Use of an anti-CCR7 antibody in combination therapies with a BTK inhibitor and/or BCL2- inhibitor for treating hematological malignancies

Abstract

The present invention provides a novel use and methods comprising antibodies, or antigen-binding fragments thereof, which bind to a CCR7 receptor for use as a novel combination therapy with a BTK inhibitor and/or a Bcl-2 inhibitor in treatment of hyperproliferative blood malignancies, preferably in B-cell lymphomas, such as CLL. The combination can be used as first line, or in naïve patients not treated before with a BTK inhibitor and/or Bcl-2 inhibitor, or in patients with a BTK-inhibitor and/or Bcl-2-inhibitor refractory/relapsed disease. The antibodies and antigen-binding fragments are capable of selectively depleting ex vivo or in vitro malignant cells expressing CCR7 and are capable of impairing/blocking migration of said tumor cells towards CCR7 ligands. These effects are not related to previous or contemporary treatments with a BTK inhibitor and/or a Bcl-2 inhibitor. Similarly, the efficacy of the antibodies is not affected in patients that have relapsed/refractory disease. The use of said antibodies as a monotherapy or as a combination with a BTK inhibitor and/or a Bcl-2 inhibitor for depleting, killing and impairing/blocking migration and activation of tumor cells expressing CCR7 cells is disclosed, thus providing an alternative therapy treating hyperproliferative blood cancers.

Claims

1.-15. (canceled)

16. A method of treating a hyperproliferative hematological disorder, the method comprising administering an anti-CCR7 to a subject in need thereof, wherein the hyperproliferative hematological disorder is at least one of: a) a disorder that is treated with at least one of a Bruton's tyrosine kinase (BTK) inhibitor and a B-cell lymphoma 2 (Bcl-2) inhibitor; b) a disorder that has relapsed after treatment with at least one of a BTK inhibitor and a Bcl-2 inhibitor; and, c) a disorder that is refractory to treatment with at least one of a BTK inhibitor and a Bcl-2 inhibitor.

17. The method of claim 16, wherein the antibody is administered simultaneously, separately or sequentially with at least one of a BTK inhibitor and a Bcl-2 inhibitor.

18. The method of claim 16, wherein the hyperproliferative hematological disorder is a disorder wherein the hyperproliferating cells are cells of the B cell lineage.

19. The method of claim 18, wherein the hematological malignancy is selected from the group consisting of acute lymphocytic leukemia (ALL), acute myeloid leukemia (AML), acute monocytic leukemia (AMoL), chronic lymphocytic leukemia (CLL), high-risk CLL, small lymphocytic lymphoma (SLL), high-risk SLL, multiple myeloma (MM), non-Hodgkin's lymphoma (NHL), mantle cell lymphoma (MCL), follicular lymphoma (FL), Waldenstrom's macroglobulinemia (WM), diffuse large B-cell lymphoma (DLBCL), marginal zone lymphoma (MZL), Burkitt's lymphoma (BL), hairy cell leukemia (HCL), Richter's transformation and T-cell prolymphocytic leukemia (T-PLL).

20. The method of claim 16, wherein the anti-CCR7 antibody has an IC.sub.50 of no more than 100 nM for inhibiting at least one of CCR7-dependent intracellular signalling and CCR7 receptor internalization, by at least one CCR7-ligand selected from CCL19 and CCL21.

21. The method of claim 20, wherein the anti-CCR7 antibody inhibits CCR7-dependent intracellular signalling without substantial agonistic effects.

22. The method of claim 16, wherein the anti-CCR7 antibody has a K.sub.d for the N-terminal extracellular domain of human CCR7 that is not more than a factor 20 higher than the K.sub.d of a reference anti-CCR7 antibody, whereby the reference anti-CCR7 antibody is a mouse anti-CCR7 antibody of which the amino acid sequence of the heavy chain variable domain is SEQ ID NO: 1 and of which the amino acid sequence of the light chain variable domain is SEQ ID NO: 2.

23. The method of claim 16, wherein the anti-CCR7 antibody is a chimeric, humanized or human antibody.

24. The method of claim 23, wherein the anti-CCR7 antibody is an antibody having the HVRs of the anti-human CCR7 antibody of which the amino acid sequence of the heavy chain variable domain is SEQ ID NO: 1 and of which the amino acid sequence of the light chain variable domain is SEQ ID NO: 2.

25. The method of claim 16, wherein the BTK inhibitor is ibrutinib, zanabrutinib or acalabrutinib, and wherein the Bc1-2 inhibitor is venetoclax or navitoclax.

26. The method of claim 16, wherein the hyperproliferative hematological disorder is a disorder in a treatment-naïve patient.

27. The method of claim 16, wherein the hyperproliferative hematological disorder is a disorder in a patient who is naïve to the treatment with at least one of a BTK inhibitor, a Bc1-2 inhibitor and an anti-CCR7 antibody.

28. The method of claim 27, wherein the hyperproliferative hematological disorder is refractory to and/or has relapsed after treatment with a chemotherapeutic agent other than a BTK inhibitor, a Bcl-2 inhibitor and an anti-CCR7 antibody.

29. The method of claim 16, wherein the hyperproliferative hematological disorder is refractory to and/or has relapsed after treatment with at least one of a BTK inhibitor and a Bcl-2 inhibitor and a chemotherapeutic agent other than a BTK inhibitor, a Bcl-2 inhibitor and an anti-CCR7 antibody.

30. The method of claim 28, wherein the chemotherapeutic agent is one or more of fludarabine, cyclophosphamide, idelalisib, an anti-CD20 antibody.

31. The method of claim 30, wherein the anti-CD20 antibody is rituximab, obinituzumab, ocrelizumab, veltuzumab or ofatumumab, or an anti-CD52 antibody, wherein preferably the anti-CD52 antibody is alemtuzumab.

Description

DESCRIPTION OF THE FIGURES

[0364] FIG. 1. CCR7 expression is maintained in patients treated with BTK inhibitors. CCR7 expression is slightly diminished (ibrutinib) or unchanged (acalabrutinib or zanabrutinib) in CLL patients treated with BTK inhibitors. Expression of CCR7 and CD20 was determined in primary CLL cells from peripheral blood samples obtained from naïve patients (n=125), patients receiving ibrutinib treatment (OT, n=44), patients with relapsed/refractory disease to ibrutinib (RR, n=16), patients receiving acalabrutinib treatments (ACALA, n=5), or patients receiving zanabrutinib treatment (ZANA, n=4). A) Expression of surface CCR7 (or CD20) was analyzed in terms of relative median intensity of fluorescence (RMIF, relative to an irrelevant isotype control). B) Expression of surface CCR7 (or CD20) was analyzed in terms of proportion of malignant cells expressing the receptor.

[0365] FIG. 2. Expression of CCR7 in malignant cells is high irrespective of current or previous treatments.

[0366] A) The graphs show CCR7 expression from one representative naïve (untreated) CLL patient, one representative CLL patient receiving ibrutinib (OT), one representative CLL patient who failed to ibrutinib (RR), and one representative CLL patient receiving venetoclax. In each patient, CCR7 expression is disclosed as frequency histograms compared to a matched irrelevant control. Marker regions were placed on base to these irrelevant controls. Each histogram shows the intensity of fluorescence for cells labelled with anti-CCR7-PE. The proportion of CCR7-positive cells (within the marker) is also shown. B) Changes in the expression of CCR7 and CD20 in four CLL patients that started treatment with the BTK inhibitor ibrutinib. For each patient, the expression of CCR7 (determined as RMIF) before (N) and after administration (Y) of ibrutinib is shown.

[0367] FIG. 3. CCR7 expression is maintained in patients treated with Bcl-2 inhibitors.

[0368] CCR7 expression is maintained in essentially all CLL cells from patients treated with venetoclax. Expression of CCR7 and CD20 was determined in CLL cells from peripheral blood samples obtained from naïve patients (n=125) or patients receiving venetoclax treatment (OT, n=11). A) Expression of surface CCR7 (or CD20) was analyzed in terms of relative median intensity of fluorescence (RMIF, relative to an irrelevant isotype control). B) Expression of surface CCR7 and CD20 was analyzed in terms of proportion of malignant cells expressing the receptor.

[0369] FIG. 4. Treatment (in vivo) with ibrutinib does not neutralize migration mediated by CCR7 in CLL cells.

[0370] A) Comparative analysis of migration indices (% of input) in CLL cells obtained from naïve untreated patients (N, n=7, black bars) versus cells obtained from CLL patients receiving ibrutinib (OT, n=10, grey bars). Migration was achieved with exposure of CLL cells to CCR7 ligands, CCL19 (1 μg/ml) or CCL21 (1 μg/ml). Migration was tested in chemotaxis assays where cells were placed on nude transwell chambers. Assays were conducted for 4 hours at 37° C. Spontaneous migration, not mediated by a chemotactic stimulus was considered as basal migration (in this point, no chemokine was added). B) Comparative analysis of migration indices (% of input) in CLL cells from naïve untreated patients (n=7) that were exposed for one hour to different concentrations of ibrutinib at final concentrations (0-vehilce/DMSO; 0.01, 0.1, 1, 10 μM) prior to exposure to CCR7 ligands CCL19 (1 μg/ml) or CCL21 (1 μg/ml). Migration was tested in chemotaxis assays where cells were placed on nude transwell chambers. Assays were conducted for 4 hours at 37° C. (Ibrutinib was present during all the migration time). Spontaneous migration, not mediated by a chemotactic stimulus, was considered as basal migration (in this point, no chemokine was added). As positive controls, cells without ibrutinib exposure where used (black bars) In A and B, bars represent mean±standard error of the mean (SEM). ns, not significant; *, p<0.05, **, p<0.01.

[0371] FIG. 5: Treatment (in vitro) with ibrutinib at 0.1 μM for 24 h does not neutralize migration mediated by CCR7 in CLL cells.

[0372] Comparative analysis of migration indices (% of input) in CLL cells obtained from naïve untreated patients (n=6) that where incubated for 24 h with ibrutinib at a final concentration of 0 (vehicle/DMSO) or 0.1 μM prior to exposure to CCR7 ligands CCL19 (1 μg/ml) or CCL21 (1 μg/ml). Migration was tested in chemotaxis assays where cells were placed on nude transwell chambers. Migration assays were conducted for 4 hours at 37° C. Spontaneous migration, not mediated by a chemotactic stimulus, was considered as basal migration (in this point, no chemokine was added). As positive controls, cells without ibrutinib exposure where used (black bars). Bars represent mean±standard error of the mean (SEM). ns, not significant; *, p<0.05.

[0373] FIG. 6. Treatment with an anti-CCR7 antibody CAP-100 neutralizes CCR7-mediated migration of CLL cells from patients treated with BTK inhibitors or Bcl-2 inhibitors.

[0374] The specific blocking of CCR7-ligand interactions, expressed as a reduction of % of migrating input cells is shown for CLL cells obtained from a patient receiving venetoclax (A) or for CLL cells obtained from a patient receiving ibrutinib (B). CLL cells were incubated with an anti-CCR7 antibody (CAP-100) at different final concentrations (100, 10, 1, 0.1, 0.01, 0 μg/ml). Subsequently, cells were placed on transwell chambers and exposed to the chemokines CCL19 (white bars) and CCL21 (grey bars) at 1 μg/ml. Migration assays on naked transwell chambers were conducted for 4 hours at 37° C. Basal migration is the spontaneous migration, not mediated by a chemotactic stimulus—in this point no chemokine or mAb is added. Maximum migration is observed when chemokines are added but no CAP-100 is present (point 0).

[0375] FIG. 7. Effect of the combination of ibrutinib with anti-CCR7 antibody CAP-100 on the migration induced by CCR7 ligands.

[0376] Comparative analysis of migration indices (% of input) in CLL cells obtained from naïve untreated patients (n=5) that were incubated with anti-CCR7 antibody (CAP-100; 10 μg/ml), with ibrutinib (0.1 μM), or with a combination of both compounds (CAP-100 at 10 μg/ml; ibrutinib at 0.1 μM) before performing chemotaxis assays towards CCR7 ligands [CCL19 (1 μg/ml) or CCL21 (1 μg/ml); in nude transwell chambers]. Spontaneous migration, not mediated by a chemotactic stimulus was considered as basal migration (in this point, no chemokine was added; white bar). As positive controls, cells without anti-CCR7 antibody or ibrutinib pre-incubation where used (black bars). Bars represent mean±standard error of the mean (SEM). ns, not significant; *, p<0.05.

[0377] FIG. 8. CCR7 expression in CLL cells from patients under treatment with BTK or Bcl-2 inhibitors can effectively induce cell death upon binding of an anti-CCR7 antibody.

[0378] ADCC activity was assayed in one patient receiving treatment with ibrutinib (A), in one patient receiving treatment with zanabrutinib (B), in one patient receiving venetoclax (C), and in one patient with relapsed/refractory disease to ibrutinib (D). In all cases, CLL cells were incubated in the presence of an irrelevant matched isotype control, or with anti-CD20 antibody (rituximab, RTX), or with anti-CCR7 antibody (CAP-100). Antibodies were tested at different final concentrations (0, 0.01, 0.1, 1, 10, 100 μg/ml). To perform ADCC, isolated PBMCs (peripheral blood mononuclear cells) were used as effector cells at a fixed E:T ratio of 10:1. The percentage of CLL cells killed by ADCC was determined by flow cytometry based on the incorporation of 7-aminoactinomycinD (7-AAD). The proportion of specific lysis induced by the antibodies is shown.

EXAMPLES

Introduction

[0379] The chemokine receptor CCR7 controls migration of certain immune cell subsets to the lymph nodes, where, in addition it contributes to immune cells organization and activation (Legler et al., Int J Biochem Cell Biol. 2014; 54:78-82). In agreement with a lymphoid origin, several blood cancers with lymph node involvement express CCR7 (LOpez-Giral et al., J Leukoc Biol. 2004; 76(2):462-71). In this diseases CCR7 expression correlates with bulky lymphadenopathy, aggressive disease, and short survival (Legler et al., 2014; supra). Specifically, in B-cell malignancies, CCR7 expression contributes to extend tumor cell residency in the LN as well as to guide tumor cells towards niches were pro-tumor cues can be achieved (Rehm et al., Blood, 2011; 118(4):1020-33).

[0380] Recently, some reports disclosed that ibrutinib treatment of CLL cells resulted in a down-regulation in surface CCR7 which, in addition, translated to a subsequent impairment in CCR7-mediated migration and CCR7-mediated adhesion (Patrussi et al., 2015; supra 75(19):4153-63; de Rooij et al., 2012; supra). As result of these works, it was proposed that one off-target effect mediated by ibrutinib was to decrease lymph node migration, partially caused by a restoration of increased surface CCR7 in CLL cells to levels normally expressed in B-cells. Similarly, a positive feedback on expression profiles has been reported for CCR7 and Bcl-2 (Kim et al., 2005; supra).

[0381] On the basis of the described findings, a combination of a BTK inhibitor, and/or a Bcl-2 inhibitor with an anti-CCR7 antibody would not be expected to improve the therapeutic inhibition of migration of malignant cells to the lymph nodes (or other SLOs). Moreover, based on the previously reported loss of CCR7 expression, a therapeutic approach aiming to target CCR7 to kill tumor cells would be precluded and would not be expected to improve individual therapeutic efficacy of BTK or Bcl-2 inhibitors.

[0382] Herein the inventors document the impact of BTK or Bcl-2 inhibitors on CCR7 expression in CLL patients and perform several approaches to investigate whether these compounds could negatively impact on CCR7-triggered functions (such as migration) as well as whether these compounds could prevent an effective target cell killing mediated by anti-CCR7 antibodies as a result of a lack of CCR7 expression on target cell surface.

Materials and methods

Samples

[0383] Leukemic cells from patients were isolated from the freshly donated peripheral blood using ficoll-paque plus density gradient centrifugation (Amersham Biosciences). Isolated cells were maintained for short-term cultures in RPMI 1640 media supplemented with 10% heat-inactivated fetal bovine serum (FBS), 2 mM L-glutamine and 100 U/mL penicillin/100 μg/mL streptomycin at 37° C. in 5% CO2. Normal peripheral blood mononuclear cells (PBMCs) were obtained from adult blood buffy coats after ficoll gradient and maintained in complete medium as described above. In all cases, patients and healthy donor signed an informed consent in accordance to the Declaration of Helsinki. Experimental procedures were approved by the Institutional review Board of the Hospital de la Princesa.

[0384] Three types of patient samples (n=196) were included in this study: [0385] 1) Naïve patients (i.e., patients not treated before with either a BTK inhibitor or a Bcl-2 inhibitor). [0386] 2) On ibrutinib (or other BTK inhibitor) treatment patients. [0387] 3) Ibrutinib R/R patients.

CCR7 Expression

[0388] Flow cytometry analysis of CCR7 and CD20 expression in CLL cells was performed with a four-colour panel of monoclonal antibodies: CD19-APC-H7 (BD Biosciences), CD3-FITC (BD Biosciences), CD5-APC (BD Biosciences) and either CCR7-PE (R&D Systems) or CD20-PE (R&D

[0389] Systems). Studies were conducted on the CD19.sup.+ CD3.sup.-CD5.sup.+ CLL population. An appropriate matched isotype control (IC) conjugated to PE was included (R&D Systems). To determine receptor expression, 10.sup.6 cells (in ˜50 μl of PB) were incubated with the antibodies for 15-20 minutes at room temperature (RT). Cells were lysed with BD FACS™ Lysing Solution (BD Biosciences) for 10 minutes at RT and centrifugated at 1.800 rpm for 2 minutes. Then, cells were washed with 2m1 of Dulbecco's Phosphate Buffered Saline (PBS) (Lonza) and, finally centrifugated at 1.800 rpm for 2 minutes. Data acquisition was performed on a BD FACSCanto™ 11 Flow Cytometer (BD Biosciences). A minimum of 10.000 CLL cells were analysed using the BD FACSDIVA™ Software. Results are expressed both as a percentage of CCR7 and CD20 positive cells [% receptor−% control] and relative median fluorescence intensity (RMFI) of CCR7 and CD20 expression compared to the IC [MIF(receptor)/MIF(control)].

Chemotaxis Assay (Migration)

[0390] PBMCs were isolated by centrifugation on Biocoll Separating Solution density gradient centrifugation (Merck Millipore). PBMC were washed twice with saline solution (Fresenius) and centrifugated at 1200 for 10 minutes. Cells were suspended in RPMI 1640 media (GIBCO) supplemented with 0.1% bovine serum albumin (BSA) at a concentration of 5×10.sup.6 cells/ml. When indicated, and prior to the chemotaxis assay, cells were pre-incubated with the followings reagents: a) Ibrutinib, 1 hour at RT; at different final concentrations; b) Ibrutinib, 24 hours at 37° C.; at different final concentrations; b) anti-CCR7 mAb, 0.5 h at RT. Chemotaxis was performed in Transwell chambers (6.5-mm diameter, 10-μm thickens, 5-μm diameter pore size, Costar). Only samples from patients with >90% of tumour cells were included. A total of 5×10.sup.5 cells suspended in RPMI-1640, 0.1% BSA were loaded in the upper chamber and chemokines CCL19 or CCL21 (1 μg/ml, Peprotech) were added to the lower well. After 4 hours at 37° C. 5% CO, cells in the lower chamber were collected, and stained with anti-CD3-PE (BD Biosciences) and anti-CD5-APC (BD Biosciencies). The migrated cells were counted for 60 seconds in a BD FACSCanto™ II flow cytometer. The percentage of migrated cells (% of input) was calculated according to the following formula: 100×(number of cells in the lower chamber/number of cells loaded in the upper chamber). Once the % of input was calculated, the % of inhibition was estimated by means the following formula: % of inhibition=[(% input without mAb−% input with mAb)×100]/[% input without mAb]. In addition, results are shown as the % of input relative to the maximum effect mediated by each chemokines (CK) which was calculated according to: %input rel. to CK=100×(% input/% input with CK).

Antibody-Dependent Cell-Mediated Cytotoxicity (ADCC)

[0391] ADCC assays were performed as described in SR-HPM-1026. Briefly: target tumour cells were incubated with media alone (RPMI+10% FBS) or in the presence of IC, rituximab, alemtuzumab, or CAP-100 antibodies, at different final concentrations, at 37° C. for 30 min. Unbound antibody was washed off (1800 rpm, 2 minutes/twice) and the cells plated at 10.sup.5 cells/well in p96 U-bottom plates. Human PBMCs from healthy donors were obtained by ficoll density gradient centrifugation. Effector cells were labelled with calcein-UV Cell Tracker (Invitrogen) according to the manufacturer's protocol, and stimulated with recombinant human IL-2 (500 UI/ml, StemCell Technologies). Different effector:target (E:T) ratios were used [10:1 for dose-response assays, and (5:1, 25:1, 50:1) for E:T assays]. After 6 hours, cells were stained with 7AAD, CD3-PE and CD5-APC and analysed by flow cytometry. The percentage of specific lysis in Cell-TrackerCD5.sup.+CD3.sup.− CLL cells was determined by incorporation of 7-AAD (BD Pharmingen) and calculated according to the following formula: % Specific Lysis=100×(ER−SR)/(MR−SR). ER, SR, and MR represent experimental, spontaneous and maximum cell death.

Statistical Analysis

[0392] For statistics, the following steps were followed: [0393] 1) Homogeneity was tested with KS and/or Shapiro-Wilk and/or D'Agostino & Pearson normality tests. [0394] 2) Bartlett test was used to check homogeneity of variance. [0395] 3) For parametric variables, ANOVA (one-way analysis of variance, followed by Dunett's multiple comparison test) or two-sample t-test were used to compare group means. [0396] 4) For non-parametric variables, Kruskal-Wallis (followed by Dunns multiple comparison test) and two-sample Mann-Whitney U tests were used to compare group medians. When paired samples were analysed, then the Wilcoxon or Friedman tests were used. [0397] 5) All data were analysed with Graph-Pad Prism 5 (GraphPad Software, San Diego, Calif.). All tests were two-sided. P<0.05 was considered to be statistically significant. Except otherwise stated, the mean and standard error of the mean (SEM) are provided for each group.

[0398] For the calculation of EC50 values, GraphPad Prism 5.0 (GraphPad Software, Inc.) was used. To this end, global non-linear regression, dose-response stimulation equation with a robust fit mode (standard slope) was selected.

Results

[0399] Surface expression of CCR7 is maintained in CLL patients treated with BTK or Bcl-2 inhibitors.

[0400] As previous works reported a substantial loss of surface CCR7 in CLL cells treated with ibrutinib, the first aim of the present study was to validate these results in a larger cohort of CLL patients. For this reason we measured surface CCR7 in 125 samples obtained from naïve patients (never treated before with either a BTK inhibitor or a Bcl-2 inhibitor), in 44 samples obtained from patients on ibrutinib treatment at the time of the determination, and in 16 samples obtained from patients who left ibrutinib treatment because they had developed relapsed/refractory disease. Strikingly, we observed that CCR7 expression was maintained during treatment with BTK inhibitors. As seen in FIG. 1-A-B and FIG. 2-A, we observed that patients receiving ibrutinib as current treatment, continued to show marked surface CCR7 expression. In general, a slight but significant down-modulation was observed in patients treated with ibrutinib, nonetheless approximately 100% of CLL cells maintained surface expression (as determined as percentage of CCR7-expressing cells), and also keep high surface levels (as determined as RMIF). Indeed, surface levels in treated patients were still over 200 arbitrary units. In other words, expression was still 200 times higher than a negative irrelevant isotype control. In patients on treatment with other BTK inhibitors such as acalabrutinib or zanabrutinib, no changes in CCR7 cells surface expression were observed. Finally, in the case of ibrutinib R/R CLL cells, CCR7 surface levels were similar to (or even higher than) those of naïve patients (FIG. 1-A-B and FIG. 2-A).

[0401] Interestingly, in the case of CD20, a receptor known to be down-modulated or lost by ibrutinib treatment, we confirmed that BTK-inhibitors led to a substantial down-regulation of CD20 surface levels. For example, treatment led to a complete loss of CD20 in some patients as determined by the reduction in the proportion of CD20-positive cells compared to naïve patients (FIG. 1-A-B). Accordingly, in FIG. 2-B is shown how ibrutinib treatment led to a consistent down-regulation of CD20 surface levels in four (4/4) patients as compared to the time they started ibrutinib treatment (Y), while in the case of CCR7 the overall picture remained essentially constant, with a strong reduction in one patient (1/4), a mild reduction in another patient (1/4), but increases in two other patients (2/4). Finally, and strikingly different from CCR7, CD20 expression in ibrutinib R/R CLL cells was even lower than in patients receiving ibrutinib (FIG. 1A and 2B).

Surface Expression of CCR7 is Maintained in CLL Patients Ttreated with Bcl-2 Inhibitors.

[0402] In CLL cells obtained from patients under treatment with venetoclax, we observed that approximately 100% of CLL cells maintained surface expression (as determined as percentage of CCR7-expressing cells), and also keep high surface levels (as determined as RMIF). As with ibrutinib, surface levels in venetoclax-treated patients were still about 150 - 250 times higher than a negative irrelevant isotype control (FIG. 2-A and FIG. 3-A-B.

Treatment with BTK Inhibitors Does Not Neutralize Migration Mediated by CCR7

[0403] Once it was determined that BTK inhibitors did not induce a meaningful loss of CCR7 on CLL cells surface we aimed to confirm that these compounds could have a negative impact on functions mediated by CCR7 through additional mechanism that could impair migration induced by CCR7 ligands. To this end, we performed a comparative analysis of the in vitro chemotactic response of CLL cells obtained from naïve patients (N) versus patients treated with ibrutinib (OT) (FIG. 4-A). In both groups of patients, basal migration indices were similar and, in both groups, CLL cells migrated in a significant way towards CCR7 ligands. Although a slight reduction was observed in the migration indices of the OT group, these values did not significantly differ from the N group, indicating that BTK-inhibitors had only a marginal effect on CCR7 induced migration. Moreover, migration indices in the OT group did not reach basal levels indicating that BTK inhibitor treatment does not completely neutralize migration induced by CCR7.

[0404] To further confirm this, we performed in vitro chemotaxis assays in transwell chambers. In these settings, CLL cells from naïve patients were incubated for one hour with increasing concentrations of ibrutinib (0; 0.01; 0.1; 1; 10 μM) before exposure to CCR7 ligands (black bars). As seen in FIG. 4-B, migration indices with ibrutinib pretreatment were comparable to control migration induced by CCR7 ligands. Only when administrated at 0.1 μM, ibrutinib showed a moderate (though not significant) effect on migration towards CCL21.

[0405] As the lack of ibrutinib inhibition could be associated to the short incubation times used (1 h prior to the chemotaxis+4 additional h during the chemotaxis assay) we decided to conduct new experiments where naïve CLL cells were incubated with ibrutinib at 0.1 μM for 24 h before testing chemotaxis. Again, as depicted in FIG. 5, no effect of ibrutinib was seen in CCR7-mediated migration of CLL cells towards CCL19, and a slight, not significant effect was observed in migration indices towards CCL21.

[0406] All together, these results indicated that CLL cells migration induced by CCR7 was not significantly affected by previous in vivo or in vitro treatments with ibrutinib. In other words, effects mediated by BTK inhibitors proved to be insufficient to abrogate CCR7-mediated migration, and agents blocking CCR7 are necessary to achieve a complete neutralization of CCR7-mediated migration to LNs. Nonetheless, the utility of anti-CCR7 as a blocking agent in patients receiving ibrutinib remained unaddressed. Moreover, as seen in FIG. 1-A, it was likely that the slight down-modulation of CCR7 surface levels could have a negative impact on the activity of CAP-100 in this group of patients on ibrutinib treatment. For these reasons we tested the neutralizing activity of CAP-100 in CLL cells obtained from 10 CLL patients treated with ibrutinib. As seen in FIG. 6, CAP-100 displayed a clear dose-response inhibitory activity on CLL cells obtained from one patient receiving venetoclax treatment (FIG. 6-A) or from one patient who failed to ibrutinib treatment (FIG. 6-B). In both cases, migration indices towards CCL19 or CCL21 reached basal levels after treatment with 10 or 100 μg/ml of antibody. These results confirmed that CLL cells from these groups of patients still respond to CCR7 ligands and that anti-CCR7 therapy probably is the best way to impair migration induced by CCR7.

[0407] We further tested whether combination of ibrutinib with an anti-CCR7 antibody such as CAP-100 could have an additive or synergistic neutralizing effect on the migration of CLL cells induced by CCR7 ligands. To this end, CLL cells from naïve patients were incubated with ibrutinib as single agent (0.1 μM), with CAP-100 as single agent (10 μg/ml), and with a combination of both compounds before exposure to CCL19 or CCL21. Selection of compound concentrations was based on previous findings on ibrutinib (FIG. 4B) and on CAP-100 (FIG. 6) as inhibitors of CCR7-mediated migration. As seen in FIG. 7, ibrutinib as a monotherapy had no effect on migration induced by CCR7, thus confirming results shown on FIG. 4B and FIG. 5. As expected, reduction of migration to basal levels was achieved with treatment with CAP-100, thus confirming that CAP-100 is a potent agent for inhibition of migration triggered by CCR7 ligands. When CCL21 was used as ligand, the combination of ibrutinib with CAP-100 showed a moderate, though not significant increase in the neutralizing activity obtained by anti-CCR7 antibody as single agent thus suggesting that a potential synergistic effect can be achieved by combining both compounds (FIG. 7). Although further validation is needed, this effect seemed to be specific for CCL21 as no similar outcome was observed when CLL cells treated with such a combination were exposed to CCL19.

CCR7 Expression in CLL Cells from Patients Under Treatment with BTK Or Bcl-2 Inhibitors can Effectively Induce Cell Death Upon Binding of an Anti-CCR7 Antibody.

[0408] In therapies based on antibodies, high target surface levels are mandatory to achieve an effective killing activity mediated by effector immune mechanisms such as ADCC, ADCP or CDC.

[0409] Indeed, proximity of two antibodies bound to target are needed to successfully complete any of the cited mechanisms [12]. Our results on expression demonstrated that surface CCR7 levels were slightly reduced after treatments with BTK inhibitors though no reduction on the proportion of tumour cells expressing the receptor was observed. For this reason, we determined whether the reduction in RMIF observed in patients treated with BTK inhibitors could have a negative impact on killing activities mediated by anti-CCR7 antibodies. To this end, we performed a set of ADCC assays, wherein target CLL cells from patients on ibrutinib treatment (FIG. 8A), on zanabrutinib treatment (FIG. 8B), on venetoclax treatment (FIG. 8C), or from ibrutinib R/R patients (FIG. 8D) were incubated with increasing concentrations of CAP-100, rituximab (used as reference therapeutic antibody), or an IC, and then, cells were incubated along with effector immune cells obtained from healthy donors at a fixed effector:target ratio of 10:1.

[0410] As seen in FIG. 8, CAP-100 demonstrated a potent ADCC activity in all four groups of patients. Remarkably, CAP-100 outperformed rituximab. These results confirm that, in the current settings, the slight reduction on surface CCR7 levels observed in patients treated with BTK or Bcl-2 inhibitors had no negative impact on immune effector mechanism mediated by antibodies such as ADCC. These results further confirm the utility of combinations based on BTK-inhibitors or Bcl-2 inhibitors with anti-CCR7 antibodies such as CAP-100.