTGGAGTGGGAGAGCAATGGGCAGCCGGAGAACAACTACAAGACCACGCCTCCCGTGCTGGACTCCGACGGCTCCTTCTTCCTCTACAGCAGGCTCACCGTGGACAAGAGCAGGT GGCAGGAGGGGAATGTCTTCTCATGCTCCGTGATGCATGAGGCTCTGCACAACCACTACACACAGA AGTCCCTCTCCCTGTCTCTGGGTAAATGA;SEQ ID NO: 38.QVQLVESGGGVVQPGRSLRLSCAASGFTFSYYGMHWVRQAPGKGLEWVAFLRYDGNNKYYSDSVKGRFTMSRDNSKNTLYLQMNSLRADDTAVYYCARGALSREYSYGLDYWGQGTLVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPPCPAPPVAGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQ PREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLT VDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK;SEQ ID NO: 39.GACATCCAGATGACCCAGTCTCCATCATCCCTGTCTGCATCTGTAGGAGACAGAGTCACCATCACTTGCCGGGCAAGTCAGGGCATTAGAAATGATTTAGGCTGGTTTCAGCAGAAACCAGGGAAAGCCCCTAAGCGCCTGATCTATGCTGCATCCAGTTTGCAAAGTGGGGTCCCATCAAGGTTCAGCGGCAGTGGATCTGGGACAGATTTCACTCTCACAATCAGCAGCCTGCAGCCTGAAGATTTTGCAACTTACTACTGTCTACAGCATAATACTTACCCTCTCACTTTCGGCGGAGGGACCAAGGTGGAGATCAGACGAACTGTGGCTGCACCATCTGTCTTCATCTTCCCGCCATCTGATGAGCAGTTGAAATCTGGAACTGCCTCTGTTGTGTGCCTGCTGAATAACTTCTATCCCAGAGAGGCCAAAGTACAGTGGAAGGTGGATAACGCCCTCCAATCGGGTAACTCCCAGGAGAGTGTCACAGAGCAGGACAGCAAGGACAGCACCTACAGCCTCAGCAGCACCCTGACGCTGAGCAAAGCAGACTACGAGAAACACAAAGTCTACGCCTGCGAAGTCACCCATCAGG GCCTGAGCTCGCCCGTCACAAAGAGCTTCAACAGGGGAGAGTGTTAG;SEQ ID NO: 40.DIQMTQSPSSLSASVGDRVTITCRASQGIRNDLGWFQQKPGKAPKRLIYAASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCLQHNTYPLTFGGGTKVEIRRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFN RGEC;SEQ ID NO: 41.CPSCPAPEFLG;SEQ ID NO: 42.CPPCPAPPVA;SEQ ID NO: 43.MEGISIYTSDNYTEEMGSGDYDSMKEPCFREENANFNKIFLPTIYSIIFLTGIVGNGLVILVMGYQKKLRSM TDKYRLHLSVADLLFVITLPFWAVDAVANWYFGNFLCKAVHVIYTVNLYSSVLILAFISLDRYLAIVHATNSQ RPRKLLAEKVVYVGVWIPALLLTIPDFIFANVSEADDRYICDRFYPNDLWVVVFQFQHIMVGLILPGIVILSC YCIIISKLSHSKGHQKRKALKTTVILILAFFACWLPYYIGISIDSFILLEIIKQGCEFENTVHKWISITEALAFFHC CLNPILYAFLGAKFKTSAQHALTSVSRGSSLKILSKGKRGGHSSVSTESESSSFHSS;SEQ ID NO: 44.MEGISIYTSDNYTEEMGSGDYDSIKEPCFREENAHFNRIFLPTIYSIIFLTGIVGNGLVILVMGYQKKLRSMT DKYRLHLSVADLLFVITLPFWAVDAVANWYFGNFLCKAVHVIYTVNLYSSVLILAFISLDRYLAIVHATNSQK PRKLLAEKVVYVGVWIPALLLTIPDFIFASVSEADDRYICDRFYPNDLWVVVFQFQHIMVGLILPGIVILSCY CIIISKLSHSKGHQKRKALKTTVILILAFFACWLPYYIGISIDSFILLEIIKQGCEFENTVHKWISITEALAFFHCC LNPILYAFLGAKFKTSAQHALTSVSRGSSLKILSKGKRGGHSSVSTESESSSFHSS;SEQ ID NO: 45.MRVKGIRRNYQHWWGWGTMLLGLLMICSATEKLWVTVYYGVPVWKEATTTLFCASDAKAYDTEVHNV WATHACVPTDPNPQEVELVNVTENFNMWKNNMVEQMHEDIISLWDQSLKPCVKLTPLCVTLNCTDLRNT TNTNNNTDNNNSKSEGTIKGGEMKNCSFNITTSIGDKMQKEYALLYKLDIVSIDNDSTSYRLISCNTSVITQ ACPKISFEPIPIHYCAPAGFAILKCNDKKFSGKGSCKNVSTVQCTHGIRPVVSTQLLLNGSLAEEEVVIRSE NFTDNAKTIIVHLNESVQINCTRPNYNKRKRIHIGPGRAFYTTKNIKGTIRQAHCNISRAKWNDTLRQIVSKL KEQFKNKTIVFNPSSGGDPEIVMHSFNCGGEFFYCNTSPLFNSTWNSNNTWNNTTGSNNNITLQCKIKQII NMWQKVGKAMYAPPIEGQIRCSSNITGLLLTRDGGEDTDTNDTEIFRPGGGDMRDNWRSELYKYKVVTI EPLGVAPTKAKRRVVQREKRAAIGALFLGFLGAAGSTMGAASVTLTVQARLLLSGIVQQQNNLLRAIEAQ QHMLQLTAWGIKQLQARVQAVERYLKDQQLLGFWGCSGKLICTTTVPWNASWSNKSLDDIWNNMTWM QWEREIDNYTSLIYSLLEKSQTQQEKNEQELLELDKWASLWNWFDITNWLWYIKIFIIIVGGLVGLRIIFAVL SIVNRVRQGYSPLSLQTRPPVPRGPDRPEGIEEEGGERDRDTSGRLVHGFLAIIWVDLRSLFLFSYHHLR DLLLIAARIVELLRRRGWEVLKYWWNLLQYWSQELKSSAVSLLNAAAIAVAEGTHRVIEVLQRAGRAILHIP TRIRQGLERALL

Claims

What is claimed is:1 . An antibody or antigen binding fragment thereof that binds specifically to an epitope of human C-X-C Motif Chemokine Receptor 4 (CXCR4), wherein the epitope comprises amino acid residues M24, E26, P27, C28, F29, R30, S178, E179, A180, D181 , D182, 1185, D187, F189, Y190, N192, and D193.

2. The antibody or antigen binding fragment thereof of claim 1 that binds to an epitope of human CXCR4 comprising amino acid residues M24, K25, E26, P27, C28, F29, R30, E32, A34, S178, E179, A180, D181 , D182, R183, 1185, D187, R188, F189, Y190, N192, D193, and V196.

3. The antibody or antigen binding fragment thereof of claim 1 that binds to an epitope of human CXCR4 comprising amino acid residues M24, E26, P27, C28, F29, R30, N176, S178, E179, A180, D181 , D182, 1185, D187, F189, Y190, N192, and D193, C274, and E275.

4. The antibody or antigen binding fragment thereof of claim 2, wherein the antibody or antigen-binding fragment thereof comprises three heavy chain complementarity determining regions (CDRs) (HCDR1 , HCDR2 and HCDR3) contained within a heavy chain variable region (HCVR) comprising the amino acid sequence of SEQ ID NO: 2; and three light chain CDRs (LCDR1 , LCDR2 and LCDR3) contained within a light chain variable region (LCVR) comprising the amino acid sequence of SEQ ID NO: 10.

5. The antibody or antigen binding fragment thereof of claim 3, wherein the antibody or antigen-binding fragment thereof comprises three heavy chain complementarity determining regions (CDRs) (HCDR1 , HCDR2 and HCDR3) contained within a heavy chain variable region (HCVR) comprising the amino acid sequence of SEQ ID NO: 18; and three light chain CDRs (LCDR1 , LCDR2 and LCDR3) contained within a light chain variable region (LCVR) comprising the amino acid sequence of SEQ ID NO: 26.

6. The antibody or antigen-binding fragment of claim 4, wherein HCDR1 - HCDR2-HCDR3-LCDR1 -LCDR2-LCDR3 comprise the amino acid sequences of SEQ ID NOs: 4-6-8-12-14-16, respectively.

7. The antibody or antigen-binding fragment of claim 5, wherein HCDR1 - HCDR2-HCDR3-LCDR1 -LCDR2-LCDR3 comprise the amino acid sequences of SEQ ID NOs: 20-22-24-28-30-32, respectively.

8. An antibody or antigen binding fragment thereof that binds specifically to C-X- C Motif Chemokine Receptor 4 (CXCR4), wherein the antibody or antigen-binding fragment comprises three heavy chain complementarity determining regions (CDRs) and three light chain CDRs contained within a heavy chain variable region (HCVR) and light chain variable region (LCVR) amino acid sequence pair selected from the group consisting of SEQ ID NOs: 2 / 10 and 18 / 269. The antibody or antigen-binding fragment of claim 8, wherein the antibody or antigen-binding fragment comprises HCDR1 -HCDR2-HCDR3-LCDR1 -LCDR2-LCDR3 domains comprising the amino acid sequences, respectively, selected from the group consisting of SEQ ID NOs: 4-6-8-12-14-16 and 20-22-24-28-30-32.

10. The antibody or antigen-binding fragment of claim 9, wherein the antibody or antigen-binding fragment comprises a HCVR / LCVR amino acid sequence pair selected from the group consisting of: SEQ ID NOs: 2 / 10 and 18 / 26.1 1 . The antibody of any one of claims 8 - 10, wherein the antibody comprises a heavy chain (HC) and a light chain (LC) forming a HC / LC pair, wherein the HC / LC pair comprise the amino acid sequences selected from the group consisting of SEQ ID NOs: 34 / 36 and 38 / 40.

12. A pair of polynucleotide molecules encoding a heavy chain variable region (HCVR) and a light chain variable region (LCVR) of an antibody or antigen-binding fragment thereof that binds specifically to C-X-C Motif Chemokine Receptor 4 (CXCR4), wherein the pair of polynucleotide molecules encode, respectively, a HCVR and a LCVR comprising six complementarity determining regions (CDRs) of a HCVR / LCVR amino acid sequence pair selected from the group consisting of SEQ ID NOs: 2 / 10 and 18 / 26.

13. The pair of polynucleotide molecules of claim 12, wherein the six CDRs, HCDR1 -HCDR2-HCDR3-LCDR1-LCDR2-LCDR3, comprise the amino acid sequences, respectively, selected from the group consisting of SEQ ID NOs: 4-6-8-12-14-16 and 20-22-24- 28-30-32.

14. A pair of polynucleotide molecules encoding a heavy chain variable region (HCVR) and a light chain variable region (LCVR) of an antibody or antigen-binding fragment thereof that binds specifically to C-X-C Motif Chemokine Receptor 4 (CXCR4), wherein the pair of polynucleotide molecules encode, respectively, a HCVR / LCVR amino acid sequence pair selected from the group consisting of SEQ ID NOs: 2 / 10 and 18 / 26.

15. A pair of polynucleotide molecules encoding a heavy chain (HC) and a light chain (LC) of an antibody that binds specifically to C-X-C Motif Chemokine Receptor 4 (CXCR4), wherein the pair of polynucleotide molecules encode, respectively, a HC / LC amino acid sequence pair selected from the group consisting of SEQ ID NOs: 34 / 36 and 38 / 40.

16. The pair of polynucleotide molecules of claim 15, wherein the pair of polynucleotides encoding the HC / LC amino acid sequence pair comprise polynucleotide sequences selected from the group consisting of SEQ ID NOs: 33 / 35 and 37 / 39.

17. The pair of polynucleotide molecules of any one of claims 12-16, further encoding an immunoglobulin heavy chain constant region of the antibody.

18. The pair of polynucleotide molecules of any one of claims 12-16, further encoding an immunoglobulin light chain constant region of the antibody.

19. A recombinant expression vector comprising the pair of polynucleotide molecules of any one of claims 12-16.

20. A recombinant expression vector comprising a polynucleotide molecule encoding for a heavy chain variable region (HCVR) comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 2 and 18.21 . A recombinant expression vector comprising a polynucleotide molecule encoding for a light chain variable region (LCVR) comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 10 and 26.

22. A recombinant expression vector comprising a polynucleotide molecule encoding for a heavy chain (HC) comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 34 and 38.

23. A recombinant expression vector comprising a polynucleotides molecule encoding for a light chain (LC) comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 36 and 40.

24. An isolated host cell comprising the antibody or antigen binding fragment thereof of any one of claims 1-11 , the pair of polynucleotide molecules of any one of claims 12- 18, or the vector of any one of claims 19-23, or any combination thereof.

25. A pair of polynucleotide molecules encoding a heavy chain variable region (HCVR) and a light chain variable region (LCVR), respectively, of an antibody or antigen-binding fragment thereof that binds specifically to C-X-C Motif Chemokine Receptor 4 (CXCR4), wherein:(a) the polynucleotide molecule encoding the HCVR comprises three polynucleotide molecules encoding three heavy chain complementarity determining regions (HCDR1 , HCDR2 and HCDR3) within the HCVR polynucleotide sequence, wherein the HCDR1 polynucleotide sequence comprises a nucleic acid sequence encoding the amino acid sequence of SEQ ID NO: 4, the HCDR2 polynucleotide sequence comprises a nucleic acid sequence encoding the amino acid sequence of SEQ ID NO: 6, and the HCDR3 polynucleotide sequence comprises a nucleic acid sequence encoding the amino acid sequence of SEQ ID NO: 8, and(b) the polynucleotide molecule encoding the LCVR comprises three polynucleotide molecules encoding three light chain complementarity determining regions (LCDR1 , LCDR2 and LCDR3) within the LCVR polynucleotide sequence, wherein the LCDR1 polynucleotide sequence comprises a nucleic acid sequence encoding the amino acid sequence of SEQ ID NO: 12, the LCDR2 polynucleotide sequence comprises a nucleic acid sequence encoding the amino acid sequence of SEQ ID NO: 14, and the LCDR3 polynucleotide sequence comprises a nucleic acid sequence encoding the amino acid sequence of SEQ ID NO: 16.

26. The pair of polynucleotide molecules of claim 25, wherein the polynucleotide molecule encoding the HCVR comprises a nucleic acid sequence encoding the amino acid sequence of SEQ ID NO: 2.

27. The pair of polynucleotide molecules of claim 25, wherein the polynucleotide molecule encoding the HCVR comprises the HCDR1 nucleic acid sequence set forth in SEQ ID NO: 3, the HCDR2 nucleic acid sequence set forth in SEQ ID NO: 5, and the HCDR3 nucleic acid sequence set forth in SEQ ID NO: 7.

28. The pair of polynucleotide molecules of claim 25, wherein the polynucleotide molecule encoding the HCVR comprises the nucleic acid sequence of SEQ ID NO: 1 .

29. The pair of polynucleotide molecules of claim 25, wherein the polynucleotide molecule encoding the HCVR further encodes an immunoglobulin heavy chain constant region of the antibody.

30. The pair of polynucleotide molecules of claim 25, wherein the immunoglobulin heavy chain constant region is a human IgG 1 constant region.31 . The pair of polynucleotide molecules of claim 25, wherein the LCVR comprises the amino acid sequence of SEQ ID NO: 10.

32. The pair of polynucleotide molecules of claim 25, wherein the polynucleotide molecule encoding the LCVR comprises the LCDR1 nucleic acid sequence set forth in SEQ ID NO: 11 , the LCDR2 nucleic acid sequence set forth in SEQ ID NO: 13, and the LCDR3 nucleic acid sequence set forth in SEQ ID NO: 15.

33. The pair of polynucleotide molecules of claim 25, wherein the polynucleotide molecule encoding the LCVR comprises the nucleic acid sequence of SEQ ID NO: 9.

34. The pair of polynucleotide molecules of claim 25, wherein the polynucleotide molecule encoding the LCVR further encodes an immunoglobulin light chain constant region of the antibody.

35. The pair of polynucleotide molecules of claim 34, wherein the immunoglobulin light chain constant region is a human kappa constant region.

36. A pair of polynucleotide molecules encoding a heavy chain variable region (HCVR) and a light chain variable region (LCVR), respectively, of an antibody or antigen-binding fragment thereof that binds specifically to C-X-C Motif Chemokine Receptor 4 (CXCR4), wherein:(a) the HCVR comprises three heavy chain complementarity determining regions (HCDR1 , HCDR2 and HCDR3), wherein the HCDR1 comprises the amino acid sequence of SEQ ID NO: 20, the HCDR2 comprises the amino acid sequence of SEQ ID NO: 22, and the HCDR3 comprises the amino acid sequence of SEQ ID NO: 24, and(b) the LCVR comprises three light chain complementarity determining regions (LCDR1 , LCDR2 and LCDR3), wherein the LCDR1 comprises the amino acid sequence of SEQ ID NO: 28, the LCDR2 comprises the amino acid sequence of SEQ ID NO: 30, and the LCDR3 comprises the amino acid sequence of SEQ ID NO: 32.

37. The pair of polynucleotide molecules of claim 36, wherein the HCVR comprises the amino acid sequence of SEQ ID NO: 18.

38. The pair of polynucleotide molecules of claim 36, wherein the polynucleotide molecule encoding the HCVR comprises the HCDR1 nucleic acid sequence set forth in SEQ ID NO: 19, the HCDR2 nucleic acid sequence set forth in SEQ ID NO: 21 , and the HCDR3 nucleic acid sequence set forth in SEQ ID NO: 23.

39. The pair of polynucleotide molecules of claim 36, wherein the polynucleotide molecule encoding the HCVR comprises the nucleic acid sequence of SEQ ID NO: 17.

40. The pair of polynucleotide molecules of claim 36, wherein the polynucleotide molecule encoding the HCVR further encodes an immunoglobulin heavy chain constant region of the antibody.41 . The pair of polynucleotide molecules of claim 40, wherein the immunoglobulin heavy chain constant region is a human IgG 1 constant region.

42. The pair of polynucleotide molecules of claim 36, wherein the LCVR comprises the amino acid sequence of SEQ ID NO: 26.

43. The pair of polynucleotide molecules of claim 36, wherein the polynucleotide molecule encoding the LCVR comprises the LCDR1 nucleic acid sequence set forth in SEQ ID NO: 27, the LCDR2 nucleic acid sequence set forth in SEQ ID NO: 29, and the LCDR3 nucleic acid sequence set forth in SEQ ID NO: 31 .

44. The pair of polynucleotide molecules of claim 36, wherein the polynucleotide molecule encoding the LCVR comprises the nucleic acid sequence of SEQ ID NO: 25.

45. The pair of polynucleotide molecules of claim 36, wherein the polynucleotide molecule encoding the LCVR further encodes an immunoglobulin light chain constant region of the antibody.

46. The pair of polynucleotide molecules of claim 45, wherein the immunoglobulin light chain constant region is a human kappa constant region.

47. A method of producing an anti-CXCR4 antibody or antigen-binding fragment thereof by culturing the isolated host cell of claim 24 under conditions permitting production of the antibody or antigen-binding fragment, and recovering the antibody or antigen-binding fragment so produced.

48. The method of claim 47, wherein the isolated host cell is a Chinese hamster ovary (CHO) cell.

49. A method to enhance hematopoietic stem cell engraftment, comprising administering to a subject in need thereof an antibody or antigen binding fragment thereof of any one of claims 1 -1 1.

50. A method of treating cancer, comprising administering to a subject in need thereof an antibody or antigen binding fragment thereof of any one of claims 1-11.51 . The method of claim 50, wherein the cancer is a leukemia or a lymphoma.

52. A method of altering B-cell localization in the bone marrow or peripheral blood supply, comprising administering to a subject in need thereof an antibody or antigen binding fragment thereof of any one of claims 1 -11.

53. A method of hematopoietic stem cell engraftment host conditioning, comprising administering to a subject in need thereof an antibody or antigen binding fragment thereof or any one of claims 1 -1 1.

54. A method of inducing cancer cell apoptosis, comprising administering to a subject in need thereof an antibody or antigen binding fragment thereof of any one of claims 1 - 1 1.

55. A method of preventing HIV infection, comprising administering to a subject in need thereof an antibody or antigen binding fragment thereof of any one of claims 1 -1 1.

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