PHARMACEUTICAL COMPOSITIONS, ASSESSMENT METHODS AND KITS FOR THE TREATMENT OF C1013G / CXCR4-RELATED GAULULINEmia IN A SUBJECT

Abstract

The present disclosure provides a pharmaceutical composition for the treatment of C1013G / CXCR4-associated macroglobulinemia in a subject, comprising an antibody or antigen-binding portion thereof that specifically binds to a CXCR4 receptor carrying a C1013G / CXCR4 mutation expressed on the surface of a macroglobulinemia cell. Also provided are methods (except for medical practices) for assessing whether macroglobulinemia Fahrenheit is likely to be suitable for treatment with an antibody or antigen-binding portion thereof that specifically binds to the CXCR4 receptor expressed on the surface of macroglobulinemia Fahrenheit cells, the method comprises evaluating whether a cell in a test tissue sample obtained from the macroglobulinemia Fahrenheit carries a C1013G mutation in the CXCR4 gene, and determining that the macroglobulinemia Fahrenheit is possibly suitable for treatment if it is determined that the macroglobulinemia Fahrenheit carries the C1013G mutation in the CXCR4 gene, and a kit for use in the treatment of C1013G / CXCR4 related macroglobulinemia of Fahrenheit. The pharmaceutical composition disclosed by the invention can be used for effectively treating the macroglobulinemia of Fahrenheit related to C1013G / CXCR4.

Description

This application is a divisional application of an application with an application date of November 6, 2014, international application number PCT / US2014 / 064320, national application number 201480072308.5, and invention name “Treating C1013G / CXCR4-related Waldenstrom’s macroglobulinemia with anti-CXCR4 antibodies”.

[0001] Throughout this application, various publications are referenced in parentheses by author name and date or by patent number or publication number. Full citations to these publications can be found at the end of the specification immediately preceding the claims. The disclosures of these publications are incorporated by reference in their entireties into this application to more fully describe the prior art known to those skilled in the art as of the date of the invention described and claimed herein. However, the citations referenced herein should not be construed as an admission that such references are prior art to the present invention. Field of the Invention

[0002] The present disclosure relates to the use of a monoclonal antibody that specifically binds to human CXCR4 in a method of treating C1013G / CXCR4-associated Waldenstrom's macroglobulinemia. Background of the Invention CXC chemokine receptor type 4 (CXCR4) plays a key role in physiological conditions during embryogenesis, such as lymphocyte production and bone marrow (BM) myelopoiesis. In the postnatal life process, CXCR4 and its ligand regulate CD341 cells to return to the BM microenvironment (BM niches) and lymphocyte transport. CXCR4 has been shown to be mutated in patients with hereditary heterozygous autosomal dominant diseases, which are characterized by abnormal functional immunity, known as warts, hypogammaglobulinemia, infection and congenital myelogranulocytosis (WHIM) syndrome, due to the presence of activating mutations of the CXCR4 gene represented by the C1013G variant (Balabanian et al., 2005; Balabanian et al., 2008). Recent evidence supports the presence of CXCR4 somatic aberrations (WM; lymphoplasmacytic lymphoma) (Hunter et al., 2014; Treon et al., 2014) in Waldenstrom's macroglobulinemia. The functional role of this variant in supporting the progression of lymphoplasmacytic lymphoma and whether it is unique to WM compared to other B-cell lymphoproliferative entities or arises during the IgM monoclonal gammopathy of undetermined significance (MGUS) stage have not been previously described.

[0004] WM is a rare B cell disorder with an incidence of about 3 cases per million people per year in the United States. About 1,000 to 1,500 people are diagnosed with WM each year in the United States. Whole genome sequencing has recently revealed molecular mechanisms that may contribute to the pathogenesis of the disease. Specifically, L265P / MYD88 has been described as a common somatic mutation in WM patients (Treon et al., 2012). In vitro studies have confirmed that the L265P / MYD88 variant can lead to increased tumor cell proliferation (Yang et al., 2013); this can be explained at least in part by the activation of MYD88-dependent nuclear factor κB (NF-κB), a signaling pathway known to mediate tumor B cell survival, growth and resistance to treatment (Leleu et al., 2008). These observations are also consistent with previous findings that overexpression of oncogenic microRNA-155 in cloned WM cells leads to activation of NF-κB in primary WM cells. Indeed, loss-of-function studies of microRNA-155 resulted in inhibition of NF-κB and reduction of tumor growth in vitro and in vivo (Leleu et al., 2008; Roccaro et al., 2009). However, MYD88 mutations did not predict progression or resistance to therapy in several published studies, suggesting that other genetic alterations may be critical for tumor progression and spread to distant organs.

[0005] Among low-grade B-cell lymphomas, WM represents a lymphoplasmacytic subtype characterized by BM-infiltrating lymphoplasmacytic cells and secretion of serum monoclonal immunoglobulin M (IgM) proteins, which can lead to complications of hyperviscosity, bleeding, and peripheral neuropathy (Ghobrial et al., 2003; Vijay and Gertz, 2007). Evidence of extensive BM involvement at diagnosis suggests cellular trafficking of clonal B cells to the BM. In this context, one of the major regulators of tumor B cell return to the BM is represented by CXCR4 through its associated ligand stroma-derived factor-1 (SDF-1 / CXCL12).

[0006] Initial reports of complete genome sequencing have indicated that CXCR4 may be mutated in 29% (16 / 55) of patients with WM (Cao et al., 2012). The presence and role of the C1013 / CXCR4 variant was therefore investigated in patients with WM and different B-cell lymphoproliferative disorders in order to determine the in vivo functional role of this variant in WM and in the response of C1013 / CXCR4-associated WM patients to treatment with anti-CXCR4 antibodies.

[0007] Anti-CXCR4 monoclonal antibodies that demonstrate therapeutic efficacy in treating cancer have previously been described in PCT Publication Nos. WO2008 / 060367 and WO 2013 / 071068. The disclosures of both applications are therefore incorporated herein by reference in their entirety. The fully human monoclonal antibody ulocuplumab (named F7 in WO 2008 / 060367, previously also referred to as BMS-936564 or MDX-1338, all four nomenclatures are used interchangeably herein) exhibits unexpectedly favorable anti-tumor properties in preclinical studies of solid tumors and hematological cancers. Ulocuplumab is currently undergoing a Phase I clinical study in patients with relapsed / refractory B-cell malignancies (NCT01120457; see Clinical Trials Website, http: / / www.clinicaltrials.gov). SUMMARY OF THE INVENTION The present disclosure provides a method for treating a subject with C1013G / CXCR4-associated Waldenstrom's macroglobulinemia, the method comprising administering to the subject a therapeutically effective amount of an antibody or an antigen binding portion thereof that specifically binds to a CXCR4 receptor expressed on the surface of a WM cell. In a preferred embodiment, the subject is a human and the antibody or its antigen binding portion binds to a human CXCR4 receptor expressed on the surface of a cell, preferably a human CXCR4 carrying a C1013G mutation. In certain embodiments, the antibody or its antigen binding portion is a chimeric, humanized or human antibody or its antigen binding portion. Preferably, the antibody or its antigen binding portion is a human antibody or its antigen binding portion.

[0009] The present disclosure also provides a method for treating a subject having C1013G / CXCR4-associated WM, the method comprising: (a) selecting a subject as a suitable candidate for treatment, the selection comprising: (i) optionally providing a test tissue sample obtained from WM tissue in the subject; (ii) performing an assay to determine whether cells in the WM test tissue sample carry the C1013G mutation in the CXCR4 gene; and (iii) selecting the subject as a suitable candidate based on determining that the WM cells carry the C1013G mutation in the CXCR4 gene; and (b) administering to the selected subject a therapeutically effective amount of an antibody, or an antigen-binding portion thereof, that specifically binds to a CXCR4 receptor expressed on the surface of C1013G / CXCR4-associated WM cells.

[0010] The present disclosure further provides methods for treating a subject having C1013G / CXCR4-associated WM, the methods comprising administering to the subject a therapeutically effective amount of an antibody, or an antigen-binding portion thereof, that specifically binds to a CXCR4 receptor expressed on the surface of C1013G / CXCR4-associated WM cells, the subject having been selected based on a determination that the subject's WM cells carry the C1013G mutation in the CXCR4 gene.

[0011] The present disclosure also provides a method for selecting a WM patient for treatment with an antibody, or an antigen-binding portion thereof, that specifically binds to a CXCR4 receptor expressed on the surface of a WM cell, the method comprising: (a) providing a test tissue sample obtained from a WM tissue in a patient; (b) performing an assay to determine whether cells in the WM test tissue sample carry a C1013G mutation in the CXCR4 gene; and (c) selecting a patient for treatment based on determining that the WM cells carry the C1013G mutation in the CXCR4 gene.

[0012] In addition, the present disclosure provides a method for determining a treatment regimen comprising an anti-CXCR4 antibody or an antigen-binding portion thereof for treating C1013G / CXCR4-associated WM in a subject, the method comprising: (a) providing a test tissue sample obtained from a WM tissue in a subject; (b) performing an assay to determine whether cells in the WM test tissue sample carry a C1013G mutation in the CXCR4 gene; and (c) determining a treatment regimen comprising an anti-CXCR4 antibody or an antigen-binding portion thereof based on determining that the WM cells carry the C1013G mutation in the CXCR4 gene. In one aspect, the present disclosure also provides a treatment regimen for treating a patient suffering from C1013G / CXCR4-associated WM, the regimen determined by the present method.

[0013] In certain embodiments of any of the above methods, C1013G / CXCR4-mutant cells in WM patients exhibit resistance to commonly used anti-WM agents, including mammalian target of rapamycin (mTOR), Bruton's tyrosine kinase (BTK), and / or phosphatidylinositol 3-kinase (PI3K) inhibitors. In certain preferred embodiments, C1013G / CXCR4-mutant cells are resistant to the BTK inhibitor ibrutinib. is resistant. Thus, in certain embodiments, after a subject with C1013G / CXCR4-associated WM has failed treatment with an mTOR, BTK and / or PI3K inhibitor, preferably after failure of treatment with ibrutinib, a therapeutically effective amount of an anti-CXCR4 antibody or an antigen-binding portion thereof is administered to the subject. In other embodiments, the subject is treated with a combination of a therapeutically effective amount of an anti-CXCR4 antibody or an antigen-binding portion thereof and a therapeutically effective amount of an mTOR, BTK or PI3K inhibitor. In certain preferred embodiments, the subject is treated with a combination of an anti-CXCR4 antibody, ulocuplumab and a therapeutically effective amount of the BTK inhibitor ibrutinib.

[0014] In certain embodiments of any of the above methods, the anti-CXCR4 antibody, or antigen-binding portion thereof, comprises: CDR1, CDR2, and CDR3 domains in the heavy chain variable region comprising consecutively linked amino acids, the sequence of which is described in SEQ ID NO:25; and CDR1, CDR2, and CDR3 domains in the light chain variable region comprising consecutively linked amino acids, the sequence of which is described in SEQ ID NO:29.

[0015] In other embodiments, the anti-CXCR4 antibody, or antigen binding portion thereof, comprises: a heavy chain variable region comprising consecutively linked amino acids whose sequence is set forth in SEQ ID NO:25; and a light chain variable region comprising consecutively linked amino acids whose sequence is set forth in SEQ ID NO:29.

[0016] In a further embodiment, the anti-CXCR4 antibody, or antigen-binding portion thereof, comprises: a heavy chain variable region CDR1 comprising consecutively linked amino acids of the sequence described in SEQ ID NO: 1, a heavy chain variable region CDR2 comprising consecutively linked amino acids of the sequence described in SEQ ID NO: 5, a heavy chain variable region CDR3 comprising consecutively linked amino acids of the sequence described in SEQ ID NO: 9, a light chain variable region CDR1 comprising consecutively linked amino acids of the sequence described in SEQ ID NO: 13, a light chain variable region CDR2 comprising consecutively linked amino acids of the sequence described in SEQ ID NO: 17, and a light chain variable region CDR3 comprising consecutively linked amino acids of the sequence described in SEQ ID NO: 21. In a preferred embodiment, the antibody is ulocuplumab.

[0017] In another aspect, the disclosure provides a kit for treating C1013G / CXCR4-associated WM in a subject, the kit comprising: (a) a dose of an anti-CXCR4 antibody or an antigen-binding portion thereof; and (b) instructions for using the anti-CXCR4 antibody in any of the above methods. Exemplary anti-CXCR4 antibodies for use in the kit include human monoclonal antibodies F7 (ulocuplumab), F9, and D1, which are described in WO 2008 / 060367. In a preferred embodiment, the anti-CXCR4 antibody in the kit is ulocuplumab.

[0018] Other features and advantages of the present disclosure will become apparent from the following detailed description and examples, which should not be construed as limiting. The contents of the articles, patents, and published patent applications are expressly incorporated herein by reference. BRIEF DESCRIPTION OF THE DRAWINGS Figure 1 shows the nucleotide sequence (SEQ ID NO: 33) and amino acid sequence (SEQ ID NO: 25) of the heavy chain variable region (A) of the F7 (ulocuplumab) human monoclonal antibody. The heavy chain CDR1 (SEQ ID NO: 1), CDR2 (SEQ ID NO: 5) and CDR3 (SEQ ID NO: 9) regions are depicted and show V, D and J germline derivation. The nucleotide sequence (SEQ ID NO: 37) and amino acid sequence (SEQ ID NO: 29) of the light chain variable region (B) of F7 are also shown. The light chain CDR1 (SEQ ID NO: 13), CDR2 (SEQ ID NO: 17) and CDR3 (SEQ ID NO: 21) regions are depicted and show V and J germline derivation.

[0020] Figure 2 Identification of the somatic C1013G / CXCR4 variant in WM patients was shown. Primary WM samples carrying the C1013G / CXCR4 variant presented with higher CXCR4 surface expression compared to wild-type (WT) primary WM samples, as shown by flow cytometry on CD191BM-derived WM cells.

[0021] Figure 3 To demonstrate the efficacy of CXCR4 overexpression in WM cells, BCWM.1 cells were transfected with precise LentiORF / CXCR4 / GFP (CXCR4 +) or infected with empty vector / RFP control. Human CXCR4 expression levels were evaluated by qRT-PCR, using the ΔΔCt method, and GAPDH was normalized. P represents P value.

[0022] Figure 4 shows the enhanced ability of WM cells overexpressing CXCR4 to spread in vivo. BCWM.1 cells were precisely expressed with LentiORF / CXCR4 / GFP (CXCR4 + ) or infected with empty vector / RFP (control) and injected intravenously into SCID / Bg mice (n=5 / group). After 3 weeks, mice were euthanized and organs were harvested. Hematoxylin-eosin immunohistochemistry staining of human CD20 and human CXCR4 was performed on femoral BM (A), liver (B) and kidney (C). Quantification of CD20 and CXCR4 staining showed a higher number of CXCR4 / CD20+ cells in mice injected with CXCR4+ cells compared to mice injected with control cells. P represents P value. (D) Mice were euthanized and serum was harvested in the third week, and human IgM levels were tested by ELISA. (E) Human CXCR4 levels were detected in cells isolated from BM femur ex vivo by qRT-PCR (ΔΔCt method). Uninjected mice (n=3) and mice injected with combined control cells (n=4) were used as controls. P represents P value. Column represents standard deviation.

[0023] Figure 5 shows that overexpression of CXCR4 in WM cells leads to decreased survival and enhanced adhesion properties and growth in vitro. Compared to the relevant empty vector infected cells, cells overexpressing CXCR4 (CXCR4 + ) showed a more aggressive ability to spread to the BM (A), liver (B), and kidney (C) (hematoxylin eosin X10, antibody staining X20 and X10, magnification). Quantification of human CD20 and CXCR4 staining is shown in Figure 4. (D) Kaplan-Meier curves show that compared with empty vector cell-injected mice, the CXCR4 + =Decreased survival rate in mice injected with CXCR4 cells (n=7 / group). P represents P value (log-rank test). (E, F) CXCR4 overexpression leads to increased WM cell adhesion and increased cell proliferation to primary bone marrow mesenchymal stem cells (BM-MSCs) when compared to CXCR4-silenced WM cells (CXCR4-KD) where decreased adhesion and decreased cell proliferation to BM-MSCs were observed. Columns represent standard deviations. DAPI, 4',6-diamidino-2-phenylindole; NS, not significant.

[0024] Figure 6Figure 2 shows the efficacy of CXCR4 knockdown in infected WM cells. BCWM.1 cells were infected with CXCR4-shRNA / GFP (CXCR4K.D.) or scramble probe / GFP as a control. Human CXCR4 expression levels were evaluated by qRT-PCR using the ΔΔCt method and normalized to GAPDH. P represents P value.

[0025] Figure 7 shows the effects of CXCR4 loss-of-function and gain-of-function on the adhesion properties of WM cells. BCWM.1 cells were treated with LentiORF / CXCR4 (CXCR4 + ) (A) or CXCR4 shRNA (CXCR4K.D.) (B) and respective scrambled probes were stably infected. Adhesion was tested using BSA-coated (negative control), poly-D-lysine-coated (positive control) and fibronectin-coated wells. Columns represent standard deviations.

[0026] Figure 8 shows the efficiency of mutagenesis in infected WM cells and the lack of brain damage in mice injected with C1013G / CXCR4-mutated WM cells. Genomic DNA (gDNA; A) and cDNA (B) were isolated from infected BCWM.1 cells. Cells infected with empty / control vectors were used as controls (control cells). Detection of the C1013G allele was evaluated by using allele-specific PCR. P represents P value. Column represents standard deviation. (C) Detection of the C1013G mutation was evaluated using Sanger sequencing. (D) SCID / Bg mice were intravenously injected with BCWM.1 cells or control cells carrying C1013G / CXCR4 mutations. (E) Hematoxylin-eosin immunohistochemical staining (HE) of the brain for human CD20 and human CXCR4 showed the absence of tumor cell infiltration. (4X magnification is provided).

[0027] FIG9 shows the effect of the CXCR4 / C1013G mutation on in vivo proliferation and survival of WM cells. WM cells carrying the CXCR4 / C1013G variant have altered mRNA levels along with increased cell adhesion and cell proliferation. SCID / Bg mice injected intravenously with WM cells carrying the CXCR4 / C1013G variant have significant lesions in (A) BM, (B) lymph nodes, (C) liver, (D) lungs, and (E) kidneys (X4, X10, and 20X magnification). Quantification of human CD20 and CXCR4 staining is shown in Fig.10 (F) Kaplan-Meier curves show decreased survival in C1013G / CXCR4 cell-injected mice (left panel) compared to control vector cell-injected mice (right panel) (n=7 / group). Death was observed in control vector cell-injected mice on days 37, 48, and 62. P represents P value (log-rank test).

[0028] Fig.10 Quantification of human CD20 and CXCR4 expression in tissues harvested from mice injected with WM cells of C1013G / CXCR4 mutations is shown. SCID / Bg mice were intravenously injected with BCWM.1 cells carrying C1013G / CXCR4 variants or cells infected with empty / control vectors (control cells). After 3 weeks, mice were euthanized and organs were harvested. Hematoxylin-eosin immunohistochemical staining (Nikon, Melville, NY) was performed on femur BM, lymph nodes, liver, lungs and kidneys to human CD20 and human CXCR4. Quantification (NIS Elements software, Nikon, Melville, NY) of CD20 and CXCR4 staining was performed. P represents P value. T test was used to analyze femur, liver, kidney and lung, and Anova test was used to analyze lymph nodes.

[0029] Figure 11 shows the effect of CXCR4 / C1013G mutation on adhesion and proliferation of WM cells in vivo. In SCID / Bg mice injected intravenously with C1013G / CXCR4-WM cells, the C1013G / CXCR4 variant increased the adhesion (A) and proliferation (B) of WM cells alone or in a BM-MSC background. (C, D) The C1013G / CXCR4 variant increased the adhesion and proliferation of MWCL1 cells alone or in a BM-MSC background. Cells infected with empty / control vectors were used as controls. Columns represent standard deviations. P represents P value.

[0030] Figure 12 shows that the cells of C1013G / CXCR4 mutation are different from control cells at the gene level. (A) Gene set enrichment analysis (GSEA) enrichment curves of tumor invasiveness, cell proliferation, anti-apoptosis and oncogenic signature genes in cells infected by control vectors (control cells) in cells of C1013G / CXCR4 mutations. The drawn curve shows the enrichment score and reflects the degree to which each gene (vertical line) is represented at the top or bottom of the arranged gene list. The heat map represents the relative abundance (left to right) of genes specifically enriched in mutant cells and compared to control cells. All gene sets are enriched in C1013G / CXCR4 mutant cells, and the false discovery rate (FDR) is always <0.25. Each gene set analyzed shows standardized enrichment scores (NES) and FDR. (C) GSEA enrichment curves of up-regulated genes listed in Table 3 in C1013G / CXCR4 mutant cells compared to cells infected by control vectors (control cells). The same genes were enriched in mutant cells compared to control cells.

[0031] Figure 13 shows that WM cells carrying C1013G / CXCR4 somatic variants exhibit drug resistance. (A) GSEA enrichment curves for multidrug resistance and response to drug marker genes in C1013G / CXCR4 mutant cells and cells infected with control vectors (control cells), respectively. The drawn curves show enrichment scores and reflect the extent to which each gene (vertical line) is represented at the top or bottom of the arranged gene list. The heat map represents the relative abundance (left to right) of genes specifically enriched in mutant cells compared to control cells. It is considered that the gene set is enriched, and the FDR is always <0.25. Each gene set analyzed shows standardized enrichment scores (NES) and FDR. (BF) C1013G / CXCR4-WM cell lines (BCWM1; MWCL1) or cells infected with control vectors (control) were exposed to everolimus, ibrutinib, idelalisib, bortezomib, and carfilzomib for 48 hours. Cytotoxicity was determined by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide.

[0032] Figure 14 shows the effect of BMS-936564 on the migration of WM cells in vitro. The migration properties of WM cell lines (BCWM.1; MWCL.1) were tested using a transwell migration assay. Cells were exposed to increasing concentrations of BMS-936564 and migration was measured after 5 hours. Transwell migration assays were performed in the presence of primary WM BM-MSCs (A) or in the presence of SDF-1α (B). In both cases, BMS-936564-dependent inhibition of WM cell migration was observed.

[0033] Figure 15 shows the effect of BMS-936564 on adhesion and proliferation of WM cells in vitro. (A) WM cell lines were treated with BMS-636564 for 4 hours and then tested for adhesion to primary WM BM-MSCs. Adhesion was evaluated after 2 hours showing BMS-936564-dependent inhibition of WM cell adhesion. (B, C) WM cell lines (BCWM.1; MWCL.1) were cultured with control medium and with BMS-936564 in the presence or absence of primary WM BM-MSCs for 48 hours. [ 3 [H]-Thymidine uptake assay to assess cell proliferation. Columns represent standard deviations. P represents P value. All data represent the mean (± sd) of three experiments. The CXCR4 antagonist, AMD3110 (Sigma-Aldrich, St. Louis, MO) was used as a control.

[0034] Fig.16The effect of BMS-936564 on WM cell signaling is shown. BCWM.1 cells were exposed to BMS-936564 for 6 hours in the presence or absence of primary WM BM-MSCs. BCWM.1 cells were subsequently harvested and cell lysates were subjected to Western blot analysis using anti-phospho (p)-ERK1 / 2, anti-ERK1 / 2, anti-p-Akt, anti-Akt, anti-p-SRC and anti-SRC antibodies. BCWM.1 cells cultured in the absence of WM BM-MSCs were used as controls. Columns represent standard deviations.

[0035] Figure 17 shows that control antibodies do not affect adhesion (A) or migration (B) of WM cell lines in vitro.

[0036] Fig.18 Effects of BMS-936564 on apoptosis-related proteins in WM cells are shown. BCWM.1 cells were exposed to BMS-936564 for 14 hours. Whole cell lysates were then harvested and subjected to Western blot analysis using anti-caspase 9, anti-PARP, anti-p-β catenin, anti-β-catenin, anti-p-GSK3β, and anti-tubulin antibodies.

[0037] Figure 19 shows that BMS-936564 targets C1013G / CXCR4 mutant cells in WM cells in vivo. (AE) BMS-936564 inhibits the spread of C1013G / CXCR4 mutant WM cells in vivo. Compared to mice treated with isotype controls (untreated), human CD20 and CXCR4 staining were significantly reduced in tissues transplanted from mice treated with BMS-936564. P represents P value.

[0038] Figure 20 shows representative histochemical images of the effects of BMS-936564 on C1013G / CXCR4-mutated cells in WM cells in vivo.SCID / Bg mice were injected intravenously with BCWM.1 cells carrying C1013G / CXCR4 variants or cells infected with empty / control vectors (control cells).Mice were treated with BMS-936564 or control antibodies (10 mg / kg, intraperitoneally; x3 / 4 / week; mice: 5 / group).After 3 weeks, mice were euthanized and organs were harvested.Hematoxylin-eosin immunohistochemical staining of human CD20 and CXCR4 was performed on femur BM (A), liver (B), kidney (C), lung (D) and lymph node (E) (Nikon, Melville, NY; 4X, 10X or 20X magnification is shown).

[0039] Figure 21 shows cells with C1013G / CXCR4 mutations in BMS-936564-targeted WM cells in a BM context. Transwell migration assays were used to test the migration properties of C1013G / CXCR4-infected BCWM.1 cells. Cells were exposed to increasing concentrations of BMS-936564 and migration was determined after 5 hours. Transwell migration assays were performed in the presence of primary WM BM-MSCs (A) or SDF-1α (B). In both cases, BMS-936564-dependent inhibition of WM cell migration was observed. (C) C1013G / CXCR4-infected BCWM.1 cells were treated with BMS-636564 for 4 hours and then tested for adhesion to primary WM BM-MSCs. Evaluation of adhesion after 2 hours showed BMS-936564-dependent inhibition of WM cell adhesion. (D) C1013G / CXCR4-infected BCWM.1 cells were cultured with control medium and BMS-936564 in the presence or absence of primary WM BM-MSCs for 48 h. 3 [H]-Thymidine uptake assay to assess cell proliferation. Columns represent standard deviations. P represents P value. All data represent the mean (± sd) of three experiments. Columns represent standard deviations.

[0040] Fig. 22 The effect of BMS-936564 on survival and apoptosis-related signaling proteins in C1013G / CXCR4-WM cells is shown. C1013G / CXCR4-mutated cells were cultured for 6 hours in the presence or absence of BMS-936564, showing inhibition of phospho (p)-ERK, ERK, p-Akt, Akt, p-Src. Similarly, cells were exposed to the compound for 14 hours and the induction of apoptosis-related pathways was observed (p-GSK3β, p-β-catenin, PARP, and caspase-9).

[0041] FIG23 shows that BMS-936564 inhibits WM regression in vivo and synergistically enhances WM chemosensitivity to bortezomib in vivo and in vitro. (A, B) BCWM.1-mCherry + Cells were injected intravenously into SID / Bg mice. Mice were treated with control antibody or BMS-936564 (10 mg / kg, intraperitoneal; x3 / 4 / week); bortezomib (0.5 mg / kg, intraperitoneal x2 / week); or BMS-936564 + bortezomib (same dose as above, x2 / week). BM cells were harvested at the end of the study and evaluated using a fluorescent plate reader. Controls were intended to be mCherry identified from the BM of control antibody treated mice. +Number of cells. Serum was collected at the end of the study. Serum IgM secretion was evaluated using a human IgM ELISA kit. (C, D) WM cell lines (BWM.1; MWCL.1) were exposed to BMS-936564 or bortezomib for 48 hours, either alone or in a combination regimen. Cytotoxicity was performed by MTT. Synergy was evaluated by using Calcusyn software: isobolograms, composite indexes (CI), and fractions affected (FA) for the combination of BMS-936564 and bortezomib are shown. Columns represent standard deviations. DETAILED DESCRIPTION OF THE INVENTION The present disclosure relates to methods for modulating CXCR4 activity in patients with Waldenstrom's macroglobulinemia or otherwise treating the patient using a monoclonal antibody that specifically binds to native human CXCR4 expressed on the surface of a cell. These methods are particularly useful for WM patients carrying an activating C1013G mutation in the CXCR4 gene. In certain embodiments, an anti-CXCR4 antibody is administered as a first ("front") line of treatment, such as an initial or first treatment, as a monotherapy or as part of a combination therapy (e.g., in combination with an mTOR, BTK, and / or PI3K inhibitor). In other embodiments, an anti-CXCR4 antibody is administered as a second or third line of treatment, such as after initial treatment with the same or one or more different therapies, including after relapse and / or where the first treatment has failed. Therefore, in certain embodiments, an anti-CXCR4 antibody is administered after another anti-WM agent, such as an mTOR, BTK, and / or PI3K inhibitor, has failed.

[0043] definition In order to make the present disclosure easier to understand, some terms are first defined. As used in this application, unless otherwise expressly provided herein, each of the following terms shall have the meaning shown below. Additional definitions are described throughout this application.

[0044] "Administering" refers to any introduction of a composition comprising a therapeutic agent into a subject's body using various methods and delivery systems known to those skilled in the art. Preferred routes of administering the antibodies of the invention include intravenous, subcutaneous, intramuscular, intraperitoneal, spinal or other parenteral routes of administration, e.g., by injection or infusion. As used herein, the phrase "parenteral administration" means modes of administration other than enteral and topical administration. Administration can also be performed, e.g., once, multiple times, and / or over one or more extended cycles.

[0045] "Antibody" (Ab) shall include, but is not limited to, a glycoprotein immunoglobulin that specifically binds to an antigen and comprises at least two heavy (H) chains and two light (L) chains interconnected by disulfide bonds, or an antigen-binding portion thereof. Each H chain comprises a heavy chain variable region (abbreviated herein as V H) and the heavy chain constant region. The heavy chain constant region contains three domains C H1 , C H2 and C H3 Each light chain comprises a light chain variable region (abbreviated herein as V L ) and the light chain constant region. The light chain constant region contains a domain C L . V H and V L The V region can be further subdivided into regions of hypervariability, termed complementarity determining regions (CDRs), interspersed with more conserved regions, termed framework regions (FRs). H and V L It is composed of three CDRs and four FRs, arranged in the following order from the amino terminus to the carboxyl terminus: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. The variable regions of the heavy and light chains contain binding domains that interact with antigens. The constant region of the antibody can mediate the binding of immunoglobulins to host tissues or factors including various cells of the immune system (e.g., effector cells) and the first component (Clq) of the classical complement system.

[0046] Antibodies usually bind to their cognate antigens with high affinity and specificity. -5 -10 -11 M -1 or lower dissociation constant (K D ) to reflect. Any value greater than about 10 -4 M -1 K D Generally considered to indicate non-specific binding. As used herein, an antibody that "specifically binds" an antigen refers to an antibody that binds the antigen and substantially identical antigens with high affinity, meaning with about 10 affinity, but does not bind unrelated antigens with high affinity. -7 M or less, preferably about 10 -8 M or less, more preferably about 5x10 -9 M or less, even more preferably about 5 x 10 -10 M or less, and most preferably about 10 -9 M to about 10 -11 M or lower K D An antigen is "substantially identical" to a given antigen if it exhibits a high degree of sequence identity to the given antigen, for example, if it exhibits at least 80%, at least 90%, preferably at least 95%, more preferably at least 97%, or even more preferably at least 99 sequence identity to the given antigen sequence. By way of example, an antibody that specifically binds to human CXCR4 may also have cross-reactivity with CXCR4 antigens from certain primate species, but not with CXCR4 antigens from certain rodent species or with antigens other than CXCR4, such as the human PD-1 antigen.

[0047] Immunoglobulins can be derived from any commonly known isotype, including but not limited to IgA, secretory IgA, IgG and IgM. IgG subclasses are also well known to those skilled in the art and include but are not limited to human IgG1, IgG2, IgG3 and IgG4. "Isotype" refers to the antibody class (e.g., IgM or IgG1) encoded by the heavy chain constant region gene. "Antibodies" include, for example, naturally occurring and non-naturally occurring antibodies; monoclonal and polyclonal antibodies; chimeric and humanized antibodies; human or non-human antibodies; fully synthetic antibodies; and single-chain antibodies. Non-human antibodies can be humanized by recombinant methods to reduce their immunogenicity in humans.

[0048] When not explicitly stated, and unless the context otherwise indicates, the term "antibody" also includes the antigen binding portion of any of the above-mentioned immunoglobulins. An "antigen binding portion" or "antigen binding fragment" of an antibody refers to one or more fragments of an antibody that retains the ability to specifically bind to an antigen bound by a complete antibody. Examples of binding fragments encompassed in the term "antigen binding portion" of an antibody include Fab, F(ab')2Fd, Fv and single-chain variable fragments (scFv), divalent or divalent (divalent or bivalent) scFv, diabodies or other antibody multimers (e.g., trivalent triantibodies or tetravalent tetraantibodies), miniantibodies, and even isolated CDRs, which can display antigen binding function. All of the above-mentioned proteolytic and engineered fragments and related variants of antibodies (see Hollinger et al., 2005 for further details; Olafsen et al., 2010) are encompassed in the term "antigen binding portion" of an antibody.

[0049] The term "monoclonal antibody" ("mAb") refers to a preparation of antibody molecules of single molecular composition, i.e., antibody molecules whose primary sequences are substantially identical and which display a single binding specificity and affinity for a particular epitope. Monoclonal antibodies can be produced by hybridoma, recombinant, transgenic, or other techniques known to those skilled in the art.

[0050] "HuMAb" refers to an antibody having a variable region in which both the framework region and the CDR region are derived from human germline immunoglobulin sequences. In addition, if the antibody contains a constant region, the constant region is also derived from human germline immunoglobulin sequences. The human antibody of the present invention may include amino acid residues not encoded by human germline immunoglobulin sequences (e.g., mutations introduced by random or site-directed mutagenesis in vitro or by somatic mutations in vivo). However, as used herein, the term "human antibody" is not intended to include antibodies in which the CDR sequences derived from the germline of another mammalian species such as a mouse have been connected to human framework sequences. The terms "human" antibody and "fully human" antibody are used synonymously.

[0051] A "humanized" antibody refers to an antibody in which some, most or all of the amino acids outside the CDR domains of a non-human antibody are replaced by the corresponding amino acids derived from human immunoglobulins. In one embodiment of a humanized form of an antibody, some, most or all of the amino acids outside the CDR domains have been replaced by amino acids from human immunoglobulins, while some, most or all of the amino acids in one or more CDR regions have not changed. Small additions, deletions, insertions, substitutions or modifications of amino acids are permitted as long as they do not eliminate the ability of the antibody to bind to a specific antigen. A "humanized" antibody retains an antigenic specificity similar to that of the original antibody.

[0052] A "chimeric antibody" refers to an antibody in which the variable region is derived from one species and the constant region is derived from another species, such as an antibody in which the variable region is derived from a mouse antibody and the constant region is derived from a human antibody.

[0053] "Cancer" refers to a broad group of various diseases characterized by the uncontrolled growth of abnormal cells in the body. Unregulated cell division and growth leads to the formation of malignant tumors that invade adjacent tissues and may also metastasize to distant parts of the body via the lymphatic system or bloodstream. Cancer includes solid tumors and hematological malignancies.

[0054] Solid tumors are neoplasms (new growth of cells) or lesions (damage to anatomical structures or physiological disorders) formed by abnormal growth of body tissue cells outside blood, BM or lymphocytes. It consists of abnormal cell masses that may originate from different tissue types such as liver, colon, breast or lung, and it initially grows in the organ of its cell origin. However, solid tumors can spread to other organs through metastatic tumor growth in the late stage of the disease.

[0055] Hematological malignancies are cancer types that affect blood, BM, spleen and / or lymph nodes, and include lymphomas, leukocytes, myelomas and lymphatic system malignancies. Hematological malignancies can be derived from either of two major blood cell lineages, namely bone marrow and lymphoid cell lines. Myeloid cell lines generally produce granulocytes, erythrocytes, hemocytes, macrophages and mast cells, while lymphoid cell lines produce B, T, NK and plasma cells. Lymphomas (e.g., Hodgkin's lymphoma), lymphocytic leukemias and myelomas are derived from lymphoid cell lines, while acute and chronic myeloid leukemias (AML, CML), myelodysplastic syndromes and myeloproliferative diseases are bone marrow origins. Waldenstrom's macroglobulinemia (WM) is an example of a B cell lymphoma, and is shown herein to be amenable to treatment with anti-CXCR4 antibodies.

[0056] The term "CXCR4" ("CXC" chemokine receptor 4) includes variants, isoforms, homologs, orthologs, and paralogs. For example, an antibody specific for CXCR4 may, in some cases, cross-react with CXCR4 from species other than human. In other embodiments, an antibody specific for human CXCR4 may be completely specific for human CXCR4 and may not exhibit species or other types of cross-reactivity. The term "human CXCR4" refers to a human sequence CXCR4, such as having The complete amino acid sequence of human CXCR4 of accession number P61073 (SEQ ID NO: 51). CXCR4 is also known in the art as, for example, LESTR, fusin, or CD184. The human CXCR4 sequence may differ from the human CXCR4 of SEQ ID NO: 51 in having, for example, conservative mutations or mutations in non-conserved regions, and the CXCR4 has substantially the same biological function as the human CXCR4 of SEQ ID NO: 51.

[0057] "CXCR4-expressing cancer" or "CXCR4 + A "WM cancer" is a cancer in which the malignant cells characterizing the cancer express CXCR4, preferably high levels of CXCR4, on the cell surface. The C1013G mutation in the CXCR4 gene, which has previously been associated with WHIM syndrome, is disclosed herein as being present in 28% of WM patients but absent or present in only 7% of other B-cell lymphomas. C1013G / CXCR4-associated WM refers to WM in which the C1013G mutation is present.

[0058] "Subject" includes humans or any non-human animal such as non-human primates (e.g., monkeys), dogs, rabbits, rodents, or chickens. In preferred embodiments, the subject is a human cancer patient such as a WM patient. The terms "subject," "patient," and "individual" are used interchangeably herein.

[0059] A "therapeutically effective amount" or "therapeutically effective dose" of a drug or therapeutic agent, such as an anti-CXCR4 antibody, is any amount of the drug that, when used alone or in combination with another therapeutic agent, promotes disease regression as evidenced by a decrease in the severity of disease symptoms, an increase in the frequency and duration of disease symptom-free periods, or prevents impairment or disability due to disease affliction. A therapeutically effective amount or dose of a drug includes a "prophylactically effective amount" or "prophylactically effective dose," which is any amount of a drug that, when administered alone or in combination with another therapeutic agent to a subject at risk of developing the disease or suffering from a recurrence of the disease, inhibits the onset or recurrence of the disease. The ability of a therapeutic agent to promote disease regression can be evaluated using a variety of methods known to practitioners, such as in human subjects during clinical trials, in animal model systems predictive of efficacy in humans, or by measuring the activity of the agent in in vitro assays.

[0060] In addition, the terms "effective" and "effectiveness" with respect to treatment include pharmacological effectiveness and physiological safety. Pharmacological effectiveness refers to the ability of a drug to promote cancer regression in a patient. Physiological safety refers to the level of toxicity caused by the administration of the drug, or adverse physiological effects (adverse effects) at the level of other cells, organs and / or organisms.

[0061] "Treatment" or "therapy" of a subject refers to any type of intervention or treatment of a subject, or the administration of an active agent to a subject, with the goal of reversing, alleviating, ameliorating, inhibiting, slowing or preventing the onset, progression, occurrence, severity or recurrence of symptoms, complications, conditions or biochemical markers associated with a disease.

[0062] Use of alternatives (eg, "or") should be understood to mean any one, both, or any combination of the alternatives. As used herein, the indefinite article "a" or "an" should be understood to mean "one or more" of any recited or listed component.

[0063] The term "about", "substantially" or "substantially includes" refers to a value or composition within an acceptable error range for a specific value or composition as determined by one of ordinary skill in the art, which will depend in part on how the value or composition is measured or determined, i.e., the limitations of the measurement system. For example, "about", "substantially" or "substantially includes" can mean within 1 or within more than 1 standard deviation / practice in the art. Alternatively, "about", "substantially" or "substantially includes" can mean a range of plus or minus 20%, more typically a range of plus or minus 10%. In addition, specifically with respect to biological systems or methods, the term can mean up to an order of magnitude of a value or up to 5 times of a value. When specific values ​​or compositions are provided in the application and claims, unless otherwise stated, the meaning of "about", "substantially" or "substantially includes" should be assumed to be within an acceptable error range for the specific value or composition.

[0064] As described herein, any concentration range, percentage range, ratio range or integer range should be understood to include any integer value within the range, and, where appropriate, fractions thereof (such as tenths and hundredths of integers), unless otherwise specified.

[0065] Various aspects of the disclosure are described in further detail in the following subsections.

[0066] Therapeutic anti-CXCR4 antibodies The generation of exemplary anti-CXCR4 antibodies for use in the present treatment methods, including human monoclonal antibodies F7 (ulocuplumab; BMS-936564; MDX-1338), F9, D1, and E2, is described in detail in WO 2008 / 060367. Methods of using these antibodies to treat hematological malignancies are also described in WO 2008 / 060367 and WO 2013 / 071068. The anti-CXCR4 antibodies of the present disclosure can be chimeric, humanized, or preferably fully human antibodies, and are characterized by specific functional characteristics or properties. For example, the antibodies bind to native human CXCR4 expressed on the surface of a cell. Preferably, the antibodies of the present disclosure bind to CXCR4 with high affinity, for example, with 1 x 10 -8 M or lower K D The anti-CXCR4 antibodies of the present disclosure preferably exhibit one or more of the following characteristics that are desirable for therapeutic applications: (a) binds to human CXCR4 expressed on the cell surface; (b) inhibiting the binding of SDF-1 to CXCR4; (c) inhibiting SDF-1-induced calcium flux in cells expressing CXCR4; (d) inhibits SDF-1-induced migration of cells expressing CXCR4; (e) inhibiting capillary tube formation by human umbilical vein endothelial cells; (f) 1x 10 -8 M or lower K D Binds to human CXCR4; (g) inducing apoptosis in cells expressing CXCR4; (h) Inhibition of CXCR4 in vitro + Proliferation of tumor cells; (i) Inhibition of CXCR4 + Tumor cell proliferation and / or induction of CXCR4 in vivo + Tumor cell apoptosis; (j) Inhibition of CXCR4 + metastasis of tumor cells; and (k) Increased CXCR4 + The survival time of the tumor subject.

[0067] In a preferred embodiment, the anti-CXCR4 antibodies of the present disclosure are administered at 1 x 10 -8 M or lower K D Binds to human CXCR4, induces apoptosis in cells expressing CXCR4, and exhibits at least five of the other properties listed above. In a more preferred embodiment, the anti-CXCR4 antibodies of the present disclosure exhibit all of the properties listed above.

[0068] In certain embodiments, the anti-CXCR4 antibodies used in the methods of the present disclosure are administered at 5 x 10 -8 M or lower K D Bind to human CXCR4, at 2x 10 -8 M or lower, 5x 10 -9 M or lower, 4x 10 -9 M or lower, 3x 10 -9 M or lower, or 2x 10 -9 M or lower K D Binding to human CXCR4. In a preferred embodiment, the anti-CXCR4 antibody is ulocuplumab (BMS-936564).

[0069] In one aspect, the anti-CXCR4 antibodies of the present disclosure comprise the heavy and light chain CDR1's, CDR2's and CDR3's of F7, F9, D1 or E2, or a combination thereof. H The amino acid sequences of CDR1's are shown in SEQ ID NOs. 1-4, respectively. H The amino acid sequences of CDR2's are shown in SEQ ID NOs. 5-8, respectively. H The amino acid sequences of CDR3's are shown in SEQ ID NOs. 9-12, respectively. k The amino acid sequences of CDR1's are shown in SEQ ID NOs. 13-16, respectively. k The amino acid sequences of CDR2's are shown in SEQ ID NOs. 17-20, respectively. k The amino acid sequences of the CDR3's are shown in SEQ ID NOs. 21-24, respectively. The amino acid sequences of the heavy chain variable region, light chain variable region and six CDRs of the F7 (ulocuplumab) human monoclonal antibody are also shown in Figure 1. The CDR regions identified throughout this disclosure are described using the Kabat system (Kabat et al., 1991).

[0070] In another aspect, an anti-CXCR4 antibody of the present disclosure comprises: (a) CDR1, CDR2 and CDR3 domains in the heavy chain variable region having the sequence described in SEQ ID NO: 25 or 41, and CDR1, CDR2 and CDR3 domains in the light chain variable region having the sequence described in SEQ ID NO: 29 or 45; (b) CDR1, CDR2 and CDR3 domains in the heavy chain variable region having the sequence described in SEQ ID NO: 26 or 42, and CDR1, CDR2 and CDR3 domains in the light chain variable region having the sequence described in SEQ ID NO: 30 or 46; (c) CDR1, CDR2 and CDR3 domains in the heavy chain variable region having the sequence described in SEQ ID NO: 27 or 43, and CDR1, CDR2 and CDR3 domains in the light chain variable region having the sequence described in SEQ ID NO: 31 or 47; or (d) CDR1, CDR2 and CDR3 domains in the heavy chain variable region having the sequence described in SEQ ID NO: 28 or 44, and CDR1, CDR2 and CDR3 domains in the light chain variable region having the sequence described in SEQ ID NO: 32 or 48.

[0071] In another preferred embodiment, the anti-CXCR4 antibody of the present disclosure comprises: (a) comprising a heavy chain variable region CDR1 having consecutively linked amino acids of a sequence described in SEQ ID NO: 1 or conservative variations thereof; comprising a heavy chain variable region CDR2 having consecutively linked amino acids of a sequence described in SEQ ID NO: 5 or conservative variations thereof; comprising a heavy chain variable region CDR3 having consecutively linked amino acids of a sequence described in SEQ ID NO: 9 or conservative variations thereof; comprising a light chain variable region CDR1 having consecutively linked amino acids of a sequence described in SEQ ID NO: 13 or conservative variations thereof; comprising a light chain variable region CDR2 having consecutively linked amino acids of a sequence described in SEQ ID NO: 17 or conservative variations thereof; and comprising a light chain variable region CDR3 having consecutively linked amino acids of a sequence described in SEQ ID NO: 21; (b) comprising a heavy chain variable region CDR1 having consecutively linked amino acids of a sequence described in SEQ ID NO: 2 or conservative variations thereof; comprising a heavy chain variable region CDR2 having consecutively linked amino acids of a sequence described in SEQ ID NO: 6 or conservative variations thereof; comprising a heavy chain variable region CDR3 having consecutively linked amino acids of a sequence described in SEQ ID NO: 10 or conservative variations thereof; comprising a light chain variable region CDR1 having consecutively linked amino acids of a sequence described in SEQ ID NO: 14 or conservative variations thereof; comprising a light chain variable region CDR2 having consecutively linked amino acids of a sequence described in SEQ ID NO: 18 or conservative variations thereof; and comprising a light chain variable region CDR3 having consecutively linked amino acids of a sequence described in SEQ ID NO: 22; (c) comprising a heavy chain variable region CDR1 having consecutively linked amino acids of the sequence described in SEQ ID NO: 3 or conservative variations thereof; a heavy chain variable region CDR2 having consecutively linked amino acids of the sequence described in SEQ ID NO: 7 or conservative variations thereof; a heavy chain variable region CDR3 having consecutively linked amino acids of the sequence described in SEQ ID NO: 11 or conservative variations thereof; a light chain variable region CDR1 having consecutively linked amino acids of the sequence described in SEQ ID NO: 15 or conservative variations thereof; a light chain variable region CDR2 having consecutively linked amino acids of the sequence described in SEQ ID NO: 19 or conservative variations thereof; and a light chain variable region CDR3 having consecutively linked amino acids of the sequence described in SEQ ID NO: 23; or (d) comprising a heavy chain variable region CDR1 having consecutively linked amino acids of a sequence described in SEQ ID NO:4 or a conservative variation thereof; a heavy chain variable region CDR2 having consecutively linked amino acids of a sequence described in SEQ ID NO:8 or a conservative variation thereof; a heavy chain variable region CDR3 having consecutively linked amino acids of a sequence described in SEQ ID NO:12 or a conservative variation thereof; a light chain variable region CDR1 having consecutively linked amino acids of a sequence described in SEQ ID NO:16 or a conservative variation thereof; a light chain variable region CDR2 having consecutively linked amino acids of a sequence described in SEQ ID NO:20 or a conservative variation thereof; and a light chain variable region CDR3 having consecutively linked amino acids of a sequence described in SEQ ID NO:24.

[0072] In a preferred embodiment, the anti-CXCR4 antibody or antigen-binding portion thereof comprises: a heavy chain variable region CDR1 comprising consecutively linked amino acids having the sequence described in SEQ ID NO: 1, a heavy chain variable region CDR2 comprising consecutively linked amino acids having the sequence described in SEQ ID NO: 5, a heavy chain variable region CDR3 comprising consecutively linked amino acids having the sequence described in SEQ ID NO: 9, a light chain variable region CDR1 comprising consecutively linked amino acids having the sequence described in SEQ ID NO: 13, a light chain variable region CDR2 comprising consecutively linked amino acids having the sequence described in SEQ ID NO: 17, and a light chain variable region CDR3 comprising consecutively linked amino acids having the sequence described in SEQ ID NO: 21.

[0073] The nucleotide and amino acid sequences of the heavy chain variable region of F7 are shown in Figure 1A and SEQ ID NOs. 33 and 25, respectively. The nucleotide and amino acid sequences of the light chain variable region of F7 are shown in Figure 1B and SEQ ID NOs. 37 and 29, respectively. Analysis of F7V using the Kabat system for CDR region determination H The sequence generation is depicted as Figure 1A and the heavy chain CDR1, CDR2 and CD3 regions described in SEQ ID NOs: 1, 5 and 9, respectively, and Figure 1B and the light chain CDR1, CDR2 and CD3 regions shown in SEQ ID NOs: 13, 17 and 21, respectively.

[0074] The nucleotide and amino acid sequences for the heavy chain variable region of F9 are shown in SEQ ID NOs. 34 and 26, respectively, and for the light chain variable region of F9 are shown in SEQ ID NOs. 38 and 30, respectively. The sequences of the F9 heavy chain CDR1, CDR2 and CD3 regions are shown in SEQ ID NOs. 2, 6 and 10, respectively, and the F9 light chain CDR1, CDR2 and CD3 sequences are shown in SEQ ID NOs. 14, 18 and 22, respectively.

[0075] The nucleotide and amino acid sequences for the heavy chain variable region of D1 are shown in SEQ ID NOs: 35 and 27, respectively, and for the light chain variable region of D1 are shown in SEQ ID NOs. 39 and 31, respectively. The sequences of the D1 heavy chain CDR1, CDR2 and CD3 regions are shown in SEQ ID NOs. 3, 7 and 11, respectively, and the F9 light chain CDR1, CDR2 and CD3 sequences are shown in SEQ ID NOs. 15, 19 and 23, respectively.

[0076] The nucleotide and amino acid sequences for the heavy chain variable region of E2 are shown in SEQ ID NOs: 36 and 28, respectively, and for the light chain variable region of E2 are shown in SEQ ID NOs. 40 and 32, respectively. The sequences of the E2 heavy chain CDR1, CDR2 and CD3 regions are shown in SEQ ID NOs. 4, 8 and 12, respectively, and the F9 light chain CDR1, CDR2 and CD3 sequences are shown in SEQ ID NOs. 16, 20 and 24, respectively.

[0077] In addition, alternative forms of F7, F9, D1 and E2 were generated in which certain framework residues were replaced by germline residues and are referred to herein as F7GL, F9GL, D1GL and E2GL. H The amino acid sequences are shown in SEQ ID NOs. 41, 42, 43, and 44, respectively. V L The amino acid sequences are shown in SEQ ID NOs. 45, 46, 47, and 48, respectively.

[0078] In one aspect, an anti-CXCR4 antibody for use in the methods of the present disclosure cross-competes for binding to CXCR4 with any of the following: mAb F7 (ulocuplumab; having V values ​​as shown in SEQ ID NOs: 25 and 29, respectively; H and V L sequence), mAb F9 (having V as shown in SEQ ID NOs: 26 and 30, respectively). H and V L sequence), mAb D1 (having V as shown in SEQ ID NOs: 27 and 31, respectively H and V L sequences), and mAb E2 (having V as shown in SEQ ID NOs: 28 and 32, respectively). H and V L Sequence). Antibodies that cross-compete for binding to CXCR4 bind to the same epitope region on CXCR4 (ie, the same or overlapping epitope).

[0079] In certain embodiments, a cross-competing anti-CXCR4 monoclonal antibody comprises a polypeptide having a V region derived from a human V region as shown in SEQ ID NO: 49. H 3-48 consecutive amino acids of the germline sequence H region and / or contains a region derived from a human V region as shown in SEQ ID NO: 50 K The L15 germline sequence is a sequence of consecutively linked amino acids V LCross-competing antibodies can be identified based on their ability to cross-compete with ulocuplumab (F7), F9, D1, E2, or any other reference anti-CXCR4 antibody in a standard CXCR4 binding assay, such as flow cytometry with CEM cells, wherein the reference antibody is labeled with FITC and the ability of the test antibody to inhibit the binding of the FITC-labeled reference antibody to CEM cells is assessed.

[0080] Pharmaceutical composition On the other hand, the present disclosure provides compositions, such as pharmaceutical compositions, comprising one or a combination of monoclonal antibodies or antigen-binding portions thereof of the present disclosure (formulated together with a pharmaceutically acceptable carrier). As used herein, "pharmaceutically acceptable carriers" include any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic agents and absorption delaying agents, etc., which are physiologically acceptable. Preferably, the carrier is suitable for intravenous, subcutaneous, intramuscular, parenteral, spinal or epidermal administration (e.g., by injection or infusion). The pharmaceutical compositions of the present invention may include one or more pharmaceutically acceptable salts, antioxidants, aqueous and non-aqueous carriers, and / or adjuvants such as preservatives, wetting agents, emulsifiers and dispersants.

[0081] Dosage regimens may be adjusted to provide the optimum desired response (eg, a therapeutic response or minimal adverse effects).

[0082] For administration of human anti-CXCR4 antibodies, the dosage range is about 0.0001-100 mg / kg, preferably about 0.01-about 20 mg / kg, and more preferably 0.1-10 mg / kg of the subject's body weight. For example, the dosage can be 0.1, 0.3, 1, 3, 5 or 10 mg / kg, and more preferably, 0.3, 1, 3 or 10 mg / kg of body weight. The dosing schedule is generally designed to achieve exposure that produces sustained receptor occupancy based on the typical pharmacokinetic properties of the antibody. Exemplary treatment regimens include administration once a week, once every two weeks, once every three weeks, once every four weeks, once a month, once every 3 months, or once every 3-6 months. Considering that IgG4 antibodies generally have a half-life of 2-3 weeks, a preferred dosage regimen for the anti-CXCR4 antibodies of the present disclosure includes 0.3-20 mg / kg body weight, preferably 1-10 mg / kg body weight, administered intravenously, with the antibody being given every 7 or 14 days in a cycle of up to 6, 8 or 12 weeks until complete response or confirmed progressive disease.

[0083] Dosage and schedule can be changed during the course of treatment. For example, the dosing regimen of the anti-CXCR4 antibodies of the present disclosure includes 1, 3 or 10 mg / kg body weight administered intravenously (IV), and the antibody is administered using one of the following dosing schedules: (i) every 7 days in a cycle of up to 6 weeks; (ii) every two weeks for up to 6 doses, followed by every three months; (iii) every three weeks; (iv) 1-10 mg / kg body weight once followed by 1 mg / kg body weight every 2-3 weeks.

[0084] The C1013G / CXCR4 mutation found in 28% of WM patients plays an activating role in WM cells, as evidenced by significant tumor proliferation and spread to extramedullary organs leading to disease progression and reduced survival. Administration of the anti-CXCR4 antibody BMS-936564 resulted in significant tumor reduction and abrogation of extramedullary spread in vivo in the C1013G / CXCR4 WM model (see Examples 6 and 7). In certain embodiments of the invention, patients with C1013G / CXCR4-associated WM are treated with a treatment regimen comprising administering an anti-CXCR4 antibody, or an antigen-binding portion thereof, to the patient at a dose at least twice, at least three times, or at least five times the dose administered in treating WM patients carrying a wild-type CXCR4 gene, and / or administering the anti-CXCR4 antibody, or an antigen-binding portion thereof, to the patient at a frequency at least twice, at least three times, or at least five times the frequency administered in treating WM patients carrying a wild-type CXCR4 gene.

[0085] The actual dosage level of the active ingredient in the pharmaceutical composition of the present disclosure can be changed to obtain the amount of such active ingredient, which effectively achieves the desired therapeutic response for a specific patient, composition and mode of administration without toxicity to the patient. The selected dosage level will depend on a variety of pharmacokinetic factors, including the activity of the specific composition of the present disclosure used, or its ester, salt or amide, the route of administration, the time of administration, the excretion rate of the specific compound used, the duration of treatment, other drugs, compounds and / or materials used in combination with the specific composition used, the age, sex, weight, condition, general health and early medical history of the patient being treated, and other factors well known in the medical field. The composition of the present invention can be administered using one or more of the various methods well known in the art through one or more routes of administration. As will be appreciated by those skilled in the art, the route of administration and / or mode will be different according to the desired result.

[0086] Uses and methods of the invention The present disclosure provides methods for treating a subject suffering from WM, the methods comprising administering to the subject a therapeutically effective amount of an antibody that specifically binds to a CXCR4 receptor and exhibits certain functional properties desirable for therapeutic use.

[0087] The present disclosure also provides methods for treating a subject having C1013G / CXCR4-associated WM, the methods comprising administering to the subject a therapeutically effective amount of an anti-CXCR4 antibody.

[0088] CXCR4 is known to be expressed on many tumor cell types and is involved in tumor metastasis, HIV infection, inflammatory conditions and angiogenesis or new blood vessel formation (see, e.g., WO 2008 / 060367 and WO 2013 / 071068). Thus, the anti-CXCR4 antibodies disclosed herein can be used in a variety of clinical settings, but the present disclosure focuses on the use of these antibodies for the treatment of cancer, especially WM.

[0089] Role of CXCR4 in cancer Overexpression of CXCR4 has been demonstrated in approximately 75% of cancers, and in some cases an inverse correlation has been established between CXCR4 expression and patient prognosis or survival. Non-limiting examples of cancer types associated with CXCR4 expression or the CXCR4 / CXCL12 pathway include solid tumors such as breast cancer (Muller et al., 2001), ovarian cancer (Scotton et al., 2001), prostate cancer (Taichman et al., 2002), non-small cell lung cancer (Spano et al., 2004), pancreatic cancer (Koshiba et al., 2000), colorectal cancer (Zeelenberg et al., 2003), renal cancer (Schrader et al., 2002) and thyroid cancer (Hwang et al., 2003), nasopharyngeal carcinoma (Wang et al., 2005), melanoma (Scala et al., 2005), renal cell carcinoma (Staller et al., 2003), neuroblastoma (Geminder et al., 2001), glioblastoma (Re mpel et al., 2000), rhabdomyosarcoma (Libura et al., 2002) and osteosarcoma (Laverdiere et al., 2005) as well as hematological malignancies such as acute lymphoblastic leukemia (Crazzolara et al., 2001), acute myeloid leukemia (AML) (Mohle et al., 1998; Rombouts et al., 2004), multiple myeloma (MM) (Alsayed et al., 2007; Azab et al., 2009), chronic lymphocytic leukemia (Mohle et al., 1999; Burger et al., 1999), chronic myeloid leukemia (Jin et al., 2008), WM (Ngo et al., 2008) and non-Hodgkin's lymphoma (NHL) (Bertolini et al., 2002; Weng et al., 2003).

[0090] In addition, this pathway has been implicated in stimulating the metastatic process in multiple tumors (Murphy, 2001). In clinical studies, CXCR4 has been associated with increased metastatic propensity and decreased survival and has been identified as a prognostic indicator for AML, breast cancer, colorectal cancer, non-small cell lung cancer, ovarian cancer, and pancreatic cancer, with higher CXCR4 expression associated with disease severity (Spoo et al., 2007; Hiller et al., 2011; Ottaiano et al., 2006; Spano et al., 2004; Jiang et al., 2006; Marechal et al., 2009).

[0091] BM-MSC secretes CXCL12 (SDF-1) and interacts with CXCR4 for returning and maintaining hematopoietic stem cells in the BM microenvironment is essential (Mohle et al., 1998). Leukemia cells express high levels of CXCR4, and the pathway plays a key role in the migration of leukemia cells to BM (which then supports the survival of their growth). CXCR4 is essential for metastasis and propagation to organs such as BM in which CXCL12 is expressed. Collectively, CXCR4 plays an important role in the return and retention of hematopoietic stem cells in BM and CXCR4 antagonists mobilize stem cells into the bloodstream, as confirmed by small molecule CXCR4 antagonist AMD3100 (plerixafor; Mozobil) (which is approved by FDA for use in combination with granulocyte-colony stimulating factor for autologous transplants in NHL and MM patients). Another CXCR4 inhibitor AMD3465 shows chemotaxis induced by antagonizing CXCL12 and interstitial and inhibits activation of CXCL12 induction of prosurvival signaling pathways in leukocytes (Zeng et al., 2009). In addition, it is confirmed that AMD3465 alone or in combination with granulocyte-colony stimulating factor can induce the anti-leukocyte effect of chemotherapy and sorafenib that mobilizes AML cells and progenitor cells to enter the circulation and enhance, resulting in significantly reduced leukocyte burden and prolonged survival of animals (Zeng et al., 2009). Such findings show that the destruction of CXCR4 / CXCL12 interactions can be used to sensitize leukocytes to chemotherapy by targeting their protective BM microenvironment.

[0092] As described in WO 2008 / 060367 and WO 2013 / 071068, novel first-in-human therapeutic monoclonal antibodies against CXCR4 have been developed. These monoclonal antibodies bind to cells expressing CXCR4 with low nanomolar affinity and with low nanomolar EC 50The value blocks CXCL12 from binding to cells expressing CXCR4 and inhibits CXCL12-induced migration and calcium flux. Because CXCR4 plays a role in multiple fundamental aspects of cancer, including proliferation, migration / invasion, and angiogenesis, antagonists may have multiple ways to interfere with malignant tumors in which CXCR4 is expressed. Significantly, in addition to blocking CXCL12-induced calcium flux and migration, WO2013 / 071068 also teaches that antibody-dependent induction of apoptosis of tumor cells expressing CXCR4 is the mechanism of action of these human anti-CXCR4 antibodies. Antibody-induced apoptosis results in potent in vivo efficacy in multiple hematopoietic tumor xenograft models. In contrast, small molecule CXCR4 antagonists such as AMD3100 increase CXCR4 + Mobilization of tumor cells from the BM and thereby increased chemosensitization, but not direct killing of such tumor cells, suggests an enhanced potency of anti-CXCR4 antibodies in killing cancer cells compared to small molecule CXCR4 antagonists.

[0093] Waldenstrom's Macroglobulinemia WM, also known as lymphoplasmacytic lymphoma, is a painless (slowly growing) subtype of non-Hodgkin's lymphoma (NHL). The disease is characterized by the uncontrolled clonal proliferation of terminally differentiated B lymphocytes (i.e., leukocytes formed in BM and lymph nodes). The unique feature of the disease is that B cells produce excessive monoclonal IgM, which can cause the blood thickening called hyperviscosity. The IgM protein can cause many symptoms, including fatigue, unexplained weight loss, enlarged lymph nodes or spleen, impaired renal function, systemic amyloidosis, weakness and unexplained bleeding. B cell proliferation also interferes with the production of red blood cells, causing anemia.

[0094] WM is a rare disease, with only about 1,500 cases per year in the United States. There is no single approved treatment for WM, but treatment regimens that include combinations of biologics that stimulate the immune system and chemotherapy have provided promising results (Treon, 2009). Some examples of combination therapies currently used to treat WM patients include BDR: bortezomib Dexamethasone and rituximab (Treon et al., 2009); RCD: cyclophosphamide Dexamethasone and rituximab (Dimopoulos et al., 2007); R-CHOP: cyclophosphamide, doxorubicin Vincristine Prednisone and rituximab; CPR: cyclophosphamide, prednisone, and rituximab; VR: bortezomib and rituximab; FR: fludarabine and rituximab; and TR:thalidomide and rituximab. Objective response rates are high (>80%) but complete response rates are low (0-15%). Therapies being studied include ibrutinib Selective Bruton's tyrosine kinase (BTK) inhibitor; alemtuzumab It targets CD52 on the surface of WM cells; ofatumumab It targets the CD20 molecule on the surface of B cells; bendamustine It attacks the DNA of cancer cells and disrupts the cell division cycle; mTOR inhibitor everolimus It inhibits cell growth and division; and carfilzomib (KYPROLIS TM ), a selective proteasome inhibitor. In certain embodiments of the methods disclosed herein for treating WM or C1013G / CXCR4-associated WM, any of the foregoing agents or any combination thereof can be used in combination with an anti-CXCR4 antibody of the present disclosure. In other embodiments, an anti-CXCR4 antibody is used after progression of a WM patient to any of these anti-WM therapies (already used or in development) for the treatment of WM.

[0095] The role of CXCR4 and C1013G / CXCR4 mutations in Waldenstrom's macroglobulinemia has previously been shown to express high levels of chemokines and adhesion receptors, including CXCR4 and VLA-4, and CXCR4 is essential for the migration and transendothelial migration of WM cells induced by CXCL12 (Ngo et al., 2008). It was shown that CXCR4 knockdown or CXCR4 inhibitor AMD3100 significantly inhibited the migration of WM cells and their adhesion to fibronectin, stromal cells and endothelial cells. The reduced adhesion of WM cells to stromal cells induced by AMD3100 resulted in increased sensitivity of these cells to the cytotoxicity of bortezomib, i.e., inhibition of proliferation. It was further confirmed that CXCR4 and VLA-4 interact directly in response to CXCL12. Therefore, it has been shown that the CXCR4 / CXCL12 axis interacts with VLA-4 to regulate the migration and adhesion of WM cells in the BM microenvironment (Ngo et al., 2008).

[0096] Examination of 418 patients with B-cell lymphoproliferative disorders as described herein (see Example 1; Table 1) revealed that 28% of patients with low-grade lymphoplasmacytic lymphoma (WM) had the C1013G / CXCR4 variant. Studies were therefore conducted to analyze the functional relevance of the C1013G / CXCR4 variant, to evaluate its putative role in mediating WM cell spread and disease progression using in vivo models, and to identify novel therapeutic approaches that could target C1013G / CXCR4-mutated lymphoplasmacytic cells. These studies demonstrate that CXCR4 plays a critical role in supporting WM cell homing to the BM and WM spread to distant organs, as shown in vivo using CXCR4 gain-of-function studies (Example 2). Importantly, WM cells carrying the C1013G / CXCR4 variant produced a similar phenotype compared to cells overexpressing CXCR4 (Example 3), suggesting that the C1013G / CXCR4 variant may represent a functionally active mutation. Furthermore, C1013G / CXCR4-mutated cells showed in vivo spread to lymph nodes compared to control cells or CXCR4-overexpressing WM cells (Example 3), indicating that the generated C1013G / CXCR4 variant is able to recapitulate the clinically observed situation in patients. Significantly, C1013G / CXCR4 mutant cells are able to colonize extramedullary tissues such as lung and kidney in vivo (Example 3), reflecting the phenotype observed in patients with extramedullary WM, where the variant has been detected in all cases of lung and kidney damage.

[0097] The observed C1013G / CXCR4 variant is one of the mutations originally described in WHIM syndrome. This is a rare, hereditary, heterozygous, autosomal dominant disease characterized by abnormal functional immunity. This is caused by CXCR4 mutations, which are responsible for the truncation of the carboxyl terminal tail (talk) (C tail) of the receptor that weakens its intracellular transport, resulting in increased responsiveness to chemokine ligands and retention of neutrophils in BM (Balabanian et al., 2005; 2008; Hernandez et al., 2003; Taniuchi et al., 2005). Recent studies have confirmed the presence of WHIM-like C1013G / CXCR4 mutations in WM patients (Hunter et al., 2014), but the in vivo functional sequelae of this mutation in WM and whether somatic variants are present in other B-cell lymphoproliferative diseases (where CXCR4 mediates tumor cell transport and diffusion) have not been described before this study. The increased ability of C1013G / CXCR4 mutant cells to spread in vivo, leading to disease progression, is supported by the enrichment of genes involved in cell invasiveness, anti-apoptosis, cell proliferation, and tumorigenesis (Example 3). This results in the upregulation of pro-survival pathways in mutant cells compared to control cells. These effects were reversed when mice bearing WM injected with C1013G / CXCR4 mutant cells or control cells were treated with the anti-CXCR4 antibody ulocuplumab, as described below.

[0098] Altered sensitivity of WM cells carrying the C1013G / CXCR4-mutation to commonly used anti-WM agents Drugs commonly used to treat WM include agents such as rituximab, chlorambucil, Cladribine Fludarabine, bortezomib, bendamustine (alone or in various combinations). Additional therapies for the treatment of refractory / relapsed WM include ofatumumab (for patients allergic to rituximab), everolimus, alemtuzumab, and high-dose chemotherapy with autologous or allogeneic stem cell transplantation. Several new drugs or drug combinations are being studied in clinical trials for WM (some for refractory / relapsed disease), including carfilzomib, ibrutinib, and panobinostat (LBH-589).

[0099] Ibrutinib, an orally administered inhibitor of the enzyme Bruton's tyrosine kinase, was recently approved by the U.S. Food and Drug Administration (FDA) for the treatment of mantle cell lymphoma and chronic lymphocytic leukemia, and was granted Breakthrough Therapy designation by the FDA as a monotherapy for patients with WM in February 2013. Ibrutinib has been reported to be highly active and well tolerated in patients with relapsed or refractory WM (Treon et al., 2013), and a supplemental New Drug Application (sNDA) was submitted to the FDA in October 2014 based on data from a Phase 2 clinical trial of ibrutinib in WM patients previously treated with at least one systemic WM regimen (ClinicalTrials.gov Identifier: NCT01614821). However, the presence of WHIM-like CXCR4 mutations has been shown to confer reduced sensitivity to ibrutinib-mediated growth inhibition in WM cells (Cao et al., 2013; Treon et al., 2013), with only a 30% major response rate in patients with WHIM-like CXCR4 mutations compared to a 77% response rate in patients with wild-type CXCR4 (Treon et al., 2013).

[0100] In addition, it has been shown herein (Example 4) that the C1013G / CXCR4 somatic variant is resistant to conventionally used anti-WM small molecules including BTK inhibitors, ibrutinib, mTOR inhibitors, everolimus and phosphatidylinositol-3-kinase (PI3K) inhibitors, idelalisib 1013G / CXCR4-mutated WM cells, supporting the role of this variant in mediating drug resistance. This result suggests that mTOR, BTK and PI3K inhibitors may not be effective in treating patients whose WM cells carry C1013G / CXCR4-mutations. In contrast, C1013G / CXCR4-mutated WM cells and unmutated control cells are equally sensitive to the proteasome inhibitor drugs carfilzomib and bortezomib (Example 4), thus illustrating the possible combination regimen of ulocuplumab and proteasome inhibitors in WM patients carrying C1013G / CXCR4 somatic mutations.

[0101] Without being bound by any specific theory, it is proposed that BTK, PI3K and mTOR inhibitors are not effective for C1013G / CXCR4-mutated cells, because of the upregulation of genes associated with those specific pathways. In addition, it is confirmed that the mutated CXCR4 cells have RAF and mitogen-activated protein kinase / mitogen-activated protein kinase kinase and mTOR pathway enrichment, thus further indicating the presence of crosstalk (Downward, 2003; Lim and Counter, 2005) between RAF and mitogen-activated protein kinase kinase / ERK / mTOR pathways. On the contrary, NF-κB and NF-κB-related apoptosis genes are not differentially regulated in CXCR4 mutated cells compared to control cells (data not shown). This can (at least partially) explain the presence of BTK, mTOR and PI3K inhibitor resistance and sensitivity to proteasome inhibitors in mutated WM cells compared to wild-type cells.

[0102] Treatment of Waldenstrom's macroglobulinemia with anti-CXCR4 antibodies The anti-CXCR4 antibody BMS-936564 is shown herein to inhibit the migration of WM cell lines toward primary WM bone marrow mesenchymal stromal cells (BM-MSCs) or the CXCR4 ligand CXCL12 (see Example 5). The anti-CXCR4 antibody also inhibits WM cell adhesion to WM BM-MSCs and inhibits WM cell proliferation in the presence of WM BM-MSCs. In addition, evidence is provided that antibodies can directly induce apoptosis in WM cells (Example 5). Therefore, the present disclosure provides a method for treating a subject suffering from WM, the method comprising administering to the subject a therapeutically effective amount of an antibody or an antigen-binding portion thereof that specifically binds to a CXCR4 receptor expressed on the surface of a cell (including a WM cell). In a preferred embodiment, the antibody used in the present method is an anti-CXCR4 antibody of the present disclosure, which specifically binds to a CXCR4 receptor and exhibits certain functional properties desired for therapeutic use. Such functional properties include high affinity binding to human CXCR4 expressed on the cell surface, inhibition of SDF-1-induced calcium flux in cells expressing CXCR4 and migration of cells expressing CXCR4, and inhibition of CXCR4 in vivo. + Tumor cell proliferation and / or induction of CXCR4 + Tumor cell apoptosis and increased CXCR4 + survival time of the subject with the tumor, and / or other functional characteristics described herein.

[0103] The present disclosure also provides experimental evidence that the C1013G mutation in the CXCR4 gene is present in about 28% of WM patients and in about 20% of IgM-MGUS patients, but is rare or absent in several other B-cell cancers tested (see Example 1). Therefore, among B-cell lymphoproliferative diseases, WM patients are characterized by C1013G / CXCR4 mutations. Data showing that CXCR4 overexpression promotes WM cell growth and spread in vivo are also provided (Example 2). The presence of the C1013G / CXCR4 mutation increases the adhesion and proliferation of WM cells in the presence of primary WM BM-MSCs, outlining the role of CXCR4 overexpression in WM cells (Example 3). Therefore, the C1013G / CXCR4 mutation is an activating mutation in WM cells, as confirmed by significant tumor proliferation and spread to extramedullary organs leading to disease progression and reduced survival.

[0104] Significantly, ulocuplumab showed adhesion, migration and proliferation properties of WM cells that effectively targeted CXCR4 mutations in vitro and in vivo in the context of the BM microenvironment (see Example 6). The administration of ulocuplumab resulted in significant tumor reduction and elimination of extramedullary spread in the C1013G / CXCR4WM model in vivo. These findings confirm that CXCR4 is a key regulator of WM molecular pathogenicity and an important therapeutic target. The use of anti-CXCR4 antibodies to successfully target wild-type and C1013G / CXCR4 mutant WM cells (as described in Examples 5-7) provides a reliable basis for translating these observations into clinical trials for WM patients.

[0105] Thus, the present disclosure provides a method for treating a subject suffering from WM (including C1013G / CXCR4-associated WM), the method comprising administering to the subject a therapeutically effective amount of an antibody or antigen binding portion thereof that specifically binds to a CXCR4 receptor expressed on the surface of a cell (including a WM cell). In certain embodiments of the present method, the subject is a human WM patient, preferably a human patient suffering from C1013G / CXCR4-associated WM. In certain preferred embodiments, the antibody or antigen binding portion thereof binds to a human CXCR4 receptor expressed on the surface of a cell, such as a human CXCR4 receptor expressing on the surface of a cell carrying a C1013G mutation. In certain embodiments, the antibody or antigen binding portion thereof is of the IgG1, IgG2, IgG3 or IgG4 isotype. In certain other embodiments, the antibody or antigen binding portion thereof is of the IgG1 isotype. In other embodiments, the antibody or antigen binding portion thereof is of the IgG4 isotype. In further embodiments, the antibody or antigen binding portion thereof is a monoclonal antibody or antigen binding portion thereof. In still certain embodiments, the antibody or antigen binding portion thereof is a chimeric, humanized or human antibody or antigen binding portion thereof. Preferably, the antibody or antigen binding portion thereof is a human antibody or antigen binding portion thereof. In a more preferred embodiment, the antibody or fragment thereof is ulocuplumab or a fragment thereof that binds CXCR4.

[0106] Importantly, it has been disclosed herein that C1013G / CXCR4 mutated WM cells are resistant to three important classes of drugs for the treatment of B cell malignancies that are being actively tested for the treatment of WM, namely, BTK inhibitors (exemplified by ibrutinib), mTOR inhibitors (exemplified by everolimus), and PI3K inhibitors (exemplified by idelalisib) (Example 4). In contrast, anti-CXCR4 antibodies were shown to be effective in inhibiting the proliferation and migration of both C1013G / CXCR4 mutated and non-mutated WM cells. These results suggest that mTOR, BTK, and PI3K inhibitors will not be able to effectively treat patients whose WM cells carry C1013G / CXCR4 mutations, and highlight the utility of anti-CXCR4 antibodies for the treatment of such C1013G / CXCR4-WM variants.

[0107] It has been shown herein that Ulocuplumab targets WM cells, regardless of their mutational status, by inhibiting activation of pro-survival signaling pathways, stimulating pro-apoptotic cascades, and inhibiting WM cell spread in vivo (Examples 5 and 6). Therefore, in certain embodiments of the disclosed methods of treatment, WM patients are screened for the presence of C1013G / CXCR4 mutations, thereby identifying patients who are unsuitable for BTK, mTOR, or PI3K inhibitor drug treatment, wherein such patients are suitable for treatment with anti-CXCR4 antibodies. Such WM patients may not be good candidates for treatment with BTK, mTOR, or PI3K inhibitor drugs, and can instead be treated with the anti-CXCR4 antibodies disclosed herein as first-line monotherapy. In certain preferred embodiments of the present method, the anti-CXCR4 antibody is ulocuplumab. In other embodiments, patients with C1013G / CXCR4-WM are treated with anti-CXCR4 antibodies such as ulocuplumab and anti-WM agents such as ibrutinib, everolimus, idelalisib, carfilzomib, and / or bortezomib. In further embodiments, C1013G / CXCR4-WM patients are treated with an anti-CXCR4 antibody, alone or in combination with another anti-WM agent, as a second or third line therapy after the patient has failed one or more treatments. For example, an anti-CXCR4 antibody can be administered after a WM patient has failed first-line treatment with ibrutinib, or the antibody can be administered after a patient has failed first-line treatment with a chemotherapy BDR (bortezomib, dexamethasone, and rituximab) or RCD (cyclophosphamide, dexamethasone, and rituximab) regimen and a second line of treatment with ibrutinib.

[0108] Anti-CXCR4 antibodies for monotherapy in Waldenstrom's macroglobulinemia Exposure of WM cells to BMS-936564 resulted in an increase in caspase-9 and PARP cleavage (Example 5). Neutralization of CXCR4 with BMS-936564 also increased the levels of phospho-(p)-GSK3-β and upregulated p-β-catenin, leading to β-catenin degradation. BMS-936564 similarly increased caspase-9 cleavage and PARP cleavage in C1013G / CXCR4-associated WM cells and modulated GSK3-β / β-catenin signaling, leading to upregulation of p-GSK3-β / p-β-catenin and β-catenin degradation (Example 6). These effects of BMS-936564 are consistent with activation of apoptotic pathways in WM and C1013G / CXCR4-associated WM cells, and reflect the pro-apoptotic effects of BMS-936564 demonstrated in other B-cell malignancies (WO 2013 / 071068; Kuhne et al., 2013). Thus, in certain embodiments of these methods for treating WM patients, anti-CXCR4 antibodies or fragments thereof induce apoptosis of cells expressing CXCR4 or C1013G / CXCR4.

[0109] The apoptotic effects of the disclosed anti-CXCR4 antibodies, a property not exhibited by small molecule CXCR4 antagonists such as AMD3100, suggest that these antibodies may be used alone as monotherapy to treat patients with cancer. Previous studies of the effects of CXCR4 antagonists in in vivo AML and MM tumor models have demonstrated that these antagonists are effective in enhancing the sensitivity of tumor cells to chemotherapy (Azab et al., 2009; Zeng et al., 2009). In addition, the data presented in Example 6 herein demonstrate that ulocuplumab (administered as a monotherapy) inhibits the spread of WM in mice injected with C1013G / CXCR4 WM cells, as demonstrated by the reduction of CXCR4 in the femur, liver, kidney, and lung in treated mice compared to control antibody treated mice. + / CD20 + Similarly, Example 7 shows that ulocuplumab inhibits the spread of wild-type WM cells in vivo. These results are consistent with the significant tumor growth inhibition achieved when ulocuplumab was administered as a monotherapy in a variety of AML, NHL, and MM models (WO 2013 / 071068; Kuhne et al., 2013).

[0110] Thus, in certain embodiments of the present treatment methods for treating wild-type WM or C1013G / CXCR4-associated WM, the anti-CXCR4 antibody or fragment thereof is administered as a monotherapy. In preferred embodiments, the antibody or fragment thereof induces apoptosis of cells expressing CXCR4 or C1013G / CXCR4. Thus, the present disclosure also provides a method of inducing apoptosis of WM cells expressing CXCR4 or C1013G / CXCR4, the method comprising administering to a WM subject a therapeutically effective amount of an anti-CXCR4 antibody of the present disclosure.

[0111] Combination of anti-CXCR4 and anticancer therapy for the treatment of Waldenstrom's macroglobulinemia As described in Example 7, each of BMS-936564 and bortezomib significantly reduced the levels of tumor cells and serum IgM in the BM. However, the combination of BMS-936564 and bortezomib acted synergistically to cause even more significant reductions in tumor cells and serum IgM levels. These results are consistent with previous demonstrations that the CXCR4 / CXCL12 axis plays a major role in the return and trafficking of MM cells to the BM and that disruption of the interaction of MM cancer cells with the BM sensitizes these cells to therapeutic agents (Alsayed et al., 2007; Azab et al., 2009; Kuhne et al., 2013; WO 2013 / 071068).

[0112] In certain embodiments of the present method, the anti-CXCR4 antibody or antigen-binding fragment thereof binds to the CXCR4 receptor and inhibits the activity of the receptor. This disrupts the homing and maintenance of WM stem cells in the BM microenvironment and / or increases the mobilization of WM cells from the BM to the periphery, thereby increasing the sensitivity of WM cancer cells to one or more anti-cancer treatments such as radiation therapy or administration of chemotherapeutic agents.

[0113] Thus, in certain embodiments, the anti-CXCR4 antibodies of the present disclosure are used in combination with other cancer treatments such as surgery and / or radiation, and / or may be co-administered with one or more other therapeutic agents, such as cytotoxic agents, radiotoxic agents, or immunosuppressive agents, which enhance or increase the therapeutic effect of the anti-CXCR4 antibodies. The antibody may be linked to the agent (as an immunoconjugate) or may be administered separately from the agent. In the latter case (separate administration), the antibody may be administered before, after, or simultaneously with the agent, or may be co-administered with other known therapeutic agents, including conventional chemotherapeutic drugs and antibodies that bind to tumor-associated antigens or immunomodulatory targets.

[0114] Anticancer drugs used in these methods include, in particular, bortezomib, carfilzomib, ibrutinib, idelalisib, panobinostat, dexamethasone, rituximab, thalidomide, cyclophosphamide, lenalidomide, doxorubicin, vincristine, prednisone, fludarabine, chlorambucil, bendamustine, cladribine, alemtuzumab, ofatumumab, everolimus, cisplatin, bleomycin sulfate, carmustine, mitoxantrone, etoposide, cytarabine, ifosfamide, carboplatin and etoposide. It is confirmed herein that C1013G / CXCR4 mutated and unmutated WM cells are equally sensitive to proteasome inhibitors carfilzomib and bortezomib (Example 4). In addition, the combination of ulocuplumab and bortezomib synergizes in reducing WM tumor cells in BM and reducing serum IgM levels in vivo and inducing toxicity in WM cells in vitro (Example 7). In summary, these data strongly suggest that the combination regimen of ulocuplumab with a proteasome inhibitor such as bortezomib or carfilzomib is highly effective in treating WM patients carrying wild-type CXCR4 or C1013G / CXCR4 somatic variants. Co-administration of the anti-CXCR4 antibodies or antigen-binding fragments thereof of the present disclosure with an anti-tumor agent provides at least two anti-cancer agents that operate via different mechanisms to exert cytotoxic effects on human tumors. In addition to possible synergistic interactions of the anti-CXCR4 antibodies and other agents, such co-administration can help overcome problems arising from the development of resistance to the drug or changes in the antigenicity of the tumor cells that would render them unresponsive to treatment with the anti-CXCR4 antibodies.

[0115] Therefore, the anti-CXCR4 antibodies of the present disclosure can be used to act as chemotherapeutic agents through their ability to release malignant tumor cells from the protective environment of the BM. As described above, in addition to mobilizing WM cells and increasing their chemosensitization, these antibodies have been shown to have the additional effect of directly killing MM cells through apoptosis and are therefore effective in treating WM, including C1013G / CXCR4-related WM, when administered as a monotherapy.

[0116] dose In certain embodiments of the disclosed methods for treating WM, a therapeutically effective amount of an anti-CXCR4 antibody or an antigen-binding portion thereof comprises a dosage ranging from 0.1-20 mg / kg body weight. For example, in certain embodiments, a therapeutically effective amount of an antibody or an antigen-binding portion thereof comprises a dosage of 0.1, 0.3, 0.5, 1, 3, 5, 10 or 20 mg / kg. In a preferred embodiment, the dosage is 1, 3 or 10 mg / kg. In other embodiments, the antibody or its antigen-binding portion is administered at a dosage schedule of once a week, once every two weeks, once every three weeks or once a month. In a preferred embodiment, the antibody or its antigen-binding portion is administered at a dosage schedule of once every 7-14 days. In certain embodiments, an anti-CXCR4 antibody is administered as a monotherapy for the duration of continuous treatment. In other embodiments, an anti-CXCR4 antibody is administered weekly, as a monotherapy in cycle 1 for the first two weeks, and then combined with a chemotherapy regimen including, for example, bortezomib, dexamethasone and rituximab (BDR) or cyclophosphamide, dexamethasone and rituximab (RCD). In certain preferred embodiments, the antibody, or antigen-binding portion thereof, is administered intravenously. In other preferred embodiments, the antibody, or antigen-binding portion thereof, is administered subcutaneously.

[0117] For example, for the treatment of WM with ulocuplumab and BDR in combination, exemplary dosing regimens include: (1) ulocuplumab (1, 3, or 10 mg / kg) administered as a single 60-minute intravenous infusion on days 1, 8, 15, 22, and 29 (cycle 1) and days 1, 8, and 15 (cycle 2 and subsequent cycles); (2) bortezomib (1.3 mg / m 2 ), administered as a 3-5 second intravenous bolus on Days 15, 18, 22, and 25 (Cycle 1) and Days 1, 4, 8, and 11 (Cycle 2 and subsequent cycles); (3) dexamethasone (20 or 40 mg), administered on Days 15, 16, 18, 19, 22, 23, 25, and 26 (Cycle 1) and Days 1, 2, 4, 5, 8, 9, 11, and 12 (Cycle 2 and subsequent cycles); and (4) rituximab (375 mg / m 2 ), on Day 25 (Cycle 1) and on Day 11 (Cycle 2 and subsequent cycles) by slow intravenous infusion, e.g., over 90 minutes.

[0118] When an anti-CXCR4 antibody is used in combination therapy with other anti-WM agents, these agents are generally used at their approved or standard doses. For example, an exemplary dosing regimen with ibrutinib comprises 420 mg (three 140-mg capsules) orally once daily until unacceptable toxicity or disease progression.

[0119] In another embodiment, the dose of the anti-CXCR4 antibody is a flat-fixed dose that is fixed regardless of the patient's weight. For example, the anti-CXCR4 antibody can be administered at a fixed dose of 5, 20, 35, 75, 200, 350, 750 or 1500 mg, regardless of the patient's weight. As used herein, the terms "fixed dose," "flat dose," and "flat fixed dose" are used interchangeably and refer to a dose administered to a patient regardless of the patient's weight or body surface area (BSA). Therefore, a fixed dose or flat dose is not provided as a mg / kg dose, but rather as an absolute amount of a pharmaceutical agent (e.g., an anti-CXCR4 antibody).

[0120] Treatment of C1013G / CXCR4-associated Waldenstrom's macroglobulinemia including a patient selection step It is shown herein that the C1013G / CXCR4 mutation is an activating mutation in WM (Example 3). Despite the more aggressive phenotype of C1013G / CXCR4-mutated cells compared to wild-type WM cells, BMS-936564 was found to be equally active in targeting the adhesion, migration, and proliferation properties of CXCR4-mutated cells (Example 5). Therefore, the anti-CXCR4 antibodies of the present disclosure may have particular applicability in treating C1013G / CXCR4-associated WM, and treatment regimens may include a step for identifying patients carrying the C1013G mutation. Thus, in one aspect, the present disclosure provides a method for treating a subject with C1013G / CXCR4-associated WM, comprising (a) selecting a subject as a suitable candidate for treatment, the selection comprising: (i) providing a test tissue sample obtained from WM tissue in the subject; (ii) evaluating whether cells in the WM test tissue sample carry the C1013G mutation in the CXCR4 gene; and (iii) selecting the subject as a suitable candidate based on determining that the WM cells carry the C1013G mutation in the CXCR4 gene; and (b) administering to the selected subject a therapeutically effective amount of an antibody of the present disclosure, or an antigen-binding portion thereof, that specifically binds to a CXCR4 receptor expressed on the surface of C1013G / CXCR4-associated WM cells. In certain embodiments of the present method, the anti-CXCR4 antibody is administered in combination with an anti-WM agent such as bortezomib or carfilzomib (which is also effective for treating WM patients carrying the C1013G / CXCR4 somatic variant). In any of the present methods, the test tissue sample comprises a biopsy sample of tumor tissue or a sample of tumor cells found in the periphery.

[0121] The present disclosure also provides methods for treating a subject having C1013G / CXCR4-associated WM, the methods comprising administering to the subject a therapeutically effective amount of an antibody of the present disclosure, or an antigen-binding portion thereof, that specifically binds to a CXCR4 receptor expressed on the surface of C1013G / CXCR4-associated WM cells, the subject having been selected based on a determination that the subject's WM cells carry the C1013G mutation in the CXCR4 gene.

[0122] The present disclosure further provides a method for selecting a WM patient for treatment with an antibody of the present disclosure, or an antigen-binding portion thereof, that specifically binds to a CXCR4 receptor expressed on the surface of a WM cell, the method comprising: (a) optionally providing a test tissue sample obtained from a WM tissue in a patient; (b) evaluating whether cells in the WM test tissue sample carry a C1013G mutation in the CXCR4 gene; and (c) selecting a patient for treatment based on determining that the WM cells carry the C1013G mutation in the CXCR4 gene.

[0123] In another aspect, the present disclosure provides a method for determining a treatment regimen comprising an anti-CXCR4 antibody of the present disclosure, or an antigen-binding portion thereof, for treating C1013G / CXCR4-associated WM in a subject, the method comprising: (a) optionally providing a test tissue sample obtained from a WM tissue in the subject; (b) evaluating whether cells in the WM test tissue sample carry the C1013G mutation in the CXCR4 gene; and (c) determining a treatment regimen based on determining that the WM cells carry the C1013G mutation.

[0124] In certain embodiments of the present method, the C1013G mutation is detected by polymerase chain reaction (PCR) assay, reverse transcriptase-polymerase chain reaction (RT-PCR) assay, nucleic acid hybridization assay, or by DNA sequencing. In further embodiments, the PCR assay is a real-time allele-specific PCR (AS-PCR) assay. In further embodiments, the DNA sequencing is whole genome DNA sequencing.

[0125] In any disclosed method including screening for C1013G / CXCR4 mutations in a test tissue sample, it is understood that the step of providing a test tissue sample from a patient is an optional step. That is, in certain embodiments the method includes this step, while in other embodiments, this step is not included in the method. It is also understood that in certain embodiments, the "evaluation" step of determining whether cells in a WM test tissue sample carry a C1013G mutation is performed by a modified method of determining the presence of a mutation, such as by performing a polymerase chain reaction (PCR) assay. In other embodiments, no modified method is involved and the presence of the mutation is evaluated by, for example, referring to a report of the test results from a laboratory. In certain embodiments, the steps up to and including the method of evaluating whether cells carry a C1013G / CXCR4 mutation provide an intermediate result that can be provided to a physician or other medical practitioner for selecting a suitable candidate for treatment and / or administering a therapeutic agent to a patient. In certain embodiments, the step of providing an intermediate result can be performed by a medical practitioner or someone working under the guidance of a medical practitioner. In other embodiments, these steps are performed by an independent laboratory or by an independent person such as a laboratory technician.

[0126] Another aspect of the present disclosure relates to a treatment regimen for treating a patient with C1013G / CXCR4-associated WM, the regimen being determined by the above method, the method comprising (a) optionally providing a test tissue sample of WM tissue obtained from a subject; (b) evaluating whether cells in the WM test tissue sample carry the C1013G mutation in the CXCR4 gene; and (c) determining the treatment regimen based on determining that the WM cells carry the C1013G mutation. In certain embodiments, the treatment regimen comprises administering to the patient an anti-CXCR4 antibody or antigen-binding portion thereof of the present disclosure at the following doses and frequencies: (a) at least two times, at least three times, or at least five times the dose administered in treating WM patients carrying a wild-type CXCR4 gene; and / or (b) at least two times, at least three times, or at least five times the frequency administered in treating WM patients carrying a wild-type CXCR4 gene.

[0127] Preferred anti-CXCR4 antibodies for use in treating Waldenstrom's macroglobulinemia The treatment methods disclosed herein include administering an anti-CXCR4 antibody of the present disclosure to a WM patient. The antibody specifically binds to wild-type CXCR4 or C1013G / CXCR4 variant expressed on the surface of WM cells and exhibits certain structural and functional properties that are favorable for therapeutic efficacy. For example, the desired structural feature is that the antibody comprises the CDRs or variable regions of F7 (ulocuplumab), F9, D1 or E2 (as identified in WO 2008 / 060367), or any cross-competing regions of these antibodies that bind to the same epitope region of CXCR4. The desired functional features include high affinity binding to human CXCR4 expressed on the cell surface, inhibition of CXCR4 in vivo, and the ability to bind to CXCR4. + Tumor cell proliferation and / or induction of CXCR4 + In certain preferred embodiments of the disclosed methods of treatment, the anti-CXCR4 antibody or portion thereof comprises: a CDR1, CDR2, and CDR3 domain in a heavy chain variable region comprising consecutively linked amino acids, the sequence of which is described in SEQ ID NO: 25; and a CDR1, CDR2, and CDR3 domain in a light chain variable region comprising consecutively linked amino acids, the sequence of which is described in SEQ ID NO: 29.

[0128] In other preferred embodiments, the anti-CXCR4 antibody or portion thereof comprises: a heavy chain variable region comprising consecutively linked amino acids, the sequence of which is set forth in SEQ ID NO:25; and a light chain variable region comprising consecutively linked amino acids, the sequence of which is set forth in SEQ ID NO:29.

[0129] In still other preferred embodiments, according to the description of CDR sequences by the Kabat system (Kabat et al., 1991; Johnson and Wu, 2000), the anti-CXCR4 antibody or portion thereof comprises: a heavy chain variable region CDR1 comprising consecutively linked amino acids of the sequence described in SEQ ID NO: 1, a heavy chain variable region CDR2 comprising consecutively linked amino acids of the sequence described in SEQ ID NO: 5, a heavy chain variable region CDR3 comprising consecutively linked amino acids of the sequence described in SEQ ID NO: 9, a light chain variable region CDR1 comprising consecutively linked amino acids of the sequence described in SEQ ID NO: 13, a light chain variable region CDR2 comprising consecutively linked amino acids of the sequence described in SEQ ID NO: 17, and a light chain variable region CDR3 comprising consecutively linked amino acids of the sequence described in SEQ ID NO: 21. In a more preferred embodiment, the antibody or fragment thereof is ulocuplumab or a fragment thereof that binds to CXCR4.

[0130] One aspect of the disclosed invention is the use of an anti-CXCR4 antibody, or antigen binding portion thereof, of the disclosed invention for the preparation of a medicament for treating a subject having WM or C1013G / CXCR4-associated WM. This discloses an anti-CXCR4 antibody, or antigen binding portion thereof, of the disclosed invention for use in treating a subject having WM or C1013G / CXCR4-associated WM.

[0131] Treatment kit Also within the scope of the present disclosure are kits comprising an anti-CXCR4 antibody or antigen-binding fragment or composition thereof and instructions for use (e.g., instructions including a schedule of administration that allows a medical practitioner or patient to administer the antibody or composition thereof to a WM patient). Thus, the present disclosure provides kits for treating C1013G / CXCR4-associated WM in a subject, the kits comprising (a) one or more doses of any of the anti-CXCR4 antibodies or antigen-binding portions thereof of the present disclosure and (b) instructions for using the anti-CXCR4 antibody or fragment thereof in any of the treatment methods described herein. For example, in certain embodiments, the anti-CXCR4 antibody or antigen-binding portion thereof in the kit comprises CDR1, CDR2, and CDR3 domains in the heavy chain variable region having the amino acid sequence shown in SEQ ID NO: 25, and CDR1, CDR2, and CDR3 domains in the light chain variable region having the amino acid sequence shown in SEQ ID NO: 29. In preferred embodiments, the anti-CXCR4 antibody is ulocuplumab. Optionally, the kit includes multiple packages of a single dose of a pharmaceutical composition, each containing an effective amount of an anti-CXCR4 antibody for a single administration according to the methods described herein. The kit may further contain one or more additional therapeutic agents described herein, such as a chemotherapeutic agent or a radiotoxic agent, or one or more additional antibodies targeting a different antigen. In certain embodiments, the anti-CXCR4 antibody is co-packaged with one or more other therapeutic agents in a unit dosage form.

[0132] The apparatus or equipment required for administering the pharmaceutical composition may also be included in the kit. For example, the kit may include one or more syringes. In certain embodiments, one or more syringes are pre-filled with an amount of anti-CXCR4 antibody. The kit also typically includes a label indicating the intended use of the contents of the kit together with the instructions for use. The term label includes any written or recorded material provided on or with the kit or otherwise provided with the kit.

[0133] The present invention is further illustrated by the following examples, which are illustrative only and should not be construed as limiting the scope of the present disclosure in any way, as many variations and equivalents will become apparent to those skilled in the art upon reading the present disclosure. The contents of all drawings and all references, GenBank entries, patents and published patent applications cited throughout this application are expressly incorporated herein by reference.

[0134] Example 1 Screening for C1013 / CXCR4 mutations in patients with B-cell lymphoproliferative disorders Patients with various B-cell lymphoproliferative disorders are screened for the presence of the C1013 / CXCR4 variant.

[0135] Clinical samples Tumor samples were collected from 418 patients with different B-cell lymphoproliferative disorders (B-LPDs), with the following distribution: WM (n=131); IgM MGUS (n=40); diffuse large B-cell lymphoma (n=75); splenic marginal zone lymphoma (n=14); B-cell chronic lymphocytic lymphoma (n=37, including 16 with M component); hairy cell leukemia (n=35); MM (n=36, 3 in IgM isotype); IgA / IgG MGUS (n=22); lymphoplasmacytic lymphoma without WM criteria (n=13); amyloidosis (n=6); and B-LPD not otherwise specified (n=9). Diagnosis was made according to the latest WHO classification of tumors of hematopoietic and lymphoid tissues (Campo et al., 2011). Thirty-two healthy volunteers were also included in the study as controls.

[0136] Immunophenotyping and clonal cell quantification in diagnostic tissues Immunophenotyping was routinely performed using a previously described monoclonal antibody panel (Paiva et al., 2013) and in accordance with the general recommendations of the EuroFlow group for immunophenotyping of hematological malignancies (van Dongen et al., 2012). These cases were immunophenotyped using a 4-color combination of up to 20 different antigens, including surface IgM (sIgM) and cytoplasmic Igλ and κ (cyIgλ and cyIgκ). The percentage of clonal cells was counted using a test tube including sIgM / CD25 / CD19 / CD38MoAb. Data acquisition was performed on a FACSCalibur flow cytometer (Becton Dickinson Biosciences [BDB] San Jose, CA) using FACSDiva software (version 6.1; BDB) and a two-step acquisition program for all events and only CD19+-events. Data analysis was performed using Infinicyt software (Cytognos; Salamanca, Spain). After immunophenotyping and clonal cell quantification in diagnostic tissue, detection of the C1013G / CXCR4 variant was performed using real-time allele-specific PCR (AS-PCR). Ten cases of extramedullary WM were also included in these studies. Permission was obtained for these studies from the Dana-Farber Cancer Institute and the University Hospital of Salamanca Institutional Review Boards. Informed consent was provided in accordance with the Declaration of Helsinki.

[0137] Real-time AS-PCR for identification of CXCR4C1013G mutation A real-time AS-PCR assay was developed based on the use of two reverse primers that differ in the last two nucleotides (at the 3' position) so that each primer is specific for either the wild-type or mutant allele, as described (Paiva et al., 2013). To prevent amplification of unmatched primers (thereby increasing specificity), additional nucleotide mismatches (C>G) were introduced immediately adjacent to the mutant nucleotide. The specific length and position of the primers were calculated using Oligo 6.0 software (Molecular Biology Insights, Cascade, CO). The reverse primers were: 5'-GACTCAGACTCAGTGGAAACAGATG-3' (reverse wild-type primer) and 5'-GACTCAGACTCAGTGGAAACAGAAC-3' (reverse mutant primer). The universal forward primer was 5'-TTTCTTCCACTGTTGTCTGAACC-3', which was used to generate a 165 bp PCR product. Software v3.0.1 (Applied Biosystems, Foster City, CA, USA) was used to design specific Probe, which produces the following reporter, sequence and quencher: 6FAM: 5'-TATGCTTTCCTTGGAGCCA-3': NFQ-MGB.

[0138] For real-time AS-PCR development, two different PCR reactions were required for each experiment: one for detecting the CXCR4C1013G mutation (using the mutant reverse primer) and another as a control for DNA quality (using the wild-type reverse primer). Each reaction was performed in a final volume of 20 μl, containing 300 nM of each primer, 200 nM of the probe, 1x Universal PCR Master Mix (Applied Biosystems, Foster City, CA, USA) and 20 ng of genomic DNA. Real-Time PCR System (Applied Biosystems) was performed and consisted of an initial denaturation step at 95°C for 10 min, followed by 50 cycles of 95°C for 15 s and 60°C for 60 s. TM Data were analyzed using ELISA® Software v2.1 (Applied Biosystems), which provides CT values ​​considered to be the cycle where fluorescence begins to differ from background.

[0139] result Presence of the C1013G / CXCR4 variant in patients with WM The C1013G / CXCR4 mutation was detected in 28.2% of WM patients (37 / 131) and in 20% of IgM-MGUS patients (8 / 40). See Table 1. In addition, the variant was detected in 7% of patients with splenic marginal zone lymphoma (1 / 14) and 1.3% of patients with diffuse large B-cell lymphoma (1 / 75), while it was absent in B-cell chronic lymphocytic leukemia, hairy cell leukemia, MM and IgG / IgA MGUS (Table 1), indicating the potential role of this variant in supporting the molecular pathogenicity of WM and the precursor stage of IgM-MGUS. The C1013G / CXCR4 variant causes a single nucleotide change C→G in CXCR4, resulting in a predicted stop codon replacing serine at amino acid position 338 (S338X). This has been previously linked to truncation of the C-terminal domain of CXCR4 (Alapi K et al., 2007), resulting in its impaired intracellular translocation.

[0140] To further confirm that the observed variant may indeed induce CXCR4 alterations on the surface of WM cells, CXCR4 surface expression was evaluated on independent primary WM patient BM-derived CD191 cells carrying the variant (n=3) or not carrying the variant (n=8), and higher surface expression was found in patients carrying the C1013G / CXCR4 variant compared to wild-type / CXCR4 patients ( Figure 2 In addition, 10 patients with a confirmed histological diagnosis of extramedullary WM were also evaluated for the presence of the somatic variant: patients with lung lesions (3 / 3) and kidney lesions (1 / 1) had the mutated C1013G / CXCR4, but this mutation was absent in the small intestine (2 / 2) and soft tissues (4 / 4) as detected by AS-PCR on genomic DNA (Table 2), suggesting that this mutation may be more prevalent in patients with extramedullary involvement.

[0141] Table 1: C1013G / CXCR4 somatic mutations in patients with WM

[0142] The C1013G / CXCR4 variant is present in 28% of patients with WM and is either absent or present in a minority of patients with B-cell lymphoproliferative disorders.

[0143] B-CLL, B-cell chronic lymphocytic leukemia; MGUS, monoclonal gammopathy of undetermined significance; NOS, not otherwise specified.

[0144] Table 2: The C1013G / CXCR4 variant is present in lung and kidney tissues of patients with extramedullary WM disease

[0145] Example 2 Effects of CXCR4 overexpression on the growth and spread of WM cells To address the in vivo functional relevance of the C1013G / CXCR4 variant in supporting WM biology, we overexpressed CXCR4 (GFP + ) of WM cells (CXCR4 + ) first provides an exploration of the role of CXCR4 in WM in vivo.

[0146] Immunohistochemistry Murine tissues (BM, liver, kidney, lung and lymph nodes) were analyzed and the expression of human CD20 and human CXCR4 was quantified. The tissues were fixed with formalin 10% and ethanol 70% was used as a storage solution; and embedded in paraffin. Paraffin sections were incubated with anti-human CXCR4 (Abcam, Cambridge, MA) and anti-human CD20 (Dako, Carpinteria, CA) monoclonal antibodies using the Leica Bond III automated staining platform (Leica Biosystems, Buffalo Grove, IL). Secondary antibodies were provided as part of the Bond III Refine Detection Kit (Leica Biosystems) and sections were incubated with diaminobenzidine (DAB)-peroxidase substrate provided in the Bond III Refine Detection Kit, counterstained with hematoxylin-eosin, dehydrated, mounted and digitally photographed (Nikon Eclipse 80i, Nikon, Melville, NY). Quantification of CD20 and CXCR4 staining was performed using NIS Elements software (Nikon, Melville, NY).

[0147] Tissue immunofluorescence Tissue immunofluorescence imaging was performed to evaluate overexpression of GFP in femurs, livers, and kidneys harvested from mice. + / CXCR4 WM cells or RFP + / scrambled WM cells as described (Hsieh et al., 2012). Target cells were BCWM.1-GFP+ or BCWM.1-RFP+. Nuclear dye DAPI was added to each slide. Slides were analyzed using a fluorescence microscope (NikonEclipse 80i; objective 40X plan fluor 0.75NA). MM.1S-GFP+ cells were counted from 4 separate areas / slide. Images were acquired using a Hamamatsu OrcaER camera and NISElement software. Two different channels were merged using Image J.

[0148] ELISA Human serum IgM levels were determined by using a human IgM ELISA assay (ZeptoMetrix, Buffalo, NY) according to the manufacturer's protocol.

[0149] In vitro cell adhesion, migration and proliferation studies Adhesion of WM cells to primary WM BM-MSCs was assessed by an in vitro adhesion assay using calcein AM-labeled MM cells. The degree of fluorescence was measured using a spectrophotometer (485-520) as previously described (Roccaro et al., 2010). Adhesion of WM cells to fibronectin was assessed by using an in vitro adhesion assay with fibronectin according to the manufacturer's recommendations (EMD Biosciences, San Diego, CA), as described (Roccaro et al., 2013). Migration of WM cell lines toward SDF-1α or primary WM BM-MSCs was assessed using transwell migration plates (Corning Life Sciences, Tewksbury, MA), as previously described (Roccaro et al., 2010; Sacco et al., 2011; Azab et al., 2012). WM cell proliferation was assessed by evaluating DNA synthesis after 48 h of co-culture with primary BM-MSCs, as measured by [ 3 H]-thymidine ([ 3 H]-TdR; Perkin Elmer, Boston, MA) uptake was measured as previously described (Roccaro et al., 2010; Sacco et al., 2011; Azab et al., 2012). Cytotoxicity was assessed by measuring 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (Chemicon International, Temecula, CA) as described (Roccaro et al., 2010; Sacco et al., 2011; Azab et al., 2012).

[0150] Loss- and gain-of-function studies CXCR4 gain or loss of function studies have been performed using WM cell lines (BCWM.1; MWCL1) either alone or in the context of primary WM BM-MSCs, as previously described (Roccarro et al., 2010; Sacco et al., 2011). Briefly, CXCR4 was knocked down in WM cells using short hairpin RNA (shRNA); clones 171, 649) and lentiviral-mediated infection; scrambled probes were used as controls containing target sequences (clones 171, 649) or scrambled controls, according to the manufacturer's instructions (ThermoScientific, Waltham, MA). Transduction efficiency was verified by quantitative reverse transcription PCR (qRT-PCR) and by evaluating the detection of cells expressing GFP using a fluorescence microscope (Nikon Eclipse 80i, Nikon, Melville, NY).

[0151] CXCR4 overexpression in WM cells using precision LentiORF / CXCR4 / GFP (CXCR4 + ) or scrambled probe / RFP (Thermo Scientific) used as a control. Overexpression efficiency was confirmed by qRT-PCR and by detecting cells expressing GFP and RFP using a fluorescent microscope (Nikon Eclipse 80i, Nikon, Melville, NY).

[0152] statistics Certain statistical methods described are also used in other embodiments. The P values ​​provided by in vitro assays are based on T tests (two-tailed; α 0.05) or ANOVA. Each picture provides a P value. Drug synergy was analyzed by isobologram analysis using CalcuSyn software program (Biosoft, Ferguson, MO), as described in 25-27. Kaplan-Meier curves were obtained using GraphPadPrism and P values ​​were calculated based on log-rank tests (Roccaro et al., 2010; Sacco et al., 2011; Roccaro et al., 2008).

[0153] result Functional relevance of CXCR4 in WM in vivo CXCR4 + / GFP + Cells and empty vector / RFP + The cells were injected into SCID / Bg mice. The injection efficiency was measured by qRT-PCR ( Figure 3 ) and immunofluorescence imaging (data not shown; Roccaro et al., 2014). + / GFP + Cells and empty vector / RFP + The cells were injected into SCID / Bg mice. Mice were euthanized after 3 weeks due to disease progression, demonstrating that overexpression of CXCR4 in WM cells resulted in a more aggressive phenotype, as shown by the expression of CXCR4 in mice compared to mice injected with empty vector cells. + Significantly higher damage to organs in cell-injected mice (P < 0.01; Figure 4A -C).

[0154] In vitro and in vivo studies have confirmed that CXCR4 + WM cells spread and proliferated faster in the BM and other organs including the liver and kidney ( Fig. 4 ; Figure 5A-C ). These findings were also confirmed by immunofluorescence imaging, which showed that compared with RFP + Cells, GFP + Higher wetting, RFP +Cells and GFP + The cells represent empty vector-infected cells and CXCR4 + Importantly, in cells carrying CXCR4 + Decreased survival was observed in mice injected with cells compared with mice injected with empty vector cells (P = 0.02; Figure 5D ). In addition, compared with uninjected or empty vector-injected mice, mice injected with CXCR4 + Mice with β-cells had increased serum IgM secretion (P = 0.02; Figure 4D ). In vitro and in vivo CXCR4 + The presence of cells was also determined by the presence of + qRTPCR was performed on BM cells from the femurs of mice injected with either cells or empty vector to confirm ( Figure 4E ).

[0155] Since the in vitro sequelae of CXCR4 gain of function in WM cells were also investigated by comparing CXCR4 + Cells and CXCR4 knockdown cells were described. Infection efficiency was measured at the mRNA level ( Figure 6 ) and confirmed by immunofluorescence imaging (data not shown; Roccaro et al., 2014). The ability of WM cells to adhere to primary WM BM stromal cells (BM-MSCs) and proliferate when in a BM-MSC context was shown to be expressed in CXCR4 + This was enhanced in WM cells and suppressed in WM cells with CXCR4 knockdown ( Figure 5E , F). Similarly, CXCR4 overexpression resulted in increased adhesion of WM cells to fibronectin, in contrast to CXCR4-silenced cells, which had suppressed WM cell adhesion to fibronectin ( Fig. 7A , B). Taken together, these findings suggest that CXCR4 is critical for promoting WM cell growth and spread in vivo.

[0156] Example 3 The role of C1013G / CXCR4 mutation in the pathogenesis of WM This study addressed the question of whether the C1013G / CXCR4 variant actively supports WM pathogenicity leading to disease progression.

[0157] C1013G / CXCR4 mutagenesis C1013G / CXCR4 variants were generated in WM cell lines (BCWM.1; MWCL1) by site-directed mutagenesis using the QuickChange XL site-directed mutagenesis kit (Stratagene, Agilent Technologies, Santa Clara, CA) according to the manufacturer's instructions. Cells infected with the control vector were considered controls and are described herein as "control cells." Detection of C1013G / CXCR4 in WM infected cells was evaluated by performing allele-specific PCR on genomic DNA or cDNA isolated from infected cells. (Forward primer: 5'-TTTCTTCCACTGTTGTCTGAACC-3'; Reverse wild-type primer: 5'-GACTCAGACTCAGTGGAAACAGATG-3'; Reverse mutation primer: The data were further confirmed by DNA sequencing.

[0158] Gene expression studies Gene expression profiling was performed on C1013G / CXCR4-mutated BCWM.1 cells using HG-U133 plus 2 assays (GSE50683). RNA was isolated using the RNeasy kit (Qiagen). Data were normalized using d-Chip software. Differentially expressed gene tags were analyzed using Gene Set Enrichment Analysis (GSEA) as previously reported (Subramanian et al., 2005). Data sets were downloaded from publicly available sources (Broad Institute, Cambridge, MA).

[0159] result Stable C1013G-mutated WM cells (C1013G / CXCR4-WM) were generated and control vector-infected WM cells were used as controls. The mutagenesis efficiency was confirmed at the genomic DNA and complementary DNA (cDNA) levels by quantitative PCR (qPCR) and qRT-PCR, respectively, and further verified by Sanger DNA sequencing ( Fig. 8A -C).

[0160] The in vivo phenotype induced by WM cells carrying the mutation was then examined. Mice injected with C1013G / CXCR4-WM cells had significant tumor cell dissemination to distal organs and increased serum IgM secretion ( Fig.8D ), thus recapitulating the effects of injection of cells with overexpression of CXCR4 +The effects observed in mice injected with WM cells of C1013G / CXCR4-WM cells were consistent with those observed in mice injected with WM cells of C1013G / CXCR4-WM cells. In addition to the confirmed lesions in the liver, kidneys, and BM, mice injected with C1013G / CXCR4-WM cells also had enlarged lymph nodes and lung lesions. No spread to the central nervous system was observed. Histopathological analysis showed the presence of CXCR4 and CD20 positive cells in all tissues examined, with the exception of the brain ( Fig. 8E ; Figure 9A-E ), and the CXCR4 and CD20 positivity rates were higher in C1013G / CXCR4-WM cell-injected mice compared with those in control cell-injected mice (P < 0.05; Fig.10 Importantly, mice injected with C1013G / CXCR4-WM cells had decreased overall survival (P = 0.003; Fig.9F ).

[0161] Further characterization of C1013G / CXCR4-WM cells (BCWM.1) in vitro showed increased adhesion and cell proliferation in the presence of primary WM BM-MSCs, thus confirming that the C1013G / CXCR4 variant is able to reproduce the effects of CXCR4 overexpression in WM cells ( Fig.11A , B). Similar findings were confirmed using different WM cell lines (MWCL1; Fig. 11C , D).

[0162] Table 3: Upregulated genes in C1013G / CXCR4-WM patients

[0163] Upregulation of genes in WM patients carrying C1013G / CXCR4 mutation (n=10) compared to CXCR4 / wild-type WM patients (n=30).

[0164] To better explain the ability of the C1013G / CXCR4 variant to promote the spread of WM cells in vivo, the mutant cells were characterized at the mRNA level. Using GSEA, genes involved in invasiveness, cell proliferation, anti-apoptosis and tumorigenicity (Rizki A et al., 2008; Wang et al., 2007) were enriched in C1013G / CXCR4-WM cells compared to cells infected with the WM control vector, which is consistent with the more invasive phenotype of the mutant cells compared to the control cells ( Fig. 12A These in vivo observations together with the changes observed at the mRNA level suggest that the C1013G / CXCR4-WM variant functions as an activating mutation in WM cells.

[0165] WM patients were profiled at the mRNA level using qRT-PCR, and several genes involved in the mitogen-activated protein kinase BTK and PI3K pathways, which are known to support WM biology (Chng et al., 2008; Gutiérrez et al., 2007), were found to be significantly upregulated in C1013G / CXCR4 mutant WM patients (n=10) compared to CXCR4 / wild-type WM patients (n=30) (Table 3). Importantly, those changes were also enriched in C1013G / CXCR4 mutant BCWM1 cells compared to control vector-infected cells, thus further confirming that C1013G / CXCR4 engineered BCWM.1 cells are representative of the results observed in primary WM tumor cells ( Fig. 12B ).

[0166] Example 4 Resistance of the C1013G / CXCR4-WM variant to conventional anti-WM agents The sensitivity of WM cells carrying the C1013G / CXCR4 variant to commonly used anti-WM small molecules was investigated.

[0167] Materials and methods Reagents Everolimus, idelalisib, ibrutinib, bortezomib, and carfilzomib were purchased from Selleck Chemicals (Houston, TX). Drugs were dissolved in dimethyl sulfoxide and then diluted in cell culture medium (10% fetal bovine serum) immediately before use. The maximum final concentration of dimethyl sulfoxide (0.1%) did not affect cell proliferation and did not induce cytotoxicity to the cell lines tested.

[0168] BMS936564 / MDX-1338 and control human IgG4 isotype were obtained from Bristol-Myers Squibb (Redwood City, CA).

[0169] Gene set enrichment analysis GSEA was performed as described in Example 3.

[0170] result GSEA studies showed that genes involved in drug resistance were enriched in WM cell lines carrying mutations, while genes involved in drug responsiveness were enriched in WM control cells ( Fig.13A This has prompted the study of whether novel anti-WM agents can alter their antitumor efficacy in the context of CXCR4-mutated WM cells. WM cells carrying C1013G / CXCR4 somatic mutations were found to be resistant to BTK as well as PI3K and mTOR inhibitors ( Fig. 13B-D). These data support recent data showing that WM patients with WHIM-like mutations in CXCR4 are resistant to ibrutinib-based therapy (Cao et al., 2013; Treon et al., 2013). Importantly, proteasome inhibitors were equally effective in exerting toxicity against C1013G / CXCR4 mutant WM cells and control cells ( Fig.13E -F).

[0171] Example 5 Targeting WM cells by the anti-CXCR4 antibody BMS-936564 Based on the biological relevance of CXCR4 supporting WM progression, the activity of anti-CXCR4 human monoclonal antibody BMS-936564 (designated F7 in WO 2008 / 060367; also known as ulocuplumab or MDK-1338) targeting WM cells was tested. Assays to measure adhesion, migration and proliferation of WM cells were performed as described in Example 2, and gene expression studies were performed as described in Example 3.

[0172] Immunoblotting Protein lysates were obtained from WM cells by homogenization under non-denaturing conditions in lysis buffer supplemented with 5 mM NaF, 2 mM Na3VO4, 1 mM PMSF (polymethilsulfonyl fluoride), 5 μg / mL leupeptine, and 5 μg / mL aprotinin. Whole cell lysates were subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to polyvinylidene fluoride (PVDF) membranes (Bio-Rad Laboratories, Hercules, CA). Antibodies used for immunoblotting included: anti-phospho (p)-ERK, anti-ERK, anti-p-Akt, anti-Akt, anti-p-Src, anti-Src, anti-caspase-9, anti-PARP, anti-p-β-catenin, anti-β-catenin, anti-p-GSK3, anti-GAPDH, and anti-α-tubulin antibodies (Cell Signaling, Danvers, MA).

[0173] result BMS-936564 was shown to inhibit the migration of WM cell lines towards primary WM BM-MSCs or SDF-1 ( Fig.14A , B).

[0174] Similarly, BMS-936564-dependent inhibition of WM cell adhesion to WM BM-MSCs and inhibition of WM cell proliferation in the presence of WM BM-MSCs was also demonstrated ( Fig.15A -C).

[0175] To resolve the possible role of BMS-936564 in affecting WM signaling, WM cells were cultured in the absence or presence of primary WM BM-MSCs with increasing concentrations of anti-CXCR4 antibodies. Drug-dependent inhibition of p-Akt, p-ERK, and p-Src was observed in WM cells cultured in the context of BM-MSCs, indicating the ability of BMS-936564 to target WM cells even in the context of a BM environment ( Fig.16 ).

[0176] Isotype control had no effect on the adhesion and migration of WM cells in vitro ( Fig.17A , B).

[0177] BMS-936564 can exert pro-apoptotic effects in B-cell malignancies (WO 2013 / 071068; Kuhne et al., 2013). Thus, WM cells were exposed to BMS-936564, which resulted in an increase in caspase-9 and PARP cleavage ( Fig.18 ).

[0178] The interaction between SDF1 / CXCR4 and β-catenin has been shown before in models of metastatic solid tumors (Wang et al., 2011). In addition, phosphorylated GSK3β is known to promote β-catenin degradation (Wang et al., 2011; Polakis, 2001). The effect of BMS-936564 on β-catenin was therefore studied. It was found that by neutralizing CXCR4 with BMS-936564, WM cells had increased phosphorylation (p)-GSK3-β and upregulation of p-β-catenin, leading to β-catenin degradation (Figure 19). These findings can (at least partially) explain the possible mechanism of apoptosis induced by BMS-936564 in WM cells.

[0179] Example 6 Effects of BMS-936564 on C1013G / CXCR4-WM cells in vitro and in vivo In vivo studies SCID / Bg mice (n=5 / group) were injected with precise LentiORF / CXCR4 / GFP (CXCR4 +) or BCWM.1 cells infected with empty vector probe / RFP (control). After 3 weeks, the mice were euthanized and the organs were harvested. The transplanted organs were stained with hematoxylin-eosin and immunohistochemistry for human CD20 and CXCR4. Independent experiments were performed to evaluate survival differences (SCID / Bg mice, n=7 / group). Similar studies were performed using cells with C1013G / CXCR4 mutation and cells infected with control vector (control cells). Using BCWM.1-mCherry + Cells were subjected to in vivo homing studies: cells were injected intravenously into SID / Bg mice and treated with BMS-936564 or control antibody (10 mg / kg, intraperitoneally; 3-4 times per week for 3 weeks). The ability of BMS-936564 to modulate WM homing to BM, spleen, and lymph nodes was evaluated ex vivo on transplanted tissues using a fluorescence microscope (Nikon Eclipse 80i, Melville, NY) at week 3 (Roccaro et al., 2013).

[0180] The activity of BMS-936564 was tested in SCID / Bg mice injected with C1013G / CXCR4 mutant cells. Mouse controls were treated with isotype control antibody (n=5 mice / group; BMS-936564 or control antibody (10 mg / kg, intraperitoneal; 3-4 times per week for 3 weeks).

[0181] result The effect of BMS-936564 on CXCR4-mutant WM cells was examined in vivo. CXCR4 was expressed in the femur, liver, kidney, and lung of treated mice compared to mice treated with a control antibody. + / CD20 + BMS-936564 inhibited WM proliferation in mice injected with C1013G / CXCR4 WM cells, as shown by a significant reduction in cell infiltration ( Fig.19A -E; Fig. 20A -D). Importantly, lymph node lesions were absent in mice treated with BMS-936564 ( Fig.20E ).

[0182] These findings were further confirmed in vitro. BMS-936564 was found to be equally active in targeting the adhesion, migration, and proliferation properties of CXCR4-mutated cells in the context of the BM microenvironment ( Fig.21A -D).

[0183] Importantly, BMS-936564 increased caspase-9 cleavage and PARP cleavage in CXCR4-mutant WM cells; and also modulated GSK3-β / β-catenin signaling, leading to upregulation of p-GSK3-β / p-β-catenin and β-catenin degradation ( Fig. 22 ). In addition, BMS-936564 induced a dose-dependent inhibition of p-ERK, p-Akt, and p-Src in WM cells carrying the C1013G / CXCR4 mutation ( Fig. 22 ).

[0184] Example 7 Effects of BMS-936564 on wild-type WM cells in vivo The anti-WM activity of BMS-936564 was also tested in vivo using wild-type WM cells. + Cells were injected intravenously into SCID / Bg mice and mice were treated with BMS-936564 or control antibody. Immunofluorescence imaging of harvested BM, spleen, and lymph nodes confirmed the ability of BMS-936564 to inhibit WM cell spread ex vivo (data not shown; Roccaro et al., 2014).

[0185] Since CXCR4 inhibition is thought to induce mobilization of stem cells and tumor cells, it was hypothesized that BMS-936564 might also induce mobilization of WM cells, leading to increased chemosensitivity to anti-WM agents. Therefore, the effect of BMS-936564 as monotherapy or in combination with bortezomib in regulating tumor progression and dissemination of WM cells to the distant BM environment in vivo was evaluated. (Bortezomib was obtained from Hospital Pharmacy, diluted in DMSO and stored at -20°C until use, and then diluted in culture medium just before use. The maximum final concentration of DMSO (<0.1%) did not affect cell proliferation and did not induce cytotoxicity to the cell lines tested (data not shown).) BMS-936564-treated mice had a significant reduction in tumor cells in the BM, with similar effects observed in bortezomib-treated mice, and more significant inhibition in mice treated with the combination of BMS-936564 and bortezomib (P < 0.05; Table S1). Fig.23A Similarly, serum IgM levels were significantly reduced when mice were treated with BMS-936564 or bortezomib, but were even more significantly reduced in mice exposed to both BMS-936564 and bortezomib (P<0.05; Fig. 23B Further examination of the combined effect of BMS-936564 and bortezomib in inducing toxicity in WM cells confirmed a synergistic interaction in vitro ( Fig.23C , D). References Alapi K et al. (2007) Recurrent CXCR4 sequence variation in a girl with WHIM syndrome. Eur J Haematol. 78(1): 86-8. Alsayed Y et al. (2007) Mechanisms of regulation of CXCR4 / SDF-1(CXCL12)-dependent migration and homing in multiple myeloma. Blood 109(7): 2708-17. Azab AK et al. (2009) CXCR4 inhibitor AMD3100 disrupts the interaction of multiple myeloma cells with the bone marrow microenvironment and enhances their sensitivity to therapy. Blpod 113: 4341-51. Azab F et al. (2012) Eph-B2 / ephrin-B2 interaction plays a major role in the adhesion and proliferation of macroglobulinemia. 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Claims

1. A pharmaceutical composition for treating C1013G / CXCR4-associated Waldenstrom's macroglobulinemia in a subject, the composition comprising an antibody or an antigen-binding portion thereof that specifically binds to a CXC chemokine receptor type 4 (CXCR4) receptor expressed on the surface of Waldenstrom's macroglobulinemia cells carrying the C1013G / CXCR4 mutation.

2. The pharmaceutical composition of claim 1, wherein The subject is a human and the antibody or antigen-binding portion thereof binds to a human CXCR4 receptor expressed on a cell surface.

3. The pharmaceutical composition of claim 1 or 2, wherein: (a) the antibody or antigen-binding portion thereof is of IgG1 or IgG4 isotype; (b) the antibody or antigen-binding portion thereof is a monoclonal antibody or antigen-binding portion thereof; (c) the antibody, or antigen-binding portion thereof, is a chimeric, humanized or human antibody, or antigen-binding portion thereof; and / or (d) the subject is a human and the antibody or antigen-binding portion thereof is a human antibody or antigen-binding portion thereof.

4. The pharmaceutical composition of any one of claims 1 to 3, wherein the antibody or antigen-binding portion thereof induces apoptosis of cells expressing C1013G / CXCR4.

5. The pharmaceutical composition of any one of claims 1-4, wherein the treatment comprises administering the antibody or antigen-binding portion thereof to the subject as a monotherapy.

6. The pharmaceutical composition of any one of claims 1 to 4, wherein the antibody or antigen-binding portion thereof inhibits the activity of the CXCR4 receptor and increases the sensitivity of WM cells to anticancer agents.

7. The pharmaceutical composition of claim 6, wherein the treatment further comprises administering at least one anti-cancer agent in combination with the antibody or antigen-binding portion thereof.

8. The pharmaceutical composition of claim 7, wherein the at least one anticancer agent is rituximab, bortezomib, carfilzomib, ibrutinib, idelalisib, panobinostat, dexamethasone, rituximab, thalidomide, cyclophosphamide, lenalidomide, doxorubicin, vincristine, prednisone, fludarabine, chlorambucil, bendamustine, cladribine, alemtuzumab, ofatumumab, everolimus, cisplatin, bleomycin sulfate, carmustine, mitoxantrone, etoposide, cytarabine, ifosfamide, carboplatin and / or etoposide.

9. The pharmaceutical composition of claim 7, wherein the at least one anticancer agent is ibrutinib.

10. The pharmaceutical composition of claim 9, wherein the treatment further comprises administering oral ibrutinib at a dose of 420 mg once daily.

11. The pharmaceutical composition of claim 7, wherein the at least one anticancer agent is bortezomib or carfilzomib.

12. The pharmaceutical composition of any one of claims 1-11, wherein the treatment comprises administering the antibody, or antigen-binding portion thereof, to the subject after failure of prior treatment for Waldenstrom's macroglobulinemia, optionally wherein the prior treatment was with an agent selected from a mammalian target of rapamycin (mTOR) inhibitor, a Bruton's tyrosine kinase (BTK) inhibitor, and a phosphatidylinositol 3-kinase (PI3K) inhibitor.

13. The pharmaceutical composition of claim 12, wherein the previous treatment is (a) First-line treatment with ibrutinib; (b) first-line treatment with BDR (bortezomib, dexamethasone, and rituximab) and second-line treatment with ibrutinib; or (c) First-line treatment with the RCD (cyclophosphamide, dexamethasone, and rituximab) regimen and second-line treatment with ibrutinib.

14. The pharmaceutical composition of any one of claims 1-13, wherein the treatment comprises administering the antibody or antigen-binding portion thereof at a dose ranging from 0.1-20 mg / kg body weight.

15. The pharmaceutical composition of any one of claims 1-14, wherein the treatment comprises a dose of the antibody, or antigen-binding portion thereof, of 0.1, 0.3, 0.5, 1, 3, 5, 10, or 20 mg / kg body weight administered on a weekly, biweekly, triweekly, or monthly dosing schedule.

16. The pharmaceutical composition of claim 15, wherein the dosage is 3 mg / kg body weight or 10 mg / kg body weight.

17. The pharmaceutical composition of any one of claims 1-13, wherein the treatment comprises administering the antibody, or antigen-binding portion thereof, as a fixed dose.

18. The pharmaceutical composition of claim 17, wherein the fixed dose is 350, 750 or 1500 mg.

19. The pharmaceutical composition of claim 14 or 18, wherein the treatment comprises administering the antibody, or antigen-binding portion thereof, on a dosing schedule of once per week, once every two weeks, or once every three weeks.

20. The pharmaceutical composition of claim 14 or 18, wherein the dosing schedule is once every 14 days.

21. The pharmaceutical composition of any one of claims 1-20, wherein the treatment comprises administering the antibody or antigen-binding portion thereof intravenously or subcutaneously.

22. The pharmaceutical composition of any one of claims 1 to 21, wherein the treatment comprises: (a) selecting a subject who is a suitable candidate for treatment, said selection comprising: (i) evaluating whether cells in a test tissue sample obtained from a Waldenstrom's macroglobulinemia tissue carry a C1013G mutation in the CXCR4 gene; and (ii) selecting the subject as a suitable candidate based on determining that the WM cells carry the C1013G mutation in the CXCR4 gene; and (b) administering to the selected subject a therapeutically effective amount of an antibody or antigen-binding portion thereof that specifically binds to a CXCR4 receptor expressed on the surface of C1013G / CXCR4-related WM cells.

23. The pharmaceutical composition of any one of claims 1-21, wherein the subject is selected based on a determination that the WM cells in the subject carry the C1013G mutation in the CXCR4 gene.

24. The pharmaceutical composition of claim 22 or 23, wherein the C1013G mutation is detected by polymerase chain reaction (PCR) assay, reverse transcriptase-polymerase chain reaction (RT-PCR) assay, nucleic acid hybridization assay or by DNA sequencing.

25. The pharmaceutical composition of claim 24, wherein: (a) the PCR assay is a real-time allele-specific PCR (AS-PCR) assay; and / or (b) The DNA sequencing is whole genome DNA sequencing.

26. A method (other than medical practice) for evaluating whether Waldenstrom's macroglobulinemia may be suitable for treatment with an antibody or an antigen-binding portion thereof that specifically binds to a CXCR4 receptor expressed on the surface of a Waldenstrom's macroglobulinemia cell, the method comprising evaluating whether cells in a test tissue sample obtained from the Waldenstrom's macroglobulinemia carry a C1013G mutation in the CXCR4 gene, wherein determining that the Waldenstrom's macroglobulinemia cells carry the C1013G mutation in the CXCR4 gene indicates that the Waldenstrom's macroglobulinemia may be suitable for treatment.

27. The method of claim 26, wherein the C1013G mutation is detected by a polymerase chain reaction (PCR) assay, a reverse transcriptase-polymerase chain reaction (RT-PCR) assay, a nucleic acid hybridization assay, or by DNA sequencing.

28. The method of claim 27, wherein: (a) the PCR assay is a real-time allele-specific PCR (AS-PCR) assay; and / or (b) The DNA sequencing is whole genome DNA sequencing.

29. The pharmaceutical composition of any one of claims 1-25, wherein the antibody or portion thereof comprises CDR1, CDR2, and CDR3 domains in a heavy chain variable region having the amino acid sequence shown in SEQ ID NO: 25, and CDR1, CDR2, and CDR3 domains in a light chain variable region having the amino acid sequence shown in SEQ ID NO:

29.

30. The pharmaceutical composition of any one of claims 1-25, wherein the antibody or portion thereof comprises: A heavy chain variable region comprising consecutively linked amino acids having the sequence described in SEQ ID NO:25, and a light chain variable region comprising consecutively linked amino acids having the sequence described in SEQ ID NO:

29.

31. The pharmaceutical composition of any one of claims 1-25, wherein the anti-CXCR4 antibody or portion thereof comprises: A heavy chain variable region CDR1 comprising consecutively linked amino acids having the sequence described in SEQ ID NO: 1, a heavy chain variable region CDR2 comprising consecutively linked amino acids having the sequence described in SEQ ID NO: 5, a heavy chain variable region CDR3 comprising consecutively linked amino acids having the sequence described in SEQ ID NO: 9, a light chain variable region CDR1 comprising consecutively linked amino acids having the sequence described in SEQ ID NO: 13, a light chain variable region CDR2 comprising consecutively linked amino acids having the sequence described in SEQ ID NO: 17, and a light chain variable region CDR3 comprising consecutively linked amino acids having the sequence described in SEQ ID NO:

21.

32. The pharmaceutical composition of any one of claims 1-25, wherein the antibody or portion thereof is ulocuplumab or portion thereof.

33. The method of any one of claims 26-28, wherein the antibody or portion thereof comprises CDR1, CDR2, and CDR3 domains in a heavy chain variable region having the amino acid sequence shown in SEQ ID NO:25, and CDR1, CDR2, and CDR3 domains in a light chain variable region having the amino acid sequence shown in SEQ ID NO:

29.

34. The method of any one of claims 26-28, wherein the antibody or portion thereof comprises: A heavy chain variable region comprising consecutively linked amino acids having the sequence described in SEQ ID NO:25, and a light chain variable region comprising consecutively linked amino acids having the sequence described in SEQ ID NO:

29.

35. The method of any one of claims 26-28, wherein the anti-CXCR4 antibody or portion thereof comprises: A heavy chain variable region CDR1 comprising consecutively linked amino acids having the sequence described in SEQ ID NO: 1, a heavy chain variable region CDR2 comprising consecutively linked amino acids having the sequence described in SEQ ID NO: 5, a heavy chain variable region CDR3 comprising consecutively linked amino acids having the sequence described in SEQ ID NO: 9, a light chain variable region CDR1 comprising consecutively linked amino acids having the sequence described in SEQ ID NO: 13, a light chain variable region CDR2 comprising consecutively linked amino acids having the sequence described in SEQ ID NO: 17, and a light chain variable region CDR3 comprising consecutively linked amino acids having the sequence described in SEQ ID NO:

21.

36. The method of any one of claims 26-28, wherein the antibody or portion thereof is ulocuplumab or portion thereof.

37. A kit for treating C1013G / CXCR4-associated Waldenstrom's macroglobulinemia in a subject, the kit comprising: (a) one or more doses of an anti-CXCR4 antibody, or antigen-binding portion thereof; and (b) instructions for using the anti-CXCR4 antibody in any one of claims 1-15 and 29-32.

38. The kit of claim 37, wherein the anti-CXCR4 antibody, or antigen-binding portion thereof, comprises: CDR1, CDR2, and CDR3 domains in a heavy chain variable region having the amino acid sequence shown in SEQ ID NO: 25, and CDR1, CDR2, and CDR3 domains in a light chain variable region having the amino acid sequence shown in SEQ ID NO:

29.

39. The kit of claim 37, wherein the anti-CXCR4 antibody, or antigen binding portion thereof, is ulocuplumab or a portion thereof.

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